Some chemicals have testicular toxicity to animals and humans. However, their primary toxic targets are different each other, and their detailed mechanisms of toxicity are unclear. In addition, comprehensive protein expression analysis (proteomics) using liquid chromatography-tandem mass spectrometry (LC-MS/MS) is useful for understanding biological reactions in the tissue, and its use has been increasing in recent years. Therefore, in this study, we analyzed the changes in protein expression during repeated administration of testicular toxicants with different toxic targets ethylene glycol monomethyl ether (EGME), 1,3-dinitrobenzene (1,3-DNB), dibromoacetic acid (DBAA), and carbendazim (CBZ), and proposed their respective toxic mechanisms and related biological responses. Test substances were administrated once a day to 8-week-old male SD rats for up to 14 days, and the testes collected from rats were subjected to organ weight measurement, histopathological examination, and proteomics by LC-MS/MS. Pathway analysis were performed for these expression data by Ingenuity Pathway Analysis (IPA) (QIAGEN Bioinformatics). Protein expression analysis revealed different or common test substance-specific changes in pathways or proteins associated with energy production, sperm maturation, Sertoli cell phagocytosis, and testosterone synthesis. Some of these proteins are testis-specific or highly expressed in testis. In this presentation, we would like to discuss the relationship between toxic target differences and variation of pathway patterns.

Bohai Bay is a semi-closed bay linked to Yellow Sea in China, and has been polluted by discharge of industrial sewage that contains dioxin-like chemicals (DLCs). We used zebrafish embryo/larva as in vivo model system to evaluate the pollution in the Bohai Bay using CYP1A as a marker. We collected sediment samples at the sewage outlet of a factory in Bohai Bay (P1), at a radius of 1,000 meters (P2) and 2,000 meters (P3) from the estuary. The ethanol extracts of sediment samples were used for chemical analysis, and biological evaluation. The embryos/larvae were exposed to extracts and 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD), a positive control of DLCs from 4 hours post fertilization (hpf) until observations. Similar to TCDD, P1 extracts caused an concentration-dependent increase of fluorescence intensity in Tg(CYP1A:mCherry) zebrafish at 72 hpf, while it became weaker with the increase of the discharge radius. Immunoblotting study showed marked induction of CYP1A protein by P1 extracts and TCDD. CYP1A mRNA expression was remarkably induced by P1 extract and TCDD at 48 hpf, as revealed by RT-qPCR. Also we confirmed CYP1A mRNA expression in the gills of zebrafish embryos exposed to P1 extract and TCDD by in situ hybridization. The chemical analysis showed that the main components of P1 extract were H7CDF, O8CDF and O8CDD dioxins, which were consistent with biological assessment methods. The results highlight the usefulness of transgenic zebrafish line as a rapid, sensitive and economical method, which may be used in the initial high-throughput screening of DLCs.

[Aim]

Evaluation of miR-216a-5p and miR-217-5p, known to be pancreas-specific miRNAs, as biomarkers of pancreatic toxicity were performed using L-arginine-induced pancreatitis model in mice.

[Method]

L-arginine HCl was administered intraperitoneally once to male C57BL/6J mice. Necropsy was conducted after 1, 2, 3, 5 and 7 days from administration. Histopathological examination of pancreas, and biomarker measurement including plasma miR-216a-5p and miR-217-5p were performed.

[Result and discussion]

In histopathological examination, pancreatic injury (edema, inflammatory cell infiltration, acinar cell necrosis/degeneration) was observed and their total score was peaked on 3 day of administration. After 5 day of administration, pancreatic injury was decreased, and instead, replacement of inflammatory cells and fibrotic cells appeared. Plasma miR-216a-5p and miR-217-5p was increased corresponding to pancreatic injury and peaked on 3 day after administration. The increase in miRNAs were earlier, greater magnitude, and persisted longer compared with serum amylase or lipase. According to analysis of relation to histopathological score, amylase and lipase didn’t change when acinar cell necrosis/degeneration was relatively weak, while the miRNAs elevated clearly.

In conclusion, miRNAs can identify pancreatic toxicity earlier, and are more sensitive, greater increased and persistent biomarkers than classical biomarkers.

Various urinary biomarkers (BMs) are used to detect drug nephrotoxicity invasively. Urine for BMs are usually sampled overnight, stored at cold. To investigate 4-hour fresh urine utility for nephrotoxicity BM, GM (Gentamicin) or CDDP (Cisplatin) was administered to rats, and urine/blood BMs and renal histopathology were examined.

The increases in urinary BMs (Kim-1, NGAL, etc.) were noted in fresh urine (ice-cold storage), and had correlation with renal histopathologic changes of proximal tubule. In conclusion, the flesh urine would be applicable to detect nephrotoxicity through urinary BMs.

