We aimed to examine the effects of PM2.5 exposure on the prognosis of ischemic stroke in mice. Mice were intranasally administered PM2.5 for 7 days and then subjected to focal cerebral ischemia. The prognosis was noticeably aggravated by PM2.5 exposure compared with vehicle-treated mice. Polycyclic aromatic hydrocarbons (PAHs) induced inflammatory reaction in macrophages and PM2.5 core, by which PAHs were removed, did not affect the prognosis. Together, we have demonstrated for the first time that neuroinflammation induced by PM2.5 exposure can aggravate the outcome of ischemic stroke.

Neonicotionoid pesticides are insecticides which have been thought to be one of the causes of Colony Collapse Disorder. They were considered to be less toxic to mammals than insects, but some of the recent studies suggested their association with disruption of higher brain fuction in mammals. Nevertheless, it is still unclear how neonicotionoids affect on the central nervous system. Here, we propose the use of in vivo Ca²⁺ imaging with two-photon microscope to detect abnormal activity of neuronal circuits in brain with the application of neonicotionoids.

In this study, a less than the no-observed-adverse-effect level (NOAEL) of acetamiprid (20 mg/kg bw); and a tenth or half of the median lethal doses of nicotine (0.33 or 1.65 mg/kg bw respectively) were orally administered to mice. They were subjected to elevated plus maze test and Ca²⁺ imaging by two-photon microscope in the somatosensory cortex. We further detected acetamiprid and metabolites in brain and blood an hour after the administration.

Mice exposed to acetamiprid or nicotine exhibited an increase in anxiety-like behavior that associated with the altered activities of the neuronal population in the somatosensory cortex. Although the dose of acetamiprid used in the current study was below the NOAEL, both acetamiprid and nicotine affected the behavior and the neuronal activity in the somatosensory cortex. The results suggested that in vivo Ca²⁺ imaging using two-photon microscope enabled highly sensitive detection of brain neurodisruption by neurotoxicants.

Seizure is a severe adverse effect in neurotoxicity. In particular, seizure has a huge impact on drug development. Therefore, detection of seizure potential risk is the most important subjects in non-clinical studies. Electroencephalography (EEG) is a useful tool to monitor neuronal activity, and characteristic EEG waveform of seizure has been reported in both animals and humans, but there are few reports of EEG waveform for evaluation of seizure potential. In this study, we established a novel analytical method for detection of seizure potential from different perspective using EEG waveform in pre-seizure state.

Detection of seizure auras is effective in improving the predictive accuracy of seizure liability of drugs. Whereas electroencephalography has been known to be effective for the detection of seizure liability, no established methods are available for the detection of seizure auras. In this study, we developed a method for detecting seizure auras through artificial intelligence (AI) using frequency-characteristic images of electroencephalograms (EEG).

Histograms of frequency–intensity distribution prepared from EEG of rats analyzed during seizures induced with 4-aminopyridine (4-AP, 6 mg/kg), strychnine (3 mg/kg), and pilocarpine (400 mg/kg), were used to create an AI system that learned the features of frequency-characteristic images during seizures. The AI system detected seizure states learned in advance with 100% accuracy induced even by convulsants acting through different mechanisms, and the risk of seizure before a seizure was detected in general conditions of observation. Moreover, the AI system detected seizure liability even in EEG data associated with the use of 4-AP (3 mg/kg), strychnine (1 mg/kg), and pilocarpine (150 mg/kg), which did not induce seizures detectable in general conditions of observation. No seizure risk was detected during the entire measurement period after administration of vehicle. The AI system developed herein is an effective means for electroencephalographic detection of seizure auras, raising expectations for its practical use as a new analytical method that allows for the sensitive detection of seizure liability of drugs that has been overlooked previously in preclinical studies.

We constructed a hepatic vascular model for predicting hepatotoxicity by using our unique tissue-engineering method. Briefly, we created a viscous body by suspending cells with heparin and collagen. The viscosity can induce cell-cell adhesion suitable for constructing a hepatic tissue. We constructed the vascularized tissue whose hepatocytes ratio is 65% (close to in vivo). We confirmed the predictive performance of DILI by treating typical DILI compounds. Notably, we could predict the toxicity of Monocrotaline by vessel analysis with higher sensitivity than usual viability assay.

