Advancing Chemical Risk Evaluations
Through Use of New Approach Methods(NAMs): Challenges and Opportunities

20-21 June 2022 | Yokohama, Japan1

Introduction

The ICCA LRI & NITE workshop “Advancing Chemical Risk Evaluations Through Use of New Approach
Methods (NAMs): Challenges and Opportunities” was held June 20-21, 2022, in Yokohama, Japan. The
workshop was co-organized with the Japanese National Institute of Technology and Evaluation (NITE)
and hosted by the Japan Chemical Industry Association (JCIA).
The conference was organized as a hybrid workshop to permit in person participation from those who
were able to travel and to permit internet participation for those who could not attend in person. This
structure maximized participation and fostered meaningful interactions face-to-face and throughinternet exchanges. Figure 1 shows the diversity of attendees organized by industry and job title.
As shown in Figure 1, 379 people registered for the workshop, with roughly 2/3 of the registrants
from Japan, and 1/3 of the registrants from other countries around the world.

Fig 1. The geographic distribution of workshop registrants is displayed in the chart above.

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Fig. 2. The breakdown of attendees responding to the survey by A) industry, and B) job title, isdisplayed in the charts above (n=71).

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BackgroundSignificant investments in research over many years by governments and the private sector to improve
our understanding of how chemicals can cause toxicity have generated new methods to predict andidentify hazards of chemicals. While considerable progress has been made, this effort is still halfway to
the goal. Energized by the 2008 publication of the U.S. National Research Council (NRC) report Toxicity
Testing in the 21st Century: A Vision and a Strategy, alternative approaches that use computational
profiling and measurements of biological activity in cellular pathways have emerged. These approaches
have rapidly advanced our understanding of biological pathways in chemical toxicity.
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The European Chemicals Agency (ECHA) has encouraged the use of alternatives to animal tests
in REACH (Registration, Evaluation, Authorization and Restriction of Chemicals). ECHA also
prepares a bi-yearly report on progress made since 2007.
In the U.S., amendments to the Toxic Substances Control Act (TSCA) enacted in 2016 required
the Environmental Protection Agency (U.S. EPA) to develop, evaluate, and use alternatives toanimal tests.
o In 2019, the U.S. EPA administrator issued a directive pledging to reduce EPA’s requests
for, and funding of, mammalian studies by 30% by 2025 and to eliminate all mammal
study requests and funding by 2035.
o U.S. EPA then issued a NAMs workplan with specific objectives, deliverables, andtimelines to achieve these goals over the ensuing years.
Currently, the European Food Safety Authority (EFSA) is also developing its “EFSA Strategy
2027”, which includes development and refinement of scientific methodologies and new
approaches and tools for risk assessment that are in line with the 3Rs principle (Replacement,
Reduction, and Refinement of animal testing).
Japanese ministries (e.g., MAFF, METI, and MHLW) have also identified this challenge andcontinue to support and encourage public and private sectors to develop new methods.

Integration of information and knowledge from diverse methods is needed to ensure that a solid
scientific foundation underlies the use of results from these new approaches for chemical safetyassessment. Examples of these methods include computational methods, molecular and cellularpathways, pathogenesis of adverse effects, exposure science, and epidemiology.
Several recent initiatives provide important ideas and lessons that can be applied to accelerate the useof new approach methods in chemical risk assessments. This workshop was developed to share
information on NAMs for chemical risk assessment and their pilot applications to help all stakeholders
take stock of where the regulatory science community is at today. Additionally, the workshop will help
define the paths forward to accelerate the development and application of NAMs for specific decisioncontexts, explore knowledge gaps, and identify the challenges and opportunities for future research indeveloping and applying NAMs in chemical safety assessment.

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Conference Sessions

The conference commenced with opening remarks from Mr. Hideo Shindo (Director General, JCIA) and
Dr. Fumihiko Hasegawa (President of NITE).
See Appendix 1 for the complete agenda including abstracts for each presentation.
NOTE: presentation materials will be available through September 30, 2022 on the workshop website here1.

Session I: Setting the Stage

Session I consisted of four speakers who provided an overview on their perspectives of challenges
faced in transitioning to NAMs from hazard evaluation and risk assessments based on traditional invivo toxicity testing. These talks summarized numerous initiatives launched by their organizations,progress made to date, and strengths and limitations of NAMs. These speakers also highlighted
additional research that is needed to help develop the scientific confidence to enable NAMs to be usedin lieu of animal toxicity tests in chemical evaluations.
•Dr. Kathleen Plotzke (The Dow Chemical Company, USA) Developing and Implementing a
Scientific Confidence Framework for New Approach Methodologies (NAMs) – Proposed aflexible Scientific Confidence Framework (SCF) consisting of seven components to evaluate
NAMs and gave suggestions to make the framework semi-quantitative.

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Mr. Mitsuho Miyahara (Ministry of Economy Trade and Industry, Japan) Outlook on Chemical
Risk Assessment with New Approach Methodologies under the Regulation in Japan – Gave
Japan’s perspective on the current situation and future outlook on chemical risk assessmentwith NAMs using Chemical Substances Control Law (CSCL).

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Dr. Russell Thomas (U.S. Environmental Protection Agency) The Development, Evaluation, andApplication of New Approach Methods at USEPA – Provided a seven-step plan for application of
NAMs to regulatory decision making and finding practical solution for applying NAMs inquantitative risk evaluations.

•Dr. Elisabet Berggren (Joint Research Centre (JRC))- European Commission) Advancing Chemical
Risk Evaluations through Use of New Approach Methods – Gave the EU’s perspective on
advancing chemical risk evaluations to reach sustainable development goals by confidently
implementing NAMs.

Session II: Poster Session

Thirty poster presentations were included in the conference. These presentations covered a wide
range of topics and focus areas including NAMs for HH1 (6packs), HH2 (HH assess), HH3 (HH
predictions), ecotox, and exposures. Posters were presented both in-person and virtually. Using a web
platform, virtual participants were able to pose questions and discuss with poster authors.
Poster abstracts are included in Appendix 2.

1 https://web.cvent.com/event/d4366e52-35c6-42a9-8dcb-2ec58b08b85d/websitePage:499a9959-269f-420d-9a05-6deeabd693e6

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Session III: Using New Approach Methods to Predict Repeat-Dose and Complex ApicalEndpoints

In this session, three speakers presented their research on developing NAMs to predict complex in vivo
endpoints. These speakers covered such topics as chronic toxicity, liver toxicity, kidney toxicity, anddevelopmental neurotoxicity for human health safety assessments. In addition, a fourth speaker in thissession described the development and use of the KAshinhou Tool for Ecotoxicity.
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Dr. Kimito Funatsu (Nara Institute of Science and Technology, Japan) Development of safety
prediction system (AI-SHIPS) for industrial chemical compound using AI by METI – Outlined thedevelopment of mechanism-based toxicity-prediction system (AI-SHIPS), including Three-Layers
Model for constructing predictive model for toxicity.

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Dr. Ellen Fritsche (IUF Leibniz Research Institute for Environmental Medicine, Germany) An in
vitro battery for developmental neurotoxicity evaluation: from basic science to a regulatory
paradigm shift in 21st century Toxicology – Discussed developmental neurotoxicity in vitro
battery (DNT IVB) as a valuable tool for hazard characterization and prioritization from its
setup, applicability domains, and gaps perspective.

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Dr. Corie Ellison (The Procter & Gamble Company, USA) Internal Threshold of Toxicological
Concern (iTTC): Where We Are Today and What Is Possible in the Near Future – Shared thecurrent Internal Threshold of Toxicological Concern (iTTC) project in terms of comparing human
exposure, selecting chemicals, reviewing preliminary model output, and usage of iTTC forhuman PK data.

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Dr. Hiroshi Yamamoto (National Institute for Environmental Studies (NIES), Japan) Potential useof in silico methods for the ecological risk assessment in the chemical management in Japan –Described the performance of KAshinhou Tool for Ecotoxicity (KATE) and the limitations of
ecotoxicity QSAR systems in the chemical management in Japan.

Key Points from Session III Presentations and Discussions
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For prediction of repeated-dose toxicities, it is critical for AOPs to clearly establish the causal
relationships that link chemical information to specific key events and the adverse effect.
In silico methods have great promise, but it is important for stakeholders to understand theadvantages and the limitations of in silico methods to ensure appropriate regulatory andproduct stewardship use.
One to one replacement of a repeat dose animal study with a NAM is not realistic. Therefore,
development and use of a battery of in silico and in vitro NAMs that evaluate Molecular
Initiating Events and Key Events is a pragmatic way to address this challenge. Considerationneeds to be given to use of a battery that encompasses sufficient biological pathways.
While use of in silico and in vitro NAMs for hazard identification appears possible today forcertain mechanisms of action, there are many challenges encountered when addressing
complex endpoints, such as developmental neurotoxicity (DNT).
o There is ongoing work at OECD to develop and validate a NAMs-based DNT battery, withthe goal to eventually deploy this battery for regulatory and product stewardship uses,
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but many questions remain.
o Research to address complex endpoints needs to include, for example:
 Consideration of the number and types of AOPs that need to be developed andused, not just for specific pathway modes of action, but also for generalized
systemic toxicity and how these AOP’s should be deployed for regulatory use.
 Consideration of the range of chemical concentrations to use to develop dose
response data; to include a rationale for the selection of the highest in
vitro concentration that will be tested.
 Consideration of methods that can be used to evaluate and distinguish pathway
specific responses from non-specific responses.
 Development and use of methods such as in vitro to in vivo extrapolation PBPK
modeling to enable in vitro concentrations to be related to realistic exposure
conditions in-vivo, and to assist in equating in vitro concentrations to in vivo
limit doses.
The iTTC (internal Threshold of Toxicology Concern) holds considerable promise to ground truth
in in vitro NAM results and to interpret human biomonitoring studies.
o The iTTC approach can be deployed to evaluate aggregate human exposures.
o Further research to expand and refine the use of iTTC for risk evaluations for dermal andinhalation exposures is anticipated.
o For regulatory use of the iTTC, could one conclude that a chemical which only produces
bioactivity in vitro at a concentration above the iTTC poses no appreciable risks tohuman health?
o What battery of NAMs would be needed to confirm that a chemical has no (or very low)bioactivity, such that an iTTC can be used with confidence?
Use of the KATE in silico model in ecological risk assessment is useful, particularly forprioritization of chemicals, to identify those that require further evaluation, and forcontributing to the weight of evidence approach for the determination of PNECs for regulatory
use. However, the extent of available data is limited for constructing prediction modeling ofchronic exposure effects.

Session IV: Integrating Exposure Assessment NAMs (Internal & External) into Risk
Evaluations

Three speakers presented their research on developing PBPK models to improve quantitative
derivation of exposures for use in risk evaluations. In addition, a presentation was given describing
EAS-E suite, an online platform containing a compendium of exposure models.
•Dr. Shino Kuwa (National Institute of Technology and Evaluation, Japan) Practical Use of
Toxicokinetics Information for a Read-Across of Repeated Dose Toxicity – A Case Study Using
Halogenated Aliphatic Compounds Hepatoxicity Category – Presented a case study aimed at
utilizing physiologically based kinetic (PBK) model and showing practical use of toxicokinetics
information for a read-across of repeated dose toxicity.

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Dr. Jun Abe (Sumitomo Chemical Co., Ltd., Japan) A case study of human risk assessment of a
substance using a PBPK modeling technique – Demonstrated the feasibility of approach of using
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chimeric mice to obtain human hepatic parameters to accurately predict the pharmacokinetics
using a PBPK modeling technique.
•Dr. Katharina Schwarz (Fraunhofer Institute for Toxicology and Experimental Medicine (ITEM),
Germany) In vitro Data to Parameterise PBPK Models For Inhalation Exposure (Cefic LRI projectB21) – Described the results of inhalation-focused physiologically based pharmacokinetic(PBPK) model (lung PBPK model) for humans and discussed future steps of this model.

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Dr. Jon Arnot (ARC Arnot Research & Consulting, Canada) Development and Evaluation of a
Holistic and Mechanistic Modeling Framework for Chemical Emissions, Fate, Exposure, and Risk
– Explained a new modeling framework called PROTEX-HT (PROduction-To-Exposure HighThroughput model), which simulates aggregate human exposure and ecological exposure, and a
new online platform to aid evaluations for ecological and human health assessment called EASE Suite (Exposure And Safety Estimation Suite).

Key Points from Session IV Presentations and Discussions
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Exposure is one-half of the equation for determining risk and must be addressed intransforming risk assessment to use of NAMs.
The Age-dependent Biologically Effective dose Evaluation Tool (ABEET) incorporates a generichuman PBPK model.
o ABEET holds great promise to provide quantitative ADME information for read-across of
repeated dose toxicity for substances that lack sufficient hazard data.
Chemicals with complex kinetics can pose significant challenges for the development oflaboratory animal PBPK models and human PBPK models.
o For chemicals with simple kinetics, PBPK parameters (e.g., hepatic clearance) can beobtained from in vitro studies using rodent and human hepatocytes.
o However, for chemicals with complex kinetics, a chimeric mouse model with humanizedliver is an approach that can overcome many of the challenges.
Many exposures in the workplace, in homes, and in the ambient environment occur via
inhalation. An inhalation-focused PBPK has been developed and shown to successfully predict
respiratory uptake and bioavailability from gases, vapors, aerosols and solid particles for avariety of exposure durations.
o A web-based platform is under development, and once released will enable widespreaduse of this model.
The PROduction-To-Exposure High Throughout (PROTEX-HT) model enables efficient calculationof screening-level exposure estimates based on input information of chemical structure andproduction volume. These exposure estimates can be used for premanufacture predictions aswell as existing chemicals.
o Exposure estimates can be integrated with toxicity values to enable risk-basedprioritization and decision making.
o PROTEX-HT and other models are freely available to all user through the EAS-E suite
platform https://arnotresearch.com/eas-e-suite/.

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Session V: The challenges towards improved risk evaluations - Quantitative Human
Risk Assessment

Four speakers described their research in applying NAMs in developing quantitative risk evaluations.
•Dr. Leslie Recio (ScitoVation LLC, Durham, NC USA) Integrating New Approach Methodologies
into Quantitative Risk Assessments – Explained toxicogenomics and presented a case study onin vivo acrylamide toxicogenomics study looking at mode of action and benchmark dose.

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Dr. Satoki Fukunaga (Sumitomo Chemical Co. Ltd., Japan) Evaluating the human relevance ofchemically induced liver tumors in rodents – Quantitative risk assessment based on the mode ofaction – Presented quantitative assessment and human relevance analysis of chemicallyinduced liver tumors in rodents based on MOA using Compound X.

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Dr. James Sherman (Celanese Corporation, USA) Chemical Risk Assessment: Formaldehyde as aCase Example Use of SILMS to Inform Toxicology Testing Needs and Quantitative Risk
Evaluations – Gave an overview of SILMS (Stable Isotope Labeled Mass Spectroscopy) using
example of formaldehyde to inform toxicology testing needs with molecular dosimetry data
generated by SILMS and incorporating it into quantitative risk evaluations.

•Dr. Carl Westmoreland (Unilever, UK) Opportunities for NAMs in an EU regulatory context –Talked about Unilever’s tiered and exposure-driven approaches to assess safety of theirproducts to consumers and workers using newer techniques such as Next Generation Risk
Assessment (NGRA), which integrates NAM data from various sources into safety decisionmaking process.

Key Points from Session V Presentations and Discussions•••

In vitro to in vivo extrapolation (IVIVE) modeling is a critical tool for deployment with in vitro
NAMs.
o By using IVIVE to calculate equivalent in vivo doses, one can compare toxicity points ofdeparture derived from traditional animal toxicity tests to PODs derived from in vitro
NAMs.
o Good correlations have been shown for a number of test compounds using liver cellassays and lung cell assays (that use exposure via the air liquid interface), showing that
NAMs can be predictive and protective
Mechanistic NAMs (in silico and in vitro) are critical for use in quantitative assessment forinterspecies relevancy in carcinogenesis.
o NAMs can be used quantitatively for evaluating a range of postulated Modes of Actionand establishing human relevancy of animal study results.
o When coupled with in vivo results, these NAMs can be used to establish quantitative
species differences.
The Stable Isotope Labeled Mass Spectroscopy (SILMS) is an extremely sensitive and specificanalytical NAM for measuring biomarkers of exposure.
o For the formaldehyde case study, SILMS shows that there is a clear threshold dose for
exposure to exogenous formaldehyde.
o Integrating these NAM results with knowledge of the mode of action shows that the9•way formaldehyde causes cancer is through cytotoxicity and regenerative proliferationand that this mode of action only operates when exogenous exposures exceed athreshold level.
The Next Generation Risk Assessment (NGRA) approach is an exposure-led, hypothesis-driven
risk assessment approach that integrates NAMs for evaluation of chemical product safety for
ingredients in cosmetic and consumer products without relying on animal toxicity tests.
o Case examples have been developed using the NAMS to generate bioactivity points ofdeparture. These are then integrated with exposure assessment calculations ormeasurements to calculate bioactivity to exposure ratios or margins of safety.
o Research is showing that the NGRA approach can applied today for many types ofchemical products, and research is underway to extend the approach to cover otherroutes of exposures and effect endpoints.

Organizing Committee

The organizing committee was responsible for deciding the theme and planning the program for the
conference. The committee was comprised of scientific experts and organizational members from theJapan Chemical Industry Association (JCIA), the National Institute of Technology and Evaluation (NITE),
the American Chemistry Council (ACC), the European Chemical Industry Council (Cefic), and the
National Institute of Health Sciences (NIHS), and Together, their efforts ensured a well-balanced, highquality technical program.
Appendix 3 contains a complete list of the 2022 ICCA-LRI & NITE Workshop committee members.