Endogenous digitalis-like factor (EDLF) obtained from maternal and umbilical cord plasma at delivery were measured by fluorescence polarization immunoassay (FPIA). In each sample, concentrations of dehydroepiandrosterone, dehydroepiandrosterone sulfate, estradiol, estriol, hydrocortisone, progesterone, and testosterone were measured by radioimmunoassay, and cross-reaction tests of digitalis-like immunoreactive substances (DLIS) with these substances were performed. By FPIA, the concentration of DLIS in umbilical cord plasma (0.55±0.22 ng/mL) was significantly higher than that in maternal plasma (0.23±0.11 ng/mL). In the cross-reaction tests, when the concentration of dehydroepiandrosterone sulfate was higher than 1.0 μg/mL or that of progesterone was higher than 0.5 μg/mL, DLIS were detected by FPIA. By radioimmunoassay, there was no significant difference in the dehydroepiandrosterone sulfate concentration between the maternal plasma (2,917±1,001 ng/mL) and the umbilical cord plasma (1,957±376 ng/mL). The progesterone concentration in the umbilical cord plasma (310.0±85.7 ng/mL) was significantly higher than that in the maternal plasma (126.4±38.5 ng/mL). These results suggest that dehydroepiandrosterone sulfate in maternal plasma and progesterone in maternal and umbilical cord plasma can be measured as digoxin by FPIA, which might be EDLF.

Recently, the endogenous compounds 4β-hydroxycholesterol (4β-OH-CHO) and 6β-hydroxycortisol (6β-OH-COR) have been found to be biomarkers of CYP3A in human and monkey. In this study, we developed a method for determining 4β-OH-CHO, cholesterol, 6β-OH-COR, and cortisol concentrations in monkey plasma. After a sensitive LC-MS/MS method was developed and validated, the individual monkey plasma was measured. We will introduce information on the endogenous biomarkers of CYP3A activity in individual monkey plasma and discuss basic information on this evaluation method.

Hepatic phospholipidosis (PL) is one of storage disorders in the liver. Biomarkers (BMs) that reflect progression of PL are very limited. To search for BM candidates for PL, male SD rats was dosed with 150 mg/kg/day amitriptyline for 14 days. Phospholipids in liver and plasma were measured by mass spectrometry. In the results, phosphatidylcholine (PC) (36: 1), PC (36: 4) and PC (38: 5) were increased in liver, and decreased in plasma conversely. It was considered that the decreased phospholipids in peripheral blood might be related to the accumulation of phospholipids in the liver.

We investigated the effects of the microsampling (MS) method on organisms and toxicity assessment of mice. Firstly, Crl:CD1(ICR) mice with their blood samples collected by the MS method over time were compared with those without blood collection. Secondly, Crl:CD1(ICR) mice with blood samples collected by the MS method after administration of 1-naphthyl isothiocyanate (ANIT) were compared with those without blood collection. The results showed that although the MS method affected the hematological parameters of the mice, it did not affect the evaluation of acute hepatotoxicity caused by ANIT.

Pulse oximeters can be a powerful tool to measure heart rate (HR) noninvasively, but their accuracy is still unclear. Therefore, we investigated the accuracy by comparing HR between the pulse oximeter (MouseOx Plus^®) and the telemetry method in telemetered conscious rats administered vehicle, milrinone, or atenolol. As a result, there was a good correlation in the HR with the range from 240 to 435 bpm (R²=0.9996). The significant changes in HR noted in the telemetry method were well noted also in the pulse oximeter. The pulse oximeter can be brief but accurate method for the evaluation of HR.

The potency of mitochondrial dysfunction and BSEP inhibition is an useful factor to predict liver injury in the preclinical stage. However, hepatocellular functions are rapidly decreased using polystyrene plate, which is the lack of oxygen supply. So, conventional method is difficult to detect the risk of liver injury adequately. We newly developed an oxygen-permeable plate with low drug adsorption, called “T plate”. So, we evaluated hepatocellular function and the sensitivity of cell death in primary rat hepatocyte cultured by T plate.

Adeno-associated virus (AAV) is one of the most promising gene delivery vectors for gene therapy owing to its non-pathogenicity. Because the presence of anti-AAV antibodies may affect toxicity and efficacy, it is important to test for anti-AAV antibodies in toxicity and pharmacology studies of AAV in non-human primates. We measured anti-AAV antibodies in cynomolgus monkeys and calculated the positive rates. The high percentage of antibody-positive animals highlights the importantance of testing for anti-AAV antibodies prior to AAV administration in the studies of AAV.

We attempted to develop the method for detecting cardiotoxicity in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) without dedicated devices. The toxicity of cardiotoxic compounds (sotalol, moxifloxacin, lidocaine, verapamil, pentamidine) were tested in spheroids of GCaMP-transfected hiPSC-CM by measuring calcium transient with a combination of commonly used imaging plate reader and open-sourced imaging software. The toxicity of hERG, Nav and Cav ion channel inhibitors, beta blocker and hERG channel trafficking inhibitor were able to be detected with our method.