Micro-computed tomography (micro-CT) is an imaging modality that enable us to non-invasively obtain 3D images of organs in laboratory animals. By using micro-CT, internal organs can be monitored repeatedly, which leads to more precise evaluation and reduction in the number of animals used. In this study, we evaluated time-course of chemically-induced hepatomegaly in mice using micro-CT.

The liver volume calculated from CT images was highly correlated to the liver weight. Therefore, it was considered that CT imaging can be a useful tool for evaluating the time-course of hepatomegaly.

Obesity and type 2 diabetes mellitus have become worldwide epidemics. Evidence indicates that glucose-dependent insulinotropic polypeptide (GIP) secreted by the intestines may partially underlie these conditions, considering that GIP levels are associated with lipid deposition and fat mass expansion. However, recent studies have found that GIP is also present in other tissues, such as the liver. Notably, one study discovered through microarray analyses of livers from obese diabetic rats that the transcriptional modulation of GIP also occurred in the liver. Otsuka Long-Evans Tokushima Fatty (OLETF) rats were chosen for this experiment because previous studies have shown that thiamine (vitamin B1) could successfully decrease the tendency of the animal toward obesity and mitigate the complications of diabetes. Here, the rats were randomly assigned to either the control (non-supplemented) or thiamine-supplemented (2 g thiamine/L in drinking water) groups. For this investigation, OLETF rats were chosen for the experimental period at 93 weeks of age. In this study, hepatic GIP expression was analysed using western blotting, suggesting that GIP was present in the livers of both obese diabetic OLETF rats and obese diabetic rats that received ongoing thiamine supplementation. Results showed that hepatic GIP expression had occurred and that liver-derived GIP may exist. Moreover, results showed that ongoing thiamine supplementation modified the hepatic GIP expression and prevented additional weight gain and complications arising from obesity and diabetes.

Our recent studies have shown that the co-administration of nicotinamide (NAM), a precursor of methylnicotinamide (MNA), with S-adenosylmethionine (SAM) and hydralazine (HYD) significantly improves fatty liver in a rat model of NAFL. In this study, we conducted the in vitro metabolism studies using hepatic homogenate prepared from NAFL rats to explore some of the mechanisms of fatty liver improvement by NAM. Consequently, it was suggested that the combined administration of SAM and HYD efficiently increases the hepatic MNA exposure and led to the improvement of fatty liver in rats.

We have reported that human hepatic stellate cells were deactivated by spheroid culture. In this study, we tested culture condition of spheroids to detect activation or deactivation by morphological change. Human hepatic stellate cell line LI90 were cultured on EZSPHERE (AGC) to form spheroids. Acquired spheroids were cultured on the culture plate or low attachment culture plate.

Spheroids on the culture plate showed elongated morphology and activated gene expression pattern. This result indicated that cell morphological change would be able to evaluate stellate cell activation.

Ketoconazole (KC) rarely induces liver injury and adrenal dysfunction when orally administered. In this study, the significance of arylacetamide deacetylase (Aadac)-mediated hydrolysis of KC on above toxicities was evaluated using Aadac knockout mice. In Aadac knockout mice, oral administration of KC diminished plasma corticosterone level, inducing inflammatory response and elevation of plasma ALT level, whereas such phenomena were not observed in wild-type mice. We found that Aadac deletion caused KC-induced liver injury via inhibition of glucocorticoid synthesis in adrenal gland.

Activation of the nuclear receptor CAR induces liver cancer in rodents, and thus the information on whether a chemical compound activates CAR or not is useful for predicting its carcinogenicity. In this study, we treated rat primary hepatocytes with around 300 test compounds and found that 106 compounds increased mRNA levels of Cyp2b1, a typical CAR target gene, more than 10-fold. Among them, 18 activated the mutant CAR, which we constructed to repress its constitutive activity, in reporter assays. These methods might be useful to evaluate CAR activation of chemical compounds.

Most liver-on-a-chips are fabricated from PDMS which is a material that easily absorbs drugs. However, it is unclear that PDMS-based liver-on-a-chip could be used for drug evaluation. Here, we found that the absorption ratio of drugs into the PDMS-based device correlates with S+logD value of drugs. Additionally, we confirmed that drug responsiveness assay could be performed by using drugs with low S+logD value in the PDMS-based liver-on-a-chip. These results suggested that PDMS-based liver-on-a-chip could be useful for pharmaceutical research.

The distribution pattern of hepatocellular steatosis in pediatric non-alcoholic fatty liver disease (NAFLD) (periportal) differs from that of adults (centrilobular).