Post-Event Survey Results

Survey respondents were asked to comment on satisfaction with the sessions and presentations, andthe overall workshop. Survey respondents were also asked to provide feedback on additional topics tocover in future conferences. The survey results reported an overall workshop satisfaction of 98%.
Appendix 4 highlights the feedback from the post-event survey, including short answer responses on
future suggestions for improvements to the workshop.

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APPENDIX 1. Complete Agenda Including Abstracts

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ICCA-LRI and NITE Workshop – Yokohama, Japan –June 20-21, 2022 – InterContinental Yokohama Grand

Advancing Chemical Risk Evaluations Through Use of New Approach Methods(NAMs): Challenges and Opportunities

Day 1: Monday, 20 June 2022
Registration
11:30-12:40
(JST)
Foyer

Check-inWelcome12:40-13:00
Ballroom&Zoom

Welcome and Opening Remarks
Hideo Shindo – Director General, JCIA
Dr. Fumihiko Hasegawa – President, NITE

Session I: Setting the Stage
Session Chairs: Hideo Shindo (JCIA) & Dr. Fumihiko Hasegawa (NITE)
13:00-13:30
Ballroom&Zoom

Setting the Stage: Developing and Implementing a Scientific Confidence
Framework for New Approach Methodologies (NAMs)
Dr. Kathleen Plotzke – The Dow Chemical Company, USA
The dynamics and complexities of functional biology and homeostasis in whole organisms create significant
challenges in transitioning from lab animal-centric toxicity testing methods to assessment strategies basedon new approach methodologies (NAMs). Nevertheless, considerable progress is being made. The LRI
programs, and others, have developed approaches for using computational, in vitro, and exposure NAMs tofill data gaps, conduct risk-based prioritization, and to improve integrated approaches to testing andassessment. However, to put NAMs into use requires regulatory acceptance - demonstrating that a NAM has
achieved a requisite degree of scientific confidence for one or more specific applications before it can beused to support product stewardship or regulatory decisions. ‘Fit- for-Purpose Validation’ (FFPV) has
emerged, and while various components of such a process have been discussed, a uniform approach for
FFPV has yet to be adopted. Accordingly, we have proposed a scientifically rigorous, yet flexible framework,that can be widely applied to evaluate most, if not all, types of NAMs. This NAM Scientific Confidence
Framework (SCF), centered on explicitly applying the scientific method, consists of 7 components: 1)
problem formulation and proposition statement (description of the intended use of the NAM for a specificdecision context) 2) the biological relevance & plausibility of the NAM; 3) assay performance (documentationof sensitivity, specificity, reliability, & domain of applicability of the NAM); 4) documentation of theperformance of the inference (prediction) model imbedded in the NAM (based on the NAM response –predicted outcome response relationship); 5) dissemination of the data, inference models, etc. to support
independent replication; 6) a narrative rationale making the case that there is / is not sufficient scientificconfidence in the NAM to support the specific application for the chemistry domain of interest; and 7)
verification through independent scientific peer review. Time permitting, we will illustrate application of thisNAM Scientific Confidence Framework to the evaluation of 2) the purported key characteristics ofcarcinogens for classifying chemicals and b) to use of the Threshold of Toxicological Concern (TTC) andpoints of departure from ToxCast data for risk-based prioritization / deprioritization.

13:30-14:00
Ballroom&Zoom

Outlook on Chemical Risk Assessment with New Approach Methodologies under
the Regulation in Japan
Mitsuho Miyahara – Ministry of Economy Trade and Industry, JapanThe New Approach Methodologies(NAMs)are the technologies that have a great potential to increase quality
and efficiency in chemical assessment and management. However, there remain difficulties for further
developing NAMs and promoting practical use globally, especially introducing NAMs into regulations. In thissession, he will describe current situation and outlook on chemical risk assessment with NAMs in Japan fromthe standpoint of regulatory authority. First, he will explain overview of Chemical Substances ControlLaw(CSCL) which is one of chemical management laws in Japan. Evaluation for new chemicals and risk
assessment for existing chemicals are conducted by the authority under CSCL. NAMs such as quantitative
structure-activity relationships(QSARs)model and read-across are utilized partially for evaluation of chemical
properties such as biodegradability and bioconcentration. Next, he will introduce activities on research anddevelopment of NAMs in Japan, such as weight of evidence approaches and development of a state-of-theart hazard prediction system analyzing relationship between toxicity and chemical structure as well as othervarious factors by using AI technologies. These developments have certain achievements so far and are
expected to contribute for further development to evaluate biodegradability, bioaccumulation and toxicity ofchemicals under environment close to actuality. In addition, he will present Japan’s contribution andcooperation to the OECD which is the center of the international discussions on the NAMs. Finally, he willraise major challenges and necessary future efforts to expand the application of NAMs to regulations, suchas improving reliability, accumulating practical use cases, and creating guidelines regarding NAMs.

14:00-14:30
Ballroom&Zoom

The Development, Evaluation, and Application of New Approach Methods at US
EPA
Dr. Russell Thomas – U.S. Environmental Protection Agency, USA
Thousands of chemicals are used in commerce and are present in the environment, while more chemicals
are introduced each year. Many of these chemicals have limited data that can inform potential human healthand ecological risks. The continued development, evaluation, and application of emerging scientific andtechnological approaches are critically important to address these challenges. New Approach Methods(NAMs) is term that has been coined to capture many of these emerging approaches and they include a
broad range of in vitro and in silico approaches for evaluating potential chemical hazards, identifying modes
or mechanisms of action, extrapolating between internal and external doses, and estimating exposure. In
many cases, the endpoints and uncertainties associated with these new methods are qualitatively andquantitatively different than the traditional approaches. The inherent differences have required new efforts tocharacterize the uncertainties in both the NAMs and traditional methods while developing new frameworks tobuild confidence in the approaches for application to a range of decision contexts. The US EPA continues toinvest in NAM research and has released an updated Agency Work Plan outlining broad objectives fordeveloping and applying NAMs as well as long-term and short-term strategies to achieve those goals. This
presentation will provide an overview of the ongoing efforts at US EPA to develop and improve NAMs related
to toxicology, toxicokinetics, and exposure; evaluate NAMs for fit-for-purpose regulatory application throughcase studies and scientific confidence frameworks; and outline a path forward for increasing integration into
regulatory decisions. *The contents of the abstract and associated presentation do not necessarily reflect
U.S. EPA policy.

14:30-15:00
Ballroom&Zoom

Advancing Chemical Risk Evaluations through Use of New Approach Methods(NAMs): Challenges and Opportunities
Dr. Elisabet Berggren – Joint Research Centre (JRC) - European Commission
In the context of the EU Chemicals Strategy for Sustainability towards a toxic-free environment, aiming tobetter protect citizens and the environment and boost innovation for safe and sustainable chemicals, thediscussion on how and when NAMs can be used in chemicals safety assessment has been further
emphasised within the EU. Looking at the assessment of systemic health effects and long-term effects in theenvironment, there are many challenges how to confidently implement New Approach Methods. What levelof validation or standardisation is needed to accept a NAM? We need to ensure that we maintain or higher
the current level of safety while guaranteeing sustainability in use and exposure to chemicals, but still
accepting that a one to one replacement of traditional animal methods is not possible. The NAMs can neither
be used only to trigger further animal testing to confirm suspected adverse outcome, which could lead tomore intense use of animal testing. In addition, to achieve safe and sustainable use of chemicals, theassessment need to be more efficient to cover the large number of chemicals on the market and not only afraction. There is a need for common understanding and trust among stakeholders to progress thedevelopment of new approaches to testing and assessment for safer use of chemicals.

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Session II: Poster Session – Development of NAMs and Case Studies with NAMs
Session Chair: Dr. Tokuo Sukata (JCIA)
15:00-15:10
Ballroom&Zoom

Overview of Poster Session
Dr. Tokuo Sukata - JCIA

15:10–15:30

Afternoon Break

15:10-18:15
Foyer
&Box

Poster Session

18:15

Adjourn Day 1

Day 2: Tuesday, 21 June 2022
Registration
8:00-8:30
(JST)
Foyer

Check-in & Coffee

Welcome8:30-8:40
Ballroom&Zoom

Welcome and Agenda Review
Taketoshi Fujimori - JCIA

Session III: Using New Approach Methods to Predict Repeat-Dose and Complex ApicalEndpoints Session Chairs: Dr. Yuki Sakuratani (NITE) & Dr. Bruno Hubesch (Hubesch Consult)

8:40-9:05
Ballroom&Zoom

Development of safety prediction system (AI-SHIPS) for industrial chemicalcompound using AI by METI
Dr. Kimito Funatsu – Nara Institute of Science and Technology, JapanThe official name of this project is Development of next generation integrated chemical safety prediction
system by using A.I. and chemical toxicity related big data. Backgrounds for establishment of this project are
1. Strengthen competitiveness of Japanese material/chemical industry, 2. Changes in the global competitive
situation of business (the customer's request level is high (safety, SPEC), and the speed is particularly
required), and 3. Trend of reducing, replacement and refinement animal testing (3R).
In this project, we developed in silico prediction system for 28-day repeated dose toxicity test. As a wayof proceeding, it was decided to develop a prediction system for hepatotoxicity in the first stage and a
prediction system for hematological toxicity and nephrotoxicity in the second stage. Those three toxicities EP
were chosen because most NOELs of those environmental chemicals are observed on these toxicities in the
28-day repeated dose study.
The basic development policy and strategy for the construction of this toxicity prediction system AISHIPS are the following four points. First, in the previous SAR system, the relationship between chemicals
information and prediction endpoint was a black box, but in this system, a prediction method was developed
based on the toxicity expression mechanism, that is, AOP and mode of action. 2nd point is that the system
incorporates environmental chemical PBPK predicting system. 3rd point is that we have prepared a data
matrix including the latest toxicity research information and developed predictive models using the latest
computational science methods such as machine learning.
Finally, we also aimed to improve the accuracy of the prediction system by reflecting appropriate in vitro
assay results such as the signal transduction mechanism etc.

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As a feature of this system development, we use high-quality, uniform about 2000 GLP-level 28 days
repeated-dose toxicity test HESS, CSL and REACH EP information as training data sets.
Furthermore, ”Funatu‘s three-layers model; chemical substanceà in vitro à in vivo” is adopted as basic pillar
for development of the in vivo prediction model.
Finally, an in vivo prediction model is constructed using the in vitro predicted values obtained by in vitro
prediction models. In other words, in order to construct a systemic toxicity prediction system based on the
toxicity onset mechanism, we emphasize the relationship information between chemical substances and MIK
and KE, and build a system to predict endpoints at the in vivo level by making full use of this information.
Therefore, the basic concept of Funatsu's three-layers model was established. This has made it possible tofully utilize the reactivity information of chemical substances and living protein such as enzymes, receptors
for the construction of more accurate prediction methods.

9:05-9:30
Ballroom&Zoom

An in vitro battery for developmental neurotoxicity evaluation: from basic science
to a regulatory paradigm shift in 21st century Toxicology
Dr. Ellen Fritsche – IUF Leibniz Research Institute for Environmental Medicine,Germany
Testing for developmental neurotoxicity (DNT) is currently performed in rats according to OECD/US-EPA
guidelines. These methods are very costly, take a long time and have uncertainties in their interpretation. To
overcome these issues and allow a large number of chemicals to be tested for DNT, a DNT in vitro testing
battery (DNT IVB) has been assembled under the guidance of the European Food Safety Authority (EFSA)
in collaboration with the Danish- and US-Environmental Protection Agency and under the umbrella of theOrganisation for Economic Cooperation (OECD). Despite the large coverage of neurodevelopmental key
events in the DNT IVB, some gaps were already identified. These concern radial glia, astrocyte andmicroglia-related endpoints.
The DNT IVB consists of test methods based on primary human neural progenitor cells (hNPC), humaninduced pluripotent stem cell (hiPSC)-derived neural crest cells and neurons, as well as LUHMES cells andmodel the key neurodevelopmental processes hNPC proliferation, migration and differentiation into neurons
and oligodendrocytes, neurite morphology, neural crest cell migration and neurite outgrowth. This IVB waschallenged with 120 chemicals. In addition, a hiPSC-based test method for human neuronal network
formation was set up and challenged with 28 pesticides. To close the identified IVB gaps, we started setting
up methods concerning radial glia, astrocytes and microglia within the LRI AIMT11 project.
Concentration-response curves reveal benchmark concentrations (BMCs) for the 120 compounds in theindividual test methods. Classification models for data interpretation were applied. For interpretation ofcompound results across the whole battery, respective most sensitive endpoints (MSEs) were determined.
Battery results were used in two case studies, i.e. hazard characterization of deltamethrin and flufenacet in
an Adverse Outcome Pathway-informed Integrated Approach for Testing and Assessment (by EFSA) andflame retardant prioritization (by the US-National Toxicology Program). For the newly developed methods onradial glia, astrocytes and microglia, we established respective marker expression over development andresponses to first compounds.
These data demonstrate the successful set-up of a DNT-IVB that can be used for different regulatory
purposes. An OECD guidance document has been prepared (Crofton & Mundy 2021) that informs on use
and interpretation of the DNT-IVB for regulatory application and highlights DNT ICB gaps. Increasing trust inbattery performance by closing these gaps, testing more chemicals and lab-to-lab transfer will aid its
implementation into regulation.

9:30-9:55
Ballroom&Zoom

Internal Threshold of Toxicological Concern (iTTC): Where We Are Today andWhat Is Possible in the Near Future
Dr. Corie Ellison – The Procter & Gamble Company, USA
The Threshold of Toxicological Concern (TTC) is an important risk assessment tool that establishes
acceptable low-level exposure to chemicals with limited toxicological data. Traditionally, the TTC has beenused in food safety and consumer product safety evaluations for chemicals with low exposure potentials.
However, in cases where the scientific confidence of the TTC approach is appropriate for a given decisioncontext, the TTC can be applied more widely. One of the next steps in the continued evolution of TTC is todevelop the concept further so that it is representative of internal exposures. This refinement of TTC basedon plasma concentrations referred to as internal TTC (iTTC) requires a significant amount of data andcomputational tools that can be used to convert the chemical-specific external dose NOAELs in the TTC
database to an estimated internal exposure for each chemical. To achieve an iTTC that is suitable for humansafety risk assessment, a multi-stakeholder collaboration between Cosmetics Europe Long-Range Science
Strategy program, American Chemistry Council Long-Range Research Initiative, and the Research Institutefor Fragrance Materials (RIFM) was established. The chemicals selected to generate the iTTC databasecome from multiple existing TTC/chemical databases, including Munro et al. (1996), COSMOS (Yang et al.,2017) and the RIFM database (Patel et al., 2020). For the 1,251 chemicals in the combined database, aliterature search was conducted to identify existing in vivo and in vitro pharmacokinetic (PK) data.

4

Additionally, chemical space analysis was conducted for the 1,251 chemicals using molecular and PK
descriptors so that the combined database could be reduced to approximately 300 chemicals for final
inclusion in the iTTC database. In vitro oral absorption data for each chemical in the iTTC database and invitro hepatocyte clearance for chemicals lacking literature values are being generated. Iterative batchprocessing physiologically based pharmacokinetic (PBPK) modeling is being used for the iTTC databasechemicals to establish the best conditions for PBPK modeling and to make quantitative comparisons
between the PBPK model simulations and the existing PK data. In addition to this work bringing us closer
towards establishing the final iTTC values, it has provided an opportunity to generate new structured datarepositories for PK data and has helped inform approaches for in vitro to in vivo PBPK modeling for a wide
range of chemicals. The current presentation will share the status of the iTTC project and will provide arepresentative example use scenario for iTTC.

9:55-10:20
Ballroom&Zoom

Potential use of in silico methods for the ecological risk assessment in thechemical management in Japan
Dr. Hiroshi Yamamoto – National Institute for Environmental Studies (NIES), Japan
In Chemical Substances Control Law (CSCL), there has been long needs from industry side for the use of insilico methods not only for bioaccumulation and biodegradation tests but also for ecological risk assessment.
KAshinhou Tool for Ecotoxicity, QSAR-based ecotoxicity prediction system researched and developed by
National Institute for Environmental Studies (NIES) under the contract with Ministry of the Environment(MOE) Japan, first released in 2008, and later updated for several times before the English version(KATE2017) was first released in 2018. The latest version of KATE2020 ver.3.0(https://kate.nies.go.jp/onnet2020-e.html) was released in March 2022.
The main features of KATE2017 and 2020 are the categorization of chemicals based on substructure andthe reactivity, and the toxicity values for input chemical can be predicted for fish, daphnid and alga for bothacute (EC50 or LC50) and chronic (NOEC) using linear regression with the explanatory variable, octanolwater partitioning coefficient (log P). Training dataset used was over 1000 chemicals mainly those obtainedby MOE in GLP labs with relatively high reliability in conformity with OECD guideline for testing of chemicals
Nos. 201, 202, 203, 210, and 211. Only for fish acute toxicity tests, additional training dataset was provided
from US Environmental Protection Agency (USEPA) from Fathead Minnow Database. The external
validation with test dataset from OECD SIDS database, we found that the relatively better prediction
accuracy with nearly 80% are within the range of one order of magnitude difference from the measured
value while ECOSAR of USEPA showed relatively less accuracy with large applicability domain.
As for the use of in silico methods such as QSAR (including KATE and ECOSAR) and category approach in
CSCL, KATE and ECOSAR have long been used only as a reference for the registration of new chemicals
while these predictions can be better used in the designation of priority assessment chemicals after theproper guidance is established. For the detailed risk assessment of the priority assessment chemicals, theuse of in silico predictions for the weight of evidence approach for the determination of PNEC.
In the other chemical risk assessment framework, such as Initial Environmental Risk Assessment ofChemicals, which has been conducted since 2002, even vigorous use of in silico methods is expected to fillin the gap of test data. Since this is the screening level risk assessment for the prioritization of air and water
quality standards (or guidance values) and emerging chemicals of concern such as pharmaceuticals andpersonal care products (PPCPs), further detailed risk assessment and management should be conducted in
the higher tier risk assessment, anyway.

