Mitochondria are multifunctional organelles that have critical roles not only in energy production by oxidative phosphorylation but also in various cellular signaling pathways. Mitochondria dynamically change their structure with active membrane fusion and fission in response to cellular signaling and differentiation. Dynamin-related GTPase Drp1 plays a key role in mitochondrial fission, and several Drp1 receptors, such as Mff, is an OM-anchored proteins that recruit Drp1 to the mitochondrial fission sites. Mitochondria have their own DNA, mtDNA, which is essential for the respiratory function. We found that distribution of mtDNA was altered in mitochondrial fission deficient cells, which should be related to maintenance of mitochondrial functions. Recently we also found that mitochondrial fission factors play critical roles in cellular signaling in cultured cells and differentiation in vivo. Here we would like to discuss physiological roles of mitochondrial dynamics.

We are exposed to a variety of environmental factors in our daily lives. A wide variety of epidemiological studies have suggested that environmental electrophiles such as methylmercury (MeHg) and cigarette sidestream smoke (CSS) disrupt our homeostasis and increase the risk of cardiovascular disease. However, the underlying mechanism is unclear. We previously identified that aberrant activation of Dynamin-related protein 1 (Drp1), a mitochondrial fission-accelerating protein, is a key determinant of cardiac remodeling and fragility. Here we show that supersulfides that are redox-active sulfur metabolites have a critical role in the mitochondrial quality and cardiac homeostasis and exposure to environmental electrophiles such as MeHg and CSS increased cardiac risk by disrupting supersulfide-mediated mitochondrial quality.

Supersulfides highly exist in healthy cardiomyocytes and maintained the proper mitochondrial fission and fusion balance by regulating Drp1 activity through Cys⁶⁴⁴ polysulfidation. In a failing heart, supersulfides were catabolized to hydrogen sulfides, and this change in sulfide metabolism promoted Cys⁶⁴⁴ depolysulfidation of Drp1, which led to dysfunctions of mitochondria and cardiomyocytes. Supersulfide administration reversed electrophile-evoked mitochondrial dysfunction and improved the cardiac function of heart failure model mice. These results suggest that depolysulfidation of Drp1 at Cys⁶⁴⁴ by environmental stress such as MeHg increases cardiac fragility.

Cardiotoxity is a severe adverse effect associated with anti-cancer drug treatment. Many research focus on myocardial excitation, the cytotoxicity to cardiomyocyte is also important. This toxicity seems to be assessed by simple in vitro experiments, however, such challenges are limited. A reason for this may be that cellular sensitivities differ by culture environment, which affect gene expression profile. Therefore, we searched gene features essential to reproduce cytotoxicity. To consider as many toxicity mechanisms as possible, we utilized data in the Genomics of Drug Sensitivity in Cancer project. We can access drug sensitivity data for about 1000 cancer cells to about 300 anti-cancer drugs together with gene expression profiles for each cell. Using these data, supervised machine learning was performed to construct a neural network model, in which sensitivities such as EC₅₀ are returned when gene expression profile and drug property are input. Using this model, we extracted genes having high impacts in the model calculation when cardiomyocyte gene expression profile is input in combination with various drugs. The impact was evaluated by calculating partial derivatives to the model output, thus one can obtain a gene number-length vector. We clustered the drugs using the impact vectors and extracted high impact genes for each cluster. By gene ontology analyses, the extracted genes were confirmed to be associated with cytotoxicity. Further validation is required, but this result suggests that the extracted genes are key features to assess various types of toxicities.

Recent development of anticancer drugs, including molecular-targeted agents, the number of long-term cancer survivors, both pediatric and adult, has increased and life expectancy has improved significantly. On the other hand, it has become clear that anticancer drugs increase the risk of cardiovascular disorders in patients, affecting their life expectancy and quality of life. In particular, functional toxicity and cell damage to cardiomyocytes are known to be caused by anthracyclines as well as HER2 and tyrosine kinase inhibitors.

Based on the clinical background of anticancer drug cardiotoxicity, we have developed a prediction method for anticancer drug cardiotoxicity using human iPS cells. Furthermore, based on the possible mechanism, such as mitochondrial toxicity and contractility and cell damage, we have conducted an international validation study using iPSC cardiomyocytes and are now working on the analysis of the large datasets. Thus, we expect that our method will contribute to the development of anti-cancer drugs and patient safety.

DX is one of the ways to achieve the targeted goal. The goal of pharmaceutical companies to promote DX is to provide high quality drugs/services to patients as quickly as possible, and major changes are taking place around operations. Particularly in the field of clinical trials, DX is actively promoted mainly in Europe and the U.S., and the way clinical trials are conducted is changing dramatically, and the wave of this change is sweeping over Japan. One of these is the decentralized clinical trial (DCT), which decentralizes clinical trial operations by focusing on patient centricity, thus enabling patients to participate in clinical trials without depending on visits to the hospital. The development of digital biomarkers is also being activated, making it possible to collect continuous data for efficacy or partial safety evaluation without the patient having to visit the hospital. In addition, there have been reports of cases where Real World Data (RWD) has been used to obtain drug approvals, especially for rare cancers, and cases where the effectiveness of vaccines against Covid-19 has been verified using RWD outside of Japan. The environment for utilizing medical information data is beginning to progress in Japan. These are all considered to be examples that lead to the goal of promoting DX by utilizing digital technology and RWD, i.e., by promoting DX. The JPMA is actively conducting case studies, improving the environment, and making policy proposals to ensure that these methods are used without lagging behind overseas. In this symposium, I will introduce these cases, discussing the future prospects as well as exchanging opinions with researchers in non-clinical fields for the future activities.

The concept of digital transformation (DX) was proposed in 2004, and in 2018, the Ministry of Economy, Trade and Industry (METI) announced the “DX Promotion Guideline” which various industrial fields have been required to implement. The pharmaceutical industry is not exempt, and the frequency of seeing the keyword “DX” in our industry news is in fact increasing. The single word “DX” in the pharmaceutical industry covers many types of data ranging from manufacturing (plant) to marketing (sales). Furthermore, research data on this topic includes both human (clinical) and animal (non-clinical) data.

In this presentation, I will introduce the concepts and some practical ideas as an overview of data utilization in the non-clinical field. We will look at the general flow of data utilization through case studies using SEND data, a non-clinical CDISC standard. In addition, I would like to introduce the discussion that our team at JPMA has had regarding the possibility of applying accumulated SEND data to the management of background values and virtual control groups.

Recently in clinical settings, studies in which it is difficult to recruit subjects, such as for rare diseases, or in which it is difficult to set up a control group from an ethical standpoint, are being conducted using historical controls generated from past clinical study data or real-world data. On the other hand, in non-clinical settings, as it is easier to set up control groups than in clinical studies and guidelines clearly state the necessity of setting up a negative control group, no attempt appears to have been made to set a control group (virtual control group: VCG) designed based on facility background data. However, from an ethical point of view toward test animals and issues in animal supply, introduction of VCG may contribute to reduction in the number of animals used. In this study, we examined the issues that arise when VCG is introduced into nonclinical toxicity studies. Specifically, we replaced the control group data in a previous repeated-dose toxicity study with VCG generated from the background data for additional statistical analysis, and interpreted the results obtained. This presentation shows the results of our interpretation and discusses the differences from studies in which normal control groups were used and points to take note of for each animal species when VCG is introduced into studies. Furthermore, we present our views on the introduction of VCG in qualitative data such as in histopathological examination. We also discuss issues that arise with the introduction of VCG for studies other than general toxicity studies.