We pathophysiologically analyzed the fatty liver in rats induced by a low-protein and low-potassium (LPLP) diet to explore the possibility of developing animal models of pediatric NAFLD. In this study, macrovesicular periportal hepatocellular steatosis was seen histopathologically. Biochemically, increased LDL, LDL/HDL ratio and leptin levels and decreased adiponectin/leptin ratio were observed.

These results suggest that LPLP diet may produce a pediatric NAFLD animal model.

The present study aimed to clarify whether a bile ductular reaction induces liver fibrosis in the dietarily induced mouse NASH model, using the expression of SRY-box9 (Sox9) as a biomarker. In the liver of mice fed a special CDAHFD-0.1 diet for 14 days, steatosis became remarkable with slight fibrosis, and the extra-bile duct Sox9 expression was observed. At the end of week 13, fibrosis became prominent, and the Sox9 expression was marked and diffuse. In conclusion, the Sox9 expression may not be a consequence of NASH-derived liver fibrosis, but involved in its development and progression.

We determined the role of selective autophagy of endoplasmic reticulum (ERphagy) in the preneoplastic lesions in steatosis-related early hepatocarcinogenesis model of rats. The increase of the preneoplastic lesions in the HFD or PB groups might be related to inhibition of ERphagy as shown by increased expression of LC3B and FAM134B, and the decrease of the lesions in the HFD-PB groups might be caused by the induction of ERphagy.

NASH is one of the most serious liver disease worldwide, but onset mechanism of NASH is still unknown. Six-week-old male F344 rats were fed a CDAA diet or a normal diet (SD) for 1 to 2 weeks. In the diet switching group, the CDAA diet was fed for 1 week, then switched to SD and fed for 1 week. Most of the changes expressed by feeding the CDAA diet for 2 weeks were ameliorated by switching the diet, however, the expression of some genes was not attenuated. By comparing this result with the previous NGS analysis obtained in 2 weeks study, it will be possible to identify the target factors involved in the progression of NASH from an early stage.

To investigate effect of high fat diet on liver in obese type 2 diabetes model, 5-week-old female db/db and KK-Ay mice were fed CE-2 (Basal Diet), Quick Fat (QF:High fat diet) or Western Diet (WD: QF+2% cholesterol added) ad libitum for 8 weeks. C57BL/6J mice were fed by the same diets as a physiological control. At 13-week-old of age, mice were dissected, collected the blood and organ and were performed various analysis.

In both diabetic strains, the induction of fatty liver by QF was more advanced in KK-Ay. In addition, WD was induced liver fibrosis, and it was more prominent in db/db mice.

Antibiotics (ABXs) are one of the major drugs which induce liver injury frequently, so, it is important to clarify the mechanism of ABXs-induced liver injury. We focused the alteration of bile acid composition by ABXs treatment. Bile acids are important to maintain vital functions, but dysregulation of bile acid homeostasis increases the risk of developing liver injury. Among them, ABXs dramatically change bile acids composition, especially increasing conjugated primary bile acid level. Present study constructed the mice model and investigated the relation liver injury and alteration of bile acid homeostasis using flucloxacillin.

[Introduction]

The liver is a major target organ of chemical toxicity, and understanding their molecular mechanisms is important for the development of new toxicity tests. Nuclear receptors (NRs) are involved in the regulation of major liver functions, and have also been known as critical targets for hepatotoxic chemicals. Here, we assessed the association between rat-derived NR-activating profiles of chemicals and their hepatotoxic phenotypes in in vivo rat toxicity studies to obtain useful information for understanding their mechanisms.

[Methods]

Using reporter assay systems in human HepG2 cells, the agonistic activity of 326 chemicals toward NRs was evaluated. Rat 28-day repeated-dose toxicity data was obtained from Hazard Evaluation Support System and the association between the in vitro activity and in vivo toxicity was investigated.

[Results and Discussion]

Reporter assays identified 36, 22, 9, 6 and 17 chemicals as positives for PXR, PPARα, LXRα, FXR and RXRα, respectively. Focusing on common in vivo phenotypes observed for the positive compounds, many of them were consistent with the typical findings associated with NR activation in rodents. For example, many positives for PXR, PPARα and RXRα were reported to induce centrilobular hepatocellular hypertrophy. In addition, several PXR positives were reported to increase serum total cholesterol and decrease blood glucose. Thus, assessing the association between the in vitro NR-activating profiles of chemicals and their in vivo rat hepatotoxic phenotypes may be useful for understanding their complex toxicological mechanism.