10:20-10:40
Ballroom&Zoom

Q&A and Panel Discussion
Dr. Kimito Funatsu – Nara Institute of Science and Technology, Japan
Dr. Ellen Fritsche – Leibniz Research Institute for Environmental Medicine, Germany
Dr. Corie Ellison – The Procter & Gamble Company, USA
Dr. Hiroshi Yamamoto – National Institute for Environmental Studies (NIES), Japan

10:40-11:00

Morning Break5

Session IV: Integrating Exposure Assessment NAMs (Internal & External) into Risk Evaluations
Session Chairs: Dr. Tomoya Yamada (Sumitomo Chemical) & Dr. Richard Becker (ACC)
11:00-11:25
Ballroom&Zoom

Practical Use of Toxicokinetics Information for a Read-Across of Repeated DoseToxicity – A Case Study Using Halogenated Aliphatic Compounds Hepatoxicity
Category
Dr. Shino Kuwa– National Institute of Technology and Evaluation, JapanThe study aimed to utilize the physiologically based kinetic (PBK) model to explain the variability anduncertainty of toxicokinetics between the target substance and its analogs in read-across explicitly for
subcategorization of analogs and uncertainty characterization through a case study. In this case study, readacross of compound category for hepatotoxicity by halogenated aliphatic compounds in HESS Category was
attempted and 1,3-dichloropropane (CAS RN 142-28-9) was chosen as the hypothetical target substanceTraditionally, PBK models have been developed for individual substances with enough kinetic data but the
recently published OECD guidance document for the PBK model introduces the assessment framework for“data-poor” situations. In this study, a tool incorporating a generic human PBK model, Age-dependent
Biologically Effective dose Evaluation Tool (ABEET), was modified to calculate blood and liverconcentrations in rats during repeated dose toxicity tests for the target substance and analogs without kinetic
data. The predictive capability was validated using read-across approach described in the OECD guidance
document for the PBK model. The AUC24h (Area-Under-the-Curve over 24 hours) of the liver concentration
at 1 unit dose was chosen as a toxicokinetic indicator for read-across. Substances with toxicokinetic
dissimilar characteristics from the target substance that may affect their toxicity were detected and excluded
from the analogs by characterizing the correlation between the AUC24h of liver concentration andhepatotoxicity. In this case-study, PBK model contributed to provide quantitative ADME information for readacross and to reduce and describe the uncertainty.

11:25-11:50
Ballroom&Zoom

A case study of human risk assessment of a substance using a PBPK modelingtechniqueDr. Jun Abe – Sumitomo Chemical Co., Ltd., Japan
It is generally known that interspecies differences in efficacy or toxicity occur when significant
pharmaco/toxicokinetic and pharmaco/toxicodynamic variations arise among species. The use of kinetic
data to describe the internal dosimetry of non-pharmaceutical compounds is becoming more important;however in most cases, it is impossible to obtain kinetic data experimentally in humans. To overcome thisissue, physiologically based pharmacokinetic (PBPK) model has been being utilized. In the kinetic researchin humans using PBPK models, it is common to initially develop an animal PBPK model using the available
in vitro and in vivo kinetic data from the same animal species, and then to convert the animal model to ahuman model by applying human in vitro data as the input parameters. For the compounds which only have
simpler elimination route, e.g. hepatic metabolism, many examples are reported to successfully predict thehuman kinetics thus PBPK has been regarded in the practical level. However, when the target compound
exhibits complicated kinetics, it is difficult to reproduce its kinetics only by the combination of in vitro
experiments.
Here, we present a case study that the reliable human exposure assessment was achieved for a compound
which has very complicated kinetics. It has already been revealed that the compound immediately
metabolized in mammals and its major metabolite (Metabolite A) exhibits quite complicated kinetic profiles;
e.g. active hepatic uptake via transporters followed by hepatic metabolism and biliary excretion. For theaccurate prediction of the kinetics of Metabolite A in humans, we adopted the use of chimeric mice withhumanized liver alongside the in vitro hepatocyte uptake assay, and obtained some key human parameters,i.e., hepatic uptake intrinsic clearance and hepatic elimination intrinsic clearance via biliary excretion andmetabolism. Finally, all human parameters which were determined to have higher influence by sensitivity
analysis were obtained without applying simple extrapolation without biological basis (i.e. assuming no
species difference between humans and mice), and the exposure of Metabolite A in human could be
evaluated.
We believe this approach which utilizes the chimeric mice to obtain human hepatic parameters canaccurately predict the pharmacokinetics, but further proof of this concept was still desired. In order todemonstrate the feasibility of the concept, we applied the similar approach to predict the rat kinetics. The
chimeric mice with rat liver were prepared and the rat hepatic parameters were obtained. Finally, the kineticsof Metabolite A in rats were predicted without any rat kinetic parameters obtained from in vivo rat
experiment. The data showed that the kinetics of Metabolite A in rats in vivo were well reproduced with the
PBPK model referring the parameters obtained from the chimeric mice, suggesting the concept of thisapproach is well validated.

6

11:50-12:15
Ballroom&Zoom

In Vitro Data To Parameterise PBPK Models For Inhalation Exposure (Cefic LRI
project B21)
Dr. Katharina Schwarz – Fraunhofer Institute for Toxicology and Experimental Medicine(ITEM), Germany
Use of chemicals in the consumer and occupational environment requires a risk assessment consideringboth the hazard of and exposure to a substance. In both cases, the inhalational route is of major importance.
Usually the internal dose is not known due to absence/lack of in-vivo data describing systemic uptake. Up toknow, the inhaled dose, or the dose deposited in the respiratory tract is typically considered as the relevant
dose for the risk assessment process. This is a pragmatic approach which, however, often results inuncertainties and conservative assumptions.
To bridge the gap between external exposure concentrations to internal concentrations inside the body, an
inhalation-focused physiologically based pharmacokinetic (PBPK) model for humans has been developed toprovide a more refined determination of the systemic available dose. This PBPK model was aimed to be
publicly available and to deliver information relevant for regulatory needs.
The new model divides the lung into several sub-compartments due to its heterogenic structure and different
local clearance / uptake mechanisms. It considers deposition and dissolution, mucociliary clearance andmacrophage uptake as well as absorption into blood. The attached systemic part consists of a generic stateof-the-art design that includes various tissues. Substance-specific respiratory input parameters such as lungpermeability, particle dissolution and gas uptake, can be generated in vitro. Transfer rates/permeability
coefficients across lung epithelial cells are determined in an vitro inhalation testing with suitable human celland tissue barrier models for the pulmonary and tracheobronchial regions utilizing the P.R.I.T.® ExpoCube®.
Dissolution rates of poorly soluble particles are measured in simulated biological fluids. For integration ofsubstance-unspecific clearance processes like mucociliary and macrophage-mediated clearance existing
literature data are used. Systemic parameters can be mostly obtained using established in-silico tools.
Several substances were used to investigate the applicability of this model: highly soluble nicotine, poorly
soluble toluene (vapor), and bio-inert iron oxides and polystyrene (aerosol). Predicted lung retention or
blood kinetics for these substances were compared to available clinical data and show very good agreementwith both the quantitative concentration levels as well as the qualitative concentration or lung burden
profile.
A particular focus has been on the analysis of the sensitivity of the input parameters and their individual
relevance regarding the systemic uptake kinetics/lung retention for the different substance classes. The
identification of the key determinants is aimed at minimizing the number of input parameters required to
optimize usability of the PBPK model.
The results indicate that the lung PBPK model has a wide applicability domain and can successfully predict
respiratory uptake and bioavailability from gases and vapors to liquid and solid particles for exposure
durations of a single or a couple of breaths to several hours and clearance for up to several months.
Future steps of the lung PBK model comprise the inclusion of different human subpopulations, lungmetabolism, the diseased lung, and application for rats, all of which were already explored in the project. A
user-friendly application for the PBPK model has been developed and will be made publicly available
shortly.

12:15-12:40
Ballroom&Zoom

Development and Evaluation of a Holistic and Mechanistic Modeling Framework
for Chemical Emissions, Fate, Exposure, and Risk
Dr. Jon Arnot – ARC Arnot Research & Consulting, Canada
Thousands of new and existing chemicals are subject to ecological and human health risk assessment;
however, extensive data gaps for exposure estimation in particular present obstacles to addressing
regulatory objectives. The Exposure And Safety Estimation (EAS-E) Suite freely accessible on-line platform(www.eas-e-suite.com) facilitates regulatory objectives and the safe and sustainable production and use ofchemicals in society. EAS-E Suite contains databases of chemical information and various models and tools
to support chemical evaluations (i) retrospectively for existing chemicals and use, and (ii) prospectively(forecasting) for new chemicals and for proposed changes of use of existing chemicals. The PROduction-ToEXposure High Throughput (PROTEX-HT) in EAS-E Suite quantifies relationships between production
volumes, chemical emissions throughout the lifecycle, fate, and transport in natural and manufactured
environments (e.g., homes), persistence, bioaccumulation, toxicokinetics in ecological receptors andhumans and aggregate human and ecological exposures to estimate the potential for effects. PROTEX-HT
requires only two chemical-specific pieces of information as input parameters: production volume andstructure. The PROTEX-HT model predictions are evaluated using 95 chemicals for which humanbiomonitoring data are available. Seventy-nine percent and 97% of the PROTEX-HT human exposure
predictions were within one and two orders of magnitude, respectively, of independent human exposure
estimates inferred from biomonitoring data from the US population. The results show how PROTEX-HT in
EAS-E Suite can support: (i) the screening and ranking of chemicals based on various exposure and risk
metrics, (ii) setting chemical-specific maximum allowable tonnage based on user-defined toxicological7thresholds, and (iii) identifying the most relevant emission sources, environmental media, and exposure
routes of concern. The results also show that high chemical tonnage does not necessarily result in highexposure or health risks. PROTEX-HT enables efficient screening-level evaluations of existing andpremanufacture chemicals in various exposure- and risk-based contexts for holistic and effective chemicals
management.

12:40-13:00
Ballroom&Zoom

Q&A and Panel Discussion
Dr. Shino Kuwa – National Institute of Technology and Evaluation, Japan
Dr. Jun Abe – Sumitomo Chemical Co., Ltd., Japan
Dr. Katharina Schwarz – Fraunhofer Institute for Toxicology and Experimental Medicine (ITEM),
Germany
Dr. Jon Arnot – ARC Arnot Research & Consulting, Canada

13:00-14:15

Lunch BreakSession V: The challenges towards improved risk evaluations - Quantitative Human Risk
Assessment
Session Chairs: Dr. Hajime Kojima (NIHS) & Dr. Bruno Hubesch (Hubesch Consult)

14:15-14:40
Ballroom&Zoom

14:40-15:05
Ballroom&Zoom

Integrating New Approach Methodologies into Quantitative Risk Assessments
Dr. Les Recio– ScitoVation LLC, Durham, NC
Over the last decade, a significant shift has emerged in the safety assessment chemicals with an aim toneed to reduce reliance on animal testing and to develop highly predictive human-based biological models.
New in vitro human-relevant biological test systems integrated with in silico methods, collectively referred toas new approach methodologies (NAMs) are revolutionizing the practice of toxicology with a significant
movement away from the traditional high cost and ethical concerns of animal testing. The goals for
implementation of NAMs are to efficiently identify potential chemical hazards and to gather dose-response
data to inform human-relevant safety assessment. NAMs are gaining importance as an alternative to
traditional in vivo toxicity testing. However, replacement of traditional animal toxicity tests for toxicology
safety assessments with NAMs can encounter significant regulatory hurdles including how accurately NAMs
predict responses known to occur in traditional regulatory testing and more importantly what can occur inhumans are the biggest challenges for acceptance of NAMs in place of animal data by regulatory agencies.
To overcome this challenge, Scitovation is focused on human relevant airway and hepatocyte models
integrated with apical measures of cellular health status and genomics-based presumptive modes of action(MOA) to develop quantitative estimates of relevant dose responses to derive benchmark dose and point of
departure (POD) estimates. Significant concordance of in vivo derived POD estimates for gene expression
changes and apical measures of toxicity in animals or cancer outcomes have been observed for several
chemicals. Scitovation scientists are using NAMs-based approach with in vitro to in vivo extrapolation(IVIVE) modeling of dose-response and POD estimates “reverse dosimetry” to assess how well IVIVE aligns
with in vivo exposures expected to give similar tissue dose. A recent case study has shown that IVIVE
modeling of air:lung interface exposures to 1,3-dichloropropene and calculated rat equivalent inhaledconcentrations across the air ways (nasal to alveolar epithelium) were qualitatively in accordance with
empirically derived based air way PoD estimates. The IVIVE predicted PoD concentrations for 1,3dichloropropene were slightly higher than the PoDs observed from in vivo subchronic studies. This casestudy implementing a NAM-based chemical risk assessment approach for inhalation toxicology has provided
insight into the predictive power of this approach and opportunities to increase confidence in the predictivityof NAMs. These data and others support the use of NAMs in chemical safety testing as a more efficient andhuman relevant alternative to rodent in vivo inhalation studies.
We thank the American Chemistry Council Long-Range Research Initiative funding and PETA Science
Consortium International for donation of the Vitrocell® exposure system.

Evaluating the human relevance of chemically induced liver tumors in rodents –Quantitative risk assessment based on the mode of action –Dr. Satoki Fukunaga – Sumitomo Chemical Co. Ltd., Japan
Some chemicals are known to interfere with hepatic heme biosynthesis in rodents, mainly by inhibiting
particular enzymes of the heme biosynthesis pathway, which leads to accumulation of porphyrins in manytissues including the liver (chemically induced porphyria) and ultimately the development of liver tumors.
Compound X is not genotoxic and induces liver tumors in male mice via a porphyria-mediated cytotoxicity
mode of action (MOA) with the following key events (KEs): (#1) inhibition of mouse protoporphyrinogenoxidase (PPO); (#2) accumulation of porphyrins; (#3) hepatocellular injury; with (#4) subsequent
regenerative cell proliferation; and ultimately (#5) development of liver tumors. These key events for theporphyria-mediated cytotoxicity MOA in rodents are considered to have some similarities with human8porphyria leading to liver tumorigenesis, and therefore this carcinogenicity MOA is considered qualitatively
plausible in humans.
In the case of a carcinogenicity MOA in rodents which is qualitatively relevant to humans, quantitative
assessment between rodents and humans for each KE needs to be considered to address humanrelevance, in accordance with the International Programme on Chemical Safety (IPCS) Human Relevance
Framework. Thus, we conducted a quantitative assessment and human relevance analysis based on the
MOA, taking into account quantitative toxicokinetic (TK) and toxicodynamic (TD) differences.
Compound X is immediately metabolized and exists as Metabolite A in mammals, and is actively taken up
into hepatocytes via transporters, which corresponds to TK. In vitro TK assay using hepatocytes revealed a
6- to 13-fold lower hepatic uptake of Metabolite A in humans than in mice, which was caused by a significant
active uptake of Metabolite A via transporters in mice but with less of a contribution of transporters inhumans. Also, in vitro TD assay using hepatic PPO revealed that 14-fold weaker inhibition of PPO (KE #1)
by Metabolite A in humans than in mice.
Quantitative differences in the subsequent KEs were also assessed by in vivo studies using the chimericmouse with humanized liver (PXB mouse), where the mouse liver is repopulated by human hepatocytes at aratio of around 90%. By administration of Compound X to PXB mice in which human hepatocytes from threedonors were transplanted, slight porphyrin accumulation (KE #2) occurred, but neither hepatocyte injury (KE#3) nor subsequent regenerative proliferation (KE #4) was observed. Although there are no data on liver
tumor formation in humans (KE #5), it is highly unlikely that Compound X will induce liver tumor in humans
via this MOA since key events #3 and #4 are unlikely to occur in humans.
Overall, quantitative assessment of the MOA demonstrates that Compound X has the potential to inducemouse liver tumors through a nongenotoxic, porphyria-mediated cytotoxicity MOA with a clear threshold, andthis MOA is considered not relevant to humans based on the marked TD and TK differences between mouseand human, utilizing the IPCS human relevance framework. Consequently, a linear low-dose extrapolation
approach is not appropriate for cancer risk assessment, but instead a nonlinear, threshold margin of
exposure risk assessment is appropriate.

15:05-15:30
Ballroom&Zoom

Chemical Risk Assessment: Formaldehyde as a Case Example Use of SILMS to
Inform Toxicology Testing Needs and Quantitative Risk Evaluations
Dr. James Sherman – Celanese Corporation, USA
Recent mass spectrometry advancements have significantly enhanced the reliable characterization andquantification of biomarkers of exposure. As such, it is now possible to characterize molecular dosimetry to
the molecular target, in potential target organs, throughout the dose-response curve. For chemicals withboth endogenous and exogenous exposures, this has greatly increased the understanding of added andrelative risk, as well as helped to define thresholds for potential genotoxic and carcinogenic effects. This
presentation will provide an overview of Stable Isotope Labeled Mass Spectroscopy (SILMS) and its use inunderstanding the dose-response for formaldehyde-related nasal tumors and potential genotoxic effects ofinhaled formaldehyde at relevant levels of human exposure. Additionally, a case study of using SLIMS to
reduce uncertainty related to toxicokinetics and toxicodynamics will be discussed in relation to
directing/reducing the need for further animal testing and decreasing uncertainty when deriving acceptable
exposure limits.