As a part of the electronic data managment in GLP facilities, operational control procedures to ensure data integrity of the analytical equipment that is stand-alone and have its electronic data as raw data, the electronization of standards operating procedures and training records, and use of electronic signature system for protocols and reports will be introduced.

Predicting clinical adverse events (AEs) in preclinical studies is one of the most important challenges in drug discovery. Clinical AEs are caused not only by drug properties but also by genetic and environmental background of each patient. Therefore, nevertheless toxicities were not observed in preclinical studies, safety measures are sometimes taken in clinical trials and post-marketing. Identification of risk factors that lead to specific AEs in patients are expected to identify populations with safety risk for drug treatment, thus leading to more rapid planning and implementation of safety measures. In addition, it could be possible to develop preclinical models that incorporate these risk factors and utilize them to discover drugs without safety concerns prior to clinical studies. To explore risk factors for such patients, utilization of real-world data, that is clinical information from actual sites where drugs are used, is a promising approach. To this end, we utilize data of Biobank Japan (BBJ), which is one of the largest biobanks in Japan including information of around 270,000 patients. By using BBJ, we can access not only to clinical information such as medial and prescription history, but also to genomic information including about 900,000 single nucleotide polymorphisms (SNPs). In this symposium, we will introduce our joint research activity with BBJ and discuss how to utilize biobank data to identify risk factors for adverse events from the perspective of genetics and pharmacoepidemiology.

Many chemical substances exist in the environment, and individuals are exposed to these chemicals. In the developing brain of mammals, various neuronal signals are activated appropriately for the construction of the neural network, and exposure to chemical substances in this period can induce developmental neurotoxicity. To investigate the effects of chemical substances, we devised a comprehensive set of behavioral assays designed to evaluate these processes objectively and quantitatively, alongside techniques for gathering neuroscientific data.

In this symposium, we mainly report the results using permethrin, a pyrethroid insecticide, as a model chemical substance. We exposed mice to permethrin in drinking water during the prenatal and postnatal periods, and behavioral tests were conducted on their offspring after maturation. As a behavioral effect, impairment in learning and memory was observed in male mice, suggesting that excessive production of immature neurons and dysfunction of astrocytes in the hippocampus may be involved as the effects on the formation of neural circuit base. On the other hand, in female mice, no distinctive effects observed in male mice were found.

Based on the above results, we would like to discuss the previous efforts to detect neurobehavioral effects that manifest after maturity due to chemical exposure in early life, as well as new issues such as low-dose effects of chemical substances and sex differences.

The intestines of humans and animals contain an extremely large number of microorganisms, most of which have a symbiotic relationship with the hosts. The microbial environment in the gut has a significant impact on the health of the host, and disturbances have a negative impact on their heath. The brain-gut interaction indicates that the state of the brain correlates with the gut, or the state of the gut also affects the brain. Therefore, it is understandable that deterioration of the microbial environment in the gut can be a factor in causing neuropsychiatric as well as other mental disorders. In addition, the microbial environment in the gut has a relationship not only to disease but also to the immune functions. For example, the microbial environment in the intestine is closely related to the intestinal immunity. In fact, the immune function in the gut of animals raised in a normal environment is rarely observed in animals maintained in germ-free facilities. We have found that the microbial environment of the intestine also has a significant impact on maternal antibody production in breast milk. Specifically, certain microorganisms present in the gut promote immune activation in the gut, resulting in the accumulation of antibody-producing cells from the gut to the mammary glands. In this presentation, I will explain the recent understanding of the relationship between immune function and the intestinal environment, and introduce the novel research aimed at enhancing the immune function of breast milk by improving the intestinal microbial environment.

Acephate, an organophosphate insecticide, are effective against many insects, but the effects of early exposure on the central nervous system of mammals are not fully understood. In addition, in recent decades, the bidirectional interaction between the central nervous system and the gut microbiota has begun to be elucidated. Therefore, there may also be a correlation between acephate-induced behavioral effects and gut microbiota alteration. In this study, we examined the effects of chronic acephate exposure in early life on the brain functions and gut microbiota in adult mice, including acceptable daily intake (ADI) level concentrations (0.03 mg/kg/day). We exposed maternal mice to low (ADI level : 0.3 ppm), medium (10 ppm), and high (300 ppm) doses of acephate via drinking water. Dams were exposed to acephate from the gestation period (E-11.5) to lactation period when pups were 2 weeks of age. Then, behavioral tests were also conducted on 12–13-week-old mice, and fecal samples were collected from the rectum of mice dissected at 13 weeks of age, and 16s rRNA analysis was conducted. In the treated groups, significant learning and memory deficits were observed in male mice. However, no significant behavioral effects in female mice were observed. Gut microbiota analysis showed a reduced diversity and an altered microbiome composition. These results suggested that chronic exposure to acephate during development, even at ADI level concentrations, leads to neurobehavioral diseases and disruption of the gut microbiota.

The brain's formation during development is dependent on activity-dependent processes such as stem cell differentiation and synaptic pruning. Environmental chemical exposure during these critical periods can have long-lasting impacts on brain circuit function, even at low doses considered safe in adults. In this talk, we will present our work using VSD imaging to detect the impacts of chemical exposure during development and discuss the challenges in this assessment.We will introduce our method for quantitatively detecting changes in hippocampal neural circuit function, a key brain region involved in learning and memory. Our findings on the effects of bisphenol-A (BPA) and related substances (BBMTBP and MBMTBP) on the hippocampal trisynaptic response suggest that exposure to BBMTBP and MBMTBP poses higher risks to hippocampal neural circuit function than exposure to BPA.Moreover, we will present our results using VSD imaging to quantify other brain areas, including the anterior cingulate cortex, which plays a crucial role in many neuropsychiatric diseases. Our findings establish the usefulness of VSD imaging in assessing low-dose chemical exposure during critical periods of brain development.Despite the challenges in detecting the impacts of developmental chemical exposure, VSD imaging provides a promising avenue for future research. We will discuss future directions for improving VSD imaging techniques and understanding the mechanisms underlying the impacts of chemical exposure on neural circuit function during development. Our work underscores the need for continued research in this area to better understand the impacts of environmental chemicals on brain development and function.

Drug toxicology analysis, which is handled in forensic toxicology, has so far focused on the analysis of drugs of abuse, the analysis of drug abusers' urine, etc., and the analysis for diagnosing poisoning deaths in judicial autopsies. Under the Basic Law for Promotion of Investigation of Causes of Death, which came into force in April 2020, drug and toxicology analysis was positioned as a scientific investigation of cadavers for the purpose of investigating causes of death. The Cause of Death Investigation Promotion Plan is divided into (1) development of human resources, (2) development of education and research bases, (3) nationwide development of specialized institutions, and (4) improvement of the implementation system. (1) Regarding the development of human resources, it is necessary to provide education and research on the cause of death in the education of forensic toxicology and toxicology in the Faculty of Pharmacy (pharmaceutical university) in the undergraduate education. (2) Regarding the development of education and research bases, the national government will allocate a budget to universities that are making progressive efforts in education and research related to the investigation of the cause of death. (3) Nationwide development of specialized institutions and (4) Enhancement of the implementation system will involve the construction and enhancement of a drug and toxicology analysis system for investigating the cause of death at universities and other institutions that analyze and research drugs and toxins. With the enactment of the Basic Law, forensic toxicology, toxicology education and research, and drug analysis related to the investigation of causes of death at universities (graduate schools) will likely change significantly.