Centrilobular hepatocyte hypertrophy (CHH) is frequently observed after the administration of chemical substances, but its mechanism remains unknown. In this study, we investigated the mechanism using publicly available data of rat 2-year repeated-dose toxicity/carcinogenicity tests (367 pesticides). Statistical analyses have demonstrated significant associations between CHH and thyroid hypertrophy, which is associated with enzyme induction in the liver, and between CHH and fatty degeneration in hepatocytes, suggesting the contribution of enzyme induction and fatty degeneration to CHH.

To clarify the role of CD44 in acute kidney injury to chronic kidney disease transition, 6-week-old male SD rats were treated with 6 mg/kg of cisplatin and necropsied after 1 to 28 days. CD44 was expressed in dilated tubules after day 5 and dilated/atrophic tubules in fibrotic lesions after day 14. At day 28, pathway analysis indicated CD44 as inducer of fibrosis related genes, such as Fn1. Fn1 mRNA and protein were expressed in cytoplasm of dilated/atrophic tubules and stroma, respectively. These results indicated CD44 as trigger of extracellular matrix secretion in dilated/atrophic tubules.

Madder color (MC) induces rat renal tumor in outer stripe outer medulla (OSOM). This site specificity is considered to depend on distribution of its components. To clarify the role of MC components in renal carcinogenesis, we analyzed distribution of major components in the kidney of rats administered with MC using DESI-MSI. As a result, alizarin and purpurin detected in both cortex and OSOM whereas lucidin and rubiadin detected only in the OSOM, suggesting that its genotoxicity plays a direct role in MC carcinogenicity. Thus, MSI could be a powerful tool for exploring mechanisms of toxicity.

We investigated the effects of Ivabradine, a HCN inhibitor, on the b-wave trough and c-wave which are known to appear after the b-wave, using ERG in rats. Ivabradine at 12 and 40 mg/kg was subcutaneously administered to Long-Evans rats and the dark-adapted ERG with long-duration light stimuli was conducted at 1.5 hours after administration. In the Ivabradine-treated rats, the waveforms of b-wave trough were changed and the c-waves were attenuated, which implying that Ivabradine might affect the functions of any of retinal cells, photoreceptor, retinal pigment epithelium and Müller cells.

Objective: Anticancer drugs are often administered intravenously. The vascular pain caused by this administration can be said to be side effects of anticancer drugs, and in consideration of the QOL of patients, treatment that “reduce pain” is required. However, it has not been easy to predict and evaluate pain due to intravenous administration using animal model. Therefore, we tried to quantify the pain using the electromyogram generated during vascular pain in rats.

Methods: Rats (Slc: SD, male, aged 10 to 12) were anesthetized with Secobarbital Sodium (50 mg/kg, ip) and a bipolar stainless electrode was inserted into the semitendinosus muscle, ground electrode was placed subcutis of head region of rat, and the EMG signals were recorded. An indwelling catheter for drug administration was anterogradely cannulated into the femoral circumflex artery. After inserting bipolar electrode, the toes of the rat hind limbs were strongly pinched, and it was confirmed that the EMG increased. After confirming that the EMG was stable, administration of the test solution to the femoral circumflex artery was started.

Results and discussions: No increase in EMG amplitude was observed during saline administration. On the other hand, administration of gemcitabine increased the EMG amplitude. In addition, pretreatment with fentanyl suppressed the increase in the EMG amplitude after administration of gemcitabine. The results of this study greatly suggested the possibility of quantifying vascular pain, and that the method was useful assessment system.

The skin sensitization test h-CLAT was modified to detect respiratory sensitizers by co-culturing THP-1 cells with 3D human bronchial epithelium. Two skin and six respiratory sensitizers were tested in this co-culture system and results were compared with standard h-CLAT. Among the sensitizers tested, HBTU and TBTU were successfully judged positive only in the co-culture system, suggesting this co-culture system is useful for the detection of respiratory sensitizers. Further optimization is required to distinguish more accurately respiratory from skin sensitizers with this test system.