15:30-15:55
Ballroom&Zoom

Opportunities for NAMs in an EU regulatory context
Dr. Carl Westmoreland – Unilever, UK
At Unilever, our Safety and Environmental Assurance Centre (SEAC) is a team of over 160 safety andenvironmental sustainability scientists based in Bedfordshire in the UK. We use the latest techniques, deep
scientific expertise and an evidence-based approach to ensure that Unilever products are safe for
consumers and workers and better for the environment.
To assure that our products are safe for our consumers and the workers that make them, we use a tiered
and exposure-driven approach to all our human health safety assessments. We use a wide range of nonanimal approaches to assess the safety of our products.
This talk will demonstrate the non-animal approaches we use in our safety assessments concentrating onsome of the newer techniques we have developed and are applying in Next Generation Risk Assessment(NGRA). NGRA provides a way to integrate New Approach Methodology (NAM) data from various sources
into the safety decision-making process, allowing for safety assessments to be conducted without the use ofanimal data. Recently, the Internal Cooperation for Cosmetics Regulation (ICCR) outlined nine principles forthe use of NGRA to make decisions on consumer safety of ingredients in cosmetics products1. These
principles have been incorporated into the most recent update of the European Commission’s SCSS Notes
of Guidance for the Testing of Cosmetic Ingredients and their Safety Evaluation2 and used by Unilever inseveral published case studies in the areas of systemic toxicity3 and skin allergy4
Whilst the adoption of NAMs for decisions on consumer safety in the EU continues to progress, there has
been slower progress with the acceptance of NAMs for chemical registration in the EU5, though significant
opportunities exist to increase their use and acceptance6.

9

15:55-16:15
Ballroom&Zoom

Q&A and Panel Discussion
Dr. Les Recio – ScitoVation LLC, Durham, NC
Dr. Satoki Fukunaga – Sumitomo Chemical Co. Ltd., Japan
Dr. James Sherman – Celanese Corporation, USA
Dr. Carl Westmoreland – Unilever, UK

Closing: Closing Remarks and Next Steps
16:20-16:35
Ballroom&Zoom

Wrap-up Session
Dr. Tatsuhiro Niino - Mitsubishi Chemical Corporation

16:35-16:45
Ballroom&Zoom

Closing Remarks
Dr. Fumihiko Hasegawa – President, NITE
Hideo Shindo – Director General, JCIA

16:45

Adjourn

10

APPENDIX 2. Complete List of Posters Including
Abstracts

12

ICCA-LRI and NITE Workshop – Yokohama, Japan –June 20-21, 2022 – InterContinental Yokohama Grand

Advancing Chemical Risk Evaluations Through Use of New Approach Methods(NAMs): Challenges and Opportunities

Poster Session Abstracts
Poster ID

Presenting Authorand Affiliation

Poster Title

1

Yusuke Yamamoto
Fuji Film

Application of ADRA to various sensitizationassessments

2

Hideyuki Mizumachi
Kao

Reconstructed human epidermis-based skin sensitization
method called Epidermal Sensitization Assay (EpiSensA):
validation study and development of defined approach3

Takayuki Abo
Kao

The Short Time Exposure test method that the applicability
domain was expanded and the defined approach inassessing eye irritation potential with combination of in vitro
test methods as future new guideline

4

Izuru Mizoguchi
Tokyo Medical University

A novel alternative method for evaluation of sensitizing
potential by T cell

5

Etsushi Kuroda
Hyogo college of
medicine

Development of an evaluation for inflammatory particles
based on the activation of alveolar macrophages

6Hajime Kojima
NIHS

Update on Japanese projects regarding the New Approach
Method7

Yuhji Taquahashi
NIHS

A new approach to predicting lethality in acute toxicity studies;
Make vital signs simple to measure

8

Yusuke Okubo
NIHS

Establishment of developmental toxicity test based on the
integration of FGF signal disruption effects for safety
evaluation of drugs and chemicals using human iPS cells

9

Scott Slattery
ScitoVation

NAM-based prediction of point-of-contact toxicity in the lung: acase example with 1,3-dichloropropene

10

Jamie Scaglione
ScitoVation

Development and use of human 2D and 3D hepatic co-culture
models as alternatives to traditional hepatotoxicity testing

11

Sylvia Escher
Fraunhofer Institute for
Toxicology andExperimental
Medicine, Germany

Zet-O-Map- Identify principal molecular drivers of(dys)morphogenesis in zebrafish

12

Yuhei Nishimura
Mie University

Generation of a triple-transgenic zebrafish line forassessment of neuroinflammation in developmental
neurotoxicity

13

Yuko Sekino
The University of Tokyo

Proposal of a new AOP for the neurotoxicity anddevelopmental neurotoxicity assessment of glutamate
receptor binding agonists that cause learning and memory
impairment

14

Yaichiro Kotake
Hiroshima University

Searching for developmental neurological evaluation indicesfor chemical toxicity assessment

15
16

Keishi Ishida
Gifu Pharmaceutical
University
Michael Black
ScitoVation

Validation of brain neuronal differentiation reporter mice for
improved developmental neurotoxicity evaluation
Application of toxicogenomics in next-generation risk
assessment and predictive toxicology

17Mark Viant
University of Birmingham,
UK

MetAbolomics ring-Trial for CHemical groupING(MATCHING)-Assessing the Reproducibility of Metabolomics
Within a Regulatory Context Through a Multi-laboratory Ringtrial (LRI C8)18

Maiko Okamoto
Kao

Prediction of Repeated Dose Toxicity with Read-acrossBased on IATA

19

Bruno Hubesch
Hubesch Consult

Linking LRI AMBIT3 chemoinformatic system with the
IUCLID6 substance database to support read across of
substance endpoint data and category formation

20

George Daston
Procter & Gamble, US

Toxicogenomics and Read Across21

Masashi Kamo
AIST

Addressing some issues to expand the use of species
sensitivity distributions in ecological risk assessment

22

Kota Hirasawa
Sumitomo Chemical

Parameter acquisition with humanized chimeric mice, forhuman exposure prediction using a PBPK model

23

Aldert Piersma
RIVM, Netherlands

Developing a quantitative AOP for liver-mediated thyroid
modulation after prenatal exposure to a xenobiotic
compound in the rat

24

Jeremy Fitzpatrick
ScitoVation

Release of Population Life-course Exposure to Health
Effects Model (PLETHEM): An Online Tool for PBPK
Modeling version 3.2

25

Hiroaki Todo
Josai University

In silico estimation of skin permeation of chemicals with
their diffusion and partition parameters

26

Masanori Niwano
Sumitomo Chemical

An application of a 3D hydrodynamic ocean model topredict chemical behavior at waters off Niihama in Seto
Inland Sea

27

Yuriko Ishikawa
AIST

Development and application of a spatio-temporalchemical concentration estimation model for watershedrisk assessment

28

Kazuhide Kimbara
Graduate School ofIntegrated Science andTechnology, Shizuoka
University

A multi-model system for predicting biodegradation rates
and the presence of residual substances of chemicals
using chemical descriptors and 3D structures

29

Kaku Oguro
NITE

Evaluation of slow-stirring method for determining log Kow(n-octanol/water partition coefficient) of surfactants

30

Jon Arnot
ARC Arnot Research andConsulting

EAS-E Suite: A data integration and modelling framework
for chemical safety and sustainability

1 – Application of ADRA to various sensitization assessments
Yusuke Yamamoto1, Masaharu Fujita1, Toshihiko Kasahara1, Yasuhiro Katsuoka1
1

FUJIFILM Corporation, Safety Evaluation Center, Kanagawa, Japan

The Amino acid Derivative Reactivity Assay (ADRA) is an in chemico test method that evaluates thebinding of chemicals to proteins, which is a molecular initiating event (Key event 1) in the adverse outcome
pathway (AOP) of skin sensitization. In Key event 1, since sensitizers mainly bind to cysteine and lysine inproteins, ADRA uses cysteine and lysine derivatives N-(2-(1-naphthyl)acetyl)-L-cysteine (NAC) and N-(2(1-naphthyl)acetyl)-L-lysine (NAL) as nucleophiles and predicts skin sensitization from the reactivity ofthese nucleophiles with the test chemical. ADRA was adopted in the OECD test guidelines (TG 442C) in2019. The following three studies have been conducted so that ADRA can be used for various evaluations:
1) application to mixture evaluation, 2) establishment of photoallergy evaluation method, 3) establishmentof respiratory sensitization evaluation method. In this presentation, we report on these three studies.
Evaluation of the mixture by ADRA requires test conditions that do not use molecular weight for preparing
test chemical solution and are not interfered by the peaks derived from the components of the mixture in
HPLC measurements. Therefore, we attempted to evaluate the mixture by combining the evaluation
conditions using the test chemical solution prepared at the weight concentration and the measurement of
NAC and NAL by the fluorescence detection. As a result, it was confirmed that the sensitizer contained in
the mixture solution of known non-sensitizer could be detected as in the case of the sensitizer alone, andboth NAC and NAL could be quantified selectively even in the plant extract having many peaks. We are
currently applying for revision to the test guidelines that add this evaluation method.
In the early stage of photoallergy, photo-excited chemicals bind to proteins. Therefore, the test chemical
and NAC and NAL were reacted under photo and non-photo irradiation conditions, and it was verified
whether the photoallergic potential could be evaluated from the difference in depletion between the photoand the non-photo irradiation conditions. As a result, the predictive capacity vs human in 59 chemicals was
as high as 81.4% (48/59).
It is known that many respiratory sensitizers bind to lysine residues in proteins. Focusing on this feature, it
was verified whether respiratory sensitizers could be detected by selectively evaluating the reactivity to
NAL. As a result, all 29 respiratory sensitizers could be correctly classified, except for substances outside
applicability domain.
From the above, it was shown that by applying the ADRA technology, it is possible to evaluate a wider
variety of substances such as mixtures, and it may be used for toxicity evaluation other than skinsensitization.

2022 ICCA LRI & NITE Workshop Poster Presentations3

2 – Reconstructed human epidermis-based skin sensitization method called Epidermal
Sensitization Assay (EpiSensA): validation study and development of defined approach
Hideyuki Mizumachi1, Sho Suzuki1, Masaaki Miyazawa1
1

Kao Corporation, R&D safety Science Research, Tochigi, JapanThe development of strategy for non-animal safety assessment has recently been stimulated, and seveneffective alternative methods for skin sensitization have already been adopted to OECD test guideline. In
addition, testing strategies combining multiple assay results, Defined Approaches (DAs), have also been
adopted to OECD test guideline. However, there are some common limitations on existing alternative
methods. One of them is the applicability for lipophilic chemicals (e.g., LogKow >3.5) because all existing
methods are based on aqueous system such as culture medium or buffer. Furthermore, it is easy tounderstand that DAs also have the same limitation. Moreover, pre/pro-haptens can be common limitations
because existing methods are based on cell line or in chemico method. On the other hand, lipophilic
chemicals can be directly applied on reconstructed human epidermis (RhE) like animal test because RhE
have stratum corneum. In addition, RhE may be effective to detect pre/pro-haptens because they havemetabolic capability like real human skin (Oesch et al., 2018). EpiSensA, developed by Kao Corporation, is
RhE-based assay and the promising method to overcome these limitations. Regarding the method of
EpiSensA, a test chemical is exposed on the RhEs and incubated for 6 hours, and the fold inductions of four
marker genes of RhEs (ATF3, GCLM, DNAJB4, and IL-8) are measured by real-time PCR. When at least
one out of the four marker genes exceed the respective cut-off values, the test chemical is judged as positive.
In the past, 136 chemical datasets including 69 lipophilic ones and 37 pre/pro haptens have been tested by
EpiSensA, and an accuracy of 83% and broad applicability domain for various chemicals were confirmed.
The international validation study in which three laboratories participated was performed under the JaCVAM
using 27 chemicals. As a result, within- and between-laboratory reproducibility met the target criteria (85%and 80%, respectively), and the validation management team concluded that sufficient reproducibility wasconfirmed.
Moreover, EpiSensA-based DA called RhE-based Testing Strategy (RTS) for hazard and potency
identification was developed. RTS is simple score-based DA and consist of EpiSensA and the Times
Metabolism Simulator platform used for predicting Skin Sensitization (TIMES-SS, OASIS-LMC). Results of
EpiSensA and TIME-SS were converted to scores (from 0 to 3) based on the minimum estimatedconcentration at which any of four marker genes exceeds the respective cut-off (min.EC value) and potency
prediction of TIMES-SS, respectively. The hazard and potency identification of RTS was performed from thesum of scores. The predictive performance of RTS was compared to existing DAs adopted as OECD
Guideline No.497. It was confirmed that RTS had broader applicability domain than existing DAs at the 136chemical dataset. Furthermore, the hazard and potency accuracies of RTS were 85% and 74% for GHS
categorization based on LLNA results, respectively. In addition, it was confirmed that the predictive
performances were comparable to or greater than those of existing DAs such as 3 out of 3 DA or ITS v1/v2
at 136 chemicals.

2022 ICCA LRI & NITE Workshop Poster Presentations4

3 – The Short Time Exposure test method that the applicability domain was expanded and thedefined approach in assessing eye irritation potential with combination of in vitro test methods as
future new guideline.
Takayuki Abo
Kao Corporation, Safety Science Research Laboratories
The Short Time Exposure (STE) test method has been adopted as test guideline 491 by Organisation for
Economic Co-operation and Development (OECD) since 2015. The STE test method can identify
chemicals that induce serious eye damage, defined as Category 1 by the United Nations Globally
Harmonized System of Classification and Labelling of Chemicals (GHS), and chemicals that do not cause
eye irritation or serious eye damage, defined as No Category by GHS. However, any in vitro test methods
including the STE test method can’t identify chemicals that induce eye irritation, defined as Category 2 by
GHS. In addition, the STE test method had possessed two out of applicability domains.
For two applicability domains, the one of two applicability domains was highly volatile substances (6kPa, 25
degrees C). In 2020, TG 491 was updated for expansion of the out of applicability domain for highly volatile
substances. This update led that the STE test method can apply the almost all liquids. The point of updateis that all highly volatile substances employ mineral oil (CAS RN 8042-47-5) as a solvent. As a results forhighly volatile substances, the accuracy, false positive rate, false negative rate was updated from 75%(15/20), 7.7% (1/13), 57.1% (4/7) to 95.0% (19/20), 7.7% (1/13), 0% (0/7) (Abo et al., 2018).
For the classification of Category 2, two defined approaches for liquids other than surfactants were
proposed and discussed in OECD expert group. These two defined approaches would be accepted in 2022
at the earliest. The one of two defined approaches is the combination of the STE test method (TG 491) andthe Bovine Cornea Opacity and Permeability Laser-Light Based Opacitometer test method (BCOP LLBO,
TG 437). This defined approach classifies GHS No Category or Category 1 by the STE test method first,
continuously to classify GHS Category 1 or Category 2 by the BCOP LLBO test method. The accuracies
are 81.2% for Category 1, 56.3% for Category 2, and 85.3% for No Category (Alépée et al., 2019).
Furthermore, using the above defined approach, the principle was demonstrated by case studies withchemicals.
In conclusion, it is indicated that the defined approach can identify three GHS categories including
Category 2 for eye irritation potential with updated the STE test method. Thus, to identify three GHS
category can be to lead completely alternative to in vivo tests. After this defined approach has been
adopted as guideline by OECD, it should be further extended for the availability to be adopted to authority
bodies in various countries.

2022 ICCA LRI & NITE Workshop Poster Presentations5

4 – A novel alternative method for evaluation of sensitizing potential by T cellIzuru Mizoguchi1, Yasuhiro Katahira1, Shinya Inoue1, Eri Sakamoto1, Aruma Watanabe1, Yuma Furusaka1,
Ami Sekine1, Satomi Miyakawa1, Jukito Sonoda1, Miu Yamagishi1, Toshihiko Yoneto1, Toshiyuki Owaki1,
Mingli Xu1, Fumihiro Murakami1, Hideaki Hasegawa1, and Takayuki Yoshimoto1
1

Department of Immunoregulation, Institute of Medical Science, Tokyo Medical University, 6-1-1 Shinjuku,
Shinjuku-ku, Tokyo 160-8402, Japan
There are mainly two types of allergic responses: one is skin sensitization, that is an allergic response in
the skin following skin contact such as allergic contact dermatitis, and another is respiratory sensitization,that is an allergic response in the airways caused by inhalation, mostly asthma. Because a worldwide
movement emerged to limit the use of animal models in safety testing of chemicals, several in vitro assays
to predict the skin and respiratory sensitizing potential of chemicals have been developed. However, noneof these alternative methods can distinguish between chemical respiratory sensitizers and skin sensitizers.
The adverse outcome pathway (AOP) for skin and respiratory sensitization was described to accurately
develop alternative methods for evaluation of them. The AOP contains four key events (KE). All currently
developed and validated in vitro assays are based on KE1~3, and there is no validated in vitro assay
based on KE4 which is the activation of T cells so far.
To mimic the human airway upper epithelium, we recently developed a novel three-dimensional DC
coculture system consisting of upper airway epithelial cells, immature DCs, and lung fibroblast cells
cultured in individual scaffolds. In the recent study, to improve the versatility, peripheral monocyte-derived
immature DCs were similarly replaced with immature DCs derived from monocyte-derived proliferating
cells called CD14-ML established by introducing genes related to the cell cycle and survival and lungfibroblast cell lines were removed from this system. Moreover, to recapitulate in vivo activation of naive
CD4+ T cells by DCs that are stimulated with chemical sensitizers in the upper airway epithelium and thenmigrated into draining lymph nodes, we sought to establish a new 2-step DC/T coculture system by further
adding peripheral allogeneic naive CD4+ T cells onto the DCs stimulated in the DC coculture system. In
this DC/T coculture system, mRNA up-regulation of Th2 maker IL-4 in the T cells representing the KE4
was successfully used to discriminate respiratory sensitizers from skin sensitizers 5 days after the
stimulation. Finally, when allogenic Th2 cell lines established by repetitive stimulation of allogenic CD4+ T
cells with DCs were used instead of primary naive CD4+ T cells, selective mRNA up-regulation of IL-4 wasobserved by the stimulation with respiratory sensitizers 12 hours after the stimulation. Furthermore, Upregulation of IL-4 at protein level was detected by ELISA 48 hours after the stimulation. Currently, we arecontinuing to evaluate and improve this 2-step DC/T coculture system.