Forensic dentistry is a relatively new research field established in 1964 as a study specializing in "personal identification" in forensic medicine. The Basic Act on Promotion of Cause of Death Investigation, which came into force in April 2020, comprehensively and systematically promotes measures for investigating the cause of death (investigation of the cause of death and identification). Forensic dentistry is in charge of enhancing the scientific investigation of cadavers for dental identification, which is one of the basic measures of the law, and developing a database for identification. For identification by teeth, postmortem dental charts are prepared by collecting intraoral findings of unidentified corpses with the naked eye and radiographs. On the other hand, borrowed the medical records and X-ray photographs stored at the family dental clinic where we had seen patients, and created an anti-mortem dental chart. The charts are compared and collated, and if the findings match, the affirmative decision is made. If the findings do not match, it is determined whether the findings are inconsistent by considering the passage of time, etc., and finally the possibility of the same person is determined. Recently, the development and improvement of equipment have been vigorously pursued for the purpose of digitizing intraoral photographs and thorough infection control. In addition, a project is underway to register the contents of medical records as part of the construction of an anti-mortem database. In addition, the expression of toxic substances found in teeth is also effective for identification, such as mottled teeth found in areas where drinking water contains high concentrations of fluoride, and the use of tetracycline antibiotics during the period of tooth formation. Tooth discoloration is typical, and these are called” Tooth Wear”.

[in Japanese]

The department of judicial chemistry has served as a channel to bring the scientific knowledge of the pharmacy faculty back to the society. Judicial chemistry is the integrated science of drug analysis, pharmacokinetics, and toxicology and is now used almost exclusively by forensic toxicology. Meanwhile, the six-year program at the pharmacy school has resulted in considerably fewer lectures on judicial chemistry. The Ming Plans to Promote the Cause of Death Investigation calls for the enhancement of content related to the cause of death in pharmacy education. Therefore, it is necessary to steadily reflect on the "forensic toxicology approach in determining the cause of death" presented in the recently revised Model Core Curriculum 2022 in each university's curriculum. Forensic toxicology should be understood in continuity with clinical toxicology as they inevitably go through a state of drug intoxication as a preliminary step to drug intoxicated death. Furthermore, by linking with toxicology, which primarily covers the mechanism of toxicity, it is possible to consider the relationship between tissue toxicity and individual death. Therefore, I believe that the establishment of an educational system in which students learn clinical toxicology, forensic toxicology, and mechanism-based toxicology in an integrated manner will lead to the development of personnel who can investigate the cause of death from a clinical perspective. In this symposium, I would like to present my personal views on education and research relating to cause-of-death studies from this perspective.

Since oncology pharmaceuticals having developmental and/or genetic toxicity are occasionally administered to the pregnant and/or the reproductive potential patients for life-prolonging, it is important to consider those risks to patients and their next generations. Especially, with the increase of consciousness regarding the fertility preservation for cancer patients including Adolescent and Young Adult (AYA) generation, it has been required to reconsider appropriate education about the birth control and the risk communication between medical professionals and patients. The U.S. Food and Drug Administration (FDA) and European Medicines Agency (EMA) have published guidelines to minimize the potential risk of developmental and genetic toxicity associated with cancer treatments in reproductive potential patients. In Japan, a working group in Japan Agency for Medical Research and Development (AMED), set up for providing information on the proper use of pharmaceuticals in patients with reproductive potential, has prepared a Japanese guidance regarding the need of contraception in patients administered pharmaceuticals to minimize potential risks of developmental and genetic toxicities in their subsequent generations. On 16th of February, the guidance was finally published with its Q&A. In this presentation, we will give an outline of this guidance and introduce the viewpoints of reviewers based on some examples of the safety evaluation on pharmaceuticals having developmental and/or genetic toxicities.

The risk of teratogenesis in female rabbits via male rabbit semen was evaluated using a representative teratogen, i.e., thalidomide (THA). To analyze the seminal transition after an oral THA treatment of 250 mg/kg, in male rabbits, liquid chromatography-mass spectrometry was performed to detect THA and its 5-hydroxylated (major metabolite in humans), and 5’-hydroxylated (major metabolite in rodents) metabolites in the seminal and blood plasma. As a result, THA concentrations and two metabolites in the seminal plasma were equivalent to those in plasma. Based on this toxicokinetic information, we administered 0.4 mg/kg of THA, which was 100 times the maximum seminal plasma transfer concentration, was administered intravaginally to female rabbits. The findings revealed that intravaginal THA administration did not induce any teratogenic effects in rabbit fetuses. In addition, THA concentrations and two metabolites in the placenta, yolk sac, and fetus were not influenced by the implantation position in the uterus. Comparing the pharmacokinetics of intravaginal and oral administration, there was a significant difference in maximum concentration and area under the curve up to the last quantifiable time-point, i.e., differences of 2500 and 5000 times, respectively. In this symposium, we will discuss the possibility of teratogenic effects mediated by seminal plasma through our thalidomide experiments in rabbits.

Developmental toxicity in experimental animals that involves intravaginal exposure to the chemotherapeutic drug thalidomide via semen may be controlled by the drug disposition of thalidomide from the blood plasma to the seminal plasma. In our rabbit experiments, the seminal plasma concentrations of thalidomide and the primary metabolite 5′-hydroxythalidomide 4 h and 7 h after oral doses were similar to their concentrations in blood plasma under the present conditions. There was no apparent condensation of the substrate thalidomide or 5′-hydroxythalidomide from blood plasma to seminal plasma, as evidenced by the similar observed blood and seminal plasma concentrations after oral administration of the main experimental dose of 2.0 mg/kg and after the preliminary dose of 250 mg/kg in rabbits. In contrast, the observed seminal plasma concentrations of the other primary metabolite, 5-hydroxythalidomide, were lower than those in the blood plasma of rabbits. The in vitro apparent permeability constants for thalidomide were determined to be similar in both directions, which is consistent with the reported findings of no apparent active transport of thalidomide by transporters, such as P-glycoprotein. The reason for the lower concentrations of 5-hydroxythalidomide in rabbit seminal plasma than in blood plasma is not clear at present, and it should be noted that 5-hydroxythalidomide can be further cleared from blood plasma through secondary oxidation metabolism by liver cytochrome P450 3A enzymes, as identified in humanized liver mice. Drug metabolism and disposition of thalidomide and related compounds, especially from the blood to seminal plasma, in animal models and humans are discussed.

In February 2023, the Ministry of Health, Labor and Welfare of Japan issued guidance outlining the conditions under which contraception is recommended after the last dose and basic principles regarding the duration of contraception. The guidance is intended to minimize the potential for developmental and genotoxic risks in the next generation and beyond due to the administration of drugs to fertile patients. Based on non-clinical, clinical, and post-marketing information, this guidance describes the need for contraception at the time of the last dose and thereafter in male patients of reproductive potential, and the methods and duration of contraception when considering the risk of developmental and genotoxic effects of the drug. The basic idea is that for drugs with genotoxic properties, the period of time after the blood concentration of the drug has decreased sufficiently + 3 months (considered the period of time involved in spermatogenesis). In addition to the effect on the male patient's sperm, the effect on the female partner, who is exposed to the drug via semen, should also be considered. The possibility of migration in semen is also assumed, and this is a point where a barrier method is needed to avoid absorption from the vaginal mucosa. However, there is no clear evidence regarding semen migration. In the case of non-genotoxic, developmental toxicants and chromosome aneuploidy inducers, the effects of semen transfer of the drug must be considered, but it is considered acceptable to establish a contraceptive period or to determine that contraception is unnecessary based on the data on which it is based. This session will consider the concept of contraceptive needs associated with the administration of pharmaceuticals from a clinical standpoint.