In computerized tomography (CT) tests that are commonly used in clinical medicine, CT images are usually evaluated by a radiologist. However, manual interpretation can lead to differences between evaluators and lacks objectivity. In this investigation, a method for quantifying CT images that enables objective and simple evaluation of lung injury was established. A bleomycin-induced mice lung injury model was evaluated using micro-CT tests. This report presents the utility of the semi-automatically calculated CT value as a new biomarker of lung injury in non-clinical studies.

Evaluation of blood gases requires collection of arterial blood from unanesthetized animals, but is difficult in repeated dose studies in rats because cannulation is required and long-term maintenance of the catheter is difficult. We investigated the effects of 4 types of anesthesia on blood gases and hemoglobin fraction, and evaluated the effects of 7-day repeated dosing of phenylhydrazine hydrochloride on blood gases using an anesthetic method with the least effect on blood gases. Results suggest anesthesia by isoflurane is feasible in assessing blood gases in rats.

Ozone generators have been exponentially installed in medical institutions for viral inactivation of COVID-19. However, an acceptable concentration of ozone (0.1 ppm) is regulated based on the effects on the healthy people, and toxic effects in the patient with serious respiratory dysfunction is still uncertain. Therefore, the aim of this study is to compare the toxic effects of ozone exposure between a normal mouse and a mouse model of acute respiratory distress syndrome (ARDS). Ozone (0.1 ~ 0.3 ppm) exposure was performed 6 hours/day for 5 consecutive days in BALB/c mouse. ARDS was induced by intranasal administration of lipopolysaccharide 72 hours before data sampling. Percutaneous oxygen saturation (SpO2) was measured by pulse oximeter. Lung tissue, bronchoalveolar lavage fluid (BALF) and hilar lymph nodes (LN) were collected for pathological and immunological evaluations. SpO2 was significantly reduced by ozone exposure in ARDS groups, while there was no influence in normal groups. Ozone exposure also exacerbated the histological evaluation including neutrophil infiltration and epithelial cell necrosis in ARDS groups, while no significant changes were observed in normal groups. In BALF, ozone exposure significantly enhanced cytokine (IL-1β and RANTES) levels in ARDS groups, however, no change was observed in normal groups. Cytokine (IL-5 and IL-17) production in LN was also significantly upregulated by ozone exposure in ARDS groups. Our findings indicate that current acceptable concentration of ozone has a possible adverse effect in the patient with ARDS.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes coronavirus disease 2019 (COVID-19). SARS-CoV-2 entry to host cells are initiated by binding with its receptor, angiotensin-converting enzyme 2 (ACE2), and the ACE2 abundance is thought to reflect the susceptibility to infection. ACE2 is expressed in various types of cells, such as lung, heart, liver, cornea and ileum. Especially, respiratory cells are considered important in terms of clinical outcome. Thus, supplying human respiratory cells, such as airway basal, club, ciliated and alveolar epithelial type II (ATII) cells, is essential for COVID-19 research. However, differentiation methods of human ACE2-expressing respiratory cells has not been fully understood. In the present study, we have generated ATII cells from human iPSCs using two-dimensional culture method. We differentiated human iPSCs using a stepwise protocol. After making lung progenitor cells from human iPSCs, alveolar cells were induced by further culture in the presence of chemicals. RT-qPCR and immunofluorescence analysis revealed that the differentiated cells contained SFTPB-positive ATII cells and expressed ACE2. In addition, the cells were infected with SARS-CoV-2. These data suggest that the iPSC-derived alveolar cell is a good in vitro model for COVID-19 research.

The conventional methods of inhalation exposure for cigarette smoking required expensive equipment and wasted labor to induce potential toxicity. Therefore, we attempted to develop a novel method using intratracheal instillation (ITI) of whole cigarette smoke condensates (WCSC). The WCSC was prepared as a 20 mg/mL and diluted to 10 and 5 mg/mL, and was administered by ITI using an automatic video instillator. Separated two experiments were performed; study 1= ITI, 12 times, 28 days (3 time/week), 4 groups (1, 2, 3, 4 weeks), study 2 = ITI, 12 times, 12 days (everyday), 2 groups (6, 12 days). After final ITI, the bronchoalveolar lavage fluid (BALF) and lung tissue were collected. The BALF was analyzed using commercially available sandwich ELISA method. A mild histopathological change was observed in lung tissues treated with WCSC (20 mg/mL) for 28 days. On the other hand, repeated ITI of WCSC for 6 days increased the body and lung weight, and the level of neutrophils and lymphocytes compared to the control. In addition, the histopathological markers were observed in the lung tissues treated with a high dose of WCSC. An analysis of BALF from 6- and 12-day groups confirmed the inflammatory changes and altered secretion of a related cytokine, MCP-1, were observed in this study. However, no significant changes in the secretion of CXCL-10 and IL-2 were detected. In summary, ITI exposure may be a more brief and effective method to evaluate the adverse effects of WCSC via a short period of exposure less than 2 weeks.