2022 ICCA LRI & NITE Workshop Poster Presentations6

5 – Development of an evaluation for inflammatory particles based on the activation of alveolar
macrophages.
Prof. Etsushi Kuroda, PhD
Department of Immunology, School of Medicine, Hyogo Medical University.
It has been reported that inhalation of fine particles, such as PM2.5 and sand dusts, are associated withallergic airway inflammation. So far, many studies have demonstrated that some particles stimulate
immune systems to induce allergic immune response in the airway. These inflammatory particles arethought to be adjuvants, which induce or enhance antigen-specific immune responses. In general,pathogen-associated molecular patterns (PAMPs) such as LPS and bacterial DNA stimulate innate immune
cells such as dendritic cells and macrophages and then activated innate cells induce acquired immune
responses. The same way as PAMPs, several inorganic particles also function as adjuvant, however, themechanisms for the adjuvant activity of particles remains unclear.
As inhaled particles are thought to settle deep in the lungs and to engulfed by alveolar macrophages toclear them from the lungs, we investigate the mode of action of inhaled particles on the activation ofalveolar macrophages. we used aluminum salts (alum) as inflammatory particles and previously
demonstrated that alum stimulated alveolar macrophages to induce cell death and then released
interleukin-1alpha (IL-1) as dead cell-derived factor, called damage-associated molecular patterns(DAMPs). We also observed that released IL-1 was involved in induction of serum immunoglobulin E (IgE).
However, non-inflammatory aluminum oxide (Al2O3) particle did not induce cell death and IL-1. These
results suggest that alveolar macrophage death by inflammatory particles and subsequently released
DAMPs are closely linked to allergic immune responses. Based on these findings, we thought these
phenomena could be applied to an evaluation method to identify “danger” particles.
In this presentation, we will introduce our recent studies regarding the mechanisms of DAMPs release frominflammatory particle-stimulated alveolar macrophages and the application to evaluation method to identify
inflammatory particles.

2022 ICCA LRI & NITE Workshop Poster Presentations7

6 – Update on Japanese projects regarding the New Approach Method
Hajime Kojima, Ph.D.
JaCVAM, NIHS, Japan
We have ongoing projects that are developing New Approach Methods (NAMs) for systematic toxicology.
One NAM is to develop in vitro immunotoxicity test methods for the Organisation for Economic Cooperation and Development (OECD)Test Guideline (TG) and the other is to develop non-animal
development and reproductive toxicity test methods for the International Council for Harmonization ofTechnical Requirements for Pharmaceuticals for Human Use (ICH) S5(R3) guideline and OECD TG. The
development of these NAMs includes the following steps: 1) adverse outcome pathway (AOP), 2) detailed
review paper, 3) test methods based on AOP, 4)validation study of test methods for developing TGs, and5) integrated approaches to testing and assessment (IATA). I believe that the NAMs developed on thesesteps may enable risk assessment of a chemical with non-animal test methods in near future.

2022 ICCA LRI & NITE Workshop Poster Presentations8

7 – A new approach to predicting lethality in acute toxicity studies; Make vital signs simple tomeasureYuhji Taquahashi1, Shuji Tsuruoka2, Ken-ich Aisaki1, Yusuke Okubo1 and Satoshi Kitajima1
1Division of Cellular and Molecular Toxicology, Center for Biological Safety and Research, National Instituteof Health Sciences
2

Shinshu University, Research Institute for Supra-Materials

The endpoint of acute toxicity is "death", which is the ultimate toxicity, and the LD50 value, introduced by
Trevan (1927), have been used because of the convenience of showing the toxicity intensity of compounds as
a common index. On the other hand, the acute toxicity test was criticized for scientific, economic, and animal
ethical reasons (Zbinden 1981), the OECD TG401 using many animals was deleted in 2002, and an improved
method with a reduced number of animals was adopted (OECD TG420, TG423 and TG425). However, thecriticism from the viewpoint of animal welfare has not converged; the animal mortality is still used as theendpoint (TG423, TG425), or evident toxicity (TG420), which is a less objective endpoint and depending on
experimenter. Moreover, the significant shortcoming of acute toxicity study is that it is not useful for the
medical treatment of human poisoning because the cause of death, target organs, etc. are not considered atall. In order to solve these issues concerning acute toxicity study, it is great way to obtain valid result with
precisely measure and quantify the signs of toxicity over multiple items and make rational criteria for
determining the acute toxicity intensity of substances and toxic targets. This will be, however, a complicatedand expensive test compared to acute toxicity in which death is used as endpoint. In other words, it is thedevelopment of a test method that obtains vital signs from animals corresponding to tests that human acutely
poisoned patients undergo in emergencies. For modernization of acute toxicity, we are focusing on vital signs
and wearable devices with cutting-edge sensors; the method makes vital signs simple to measure. We
established the measurement of ECG and EEG using a novel material, carbon-nanotube yarn (CNT-Y) madefrom high-purity, highly crystalized, double-walled carbon nanotubes. CNT-Y is an advanced material withexcellent electrical conductivity and flexibility; hence, it could potentially be used as a novel electrode forbiopotential measurements. CNT-Y is flexible like silk thread, it functions as an electrode when sewn directly
to the skin. We obtained a clear ECG waveform from rats and a guinea pig; the QRS amplitude was ~1.4 mV.
In rats, we obtained an EEG waveform with an amplitude of ~150 µV; the peak frequency was 0.8 Hz and therange was ~3 Hz according to power spectral density analysis. In the guinea pig, we obtained an EEG
waveform with an amplitude of ~500 µV; the first peak was 0.1 Hz, the second peak was 1 Hz, and the range
was ~3 Hz. These results show that CNT-Y could be used in toxicology studies to easily and inexpensively
obtain high-resolution biological signals. The less invasiveness and easy to measure biopotential are
expected to have a ripple effect on toxicity tests. (Health and Labour Sciences Research Grant, Japan)

2022 ICCA LRI & NITE Workshop Poster Presentations9

8 – Establishment of developmental toxicity test based on the integration of FGF signal disruption
effects for safety evaluation of drugs and chemicals using human iPS cells
Yusuke Okubo1, Seiya Kanno2, Satoshi Kitajima1, Yoko Hirabayashi3, Junji Fukuda2
1

National Institute of Health Sciences, Division of Cellular and Molecular Toxicology, Center for
Biological Safety and Research, Kawasaki, Japan
Yokohama National University, Faculty of Engineering, Yokohama, Japan

2

3

National Institute of Health Sciences, Center for Biological Safety and Research, Kawasaki, JapanThe number of man-made chemicals including medicines has increased exponentially recently, and exposure
to some of them is possible to induce fetal malformations. Although the toxicities of chemicals have beentested in animals, chemicals that are not teratogenic in rodents can cause severe malformations in humans,
owing to the differences in the susceptibility to the teratogenicity of chemicals among species. One possible
cause of such species differences, other than pharmacokinetics, could be the difference in sensitivity to suchchemicals at the cellular level. To clarify this possibility, in vitro assays using human cells would be a powerfultool. In particular, the analysis of intracellular and intercellular signaling pathways in vitro may be useful as
one of the important factors that may generate differences in cellular susceptibility to chemicals among
species. Because these signaling pathways play important roles in both developmental processes and theirrobustness from intrinsic and extrinsic noises. We hypothesized that the developmental toxicity itself means aresult that the signal pathways were disrupted regardless of the causes. Then, we established a signal
reporter assay system based on real-time monitoring and the time-accumulation of the signal disruption overtime, rather than the classical endpoint detection of the signal disruption. This approach was useful for
detecting signal disruptions caused by the malformation chemicals, including thalidomide[1, 2]. The human
iPSC-based signal disruption assay could be a promising tool for the initial screening of developmental
toxicants.
References[1] Kanno, S. et al., 2022, ‘Establishment of a developmental toxicity assay based on human iPSC reporterto detect FGF signal disruption', iScience, 25, 103770, CellPress[2] Kanno, S. et al., 2022, ‘Integrated fibroblast growth factor signal disruptions in human iPS cells forprediction of teratogenic toxicity of chemicals’, Journal of Bioscience and Bioengineering, 133 (3), 291-299,
ELSEVIR
Keywords: Developmental toxicity testing, Alternative assays, Signal disruption, Human pluripotent stem cells,
FGF signaling

2022 ICCA LRI & NITE Workshop Poster Presentations10

9 – NAM-based prediction of point-of-contact toxicity in the lung: a case example with 1,3dichloropropene
Marjory Moreau, Jeff Fisher, Melvin E. Andersen, Asayah Barnwell, Sage Corzine, Aarati Ranade, Patrick D.
McMullen, and Scott Slattery.
ScitoVation, LLC, Durham, North Carolina
Time, cost, ethical, and regulatory considerations surrounding in vivo testing methods render them insufficient
to meet existing and future chemical safety testing demands. There is a need for the development of in vitro
and in silico alternatives to replace traditional in vivo methods for inhalation toxicity assessment. Exposures oforganotypic airway epithelial cultures to gases or aerosols can assess tissue responses and in vitro-in vivo
extrapolation can align in vitro exposure levels with in-life exposures expected to give similar tissue exposures.
The airway epithelium varies along its length, with different regions composed of different cell
types. Therefore, in this study a volatile compound of known toxicity, 1,3-dichloropropene (1,3-DCP), wasadministered as a vapor at the air liquid interface to five organotypic in vitro airway epithelial cell culture
models—MucilAir of nasal, tracheal, or bronchial origin, SmallAir, and EpiAlveolar—that represent five regionsof the airway epithelium—nasal, tracheal, bronchial, bronchiolar, and alveolar. Toxicity was monitored inthese cultures 24 hours after acute exposure using an assay for transepithelial electrical conductance (forepithelial barrier integrity) and the LDH release assay (for cytotoxicity). Finally, an airway dosimetry model for
1,3-DCP vapor was developed to predict in vivo external exposure scenarios that would produce toxic local
tissue concentrations as determined by in vitro experiments. Measured in vitro points of departure (PoDs) for
all tested cell culture models were similar (109—194 ppm). Calculated rat equivalent inhaled concentrations
varied by model according to position within the airway, with nasal respiratory tissues being the most proximaland most sensitive tissue (191 ppm), and alveolar epithelium being the most distal and least sensitive tissue(427 ppm). These predictions are qualitatively in accordance with empirically determined in vivo PoDs. The
predicted PoD concentrations were close to, but slightly higher than, PoDs reported for in vivo subchronic
studies.

2022 ICCA LRI & NITE Workshop Poster Presentations11

10 – Development and use of human 2D and 3D hepatic co-culture models as alternatives to
traditional hepatotoxicity testing
S. Corzine1, R. Samuel1, P. Kumar1, S. Slattery1, A. Ranade1, and J. Scaglione1
1

ScitoVation LLC, Durham, NC

New approach methodologies (NAMs) are gaining importance as an alternative to traditional in vivo
toxicity testing, as the latter involves high cost and time requirements as well as ethical considerations.
Regulatory agencies and industry are developing biologically relevant in vitro cell models for efficiently
and accurately predicting the toxicity of chemicals. The liver has been a major focus of these efforts,
yet there are currently no in vitro alternatives for hepatotoxicity testing accepted by regulators. While
hepatocytes are the primary component of the liver, the liver non-parenchymal cells (NPCs) also play
an important role in hepatotoxicity. Our goal was to develop 2D and 3D in vitro models using cocultures of cryopreserved human hepatocytes and NPCs as an alternative to traditional toxicity testingof pharmaceutical compounds and commodity chemicals. These models are characterized by thepresence of hepatocyte-specific markers (Albumin, CYP3A4) and NPC-specific markers (Desmin,
ACTA2, LYVE1) using qRT-PCR and by immunostaining for albumin, LYVE1, and Vimentin. The utilityof these 2D and 3D hepatic co-culture models to measure the cytotoxicity response of compounds wasevaluated by studying dose-response profiles of 12 compounds. As a control model for comparison,
human hepatocytes plated as mono-cultures were also assayed. The compounds tested wereacetaminophen, aflatoxin B1, tamoxifen, acetochlor, flusilazole, carbaryl, bisphenol A, triflumizole,
ethofumesate, ametryn, dimethenamid, and sucrose with 9 concentrations per compound including anappropriate media control. Cytotoxicity and viability were measured using CellTox Green Assay and
CellTiter Glo Assay, respectively. Also, the number of dead cells were measured using high content
imaging techniques. We will categorize the test compounds as having low, medium, or high cytotoxicity
using the dose-response curves and IC50 values. We also observed that the co-culture model
produces more consistent and reproducible dose-response curves, and for some compounds (aflatoxin
B1 and ametryn) presence of NPCs helps reduce the cytotoxic effect of the chemical. Species
comparison by implementing the ‘parallelogram approach’ for IVIVE comparisons is in progress using
data generated in ScitoVation’s rat 2D and 3D models. Further investigation and ongoing detailed
analysis of 2D and 3D co-culture systems seek to determine the optimal system for capturing responseto chemical exposure that is most representative of in vivo response.

2022 ICCA LRI & NITE Workshop Poster Presentations12

11 – Zet-O-Map - Identify principal molecular drivers of (dys)morphogenesis in zebrafish
Matthias Wehr1, Bruce Schultz², Dr. Hilda Witters³, Dr. Marc Jacobs², Dr. Sylvia E. Escher1,*1

Fraunhofer Institute for Toxicology and Experimental Medicine - ITEM

² Fraunhofer Institute for Algorithms and Scientific Computing - SCAI
³ Flemish Institute for Technological Research NV – VITO/HEALTH*Presenting author
The zebrafish embryo-toxicity test (ZET) is a promising alternative test for developmental toxicity, sinceabnormal developments can be directly observed microscopically in the transparent developing embryo; its
biology has shown to be relevant to humans and zebrafish cells are compatible with high throughput
sequencing (Hoffmann et al., 2021). The combination of transcriptome and morphological changes will allow
a better understanding of the mechanisms of teratogenicity, an essential prerequisite for the integration ofthis test as an alternative method in human risk assessment.
The zet-o-map project aims to identify predictive time-resolved transcriptome biomarkers for teratogenic
effects. For this purpose, published data such as peer-reviewed publications and data from the Gene
Expression Omnibus (GEO) repository were integrated into two databases:
1) The Zebrafish Teratogens database (ZeTera), which includes morphological changes per target, doselevel and timepoint. Data curation included the development of ontologies.
2) Transcriptome ZET data from array-based (e.g. Affymetrix/Agilent) as well as from high-throughput RNA
sequencing approaches (e.g. Illumina).
Overall data from various experimental settings were considered up to a treatment period of five days.
Experiments include chemical perturbations carried out at specific stages of embryonic development but also
experiments on unperturbed zebrafish embryos. All transcriptome data were reanalysed starting with raw
data files. For high throughput transcriptome, an adapted version of the recently proposed Omics Data
Analysis Framework (ODAF) workflow was used (Verheijen et al., 2022).
In total, metadata for 1523 GEO series were identified comprising data from 41 platforms (array based or
high-throughput RNA-sequencing). A subset of 392 GEO series from the 11 most frequently used platforms
were reanalysed, resulting in about 300 compounds with transcriptome data.
The comparison of 1400 chemicals with morphological effect data, collected in ZeTera, with the 300
compounds with omics data resulted in an overlap of 92 “data rich” compounds. The majority of thesechemical stressors are teratogenic compounds, which induced morphological alterations in zebrafish
embryos. For the identification of developmental-stage specific transcriptome biomarkers, we grouped datarich compounds into read-across categories with similar modes of action. A data gap analysis revealed thattranscriptome data for several relevant time points are lacking for many individual compounds as well as for
categories with similar modes of action.
The above outlined approach, as well as the spatio-temporal maps are exemplified for selected read-acrossgroups and further analyses approaches e.g. the integration of data into knowledge maps are discussed.
Key words: zebrafish embryo-toxicity test, developmental toxicity, transcriptomics, morphological alteration
Acknowledgment: This project is funded by CEFIC within the framework of the Long-Range Initiative (LRI)
as project no. LRI-C9: Zet-O-Map. This project is further supported by two experts in the field of zebrafish
research: Prof. Robyn Tanguay, Oregon State University, and Dr. Kristen Ryan, National Institute ofEnvironmental Health Sciences (NIH), leading the NIH SEAZIT project.

Verheijen, M. C. T. et al. (2022). "R-ODAF: Omics data analysis framework for regulatory application."

2022 ICCA LRI & NITE Workshop Poster Presentations

13

Regulatory Toxicology and Pharmacology 131: 105143.
Hoffmann, S. et al. (2021)."A systematic review to compare chemical hazard predictions of the zebrafish
embryotoxicity test with mammalian prenatal developmental toxicity." Toxicological Sciences 183 (1): 14-35.

2022 ICCA LRI & NITE Workshop Poster Presentations14

12 – Generation of a triple-transgenic zebrafish line for assessment of neuroinflammation indevelopmental neurotoxicity
Yuhei Nishimura1, Mizuki Yuge1, Eri Wakai1, Junko Koiwa1, Takashi Shiromizu1, and Munekazu Komada2
1

Department of Integrative Pharmacology, Mie University Graduate School of Medicine, Tsu, Mie, Japan
Department of Life Science, Faculty of Science and Engineering, Kindai University, Higashiosaka, Osaka,Japan
2

The incidence of neurodevelopmental disorders such as autism, attention deficit hyperactivity disorder, andlearning disabilities is increasing year by year and has become a major social problem. Although chemical
exposure during development has been suggested to increase the risk of developing theseneurodevelopmental disorders, the detailed mechanisms underlying developmental neurotoxicity remain
largely unclear. Various molecular initiating events are involved in the developmental neurotoxicity
mechanisms of chemical substances, but different molecular initiating events often exert toxicity through acommon key event. Developing a test method that can evaluate such a common key event is an effective
strategy that will lead to the elucidation of the adverse outcome pathway of chemical substances. Impaired
differentiation of neural stem cells into neurons and astrocytes, and neuroinflammation mediated by microglia,
which are resident immune cells of the brain parenchyma, are attracting attention as the key event common to
the developmental neurotoxicity of chemical substances. We have developed a transgenic zebrafish line
selectively expressing fluorescent proteins mVenus, mCherry, and Cerulean in microglia, astrocytes, andneurons. Using in vivo fluorescence microscopy, we have assessed the change of microglia, astrocytes, andneurons in the transgenic zebrafish developmentally exposed to neurotoxicants, especially focusing on
ethanol. In this presentation, we demonstrate how the triple-transgenic zebrafish line can be used to assess
neuroinflammation in developmental neurotoxicity.