Most cancer cells show abnormalities in chromosome number and structure (aneuploidy), which are attributed to chromosomal instability (a condition in which chromosome missegregation occurs at a high rate). Whether aneuploidy is a cause or consequence of cancer has long been debated, but recent studies have shown that aneuploidy promotes cancer progression and acquisition of drug resistance. For equal chromosome segregation, all pairs of replicated chromosomes (sister chromatids) must attach to microtubules extending from different spindle poles (biorientation), and to ensure this, there are mechanisms to correct erroneous attachments and to prevent chromosome segregation in the presence of erroneous attachments (spindle assembly checkpoint). Abnormalities in these mechanisms lead to chromosome missegregation, which results in micronucleus formation, causing rearrangement of chromosomes in the micronuclei (chromothripsis) and activation of the cGAS-STING pathway, an innate immune response. In this symposium, I will review the mechanisms of oncogenesis caused by chromosome missegregation. We have recently found that iterative motion of chromosomes on the spindle (oscillation) contributes to the equal segregation of chromosomes. Interestingly, chromosome oscillation is attenuated in cancer cells compared to normal cells, and I introduce this novel cause of chromosomal instability.

A micronucleus (MN) is a small nucleus formed from a chromosome fragment due to chromosome instability or mitotic abnormalities. Although MN has been used as a marker of chromosomal aberrations in the safety assessment of chemicals, its biological significance and the effects on the micronucleated cells remained unclear. However, recent studies have shown that MN could be the cause of cancer via chromothripsis. Chromothripsis is a phenomenon in which chromosomes are disrupted and rearranged in the MN, then leak into the cytoplasm through the rupture of the nuclear envelope and are incorporated into the primary nucleus during cell division. It is believed that the incorporated abnormal chromosomes cause drastic gene mutations, resulting in changes in the expression of several genes involved in tumor development and progression. Indeed, massive chromosomal rearrangements considered to be caused by chromothripsis have been found in a variety of human tumors by whole-genome sequencing using next-generation sequencer, suggesting that chromothripsis occurs in tumorigenesis and malignant transformation. On the other hand, such phenomena have not been reported in chemical carcinogenesis, although there are a number of clastogens and aneugens that form MNs in chemicals. Recently, we found that a rat hepatocarcinogen, acetamide, induces characteristic large MNs in the liver. In addition, molecular pathological analysis of large micronuclei and whole genome sequencing of acetamide-induced liver tumors revealed that acetamide induces chromothripsis-like chromosomal aberrations in the process of tumorigenesis. In this presentation, I will present the results of these studies and discuss the possible involvement of chromothripsis in chemical carcinogenesis.

Chromosome aberrations have long been studied as a biological marker of ionizing radiation exposure. Ionizing radiation induces DNA double-strand breaks (DSBs), which in turn cause chromosomal rearrangements, such as chromosomal translocation and inversion, via misrepair of DSBs, leading to carcinogenesis. Recent advances in next-generation sequencing technologies have enabled to comprehensively analyze the characteristics of chromosome rearrangements and information on the repair process of DSBs in radiation-induced tumors. On the other hand, uncertainty exists in radiation-induced cancer risk assessment, especially for low-dose radiation exposure, due to the inability to distinguish between spontaneous and radiation-induced tumors. Thus, we have been studying genomic aberrations observed in radiation-induced rodent tumors to identify molecular indicators of radiation-induced cancers that may be useful for cancer risk assessment.

In this presentation, as a case study of chromosomal rearrangements involved in radiation-induced carcinogenesis, we will discuss the characteristics of fusion genes, such as tyrosine kinase fusion genes, identified in radiation-induced tumors by RNA sequencing and chromosomal rearrangements and misrepair result in the generation of fusion genes, as well as genomic aberrations and chromosomal rearrangements identified by whole-genome sequencing.

Cellular senescence is a state of irreversible cell cycle arrest induced by many stressors. Senescent cells that accumulate in vivo during aging communicate with surrounding tissues through proinflammatory protein production, termed the senescence-associated secretory phenotype (SASP), which plays several physiological and pathological roles. In aged individuals, inflammatory SASP factors promote numerous age-related diseases, including some cancers. Therefore, elucidating the regulatory mechanism of SASP is vital for developing new preventive and therapeutic strategies against age-related cancer. We have previously shown that chromatin fragmentation and epigenetic alteration induce SASP gene expression through the activation of the DNA sensing pathway during cellular senescence. Furthermore, in addition to inflammatory proteins, we also found that senescent cells secrete a numerous number of small extracellular vesicles (EVs) and promote proliferation of cancer cells. Then, small EVs secreted from senescent cells may function as one of tumorigenic SASP factors in vivo. In addition, we have reported that senescent cells inhibit cell competition through a SASP factor. Recently, senolytics, which induce selective cell death in senescent cells, have attracted attention as a way to suppress the development of age-related diseases. It is expected that administering senolytic drugs can prevent and treat cancer development and progression. Therefore, we hope that new cancer treatments can be developed by analyzing the function of senescent cells in the cancer microenvironment.

The study director is defined in OECD GLP as "the single point of study control with ultimate responsibility for the overall scientific conduct of the study" in conducting toxicity studies. In addition, MHLW GLP is specifically stated that "responsible for conducting, recording, and reporting of the study". The increasing complexity of toxicity studies due to the recent diversification of drug modalities requires improvement of the ability of study directors. More specifically, study directors are required to understand the properties of the test article, to judge the appropriateness of special experimental procedures, and to scientifically discuss the obtained results. In order to ensure these points, we will present the current status of CRO's criteria for appointing and educating study directors, and will further discuss the issues to be resolved in the future.

In recent years, nonclinical safety studies, particularly GLP complied studies, of pharmaceuticals, chemical substances, and agrochemicals has become common to primarily conducted by the CROs. However, for example of pharmaceuticals, as drug discovery modalities have diversified from low molecular weight compounds to antibody drugs, nucleic acid drugs, gene therapy, and regenerative medical products. As a result, their safety evaluation has become increasingly challenging, and the level of demand for toxicologic pathologists as well as study directors has also been increasing. The level of demand for pathologists in charge of pathology evaluation as well as study directors is increasing. Under such circumstances, I will discuss the current status and challenges of toxicologic pathologists in CROs, including pathological evaluation, training and education, and the measures to new technologies/tools such as whole mount imaging (WSI) and digital pathology.