A safety pharmacological study evaluates the effect of QT interval prolongation, which has the risk of inducing lethal arrhythmia, Torsade de points (TdP), on an electrocardiogram. The effect of QT interval prolongation is evaluated by hERG current inhibition in an in vitro study and by QT interval change in an in vivo ECG study. However, since QT interval is also affected by Na and Ca current, there is a concern that useful compounds that do not cause TdP may have been omitted from drug development.

We attempted a multilateral analysis for TdP risk assessment using in vitro test systems based on the results of the study.

Cardiotoxicities associated with combination cancer therapy have become a hot topic of concerns. In this study, we developed a new assessment method to predict the onset of such cardiotoxicity. Male mice received TRZ and DOX at 20 or 3 mg/kg once a week for 10 weeks by single or combined administration. ECG, echocardiography, blood and histopathological examinations and PCR assay of the heart were performed. These results indicate that this method is highly valuable for assessing cardiotoxicity of molecularly targeted drugs, which may help figure out issues in the cardio-oncology.

In AAV gene therapy, a genomic integration into non-target cells, especially germline cells should be minimized. ICH indicate the necessity to evaluate the distribution, persistence and localization in gonadal tissues. However, details have not been specified, although EMA guidelines indicate the in-situ hybridization (ISH) as a method.

Therefore, we examined the usefulness of ISH with reproductive tissues of AAV treated cyno. monkeys. We will show the results, suggesting low germline risk in male, and report the possibility of semi-quantitative evaluation by algorithm analysis using AI.

Assessment of thyroid and thyroid-stimulating hormones (T4, T3, TSH) was added to the OECD test guideline Nos. 408, 414, 421, 422, and 443 to improve detection of potential endocrine activity. We obtained historical control data for these hormones using Crl:CD(SD) rats. The coefficients of variation of values obtained were less than 25% for T3 and T4 and less than 35% for TSH, which met the criteria specified in the guidelines. These data were considered useful to assess changes in hormone levels by chemical exposure. At the venue, the details of the methods and the results are presented.

Gingival Hyperplasia (GH) is a side effect after drug administration in the oral tissue. GH has been reported to be caused by taking antihypertensive drug. In this study, we investigated the physiological significance of Metallothionein (MT) in gingival cells after treatment with Amlodipine, it is known as an antihypertensive agent. Cd or Amlodipine treatment induced the MT expression. Amlodipine is known to be involved in the onset of GH, the MT in gingival cells could work as a multifunctional protein induced during cell proliferation as well as a biological defense reaction to heavy metals.

Chemotherapy induced neutropenia and febrile neutropenia are frequent side effects in anticancer drug treatment, and result in reduces in therapeutic effects. To establish TK / TD model which provide important information to patients, detailed data on the effects of Docetaxel, Paclitaxel, Irinotecan, or Topotecan on bone marrow in various animals (rats, mice, dogs, or monkeys) were evaluated. Comparative assessment of the differences in bone marrow toxicity among animals or drugs will be presented.

The activity test on alkaline phosphatase (ALP) was converted from the JSCC method to the IFCC method on 1 April 2020. We compared these two methods on measurement of serum and plasma ALP concentration in male rats. Following samples which three types of ALP isozyme changed were obtained: liver isozyme-changed sample, small intestine isozyme-changed sample, bone isozyme-changed sample were measured by both IFCC and JSCC methods.

The measurement results will be reported and the difference between two method will be discussed in this report.

28-day repeated oral toxicity study of acetaminophen (APAP) in microminipigs (MMP, Fuji Micra Inc.) was performed. MMPs (male, n=3) were administered APAP at 0, 80, 200 and 600 mg/kg body weight/day. Swelling of head, redness, pruritus were observed in all MMPs at 600 mg/kg and they died on day 3. Histologically, subcutaneous edema with granulocyte infiltration and fibrin deposition that suggest increased vascular permeability were observed. These findings are the features of NSAIDs intolerance, and allergies to APAP were unlikely, the involvement of the disease was suspected.