2022 ICCA LRI & NITE Workshop Poster Presentations15

13 – Proposal of a new AOP for the neurotoxicity and developmental neurotoxicity assessment ofglutamate receptor binding agonists that cause learning and memory impairment.
S Mase1,6, T Mitsuoka1,2, H Yamazaki3,6, Y Kanemura4, D Yamazaki5, N Koganezawa6, Y Sekino1,7
1

Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan
Faculty of Pharmaceutical Sciences, Hokkaido University of Science, Hokkaido, Japan
3
Faculty of Social Welfare, Gunma University of Health and Welfare, Gunma, Japan
4
Institute for Clinical Research, National Hospital Organization Osaka National Hospital, Osaka, Japan
5
Division of Pharmacology, National Institute of Health Sciences, Kanagawa, Japan
6
Graduate School of Medicine, Gunma University, Gunma, Japan
7
Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, Japan2

The purpose of our LRI project is to propose an adverse outcome pathway (AOP) in which the molecular
initiating event (MIE) is the binding of a compound to the glutamate receptors, resulting in the adverse
outcome (AO) defined as learning and memory impairment. First, we have established an algorithm for highcontent imaging to detect dendritic spines with drebrin immunoreactivity, and an image analysis method bymachine learning. Then, morphological changes of dendritic spines were quantitatively evaluated using frozen
hippocampal nerve cells from rat embryos and neurons derived from human iPS cells. We will validate thepossibility as an alternative method for developmental neurotoxicity/neurotoxicity testing, using known
compounds with learning and memory impairment.
In Japan, a study group of the Ministry of Health, Labor and Welfare has reported the hazard risks of chronicuse of cannabis during juvenile. However, the mechanisms of the risks are still unclear. In this study, we haveinvestigated if the chronic exposure to cannabis during juvenile has hazardous effects, using cultured rat
hippocampal neurons. The cultured rat hippocampal neurons in 96-well plates were prepared from frozen
embryonic rats’ hippocampal neurons (SKY neuron, AlzMed, Inc., Tokyo). At 7, 14, or 21days in vitro,
CP55940 (from 100 nM to10 μM) was applied depending on the schedule. After the immunocytochemical
staining to visualize drebrin, MAP2, and cell nucleus, the quantitative analyses of neuron number, dendrite
length, and drebrin clusters were performed using our algorithms. CP55940 significantly changed distributionof drebrin as well as increased the numbers of drebrin clusters. Further, treatment of 10 μM CP55940 at 7, 14
days in vitro significantly reduced neuron numbers. It has been discussed how cannabinoids affect thejuvenile brain, and lead to cognitive dysfunction and emotional abnormality in adulthood. Because drebrin
plays a key role in dendritic spine formation of neurons and the morphological plasticity of dendritic spines,changes in drebrin distribution might change glutamate signaling. Identification of drebrin clusters andanalysis of their numbers and distribution are promising to detect hazard risks of cannabis.
This work was supported by the Japan Chemical Industry Association (JCIA) Long-range Research Initiative(LRI) to YS , and the Regulatory Science Research Grant from MLHW(2020-2022) to YS

2022 ICCA LRI & NITE Workshop Poster Presentations16

14 – Searching for developmental neurological evaluation indices for chemical toxicity
assessment
Yaichiro Kotake1, Yuki Takehara1, Ryoga Fujihara1, Tomoki Takabayashi1, Kazuki Takeda1, Kanae UmedaMiyara1, Masatsugu Miyara1, Yasunari Kanda2
1

Grad. Sch. of Biomed. Health. Sci,, Hiroshima Univ., Japan
Div. of Pharmacol,, Natl. Inst. of Health Sci., Japan

2

Although there are standardized methods for evaluating the effects of chemical substances that areconsidered to be highly sensitive during development on the cranial nerves, they require a great deal of laborand involve the sacrifice of a large number of experimental animals, and a simpler and more accurate
evaluation method is desired. In recent years, basic research in the field of neuroscience has revealed anumber of molecules involved in neurite outgrowth and neuronal circuit reorganization, but few attempts havebeen made to evaluate chemical effects using these molecules as indicators. The purpose of this study was
to identify key molecules in neurodevelopment that can be used as better indicators to assess neurotoxicity ofchemicals during development and to clarify their usefulness in assessing neurotoxicity of chemicals during
development.
We established culture conditions for primary cultured rat cerebral cortex neurons and examined changes inmRNA expression encoding 12 molecules that play key roles in neurodevelopment. The results showed that
there were three major patterns: genes whose expression was low in early culture and increased with culture,genes whose expression was maximal in mid-culture, and genes whose expression was maximal in early
culture and then decreased with culture. Next, methylmercury, tributyltin, and acrylamide were used as known
developmental neurotoxicants, and the effects of these molecules on gene expression were examined. The
results showed that the expression of three genes, Dlg4, Syp, and Bdnf, fluctuated at specific time points. By
examining whether these genes are also altered by many other suspected developmental neurotoxicants,
useful developmental neurotoxicity markers may be identified.

2022 ICCA LRI & NITE Workshop Poster Presentations17

15 – Validation of brain neuronal differentiation reporter mice for improved developmental
neurotoxicity evaluation.
Keishi Ishida1, Yoshiki Minamigawa1, Kanoko Tatsumi1, Kazuma Mori1, Daisuke Matsumaru1, Hisamitsu
Nagase2, Yasunari Kanda3, Kazuhiro Takuma4, Tsuyoshi Nakanishi1
1

Laboratory of Hygienic Chemistry and Molecular Toxicology, Gifu Pharmaceutical University
Faculty of Pharmacy, Gifu University of Medical Science3
National Institute of Health Sciences
4
Graduate School of Dentistry, Osaka University
2

Developmental neurotoxicity (DNT) caused by environmental chemicals is a great concern because thenumber of patients with neurodevelopmental disorders has been increasing in recent years. Although the invivo DNT guideline test has been updated in 2007, it still has some disadvantages of high-cost and poor time
efficiency. To overcome the disadvantages of in vivo DNT tests, in vitro or in silico DNT tests are proposed asalternatives; however, it is still difficult that the results of these tests would reflect the central nervous system
of mammals, and may overlook the secondary effects caused by hypothyroidism, etc. Thus, a new technology
is required that can more easily detect chemical-induced DNT in in vivo. Here, we created a transgenic (Tg)mouse expressing the reporter gene (luciferase; Luc) under the control of a promoter of neuronaldifferentiation marker and evaluated its usefulness in detecting DNT. First, the chronologic changes of brain
Luc activity were evaluated using in vivo bioluminescence imaging and in vitro Luc assay of brain tissues. Luc
activity peaked immediately after birth, decreased with age and reached plateaus after the weaning period.
Because this temporal pattern of Luc activity is analogous to the generally recognized temporal changes ofthe dendritic spine numbers in the developing brain, the changes in Luc expression are thought to reflect thestate of brain neural differentiation. Rodents, prenatally exposed to sodium valproate (VPA), are established
as an animal model that exhibit some autistic spectrum disorder-like symptoms. Therefore, we administered
VPA (500 mg/kg i.p.) to pregnant mice at gestation day 12.5 and evaluated the temporal changes of in vivo
luminescence in the developing brain of Tg pups. In vivo luminescence in the brain of VPA-treated group
significantly decreased from postnatal day 4 to weaning compared to vehicle-treated group. Furthermore, we
performed Nissl staining in the prefrontal cortex at 8 weeks postpartum and confirmed that the number ofneurons in VPA-treated group was significantly decreased compared to vehicle-treated group. Consistent with
this, Luc activity in the same brain area of VPA-treated group was also decreased. These results suggest that
Luc expression in Tg mice may be a useful marker reflecting the state of brain neural differentiation. In vivo
luminescence using our Tg mice could be a potential tool for detecting chemical-induced DNT on developing
brain easily.

2022 ICCA LRI & NITE Workshop Poster Presentations18

16 – Application of toxicogenomics in next-generation risk assessment
and predictive toxicology
Michael B. Black, Alina Efremenko, Kevin Bronson, Jeremy Fitzpatrick, Patrick D. McMullen
ScitoVation. Durham, NC. USA
Transcriptomics in toxicology uses quantitative measures of cellular mRNA transcripts to measure changes ingene expression as a response to exposure to a chemical. Changes in gene expression have proven highly
informative data for determining cellular modes of action (MOA) and determining points of departure (POD)
for hazard assessment and comparative potency of congeners. Benchmark dose modeling (BMD) combinedwith conventional ontology enrichment to derive points of departure (POD) based on functional cellularpathway changes allows for the biologically relevant POD values from short term bioassays (in vivo and invitro) to increase throughput at reduced cost. In vivo toxicogenomic studies also offer a path forward to
reduce or eliminate animal testing. Transcriptomic values were used in a study of the food contaminantacetamide to derive short term POD values for risk assessment. The overall consistency of thesetranscriptomic POD values with those for increased cell proliferation based on Ki67 labeling indicate
transcriptomic POD values offer valid and useful alternatives to conventional toxicology POD estimates.
Similarly, in vitro exposures to ketoconazole and phenobarbital in 3 cancer cell lines (HepG2, MCF7 andIshikawa cells) indicated that transcriptomic POD values provide a consistent and reliable means of deriving
POD values for toxicogenomics from very short-term in vitro assays. Comparative analyses of differentiallyexpressed genes provides a mechanism for transcriptomic based read-across comparing alterations in genes
between compounds. ScitoVation developed a quantitative mechanism, the modified Jaccard index (MJI)tocompare transcriptomic derived MOA analyses between compounds. Use of the MJI offers another tool fortranscriptomic read across by quantitatively comparing the similarity of functional ontology enrichment using
differential gene expression and conventional ontology enrichment analysis. This takes the comparison ofchanges in individual genes to a comparison of cellular functional affects as well. Transcriptomic raw data isoften publicly available (e.g., NCBI’s Gene Expression Omnibus, GEO). However interactive or comparative
forms of processed data or analyses results are not. We are also building an analyses pipeline around a
MySQL database that captures both raw transcriptomic data, processed data and analysis outputs to build out
a comparative database and comparative analyses toolkit. Combined with visualization tools such as our
MoAviz interactive ontology enrichment viewer application, capturing the entirety of toxicogenomic
experiments in a relational database will provide a system for comparative transcriptomic read-across and
MOA investigation, along with POD derivation and comparison with similar compounds or compounds withsimilar cellular biological response.

2022 ICCA LRI & NITE Workshop Poster Presentations19

17 – MetAbolomics ring-Trial for CHemical groupING (MATCHING) - Assessing the
Reproducibility of Metabolomics Within a Regulatory Context Through a Multi-laboratory
Ring-trial (LRI C8)Mark R. Viant (University of Birmingham, UK), on behalf of the MATCHING team
Metabolomics has reached a critical point in determining its value to regulatory toxicology. Building on 20
years of metabolomics research, in the last few years the first metabolomics data to support grouping andread-across has been submitted to ECHA; metabolomics best-practice and reporting guidelines for various
applications including chemical grouping have been published in Nature Communications1 by the
MEtabolomics standaRds Initiative in Toxicology (MERIT) consortium; and an OECD project to develop an
Omics Reporting Framework is nearing completion.2 Given these and other examples of its increasing
relevance to chemical safety regulations, an assessment of the reproducibility of metabolomics in thecontext of chemical grouping - across multiple labs - is required. The overarching aim of the Cefic-funded
MATCHING international ring-trial is to determine whether this technology can demonstrate highreproducibility in grouping, and hence high reliability, or whether refinements in analytical or data analysis
practices are needed.
A consortium of multiple industrial, government and academic partners is undertaking this metabolomics
ring-trial, including:o

BASF Metabolome Solutions, GermanyoImperial College London, UKoUniversity of Birmingham, UKoSyngenta, UKoEnvironmental Protection Agency, USoNational Toxicology Program, USoVrije Universiteit Amsterdam, Netherlands

With the aim to demonstrate that these multiple metabolomics laboratories, each analysing and reporting
metabolomics data from a single rodent toxicity study, can arrive at the same regulatory conclusion (i.e.,the same grouping of chemicals), a series of specific objectives that were organised into three work
packages were developed:
1. Prepare quality-checked plasma samples for the ring-trial, derived from 28-day rodent tests usingeight chemicals selected from the MetaMap®Tox database by BASF SE and ECHA.
1. Conduct a blinded metabolomics ring-trial and report the findings of the grouping study, using OECD
guidelines, to ECHA.
1. Collate and evaluate all ring-trial findings, review all statistical analyses and the reporting template
used, and disseminate the project outcomes to regulators and industry including recommending any
refinements in analytical or data analysis practices.
To date, objective one has been completed, and objective two largely completed, with five of the ring-trial
partners presenting results to ECHA in June 2022. Ultimately, a demonstration of high reproducibility of thefindings from the grouping case study should help to facilitate the uptake of metabolomics by the chemicalindustry.
References: [1] Viant, M. R. et al. Use cases, best practice and reporting standards for metabolomics inregulatory toxicology. Nat. Commun. 10, 3041 (2019). [2] Harrill, J. A. et al. Progress towards an OECD
reporting framework for transcriptomics and metabolomics in regulatory toxicology. Regul. Toxicol.
Pharmacol. 125, 105020 (2021).

2022 ICCA LRI & NITE Workshop Poster Presentations20

18 – Prediction of Repeated Dose Toxicity with Read-across Based on IATA
Maiko Okamoto, Shota Nakagawa, Masayuki Yamane
Kao Corporation, Safety Science Research
To develop an alternative strategy for repeated dose toxicity, it is needed to take various factors such as
toxicokinetics and toxicodynamics into account in the strategy. Under these circumstances, Integrated
Approaches to Testing and Assessment (IATA) which makes an integrated hazard and exposure prediction
using New Approach Methods (NAMs) for predicting toxicological effects attracts attention. In the NAMs,
read-across which predicts the toxicological effect from structural analogs is predominantly used forassessing systemic toxicity in regulatory use. However, read-across has risk of error in prediction of systemic
toxicity due to its uncertainties. It is considered that the slightly structural changes might lead to change of
biological responses as the uncertainties. To reduce the uncertainty, comparing the biological similarities
based on adverse outcome pathway (AOP) with in vitro and in silico in addition to structural similarities havebeen discussed. Although it is considered that this concept is useful for predicting systemic toxicity, there isno established concrete strategy due to a lack of case studies. Therefore, we have confirmed the utility ofthis concept and extracted key considerations for developing a novel strategy through establishing the readacross case studies. In the case studies, we compared the biological similarity with in vitro and in silico
among structural similar chemicals which have in vivo data. Then we confirmed the prediction accuracy withreal data and extracted key considerations. As a results of case studies, it was confirmed that the
combination of in vitro and in silico enhance the prediction accuracy from viewpoint of quality and quantity.
The data showed that gene expression array plays the vital role for predicting putative AOP and selecting
suitable in vitro test for comparing similarity of toxicological hazard among structural similar chemicals.
Furthermore, pharmacokinetic modeling and simulation contributes to comparing the toxicological intensity
among the chemicals which have similar toxicological hazard. We considered that these key considerations
contribute to the development of new alternative strategy for repeated dose toxicity. In this presentation, we
would like to discuss the detail of case studies and key considerations of read-across based on IATA.

2022 ICCA LRI & NITE Workshop Poster Presentations21

19 – Linking LRI AMBIT3 chemoinformatic system with the IUCLID6 substance database tosupport read-across of substance endpoint data and category formation
B. Hubeschc , N. Jeliazkovaa , Q. Liba

Ideaconsult Ltd.,4, Angel Kanchev Str., 1000 Sofia, Bulgaria
Clariant Produkte (Deutschland) GmbH, Am Unisyspark 1, 65843 Sulzbach, Germanyc
CEFIC, 40 Rue Belliard, 1040, Brussels, Belgiumb

Read-across and category formation are indispensable techniques in safety assessments of chemicals. The
read-across approach is used on average in 20% of the Endpoint Study Records, while (Q)SAR is used inless than 1% of the dossiers, according to European Chemical Agency reports. Although many tools areavailable, only a limited number is capable to provide easily accessible data on substance identity,
composition together with chemical structures and high-quality endpoint data.
The AMBIT software, funded initially within the CEFIC LRI programme, provides a web service and user
friendly web interface to a chemical database, various chemical structure search facilities and toxicity
prediction models. The AMBIT data model supports substances with complex compositions and substances
experimental data which allows importing data from the International Uniform Chemical Information Database(IUCLID6) as well as other sources. The chemical structures already contained in AMBIT are automaticallylinked to constituents/impurities/additives of the imported substances. The flexible data storage andvisualization allows for user friendly presentation of study data (physicochemical properties, environmental
fate, ecotoxicological and toxicological information) and composition. Comprehensive assessment workflows
are developed for read-across and category formation based on all the data available in AMBIT. The
assessment workflow facilitates the search for target and source structures through multiple similarity
methods, generating data matrices, gap filling and generating assessment reports with predefined formats
automatically. The enhanced AMBIT facilitates drafting and improves quality for read-across and category
formation and will be a useful tool for experts responsible for substance assessments.
The current landscape of chemical databases is in order of many thousands, distributed under different
licenses, and are being continuously updated and may be of interest in different use cases. In addition to
REACH study results https://iuclid6.echa.europa.eu/reach-study-results and OpenFoodToxv2, AMBIT3 will
follow stakeholder’s recommendation and integrate additional sources as e.g. ECHA’s REACH 2018 dataset,
US EPA CompTox Dashboard, EFSA’s OpenFoodTox 2 dataset and the RepDose database.