Bisphenols are used as curing or crosslinking agents in the processing of fluorocarbon elastomers, rubber processing, and specialty polymers due to their thermal stability, chemical resistance, and compression set resistance. These properties have led to widespread use in consumer products such as food storage containers and medical devices. We have investigated the impact of oral exposure to Bisphenol AF (BPAF) on the developing immune system of Harlan Sprague Dawley (HSD) rats. Dosed feed was provided ad libitum to pregnant HSD rats at BPAF concentrations of 250, 500 and 1000 ppm from gestation day 6 (GD6) through weaning on postnatal day 28 (PND28). F1 pups were provided dosed feed through 11-12 weeks of age. No major reproductive effects were observed in F0 rats exposed to BPAF at doses up to 1000 ppm during gestation and lactation although there was a decrease in dam bodyweights at the highest dose level. Gender- and treatment-specific effects in lymphoid organ weights and hematological effects in erythroid and myeloid cell populations were observed in the F1 generation. The antibody forming cell response to sheep red blood cells was suppressed in F1 male rats treated with the highest dose of BPAF, but unaffected in similarly exposed F1 females. T cell proliferation was suppressed in F1 female rats in in the 500 and 1000 ppm exposure groups but unaffected in F1 male rats. Taken together, these studies suggest that bisphenol analogues demonstrate the ability to modulate the immune system and can both stimulate and suppress immune function. As BPA analogues are used with increasing frequency their toxicity should be investigated fully to protect public health. This research was supported by the Intramural Research Program of the NIH and performed under contract HHSN273201400017C.

Ultrafine particles (UFPs, ≤ 100 nm diameter) are a class of particulate matter pollutants ubiquitous in urban environments but generally lack explicit regulatory standards. Findings from our mouse model employing UFP exposure during pregnancy demonstrate offspring sex-specific effects on pulmonary immune responses when neonates are challenged with respiratory syncytial virus (RSV), a frequent cause of infant respiratory infection and hospitalization. We show that a lack of Nrf2-driven antioxidant signaling in response to in utero UFP exposure exacerbates pulmonary T cell skewing and induces pro-inflammatory effects and transcriptomic changes in fetal lung genes related to lipid metabolism and transport pathways, particularly in female offspring. In analogous work, we demonstrate the state of pregnancy also alters maternal pulmonary immune responses to UFPs and influenza, an important cause of respiratory viral infection in pregnant women. Results indicate UFP exposure during pregnancy increases susceptibility to and severity of influenza infection. Co-exposure results in decreased weight gain throughout pregnancy, elevation in viral titer and reduced lung inflammation, signifying immune suppression. Overall, findings emphasize common immunomodulatory effects from UFP exposure during pregnancy, on pregnant women and offspring, supporting interventions to protect these susceptible populations.

Recent changes in health are thought to involve the combined effects of lifestyle and environmental factors, including chemical exposure. Individuals differ in their sensitivity to the environmental factors. Life stages (perinatal, childhood, and elderly) and underlying diseases are important vulnerability factors. For these vulnerable populations, even low concentrations of chemicals can cause adverse health effects. Furthermore, endocrine disrupting chemicals may not be assessed for their toxicity with a simple dose-response relationship.

Immunotoxicity has attracted attention because chemicals such as bisphenol A (BPA) and per- and polyfluoroalkyl substances can disrupt immune system at low doses. This presentation will discuss the immunotoxicity assessment of chemicals considering vulnerability, focusing on the effects of bisphenols on allergic diseases.

BPA is used as a raw material for polycarbonate and epoxy resins in a variety of household products, including plastic products. In recent years, regulations on BPA have been tightened and thus the use of its substitutes has increased worldwide. However, there are also concerns about their health effects.

We have previously shown in animal models that exposure to low-dose BPA and its typical substitute, bisphenol S, during the juvenile period induces exacerbation of allergic asthma and modifications of immune cell function contributed to the pathogenesis. Our findings suggest that bisphenols may disrupt the immune system and exacerbate allergic reactions at the levels equivalent to human exposure.

Gaseous chemicals in the atmosphere such as indoor Volatile organic compounds (VOCs) have long been realized as the risk factor for several types of immune disorder including allergic asthma and chronic obstructive pulmonary disease. Gaseous chemicals are transparent and odorless, therefore, there have not been enough safety precautions so far. Our field survey inside the medical settings (indoor VOCs were measured by GC-FID and GCMS.) indicates that poor air circulation obviously increases the concentration of VOCs beyond the safety level. Recently, our group demonstrated that exposure to safety standard level ozone gas, which is one of the VOCs, significantly aggravates the symptoms of acute lung injury in a mouse model, which indicated low levels of gaseous chemicals possibly has an impact on the development of respiratory diseases. The object of this study is to examine the possible relationship between indoor VOCs and the development of several immune disorders, and we compared the development of asthma and atopic dermatitis symptoms in mice between normal room air (contained VOCs) and clean air removed VOCs by an activated carbon chemical filter. The VOCs in the clean air treated with activated carbon chemical filters were measured by the GC-FID method, and it was verified that VOCs were removed by about 90% compared to normal air. Our findings suggest that the control of VOCs may influence the development of immune disorders since VOCs at concentrations similar to those in a typical hospital setting suppress the initial stage of allergic development in mice.

We have investigated the mechanisms of genotoxicity involving proteins trapped at the ends of DNA. We found that the reduced repair efficiency of this type of DNA damage has a pivotal role in some diseases, such as breast cancer. At this symposium, we will present the induction of genotoxicity involving topoisomerases. Women having BRCA1 germ-line mutations develop cancer in breast and ovary, estrogen-regulated tissues, with high penetrance. Binding of estrogens to the estrogen receptor (ER) transiently induces DNA double-strand breaks (DSBs) by topoisomerase 2 (TOP2) and controls gene transcription. TOP2 resolves catenated DNA by transiently generating DSBs, TOP2-cleavage complexes (TOP2ccs), where TOP2 covalently binds to 5′ ends of DSBs. TOP2 frequently fails to complete its catalysis, leading to trapping TOP2ccs. We found that BRCA1 promotes the removal of trapped TOP2cc. BRCA1 thus plays a critical role in removing pathological TOP2ccs induced by estrogens. We propose that BRCA1 suppresses tumorigenesis by removing estrogen-induced TOP2ccs throughout the cell cycle. Furthermore, we focused on TOP1cc removal mechanism and found that TOP1cc is repaired via a two-step pathway involving proteasomal degradation of TOP1cc to the crosslinked peptide, followed by removal of the TOP1cc-derived peptide from DNA by tyrosyl-DNA phosphodiesterase 1 (TDP1). Our recent observations on the genotoxicity related to trapped topoisomerase will be discussed.

Genomic DNA is continuously exposed by exogenous and endogenous chemical reagents. Such chemical exposure may induce not only gene mutations and chromosomal aberrations via formation of DNA damages, but also epigenetic alterations via aberrant DNA methylation and histone modifications. Standard genotoxicity assays have been widely used to screen genotoxic reagents that potentially cause DNA damages in genomic DNA. Based on the in vitro genotoxicity test method, we developed a new assay system evaluating epigenetic alteration in the human genome. Using a human lymphoblastoid TK6 genotoxicity test cell line, “mTK6” cell line was established via DNA methylation at the promoter region of the endogenous TK gene by CRISPR/dCas9-DNMT3A system. The DNA methylation status at the TK reporter gene was bi-directionally variable, which was expected to enable to quantify the alteration of epigenetic profile. Obviously, based on the principle of TK gene mutation assay, epigenetic effects of certain reagents were quantitatively assessed by determining the number of TK revertant colonies after exposure to chemicals. Our results suggest that this method enables the evaluation of chemical-induced epigenetic effects in human cells without the use of expensive instruments. In this symposium, the usefulness of the established assay for detecting epigenetic effects and elucidating mechanisms of epi-genome maintenance will be discussed.