Objective: Last year, we reported the effects of inhalational anesthetics on hematology and blood biochemistry in rats at the 47th Annual Meeting of the Japanese Society of Toxicology. This year, we examined the effects on three strains of mice, and report the results.

Methods: Using 9-weeks-old male and female mice (Slc:ICR, C57BL/6JJmsSlc, and BALB/cCrSlc), blood was collected from posterior vena cava under anesthesia with isoflurane (induction: 3%, maintenance: 1.5 to 3%) or sevoflurane (induction: 4 to 5%, maintenance: 3 to 4%), and then hematological and blood biochemical parameters were measured.

Results and discussions: Between isoflurane group and sevoflurane group, there were differences in the following hematological parameters; RBC, WBC, Hb, Ht, and WBC differential count. There were also differences in the following blood biochemical parameters; alanine transaminase, total protein, albumin, glucose, creatinine, calcium, and electrolytes. All these differences were within the normal limit, and were considered no toxicologically significant. However, there were some parameters that differ for each strain, suggesting that the selection of strains according to the purpose of the experiments is important.

Rearing equipment for rats is changed to control environment depending on a test method. In this study, 4 sets of equipment were prepared to keep rats: a wire cage with no enrichment equipment (control), a wire cage with a tunnel “Shepherd Tube”, a plastic cage with bedding “Palsoft” and a plastic cage with bedding “Ecochip”. Slc:SD rats had been in each environment for 28 days to be taken the data of body weight, food consumption, water consumption, hematology, blood biochemistry and organ weight. Based on the results, it was examined whether the difference in equipment affected.

BioCelerate is a non-profit consortium of pharmaceutical companies in US, EU and Japan. Our projects publicly released a Nonclinical Common Protocol Template for repeat-dose toxicology studies in November 2019, and through efforts to evolve synergistic processes, a companion Nonclinical Common Report Template in November 2020. Benefits to using these common templates for sponsors and CROs may include 1) productivity (time/effort/resources) optimization in drafting these documents for each study, 2) improve overall study quality by decreasing errors due to unfamiliarity with protocol/report layout, 3) simpler management of multiple studies, and 4) streamlined review of toxicology study reports by worldwide regulatory agencies. Importantly, in preparation for the future digitized state of information exchange in toxicologic research, widespread adoption of these common templates will provide a consistent substrate with which to transition these documents into electronic format. For both common templates, streamlined layout and process instructions were defined where large variations in preferences existed. For template development, public feedback periods and webinars were held to collect stakeholder feedback. Specific to the protocol template, most stakeholders reported using multiple templates and experienced problems associated with inconsistency, inefficiencies, and reduced study execution quality. For the associated report template, the BioCelerate team collaborated with reviewers from the US FDA and subsequently hosted an open webinar to gather input from stakeholders.

BioCelerate is an industry consortium to increase efficiencies in early-stage R&D. Implementation of the SEND model represents an opportunity to apply large-scale data analytics to toxicology data. To realize this opportunity, differences in SEND implementation that make it difficult to conduct cross-study analysis must be addressed. In partnership with FDA, cross study analysis in SEND datasets that are significant drivers of variability that negatively impact cross-study analysis were identified. Herein the focus is on background-control data and approaches for improved data harmonization. The most relevant variables (eg MI, BW, LB, OM) of SEND datasets have been defined to function as robust and easy-to-use historical control databases. The FDA CDER repository of >1,800 SEND datasets was queried by FDA staff to gain insight into how SEND is being applied with respect to these parameters. Proposals for harmonization methods and test search scripts to transform data were developed to allow consistent extraction of these variables across SEND datasets. A framework for the development of proposed solutions from which a user could assemble the results for specific endpoints for all control animals in a user-defined subset of studies is provided. A BioCelerate/PHUSE collaboration is also engaged in further refinement of SEND database searching through the development of R search scripts. This R search script repository is publicly available on GitHub and can be used to provide greater context into the significance of toxicologic findings observed in toxicology studies.

Animal identification methods are ideally non-invasive, simple, and highly legible. Microchips (MCs) are one such method but are rarely used in toxicity studies in Japan. In this study, we implanted MCs in 30 Crl:CD1 (ICR) mice and investigated MC readability and effects for 13 weeks.