2022 ICCA LRI & NITE Workshop Poster Presentations22

20 – Toxicogenomics and Read AcrossShengde Wu, Corie Ellison, Jorge Naciff, Michael Karb, Cindy Obringer, Gang Yan, Yuqing Shan, Alex
Smith, Xiaohong Wang, George P. Daston*
The Procter and Gamble Company, Mason, Ohio, USA
Read across continues to be the most commonly used non-animal alternative for systemic toxicity
assessment of new chemicals. Read across guidelines indicate that in addition to chemical structuralsimilarity, supporting data should be provided to show that the biological activity of analogs is similar. It is
well established that exposure to virtually any toxicant produces characteristic changes in gene expression. It
is possible to evaluate gene expression across the genome (transcriptomics), potentially making it a usefultechnique for demonstrating toxicological similarity among analogs (toxicogenomics). Our lab has developeda large database of transcriptomic signatures for toxicants using a number of cell types that cover a broad
range of biology relevant to toxicology. This database, along with a large database (LINCS) on
pharmaceuticals and gene knockouts curated by the Broad Institute makes it possible to comparetranscriptomic profiles of new chemicals with those already in the database using a statistical approachcoined connectivity mapping (CMAP). We used this approach to characterize similarities among groups ofsimilar chemicals that target different toxicity endpoints, including phthalates (liver toxicity), nitrobenzenes andnitrotoluenes (testicular toxicity) and branched carboxylic acids (developmental toxicity). We will focus on the
results for the carboxylic acids for this poster. 2-Propylpentanoic acid (valproic acid, VPA) is a humandevelopmental toxicant. The transcriptomic profiles of several other alpha-branched carboxylic acids alongwith one branched alcohol and a straight-chain acid were compared to VPA. Profiles were generated using
the L1000 platform in four cell types – MCF7, A549, HepG2 and iCell cardiomyocytes – exposed to three
concentration of each chemical for 6 hours. Toxicogenomic profiles were similar among three of the acids:
VPA, 2-ethylhexanoic acid (a rodent developmental toxicant), and 2-propylnonanoic acid, but not for the othercompounds. Connectivity mapping mapped the profiles for these three compounds to histone deacetylase
inhibitors. Molecular docking studies on three isoforms of this enzyme show that VPA and the two acids withsimilar transcript profiles have chemical features that allow them to occupy the active site, while the othercompounds tested have features that exclude them from the active site. We generated in vitro toxicokinetic
data that indicate that all chemicals tested have similar absorption and hepatic clearance. PBPK modelingwas used to extrapolate from measured in vivo Cmax data for VPA and ethylhexanoic acid to predict Cmax
for the other compounds and to reverse calculate NOAELs for those compounds. This researchdemonstrates the utility of toxicogenomics to support read across, and of in vitro and in silico toxicokinetics tomake the read across useful for risk assessment.

2022 ICCA LRI & NITE Workshop Poster Presentations23

21 – Addressing some issues to expand the use of species sensitivity distributions inecological risk assessment
Masashi Kamo, Yuichi Iwasaki and Wataru Naito
National Institute of Advanced Industrial Science and Technology, Research Institute of Science for Safety
and Sustainability
Species sensitivity distribution (SSD) has long been discussed for its use in ecological risk assessment. SSD
is a distribution of toxicity data (e.g., no observed effect concentration and half lethal/effect concentrations)derived from ecotoxicity tests. A concentration leading to a protection of 95% species (HC5) is estimated andthe HC5 itself or HC5 divided by some magnitude of assessment factor is often used as a predicted no effectconcentration (PNEC) in ecological risk assessment.
For the statistical distribution of SSD, the log-normal or log-logistic distribution is often employed. Thesestatistical models have only two parameters (mean and standard deviation), but their accurate estimation
requires a large sample size. Such the sample size is rarely available, and there are concerns about theaccuracy, and the concern is a critical barrier for the use of SSD in practice. In this study, we aimed to resolve
the concern by two approaches. The first is to build models that estimate two parameters of the distribution
even when the data is limited. The second is to quantify the degree of uncertainties for the estimated SSD.
In the first approach, we first collected quality-assured acute toxicity data for a total of 60 chemicals, andestimated the means and the standard deviations of SSDs and HC5 values (data-derived HC5). We thendeveloped linear multiple regression models to estimate the means and standard deviations of SSDs using
readily obtainable descriptors such as logKow as predictor variables. As has been reported previously, themodel only with these descriptors had lower predictability. We then reconstructed the multiple regression
models by adding the means and standard deviations calculated from the toxicity data of three species
selected from algae, crustaceans, and fish to predictor variables. The HC5 values derived from the resulting
best models were within the 95% confidence intervals of data-derived HC5, and their ratios generally fellwithin a factor of 10. For the chronic SSD derivation, we examined the relationships between the acute andchronic SSDs. The HC5 values derived from chronic SSDs were, on average, 10-fold lower than acute ones.
We suggest that multiplication of the acute HC5 by a factor of 0.1 is a defensible way to obtain a first
approximation of the chronic HC5.
A theoretical analysis was carried out for the second approach. We examined the extent to which theestimated HC5 differed from the population HC5. The deviation between the population and the estimatedHC5 varied depending on the sample size and the estimated standard deviation. These quantitative results
allow us to equivalently compare different types of uncertainty: uncertainty due to small sample size anduncertainty due to large variability in susceptibility (large standard deviation). The results also allow us todiscuss quantitatively how large the assessment factor should be.

2022 ICCA LRI & NITE Workshop Poster Presentations24

22 – Parameter acquisition with humanized chimeric mice, for human exposure prediction using a
PBPK model.
Kota Hirasawa, Jun Abe, Sachiko Kitamoto
Environmental Health Science Laboratory, Sumitomo Chemical Co., Ltd.,
1-98, Kasugadenaka 3-chome, Konohana-Ku, Osaka 554-8558, Japan.
Human internal dosimetry of chemicals is essential in the risk assessment. While human toxicokinetics data ofchemicals are hardly obtained intendedly, the use of physiologically based pharmacokinetic (PBPK) modelinghas become important for predicting human internal dosimetry. For the reliability of the predicted human TK
data, it is important to acquire sensitive parameters by the well-validated methods (e.g. in vitro to in vivo
extrapolation), not to apply simple extrapolation based on an organ weight scaling factor without biologicalbasis (e.g. baseless assumption that there is no species difference in the parameters). However, when thecompound exhibits complicated pharmacokinetics via active uptake, metabolism, and biliary excretion in liver,
it is difficult to obtain these hepatic parameters only by the in vitro experiments. Compound X is rapidly
metabolized to Metabolite A in mammals. Metabolite A is eliminated from the systemic circulation by biliary
excretion and metabolism in liver. Although uptake of Metabolite A by transporters is observed in mouseprimary hepatocytes, significantly less of it is observed in human primary hepatocytes. We evaluated themouse-human interspecies TK difference by the use of PBPK modeling into which is incorporated themechanism-based descriptions of relevant ADME processes (i.e., absorption, distribution, metabolism, andexcretion).
First, we developed a precise mouse PBPK model using the available in vitro and in vivo TK data from mice.
The predicted Metabolite A exposure in mice was within about 0.9-fold and 1.1-fold of the actual values. Then
we converted a mouse PBPK model to a human PBPK model by applying the input parameters of humans.
To obtain human hepatic parameters, i.e., hepatic elimination intrinsic clearance via biliary excretion andmetabolism, we used chimeric mice with humanized liver as a model to reproduce the complicatedpharmacokinetics of Metabolite A in humans. The validity of this approach using chimeric mice cannot be fully
verified because human TK data for verification cannot be obtained experimentally. However, we validated
this approach by quantitatively verifying the prediction accuracy when performing rat PBPK modeling with thesame strategy using chimeric mice with rat hepatocytes. As the results of this verification, Metabolite A
exposure in rats were precisely reproduced by using such techniques, although it tended to be slightly
overestimated. This can lead a conclusion that the human TK data would also be predicted accurately andconservatively by the same methods using chimeric mice, which would preferable in the view of human risk
assessment. In this way, after verifying the prediction accuracy of the PBPK model in multiple animal species
and the parameter acquisition method, we predicted Metabolite A pharmacokinetics in humans. Comparing
the predicted Metabolite A exposure in human liver with the measured values in mice, we demonstrated aclear interspecies difference of approximately 4 times lower Cmax and AUC in humans. This is the first reportto present the approach to evaluating target tissue dosimetry of chemicals in humans by the PBPK model in
combination with the novel method using chimeric mice and the verification of this technique.

2022 ICCA LRI & NITE Workshop Poster Presentations25

23 – Developing a quantitative AOP for liver-mediated thyroid modulation after prenatal exposure
to a xenobiotic compound in the rat
A. Piersma1, M. Beekhuijzen2, A. Boelen3, S. Bressers2, E. Gremmer1, M. de Groot2, T. Hamers4, E. Hessel1,
K. van Dycke2, and M. Wenker2
1

RIVM, Bilthoven, Netherlands
CRL, Den Bosch, Netherlands
3
AUMC, Amsterdam, Netherlands
4
VU, Amsterdam, Netherlands.
2

Xenobiotic-induced hepatic metabolism induces phase I and phase II enzymes, which in turn may cause
enhanced clearance of thyroid hormones. In humans, thyroid hormone is needed for orderly brain
development during the first trimester of pregnancy, when the fetus is entirely dependent on the maternaltransfer of triiodothyronine (T3) and thyroxine (T4). This project aims to monitor relevant maternal andoffspring liver and thyroid parameters after prenatal exposure to a model liver enzyme inducer in rats using
BMD modelling in order to define quantitative relationships early in the AOP. A broad dose range up to 300
mg/kg bw/day of pregnenolone-16α-carbonitrile (PCN), a model pregnane-X-receptor agonist and UGT1A
isozyme inducer, was administered orally to female rats (Wistar Han) from gestational day (GD) 6 to GD 20.
Upon the completion of dosing on GD 20 pregnant rats in each treatment group were evenly divided into twocohorts. One cohort was terminated at GD21 and maternal and fetal parameters assessed. The other cohort
was terminated at weaning, and dam and pup parameters were assessed. Dose-response analyses were
performed using PROAST software (Slob, 2002). Pregnancy rate, body weight and food consumption were
unaffected throughout the study. Dam liver and thyroid weight were dose-relatedly reduced at GD21, and thiseffect had disappeared at weaning. In general, responding parameters showed full sigmoid dose-response
curves with effects appearing at the mid dose range and plateauing at the higher dose levels. Litter size andpup body weight gain until weaning were unaffected by treatment. At GD21, liver T3 decreased in the dams
while liver T4 concentrations were increased, accompanied by upregulation of relevant Cyp enzymes,
glucuronidases, sulfatases, and D3 activity. Serum T3 and T4 decreased at GD21, with no effect on TSH.
These parameters had all returned to control values at weaning. Fetal brain at GD21 and pup blood and brain
at weaning did not show changes in thyroid hormone concentrations. The data confirm that PCN acts as astrong phase II liver enzyme inducer in adult rats, followed by secondary modulation of the thyroid axis. Theinterpretation of possible brain developmental effects of thyroid hormone modulation (also if mediated by liver
metabolic changes) observed in this study protocol in the light of hazard and risk assessment meets withlimitations. Furthermore, effects on offspring brain development in this study cannot as yet be excluded andthis requires further analyses.

2022 ICCA LRI & NITE Workshop Poster Presentations26

24 – Release of Population Life-course Exposure to Health Effects Model (PLETHEM): An Online
Tool for PBPK Modeling version 3.2
Jeremy Fitzpatricka, Kevin Bronsona, Alina Efremenkoa, Marjory Moreaua, Patrick D. McMullenaaScitoVation, LLC, Durham, NC, USA

An outstanding challenge in the acceptance of alternatives to animal testing is the systematic incorporation ofcomputational models into risk-based decision-making pipelines. This can be achieved by linking exposure
estimation methods, physiologically based pharmacokinetic (PBPK) modeling, and computational systems
biology pathway modeling tools into a standardized framework. To that end, we have developed thePopulation Life-course Exposure to Health Effects Model (PLETHEM) suite, a modular open source modelingplatform that provides users the ability to create and run PBPK models. The platform consists of a selection ofdifferent exposures routes, ability to define chemical parameters, QSAR models, life-stage specificphysiological parameters, calculation of metabolism from different sources, uncertainty and variability
calculation, an R-based engine to perform model simulations, and an interactive user interface to define andselect parameter sets for the models. PLETHEM implements easy to use interfaces for a generic PBPK
model and a high-throughput IVIVE model. The most recent iteration of PLETHEM updates the ecotoxicology
module to increase stability and to include additional modeling output. The rapidPBPK module has been
overhauled to increase stability and global sensitivity analysis capabilities. PLETHEM is available online atwww.scitovation.com/plethem.

2022 ICCA LRI & NITE Workshop Poster Presentations27

25 – In silico estimation of skin permeation of chemicals with their diffusion and partition
parameters
Hiroaki Todo
Faculty of Pharmacy and Pharmaceutical Sciences, Josai University, 1-1 Keyakidai, Sakado, Saitama 3500295, Japan
Skin permeation experiments are very important to evaluate the safety and effects of topically appliedchemicals. Silicone membranes and three-dimensional cultured human skin models have been utilized forin vitro skin permeation experiments as alternative membranes, especially in the development ofpharmaceutical and cosmetic products. In silico models, which estimate the skin permeability coefficient (P,in cm/s or cm/h) of a chemical based on its physicochemical properties, have also been used to evaluate
efficacy and safety.
Skin concentration of chemicals (distributed in the lower layers of the stratum corneum) is important tocalculate its dermal absorption as well as measurement of P value. Therefore, many methods to measureskin concentration have been reported, such as suction blister, punch and shave biopsies, autoradiography,
tape-stripping, microdialysis, but are unfortunately expensive and time-consuming. Our research group
have already reported that skin concentration as well as skin permeation of chemicals could be predictedwith in silico model based on Fick’s law of diffusion following dermal exposure to chemicals with wide rangeof lipophilicity. In addition, this method could be applied to estimate chemical permeation and concentration
with different thickness of skin. Thus, this method should become a useful tool to assess the efficacy of
topically applied drugs and cosmetic ingredients, as well as the risk of chemicals likely to cause systemic
toxicity and skin disorder. However, permeation parameters, namely diffusion and partition coefficients,
should be obtained by the curve fitting of observed skin permeation data to two-layered and one-layered
diffusion models for whole and stripped skin to estimate skin concatenation and P values with in silico
model based on Fick’s law of diffusion. Therefore, a complete in silico model that does not rely on
experimental measurements is needed as a further development.
In the present study, diffusion and partition coefficients of chemicals in stratum corneum and viableepidermis were estimated by physicochemical properties of chemicals. And estimation of skinconcentration based on in silico model was compared with observed value following dermal exposure ofchemicals. When permeation parameters were estimated by random forest method with highest occupied
molecular orbital (HOMO), lowest unoccupied molecular orbital (LUMO) and lipophilicity of chemicals as
descriptors, each permeation parameters were well-fitted to the diffusion and partition coefficients thatobtained from in vitro skin permeation experiment with excised human skin. Further experiment should benecessary with different lipophilicities of chemicals, but this present method to estimate skin permeation
parameters would become a useful tool to assess the dermally exposed chemicals.

2022 ICCA LRI & NITE Workshop Poster Presentations28

26 – An application of a 3D hydrodynamic ocean model to predict chemical behavior at waters off
Niihama in Seto Inland Sea
Masanori Niwanoa, Yoshihide Matobaa, Fumio Horiguchib, and Yuriko IshikawabaEnvironmental Health Science Laboratory, Sumitomo Chemical Co., Ltd., 3-1-98 Kasugadenaka, Konohanaku, Osaka 554-8558, Japan.
bNational Institute of Advanced Industrial Science and Technology, 16-1 Onogawa, Tsukuba, Ibaraki 3058569, Japan.
In order to develop a chemical fate model for assessing the effect to aquatic organisms as a final goal, a highresolution hydrodynamic ocean model was applied to waters off Niihama in the middle of the Seto Inland Sea(SIS), Japan. For considering the effects of large spatial variability of salinity and water transparency oncomplex residual tidal flows in the middle SIS, the model incorporated estimated discharges of moderate size
rivers and a formulation of heating depending on water transparency. Using this model, tidal current,
temperature and salinity were firstly calculated in the area covering the middle of SIS, and then the behaviors
at the waters off Niihama were simulated in detail by using results of the first calculation. The simulation was
performed in July when concentration of released chemicals remains higher due to suppressed convective
mixing and the effect of the chemicals to aquatic organisms was highly concerned. The model well
reproduced spatial and temporal variations of tidal current, water temperature and salinity observed at thewaters off Niihama as well as the mid SIS. Results from the simulation demonstrated that the waters off
Niihama experienced fast water exchange by residual tidal currents enhanced at steep bathymetry and large
vertical water displacement due to propagation of waves generated there in comparison with the middle of the
SIS.