Several clinical holds in US in 2004 due to mutagenic impurities surprised pharmaceutical companies and triggered the greatest efforts to control the impurities. It was not realistic to examine all potential impurities with Ames test. (Q)SAR assessment was introduced instead. (Q)SAR was quickly spread in EU and US after the EMA and FDA guidelines requiring (Q)SAR assessment were published in 2006 and 2008. Because PMDA did not recommended (Q)SAR assessment before ICH M7 Step5 in 2015, we were greatly behind in using (Q)SAR. Currently, various (Q)SAR tools are available but the outcomes from any tools should be reviewed and complemented by toxicologists. We need further understanding and experiences on expert judgments. Japanese Environmental Mutagen and Genome Society (JEMS) has held yearly workshop (WS) where experts from (Q)SAR developers, pharmaceutical companies and regulatory agencies discuss to seek common understanding and improvement of risk assessment. Here, I discuss how toxicologists can review (Q)SAR outcomes based on the accumulated knowledge of the WS.

Genotoxicity has been evaluated via tests utilizing indicator genes, such as the Ames test. However, these methods do not provide sufficient qualitative and quantitative mutation data for each mutagen. The newly developed error-corrected sequencings (ECSs) enable direct detection of chemically-induced mutations by reducing the genome-sequencing error rate to ca. 1/10⁷ bp. ECSs can improve our understanding of chemical mutagenicity by generating high-resolution mutation data. We had previously developed an original ECS method (Hawk-Seq^TM) and demonstrated its efficiency in detecting mutations. Hawk-Seq^TM could detect mutations caused by various mutagens in diverse genotoxicity test models (e.g., S. typhimurium, TG mice, and cultured human cells). The mutation spectra generated by Hawk-Seq^TM reflected the structural features of each mutagen, leading to a systematic understanding of mutagenicity. Moreover, we identified that the errors derived from single DNA strand overhangs were not eliminated from the Hawk-Seq^TM data. Therefore, we developed a new method, Jade-Seq^TM, which provided a 10-fold reduction in error frequency compared to Hawk-Seq^TM by utilizing a single-strand specific nuclease. This method will help elucidate rare mutations or improve mutation analysis robustness. Finally, we started a collaborative study to evaluate the sensitivity and reproducibility of Hawk-Seq^TM in the Mammalian Mutagenicity Study Group in the Japanese Environmental Mutagen and Genome Society. Our research will promote the application of ECS to the genotoxicity evaluation of chemicals.

Although mid-molecular peptide drugs have the potential to address an unmet need for drugs, approaches to the evaluation of middle molecular peptide drugs have not yet been established at the global level. ICH S2(R1) Guidance on Genotoxicity Testing and Data Interpretation for Pharmaceuticals Intended for Human Use is considered to be a useful for genotoxicity assessment of mid-molecular peptide drugs containing non-natural amino acids. However, molecular properties such as the molecular weight should be taken into account in evaluation for genotoxicity of the drugs. Especially, since the bacterial reverse mutation assay which is known as the Ames test has been mainly used for detection of mutagenicity caused by small molecule compounds, it is also necessary to confirm whether the Ames test is accepted for genotoxicity testing for the drug development. In addition, it is important for considering the concepts for the toxicological assessment of genotoxic impurities in the drug substances. In this presentation, I would like to discuss the points to be considered for genotoxicity evaluation of the drugs in order to properly evaluate the safety of drugs based on scientific knowledge.

Next generation chemical risk assessment (NGRA) aims to replace and enhance traditional toxicity testing via in vitro new approach methodologies (NAMs). Translating in vitro points of departure (PODs) to more traditional contexts requires in vitro-in vivo extrapolation (IVIVE) based upon toxicokinetics (TK). Information needed for risk assessment – characterization of chemical hazard, exposure, and TK – are often unavailable for non-pharmaceutical chemicals. Single chemical methods for IVIVE have been thoroughly developed by the pharmaceutical industry, but higher throughput toxicokinetic (HTTK) methods are needed to accelerate the pace of chemical risk assessment via higher throughput IVIVE. While HTTK is unlikely to produce better predictions than single chemical (“bespoke”) models developed with detailed chemical-specific data, we expect HTTK to be more reproducible and more throughly statistically evaluated, with more accurately quantified uncertainty. Because of throughput and transparency, HTTK may, in some cases, be suitable to decision-making contexts. Chemical prioritization efforts based upon HTTK are under consideration at U.S. EPA, Health Canada, and the European Food Safety Authority. There are thousands of chemicals are in need of triage, prioritization, and, potentially, full assessment. HTTK is a key technology supporting next generation approaches to chemical decision making. The views expressed in this presentation are those of the authors and do not necessarily reflect the views or policies of the U.S. EPA.

To improve R&D efficiency and facilitate scientific decision making in drug discovery and development by using mathematical model of pharmacology and toxicology, quantitative systems pharmacology (QSP) is increasingly being utilized in pharmaceuticals and biotech industries.

QSP approach has emerged as a central technology in innovative framework based on “learn and confirm” cycle, which is currently called as Model-informed Drug Discovery and Development (MID3) (CPT Pharmacometrics Syst Pharmacol. 2016; 5: 93-122.). QSP approach is supporting the rational decision in drug discovery and development from exploratory research to late-stage clinical development, and enables selection/validation of novel target molecules, identification of mechanism-of-action of pharmacological/toxicological compounds, prioritization of pharmaceutical candidates, facilitating translational research, optimization of clinical trials, and others.

In this presentation, I explained summary and effectiveness of QSP approach by showing our case-studies related to safety assessment by using virtual patients, based on mechanism-of-action of compounds. These are 1: risk assessment of drug-induced proarrhythmic effect, 2: drug-induced liver injury (DILI), and other, they have been good practices in my experiences of applying QSP at pharmaceutical company.

When we humans think of the properties of a chemical, for example, a chemical has functional groups A and B, has toxicity T, and has a molecular weight of M, we recognize it as such. Although we can handle only a few dimensions in this way, we can manage higher dimensional information with a computer. Thus, for expansion of our recognition for a target with a computer, it is an indispensable operation to quantify the target as high-dimensional information comprehensively and without arbitrariness. Modeling is also indispensable for outputting the information of high-dimensionality thus expanded to a dimension that we can recognize again. In other words, numericalization and modeling enable us to handle information beyond our perception and to discover new aspects of the object of analysis.

Our group is developing research on the understanding and utilization of chemicals based on the above-mentioned principle of "Expansion with Computer and Extraction with Modeling". For instance, we have taken the approach of describing the effects of chemicals by capturing the biological responses of cultured cells treated with chemicals using transcriptome data, and of adapting a latent variable model to them for finding the common mechanisms behind the effects of the chemicals. End-to-end analysis, in which general deep learning models excel, is a sequential operation consisting of feature extraction and modeling, and is located in the same framework.

In this presentation, we focus on how to represent chemicals for toxicological studies and safety assessment. The presentation will focus on the recent progress in decomposition of chemical effects, numerization of chemical structures, and numerization of chemical effects in vivo.