MCs were readable throughout the study. No abnormalities were observed in any examination parameter except for very slight and focal changes in the subcutaneous tissue around the MC implantation site in histopathology. Therefore, we judged that MCs can be used in mice toxicity studies for up to 13 weeks.

We investigated the sudden deaths which occurred up to 6 month of examination (31 weeks of age) in total 156 of toxicity/carcinogenicity studies in Crl:CD(SD) rats between 2009 and 2019 in our facility. Sudden deaths occurred in 10 out of 3377 males and 1 out of 3252 females. Pathological examination revealed no life-threatening finding and the cause of death was unclear, though 2 males showed convulsion before the death. The incidence of sudden deaths tended to increase with the age of animals. In this meeting, we will update the data of recent years.

We investigated the occurrence of death in ICR mice (Crl: CD1 and Crlj: CD1) up to 31 weeks of age in total 29 of toxicity/carcinogenicity studies between April 2009 and March 2020. Dead/moribund sacrifice occurred in 4 out of 1200 males and 12 out of 1200 females. The presumptive cause of death was hyaline glomerulopathy (1 female), glomerulonephritis (1 male and 1 female), excoriation (1 female), osteosarcoma (1 male), malignant lymphoma (1 male and 3 females), hemorrhage from uterine polyp (1 female), anemia (1 female) and unknown cause (1 male and 4 females).

Toxicity of Na salicylate was compared by oral or IV route in cynomolgus monkeys. Three males and females were orally treated with escalating three dosages with a recovery period of 7 days; 500, 750, 1125 mg/kg. For IV route, 12 males and 12 females (3 animals per group) were treated with 3 dosages; 0 mg/kg, 138 mg/kg, 275 mg/kg, 550 mg/kg, respectively. The findings were as follows: oral emetic dose was between 500 mg/kg and 750 mg/kg, showing more than 750 mg/kg dosage did not show any clinical sign except vomiting. The IV emetic dose was about 275 mg/kg, and the approximate lethal dose for IV route was between 275 mg/kg and 500 mg/kg.

Imazalil (IMZ) and thiabendazole (TBZ) were given in the diet at levels of 0%/0%, 0.006%/0.006%, 0.006%/0.018%, and 0.006%/0.054% during the gestation and lactation periods. The time required of olfactory orientation was accelerated in higher-dose groups in female offspring on PND 14 in a dose-related manner. In the exploratory behavior, the frequency of mice with urination increased in a dose-related manner in male offspring. In spontaneous behavior, the longitudinal pattern of total distance was not parallel (different pattern) among the control and IMZ/TBZ treatment groups in adult males.

Acetyl-CoA carboxylase (ACC) catalyzes the first step of de novo lipogenesis. PF-05175157, a systemically acting dual ACC1/2 inhibitor, caused developmental toxicity in embryo-fetal development studies in both the rat and rabbit. Additional studies in rat whole embryo culture, mouse embryonic stem cells, and zebrafish developmental toxicity assay were conducted to evaluate the utility of these in vitro assays in detecting ACC-induced developmental toxicity. This work demonstrates that a battery of in vitro assays to screen compounds for developmental toxicity is more informative than any single assay.

Neonicotinoids are pesticides used the most frequently in Japan. Neonicotinoids act as agonists for the nicotinic acetylcholine receptor (nAchR) and exhibit insecticidal effects by disrupting neurotransmission. Neonicotinoids are considered to bind to only insect nAchR, but the effects of their exposure on mammals should be examined with regard to the similarity between insects and mammalian nAchR. In this study, we aim to investigate the developmental neurotoxicity of neonicotinoids using mice. Imidacloprid (IMI) and clothianidin (CLO) were administered at a dose of 0.1 mg/kg/day from gestational day 11 to postnatal day 21. IMI developmental exposure reduced survival rate 11 days after birth and eye opening was delayed. Abnormal anxiety-like, social and repetitive behaviors were observed after growth. CLO developmental exposure reduced survival rate 15 days after birth and eye opening was also delayed. Anxiety-like, repetitive and depression-like behavior were observed after growth. In the hippocampus of 10-day-old mice, the number of microglia decreased due to developmental exposure to neonicotinoids. The morphology of microglia changed from amoeboid to ramified forms, and the phagocytic ability was reduced, indicating decreased microglial activity by neonicotinoids. Taken together, neonicotinoid developmental exposure can induce abnormal behaviors after growth via decreases in and/or inactivation of microglia during developmental stage.