2022 ICCA LRI & NITE Workshop Poster Presentations29

27 – Development and application of a spatio-temporal chemical concentration estimation model
for watershed risk assessment
Yuriko Ishikawa
National Institute of Advanced Industrial Science and Technology (AIST)
Accurate chemical risk assessment in river basins requires exposure concentration data from different
watersheds affected by varied anthropogenic and meteorological conditions, and the lack of these data
makes risk assessment difficult. We have developed the AIST-SHANEL river model which can be applied to
any river basin in Japan. The model estimates spatial and temporal river flow rates and chemical
concentrations in the river water and sediment with a gridded spatial resolution of 250 m and a daily temporal
resolution by using watershed characteristic data including meteorological, land-use, population, industry, andsewerage data. Chemical concentrations were calculated using the estimated river flow rates and discharge
volumes and the dynamic mechanisms of advection, diffusion, adsorption and desorption of suspended solids(SS), settling and resuspension of SS, mass transfer between river water and sediment, and biodegradation
were considered at different points in time for each grid cell. The physical parameters for analysis were vapor
pressure, molecular weight, water solubility, organic carbon water partition coefficient, biodegradation rateestimated by the half-life of the chemical substance considering temperature dependence, and sewage
treatment removal rate. Wet and dry deposition from the atmosphere was also included. This presentationshows examples of the application of the model to two chemicals, namely linear alkylbenzene sulfonates andbisphenol A in river basins in Japan and discusses the validity and improvement of the model for future use.
The results provide useful information for understanding the potential risks predicted from temporal andspatial distribution of chemicals in river water and sediments. This also facilitates the evaluation of potential
impacts of chemical substance substitutions, emission reductions, climate change, water saving measures,and improvements to wastewater treatment facilities, including sewage. Moreover, we are continually
improving this model for watershed risk assessments for various contaminant types. For example, to predictthe diffusion of chemicals due to a spill, the model has been modified to estimate concentration changes
depending on precipitation, including localized heavy rainfall, using temporal resolutions of one minute andone hour. In another example, to evaluate the effectiveness of use of biodegradable plastics, the ability to
calculate the concentrations using the dynamics of plastic particles is being improved. Although the present
model targeted river basins in Japan, AIST-SHANEL can be applied to any river basin in the world if thenecessary watershed data can be obtained. Due to limited available measurement data it is challenging tovalidate the model; however, we attempt to achieve this by using uncertainty and sensitivity analyses.

2022 ICCA LRI & NITE Workshop Poster Presentations30

28 – A multi-model system for predicting biodegradation rates and the presence of residual
substances of chemicals using chemical descriptors and 3D structures
Kazuhiro Takeda a, Kensuke Takeuchi b, Yuki Sakuratani b, Kazuhide KimbaraaaGraduate School of integrated Science and Technology, Shizuoka University, Japan
National Institute of Technology and Evaluation, Japanb

In Japan, according to the Act on the Evaluation of Chemical Substances and Regulation of Their
Manufacture, etc., chemical substances are classified into ready biodegradable and non-biodegradable
substances by the OECD test guideline 301C modified MITI I test, based on biodegradability and thepresence of degradation residual substances. This test usually requires 28 days of continuous testing and iscostly. Therefore, it is necessary to establish a model relating chemical properties to biodegradability and thepresence of degradation residual substances in order to reduce costs. Many models have been proposed toestimate biodegradability and the presence of degradation residual substances, such as Lasso, XGboost
and deep learning neural networks, but no model can estimate biodegradability and degradation residual
substances with sufficient accuracy. Therefore, this study proposes an ensemble system including ninemodels for estimating biodegradability and eleven models for estimating the presence or absence ofdegradation residuals. Furthermore, a system is proposed to classify products into ready biodegradable andnon-biodegradable products using the estimated biodegradability and the estimated degradation residual
substances. The system used the chemical descriptors and steric structures of 4200 chemicals. Descriptors
were generated by AlvaDesc software and three-dimensional structures were constructed by Gaussian
software using the PM6 method. To obtain higher accuracy, chemicals were classified by estimating their
computation residuals. The residuals were estimated by a deep learning neural network using chemical
descriptors. The estimation accuracy of the system was 98% for the unlearned non-biodegradable products
and 38% for the unlearned ready biodegradable products.

2022 ICCA LRI & NITE Workshop Poster Presentations31

29 – Evaluation of slow-stirring method for determining log Kow (n-octanol/water partition
coefficient) of surfactants
Oguro Kaku, Odake Hiroshi, Shinozaki Hiroya, Higuchi Masatoshi, and Fujiwara Ayako
National Institute of Technology and Evaluation, Japan
Surfactants are widely used both in industry and in everyday life. Thus, determination of their distribution in
the environment is of particular importance. In environmental risk assessment of chemicals, the noctanol/water partition coefficient (log Kow) is a physicochemical property representing the hydrophobicity ofchemicals and frequently used as a key parameter to estimate environmental fate, exposure, and toxicity.
Existing experimental OECD methods for determining log Kow such as shake flask method are not suitable
for surfactants due to forming emulsion and micelle aggregation. “Slow-Stirring Method” can avoid
turbulence with n-octanol and aqueous phases and thus is suggested by European Chemicals Agency as
the best method in theory for surfactants. The slow-stirring method based on OECD TG 123 has been
demonstrated to provide reliable data to highly hydrophobic chemicals but applicability for surfactants has
not been validated. Recently, a developed method by other researchers has shown that the scope could be
extended to non-ionic/ionic surfactants under the condition of less than critical micelle concentration (CMS)at which no micelles are present. However, the test conditions have not been established as there iscurrently no internationally standardized method.
The aim of this study was to evaluate the accuracy of the slow-stirring method for determining log Kow of
surfactants using six surfactants of three types: non-ion (tetraethylene glycol monooctyl ether, octaethylene
glycol monolauryl ether), anion (sodium dodecyl sulfate, sodium dodecanoate) and, cation(dodecyltrimethylammonium chloride, hexadecyltrimethylammonium chloride). 300 mL test vessels with drain
port near the bottom were prepared. The test substances were introduced into either n-octanol or aqueous
phase and then stirred at 25℃. Samples of the two phases were taken and analyzed by LC-TOF/MS.
Generally, log Kow values showed a good agreement with literature values obtained by the slow-stirring
method provided that a certain amount of test substances was added to the test vessels. In order to assess
the influence of adding amount to the test vessels and the accuracy, log Kow values were determined over awide range of the concentration bellow the CMC. log Kow values of three surfactants (tetraethylene glycol
monooctyl ether, sodium dodecyl sulfate, and dodecyltrimethylammonium chloride) were almost identicalwithin the studied range. These results indicate that the slow-stirring method generally allows for accurate
determination of log Kow of surfactants.

2022 ICCA LRI & NITE Workshop Poster Presentations32

30 – EAS-E Suite: A data integration and modelling framework for chemical safety andsustainability
Jon Arnot1,2,3, Alessandro Sangion1, Li 4, Trevor N. Brown1, Liisa Toose1, Lauren Hughes1, James Armitage5
1

ARC Arnot Research & Consulting, Toronto, Canada
Department of Physical & Environmental Sciences, University of Toronto Scarborough, Toronto, Canada
3
Department of Pharmacology & Toxicology, University of Toronto, Toronto, Canada
4
School of Public Health, University of Nevada Reno, Reno, NV, USA
5
AES Armitage Environmental Sciences, Inc., Ottawa, Canada
2

There are significant data gaps in the information required to conduct reliable ecological and human healthassessments for thousands of existing and new chemicals. The Exposure And Safety Estimation (EAS-E)
Suite is a freely accessible on-line platform that addresses many data gaps and challenges for ecologicaland human health assessment and facilitates the safe and sustainable production and use of chemicals insociety in a cost-effective manner. EAS-E Suite is comprised of (i) chemical information databases (e.g.,physical-chemical properties, in vitro and in vivo toxicokinetics data, production volumes), (ii) quantitative
structure-activity relationships (QSARs) for predicting chemical properties, biodegradation andbiotransformation half-lives, and (iii) various mass balance models and tools for chemical hazard, exposure,and risk estimation. EAS-E Suite includes (i) the CiP-CAFE model for estimating life cycle chemical mode-ofentry and emission rates, (ii) the RAIDAR model for simulating chemical fate in the natural environment andexposures to a range of ecological receptors and far-field exposures to humans, (iii) the RAIDAR-ICE model
for simulating indoor chemical fate and near-field human exposure simulations linking external and internal
doses, and (iv) the PROTEX-HT model for aggregate human exposure and risk estimation. Other models in
EAS-E Suite are (i) the in vitro mass balance model for simulating the fate of chemicals in in vitro bioassays,(ii) the EAS-E Suite and US EPA’s high-throughput toxicokinetic models for forwards and reverse dosimetry
in humans, rats, and fish, (iii) in vitro-in vivo extrapolation (IVIVE) models, (iv) bioaccumulation models forplants, plankton, invertebrates, fish, birds and mammals, (v) various dermal exposure models, and (vi)various environmental fate models. EAS-E Suite facilitates the application of these databases and models
using either chemical name, CAS, or SMILES user supplied entry information. Furthermore, EAS-E Suite“autoparameterizes” the built-in tools and models for 70,000 discrete organic chemicals making it very easy
for their application for experts and non-experts. If desirable the user has the option to enter alternativevalues for chemical information to the defaults provided by the system. The platform aids the critical scientificanalysis and evaluation of data and models and provides knowledge transfer to multiple stakeholders
fostering collaboration and consensus building. This poster summarizes key concepts of the EAS-E Suite
platform showing how it can be applied for chemicals assessment and management objectives either
retrospectively for existing chemicals and use, or prospectively for new chemicals and for proposed changes
of use of existing chemicals, and for intelligent experimental test design.
EAS-E Suite quantifies and clearly demonstrates relationships between production volumes, chemical
emissions throughout the lifecycle, fate and transport in natural and manufactured environments (e.g.,homes), persistence, bioaccumulation, toxicokinetics and aggregate human and ecological exposures toestimate the potential for adverse effects at a screening-level. The recently developed PROTEX-HT system
in EAS-E Suite consolidates the principal elements of the production-to-outcome continuum for tens of
thousands of chemicals.

2022 ICCA LRI & NITE Workshop Poster Presentations

33

APPENDIX 3: Organizing Committee
Tokuo Sukata
Japan Chemical Industry Association (JCIA)
tsukata@jcia-net.or.jp

Yuki Sakuratani
National Institute of Technology and
Evaluation (NITE)
sakuratani-yuki@nite.go.jp

Keiko Kondo
National Institute of Technology and
Evaluation (NITE)
kondo-keiko@nite.go.jp

Hajime Kojima
National Institute of Health Sciences (NIHS)
h-kojima@nihs.go.jp

Tatsuhiro Niino
Mitsubishi Chemical Corporation
niino.tatsuhiro.mp@m-chemical.co.jp

Ryoji Ogawa
Showa Denko K. K.
ogawa.ryoji.xhpzr@showadenko.com

Tomoya Yamada
Sumitomo Chemical Co., Ltd.
yamadat8@sc.sumitomo-chem.co.jp

Taketoshi FujimoriJapan Chemical Industry Association (JCIA)
tfujimori@jcia-net.or.jp

Tomoko Mihashi
Japan Chemical Industry Association (JCIA)
tmihashi@jcia-net.or.jp

Yoshitaka MandaiJapan Chemical Industry Association (JCIA)
ymandai@jcia-net.or.jp

Sugio Nishimura
Japan Chemical Industry Association (JCIA)
snishimura@jcia-net.or.jp
Heli Hollnagel
European Chemical Industry Council(Cefic)/Dow EU
HMHollnagel@dow.com
Richard Becker
American Chemistry Council (ACC)
rick_becker@americanchemistry.com

Bruno Hubesch
Hubesch Consult
hubeschconsult@hotmail.com
Robert Barter
ExxonMobil Biomedical Sciences, Inc.
robert.a.barter@exxonmobil.com
Kathleen Plotzke
American Chemistry Council (ACC)/Dow
kathy.plotzke@dow.com

Logistical support for this conference was provided through contracts with MCI and ICF.
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APPENDIX 4: Post-Event Survey Feedback
The appendix below includes data collected from the post-event survey. Survey respondents wereasked to comment on satisfaction with the sessions and presentations and overall workshop. Survey
respondents were also asked to provide specific feedback on additional topics to cover in sessions andfuture conferences. Short answer questions captured data on attendees’ overall workshop suggestions
and feedback and all responses are included below.

Fig 3. The overall satisfaction of polled participants is reported above.

Short Answer Feedback
What did you like most about the workshop?
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Broad overview on NAMs was provided
Panel discussion of session 4
The workshop was held in-person.
Able to access it virtually
AI-SHIPS
Being able to communicate with various people.
Conference presenters and dinner
Discussion of NAMs from various viewpoints
Diversity of topics
F2F meeting at this season with these topics.
General information about NAM
Good spread of different topics and speakers, and easy to use virtual platforms
Hear about new strategies for risk study.
Held in hybrid was to promote the attendees who has some difficulty with traveling around. If it would
be continued to be held in this way for the future event, it would be nice.
Hybrid attendance, with Box and at venue.
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Under Covid-19, we had used only web meeting, however I could attend at the venue.
Hybrid participation (online and in person)
I could directly obtain the latest research trends related to the subject.
I could get the updated specialized info. from global via online (on my desk).
I liked that the sessions were held in a hybrid style, so I could join the sessions I wanted to attend
between my desk work.
I studied about development progress of NAMs new methods.
Information concerning toxic effect of particles
International/global character
It brought me up to date very quickly and focused on what developments to expect in the immediate
future
iTTC, NAMs at EPA
most NAMs topics
NAM UPDATEPBPK work
Presentations from experts and communication in person at the conference too,
Selection of presenters
Session 1; Session 3
Session 3; iTTC
Session V, 14:40-15:05
Simultaneous interpretation
Simultaneous translation
Social networking at on-site.
That the workshop was also conducted face-to-face.
The application in chemical management
The discussion with experts (both oral and poster)
The fact that it has both virtual and FTF Sessions available, and a key summary of each session
Varied topics and opinions, plus cutting-edge technology application
Very interested in iTTC
Very well organized
Very well organized, interesting topics
Web event!
Workshop Topics
Session 4; Session 5
It’s great to have a face-to-face discussion.

Do you have any other suggestions or feedback you would like to share?
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Box was inconvenient
Ensure rapid participant sharing of all the presentation decks
Hybrid meeting was a great idea and enabled a lot of people around the world to participate.
I was very thankful to have been able to participate in the web event.
It was unfortunate that government officials were not present on Day 2; it would be good to convey theconclusions of the workshop to the government officials who attended on Day 1 – this can be done bycirculating the Workshop Summary.
It would be great if university and high school students who are interested in the analysis of chemical
substances could also watch (Online) and increase the number of participants by using SNS and sending
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notifications of workshops to schools.
Maybe better not starting on a Monday, so that week-end travel could be avoided
Slides or recording should be shared for better understanding the content
Thank you for arranging the hybrid workshop. We have active time at the workshop! However, on-sitewould be better than on-line because of social networking.

How could the workshop be improved?
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Better poster presentation, more personal interaction, maybe include short poster pitches
Box does not work because of company's security
By increasing posters and time to communicate with participants.
Add translation to Day 1.
Discussion time should be a bit longer
For on-site attendee, the questions and answers from Zoom cannot be followed.
I am happy if the poster presenter could provide the video presentation.
I could not utilize BOX well.
I do not have any comments.
I hope that this workshop will help to further promote LRI research in JCIA, ACC, and Cefic.
I think that on-site discussion is mandatory.
I would like to enter the poster session more easily.
I would like to hear more from ECHA and EPA researchers.
If more speaker could be present at the venue, it would be better.
I'm already satisfied enough, but if I may say so, it would be easier to discuss if overseas members wereinvited to the site.
I'm satisfied with this event.
In person
Interactivity
It is better to include a luncheon session for FTF participants.
It was a very well organized hybrid workshop- thanks!, I had some trouble to enter the box.
It was better to have QA in Day1 presentations
It was unfortunate that the audio was sometimes disrupted.
It will be interesting if the discussion time and the question-and-answer time are a little longer. I
entered a sentence in the translation application
It would be nice if Experts from outside the country could patriciate in person. though I understand the
COVID situation make it impossible.
I don't think there are enough LRI studies published.
May need to have session on ecological risk assessment
Maybe it is possible to have a longer plenary session with discussions
More discussion
More interactive communication among panelists and government authorities to seek future
possibilities
Nothing really, except maybe to have a small round-table near the end to discuss where they think the"world is going" with respect to the science associated with chemical risk assessments withOn the first day, interpretation might be useful.
On the other hand of the above comment, we have to reconsider how to compromise the timedifference between three major LRI regions. I do not have any good solution but considering on demandbroad casting would be one possible way.
Online connection for web conferencing
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Paper handouts would be better.
Providing a place for lively discussions under travel restrictions.
Simultaneous translation is not reasonably activated, when question is in Japanese and speaker uses
English. Some kind of interruptions for translation should be notified from chair.
Slides or recording should be shared for better understanding the content
Unfortunately, some of the posters presented information too close to the bottom of the poster board.
We should have provided more specific instructions for the layout of the posters so the information
would be at or near eye level
Video of speakers would be satisfactory.
With more participants or speakers from different countries and regions
Pursuit of Hybrid Holding Technology

What other topics on Hazard Assessment would you like to see in future scientificdiscussions or workshop sessions focused on using NAMs to predict repeat-dose andcomplex apical endpoints?
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Ecotoxicology
Environmental Hazard Assessment, interspecies correlation
In silico approaches for hazard assessment
More databases to consider using
NAM applications in food risk assessments
No suggestions, the talks were good, and Rusty Thomas’ talk was fantastic!

What other topics on Exposure Assessment would you like to see in future scientificdiscussions or workshop sessions focused on integrating exposure assessment NAMs intorisk evaluations?
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Differences between occupational exposure compliance monitoring and conducting occupational
exposure monitoring for use in quantitative risk assessments
Environmental Exposure Assessment, release factors
PBPK
Update on ongoing developments
Lobbying for administrative use
Assessment by modeling the exposure environment

What other topics or areas would you like to see included in future conferences?
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Collaborative research between the US, EU, and Japan, if possible
Environmental fate and exposure of chemicals
More on databases to consider using.
Pros and Cons for Generic approach to risk management.
Capacity for building NAMs
Update on regulatory acceptance.

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