Ten years have passed since the 7th revision of the EU Cosmetics Directive, which banned the sale of cosmetics containing ingredients that have been tested on animals in the EU. While there is no standardized alternative method, especially for systemic toxicity assessment, the safety assessment of cosmetics is based more on a combination of available information and test results, as exemplified by the Next Generation Risk Assessment (NGRA) framework. When NOAEL of the substance to be evaluated (target) are not available, the next approach is considered to be read-across. Read-across, which estimates the toxicity of a target based on information analogs, has already become a standard safety assessment method with widespread use in many industries. Although read-across is one of the lower tiers in the NGRA, the lack of a database from which sufficient analogs and their toxicity information can be collected remains a challenge. Another issue is the difficulty in explaining the toxicological relevance of the selected analogs. In recent years, there has been much attention paid to the investigation of new read-across methods based on chemical structure information combined with in vitro systems such as transcriptome data. To solve these problems, we have been developing a comprehensive read-across method to extract analogs from more than 200 million compounds in the world and a method to extract analogs based on transcriptome data, aiming to expand read-across to mixtures of compounds such as extracts in the future. In this symposium, we will present the latest trends and challenges in safety evaluation methods using read-across and transcriptome data, as well as the results of our latest studies on our efforts.

Surgery, radiotherapy, and chemotherapy are the three major treatments for cancer. Among them, chemotherapy based on the administration of "anticancer drugs" has the shortest history, but it has achieved the most remarkable development in recent years. The birth of anticancer drugs dates back to the development of nitrogen mustard in 1946. Since then, various anti-cancer drugs (classical anti-cancer drugs) have been developed that exert cell-killing by inhibiting basic functions of cells, such as DNA synthesis, but they showed serious toxicity. The discovery of oncogenes in the 1980s brought rapid advances in cancer biology, bringing about a turning point in anticancer drugs. In 2001, a molecular-targeted drug (MTD), imatinib, which inhibits the oncogene product BCR-ABL, showed an amazing efficacy on chronic myelogenous leukemia. It ushered in the era of MTD, and over the past 20 years, more than 150 new drugs have been developed, transforming cancer treatment. MTDs act on targets that are specifically or more frequently expressed in cancer, so they have lower toxicity than classical anticancer drugs, but they also have unique toxicity. Remarkably, in 2015, immune checkpoint inhibitors such as anti-PD-1 antibodies appeared, and cancer immunotherapy came into the spotlight. During past 20 years, the development of antibody drugs as a modality has been remarkable, accounting for more than 30% of MTDs. The development of antibody-drug conjugates (ADCs) is also attracting attention. Furthermore, development of new modalities such as PROTAC (proteolysis targeting chimera), nucleic acid drugs, and middle-sized peptides is underway. In this presentation, I will briefly review the development of these anticancer drugs and the changes in toxicity factors.

Technologies to induce targeted protein degradation have been developed recently and provoke novel drug discovery research. Compounds that induce protein degradation were largely classified into two groups, molecular glues and chimeric compounds. Molecular glues such as lenalidomide interact with an E3 ubiquitin ligase CRBN and modulate the selectivity of the binding proteins to be ubiquitylated and subsequently degraded by proteasome. Another class of molecules, chimeric compounds represented by PROTACs (Proteolysis Targeting Chimeras) and SNIPERs (Specific and Nongenetic IAP-dependent Protein Erasers), are hybrids of an E3 ligand and a target ligand conjugated by an appropriate linker. Because of the modular structure of PROTACs and SNIPERs, compounds that degrade proteins of your interest can be rationally designed and chemically synthesized. Currently, more than a dozen chimeric degraders are under clinical trials.

In contrast to small molecule inhibitors, which exert their pharmacological effects by binding to their target proteins, PROTACs/SNIPERs catalytically degrade target proteins and thus exert a different pharmacological effect than small molecule inhibitors. These include more sustained suppression with lower concentration, higher selectivity to the target proteins, and suppression of all functions of the multifunctional proteins. However, when adverse effects are induced by degraders, they may be more difficult to control than inhibitors. Therefore, it is important to carefully pay attention to the potential adverse effects induced by degraders. In the symposium, I will introduce degraders with anti-cancer activity and overview future perspectives of the degraders as a novel modality for drug development.

A systemic anticancer chemotherapy induces a variety of side effects by affecting normal cells. Therefore, to reduce side effects and increase the efficacy, the development of DDS techniques and drugs aimed at accumulation in cancer tissues have been promoted. Antibody-Drug Conjugates (ADCs) composed of cytotoxic drugs with severe side effects and monoclonal antibodies with less drug efficacy instead of fewer side effects, which directly binds to the target antigen and delivers the drug to cancer cells leading to selectively and effectively kill cancer cells. By expanding the therapeutic dose range between the minimum effective dose and the maximum tolerated dose, it is expected to exert a strong anticancer effect compared with generally used chemotherapeutic agents. Multiple ADCs have already been launched, and in addition, many ADCs for advanced cancer are being developed in clinical trials. Trastuzumab deruxtecan (T-DXd, DS-8201), developed by Daiichi Sankyo Co., Ltd., is an ADC that binds the exatecan derivative DXd, a DNA topoisomerase I inhibitor, to anti HER2 antibody via a cleavable linker in tumor cells. Its main features are high uniformity with a high drug-antibody ratio of 8 on average, strong drug efficacy to heterogeneous tumors due to bystander effects being independent on the target molecule, and high stability in circulation enabling a wide therapeutic window. The technical features and efficacy of T-DXd will be summarized and its toxicological profile including interstitial lung disease (ILD) will be discussed both clinically and non-clinically.

Immunotoxicology research has progressed with establishment of test methods, international harmonization of evaluation procedures and mechanism-based assessment of immunotoxicity. ICH S8 guideline obviously drew attention to immunotoxicology. Weight of evidence approach is ingeniously taken considering the development process of pharmaceuticals. Immunotoxicological evaluation of new modality drugs (e.g., antibody and nucleic acid drugs), however, are not covered by the S8 guideline.

Currently, the necessity of the guideline for these new modality drugs is widely discussed around the world. Related immunotoxicity data which can be shared among the experts are now intensively discussed in the immunotoxicology community. There are three options in this matter; no guideline, revision of S8 guideline and newly-developed guideline. The last option may be beneficial and feasible.

It is extremely important to study the (immuno)toxicity of environmental chemicals to maintain the sustainable world. As for environmental chemicals, arsenic, tin, TCDD, and further in recent years asbestos, microparticulate matters, bisphenol A, phthalic esters and per- and polyfluoroalkyl substances (PFAS) have been the subject of immunotoxicity assessment due to the public concern raised. The mechanism study of the immunotoxicity is appreciated. At the same time, the significance of field works and epidemiological studies of environmental chemicals should be reconsidered.

Immunotoxicology holds an important position in the field of toxicology, because liver, renal, dermal and reproductive toxicities and carcinogenicity are sometimes closely associated with immunotoxicity. The presentation will give a chance to think about the current standpoint for immunotoxicology researchers.

The immune system functions through the complex interaction of various immune cells. It is important to evaluate immunotoxicity in the preclinical or clinical phases of drug development because unintended immune response may cause serious adverse effects due to immunosuppression or immunoenhancement. The ICH guidelines require the evaluation of immunotoxicity, and the Immunotoxicity Guideline (ICH S8) describes considerations and test methods for the weight of evidence. Immunotoxicity evaluations, so far, have focused on immunosuppression by small molecule drugs. However, in recent years, the modality of pharmaceuticals has been diversifying, and the description of ICH S8 is not always sufficient. The modalities of pharmaceuticals include small molecule drugs, antibody drugs, cell/gene therapy products, oligonucleotide drugs. In the case of antibody drugs, the cytokine release syndrome observed in the TGN1412 clinical study has raised concerns about toxicity due to immune stimulation, and although not described in the guidelines, pharmaceutical companies and CROs have established in vitro evaluation systems using human cells, and many antibody drugs were evaluated by the assay systems. It is difficult to apply a uniform test system to immunotoxicity concerns in each modality, so assay systems are selected on a case-by-case basis. In this presentation, I will introduce the current situation and regulations, and expect that my presentation will lead to the industry-government-academia cross collaboration.

The aim of this symposium is to facilitate to know immunotoxicology, and the present lecture shows the studies about immunological effects of silica and asbestos, summarize the point of those, and finally inform that immunotoxicological knowledge is useful for understanding of the diseases. Both of exposures to silica and asbestos, composed of silica dioxide in major proportions, cause pneumoconiosis and lung cancer following inflammation. However, silicosis is associated with autoimmune diseases of rheumatoid arthritis and systemic scleroderma, whereas asbestos causes a characteristic disease of malignant mesothelioma, suggesting difference in immunological effects. Therefore, we executed fundamental experiments using cell cultures upon exposure to silica or asbestos as well as functional analyses for immune cells of patients. It was found that silica exposure caused increase in CD69⁺ activated T cells and decrease in Foxp3⁺ regulatory T (Treg) cells, while patients with silicosis showed decrease in Treg function and high soluble IL-2R as an activation marker in peripheral blood. In contrast, asbestos exposure caused decreases in activating receptors on NK cells, Th1 function of CD4⁺T cells and cytotoxicity of CD8⁺ T cells as well as increase in Treg function, while some of those alterations were also found in peripheral blood of mesothelioma patients. Those indicate that silica and asbestos not only cause chronic inflammation and lung fibrosis commonly, but also affect lymphocytic functions differently, where the former leads to over activation of immune response and the latter causes suppressed tumor immunity, linked to the related diseases. Thus, it is beneficial for understanding the disease to know immunological effects. It is expected that immunotoxicological studies will grow more, resulting in more development of toxicology.

Recent studies have highlighted the strong relationship between specific human leukocyte antigen (HLA) alleles and the onset of idiosyncratic drug toxicities. For example, HLA-B*57:01 is a potential risk factor of abacavir-induced hypersensitivity. However, the precise mechanism of the onset has been still unrevealed, and we are attempting to elucidate it using HLA transgenic mice (HLA-Tg).

In order to reproduce the HLA-dependent immune reactions, a mouse-human chimeric HLA gene was transduced to mice. The resulting HLA-B*57:01-Tg exhibited proliferation and activation of lymphocytes including CD8⁺ T cells by exposure to abacavir. However, we could not observe strong toxic symptoms in the mice. We identified one of the main reasons was upregulated immunosuppressive factors, thus knockout of PD-1 gene and depletion of CD4⁺ T cells in HLA-B*57:01-Tg strongly induced immunotoxicity.

HLA-mediated drug toxicities are frequently observed in several specific tissues including skin. Our study indicated that exposure to abacavir induced endoplasmic reticulum (ER) stress in HLA-B*57:01-Tg. Especially, HLA-B*57:01-expressing keratinocytes evoked ER stress by abacavir. The relief of the ER stress by a chemical chaperon in HLA-B*57:01-Tg attenuated abacavir-induced toxicity, implying that ER stress played a crucial role in the onset of HLA-mediated drug toxicities.

Today, I would like to introduce the mechanism underlying HLA-mediated immunotoxicities from in vivo studies using HLA-Tg to in vitro analyses of HLA molecular characteristics.

Allergic contact dermatitis is a delayed hypersensitivity reaction categorized as a Type IV allergy and involves various immune cells. It greatly affects the quality of daily life because contact with various chemicals is inevitable, therefore, evaluating the potential of skin sensitization is an important safety endpoint. Regarding the mechanism of skin sensitization, the adverse outcome pathway (AOP) has been developed and is relatively simple compared with other toxicological endpoints. Therefore, multiple in vitro or in silico test methods have been developed. To date, several in vitro test methods corresponding to each key event of the AOP have been listed in the OECD test guidelines, however, they are not stand-alone methods. Therefore, a combination of multiple in vitro and in silico models was developed and became the OECD test guideline TG497, as a first defined approach method in 2021. However, in vitro and in silico tests that can be used for the guideline are limited, and any potency prediction method, which is necessary for risk assessment, has not yet been established. Therefore, the OECD is currently conducting a study aimed at expanding TG497, and the current status will be introduced in my talk. In addition, the presenter has been working on the development of a potency prediction method for skin sensitization using machine learning, and will introduce the summary of the method. Furthermore, the development of the threshold of toxicological concern concept for skin sensitization will be introduced as an application example. In addition, the presenter will also introduce an attempt to predict a potency of Bandrovsky base, which is a trimer of the oxidation dye paraphenylenediamine using the prediction model. Lastly, a quantitative risk assessment case study of Bandrovsky base without animal testing will be introduced.

The growing evidence of gender-specific differences in drug efficacy and adverse reactions has prompted urgent action in biomedical research. Because the June 2015 statement (NO-OD-15-103) was issued, there has been a great deal of recent attention to sex difference research. Our laboratory focuses on biological sex differences in the cardiovascular field. For example, the risk of delayed ventricular repolarization (QT prolongation) and the incidence of drug-induced arrhythmias are known to be more prone to women than to men. This sex difference is associated with a longer QT interval in adult women than in men of the same age. Changes during puberty and the menstrual cycle suggest a sex hormone influence. We have shown from rodent experiments that the molecular mechanism may involve

NO-mediated sex hormone signaling in cardiomyocytes. However, the translational question of how to translate the results of such basic research into an interpretation of biological responses remains unresolved. Gender differences in anti-cancer drug-induced cardiotoxicity are also an issue to be addressed in the future. In this symposium, we would like to present our original approach and discuss future issues.

While Gastrectomy is recommended worldwide as the standard treatment for gastric cancer (GC), there are regional differences in the choice of adjuvant chemotherapy. In East Asia, including Japan, capecitabine plus oxaliplatin (CapeOX) is the standard adjuvant chemotherapy. CapeOX was established in the CLASSIC trial conducted in Korea, China, and Taiwan, and demonstrated a 3-year disease-free survival (DFS) rate (HR 0.58, 95% CI 0. 47-0.72, p＜0.0001) and OS (HR 0.66, 95% CI 0.1-0.85, p0.0015). Although mean survival has improved, many patients experience serious adverse events. To optimize the medicine, prognostic factors affecting treatment need to be identified, including age and sex. Thus, we examined prognostic factors using a Cox proportional hazards model with stepwise variable selection based on individual patient information from the CLASSIC trial. The interaction of sex, age, and serum albumin for OS and tumor stage (T) and nodal status (N) for DFS were identified as significant. Other overall trends were consistent for OS and DFS. The HR for OS in female patients aged ≧55 years was 1.16 (95% CI 0.55-2.47) for albumin ＜4.0 g/dL and 2.39 (95% CI 1.15-4.94) for ≧4.0 g/dL, which seemed particularly the latter patients were found to be considerably less favorable for treatment. For patients with ≧T3 and ＜N2, the HR for DFS was 0.84 (95% CI 0.60-1.17), obscuring the benefit of treatment. Although these results are hypothetical due to the post hoc analysis, patient background should be fully considered when deciding on a treatment strategy for adjuvant therapy of GC.