HepG2, a liver cancer-derived cell line, is widely used as a human hepatocytes model. In this study, we had found that low-density cell culture induces hepatic function, so we examined the mechanism. Low-density culture increased CYPs gene expression and CYP3A4 activity in HepG2. Tight junction formation was also enhanced in low-density cultured cells, and occludin and tricellulin protein expression levels were also increased. These results suggest that low-density culture induces hepatic function via the promoting of tight junction formation.

A cellular response consequent to the interaction with NMDA receptor is useful for predicting toxicological action of chemicals. Drebrin is an actin-binding protein that governs the dendritic spine formation of CNS neurons and is responsible for the morphological plasticity of dendritic spines. The drebrin exodus from dendritic spines is available as a marker of the activation of NMDA receptors in neurons. The subcellular localization of drebrin is determined by glutamate receptor activity, and when drebrin is lost in dendritic spines, the learning and memory mechanism does not function properly. In the present study, we have observed drebrin and MAP2 distribution in neurons with a confocal quantitative image cytometer, CQ1 (Yokogawa, Japan), and developed new algorithms to quantify the glutamate receptor binding agonist-induced changes in the distribution pattern of their immunoreactivity. Cultured hippocampal neurons (21 DIV) were fixed after glutamate treatments and processed for immunocytochemistry to visualize drebrin, MAP2 and cell nucleus. After automated image acquisitions, neuron number, dendrite length, and drebrin clusters were automatically quantified with originally developed algorithms. In particular, the intensity distribution analysis of drebrin clusters is highly sensitive. Identification of drebrin clusters and analysis of their distribution are promising to detect drebrin exodus induced by a low concentration of glutamate receptor binding agonists.

Prediction of toxicity in humans is essential in pharmaceutical research and development, and various nonclinical toxicity studies are conducted using cells and animals. Even after various toxicity studies are conducted, some clinical studies are often discontinued due to unexpected toxicity. Especially, nephrotoxicity is difficult to predict due to structural complexity of the kidneys and interspecies differences between humans and experimental animals. Here we report a novel prediction system of nephrotoxicity using gene expression network in human ES/iPS cells and machine learning.

Here, we established a high-throughput in vitro assay system to predict reactive metabolite (RM) formation. First, we performed the glutathione (GSH) consumption assay to monitor GSH levels as an index of RM formation potential using HepaRG cells pretreated with 500 micro M D,L-buthionine-(S,R)-sulfoximine (BSO) and then treated with ticlopidine and diclofenac. Both drugs, under GSH-reduced conditions, significantly decreased relative cellular GSH content by 70% and 34%, respectively, compared with that in cells not pretreated with BSO. Next, we examined the correlation between GSH consumption and covalent binding assays; the results showed good correlation (correlation coefficient = 0.818). We then optimized the test compound concentration for evaluating RM formation potential using 76 validation compound sets, and the highest sensitivity (53%) was observed at 100 micro M. Finally, using HepG2 cells, PXB-cells, and human primary hepatocytes, we examined the cell types suitable for evaluating RM formation potential. The expression of CYP3A4 was highest in HepaRG cells, suggesting the highest sensitivity (56.4%) of the GSH consumption assay. Moreover, a co-culture model of PXB-cells and HepaRG cells showed high sensitivity (72.7%) with sufficient specificity (85.7%). Thus, the GSH consumption assay can be used to effectively evaluate RM formation potential in the early stages of drug discovery.

To simultaneously obtain the information for the speciation and spatial distribution of elements, we developed a novel hybrid technique consisting of quantitative imaging by laser ablation (LA)-inductively coupled plasma mass spectrometry (ICP-MS) and speciation by HPLC hyphenated with ICP-MS (LC-ICP-MS). Then, an application of this analytical technique was evaluated in vivo. Methylmercury or mercuric chloride were administered to rats, and continuous sections of the kidney were prepared. The speciation and distribution of mercury (Hg) differed, and the differences depended on Hg species.

The present study aimed to (i) develop a sensitive LC-MS/MS-based technique for simultaneous detection and quantification of Imidacloprid (IMI) and its metabolites in tissue specimens, and to (ii) determine the specific bioaccumulation trends of the IMI compounds in organs of C57BL/6J male mice; after exposure to 0.6 mg/kg bw/day of IMI (10% of NOAEL) through a powdered diet for 24 weeks. We successfully developed a method which was accurate (recoveries were ≥ 70% for most compounds), sensitive (LODs ≤ 0.47 ng/mL, LOQs ≤ 1.43 ng/mL and R2 ≥ 0.99) and precise (RSDs ≤ 20%) for routine analysis of IMI and seven of its metabolites in in blood and various tissues of mice. Upon biodistribution analysis, IMI and five of its metabolites were detected in various organs within mice. Blood, liver, mesenteric white adipose tissue and pancreas mainly accumulated IMI-olefin; brain, testis, inguinal white adipose tissue and gonadal white adipose tissue mainly accumulated IMI, whereas the kidney mainly accumulated 4-hydroxy-IMI. The N-desnitro-IMI metabolite showed unique accumulation in brain and testis by recording brain-blood and testis-blood detection ratios of 8.4 and 7.6, respectively. Cumulative levels of the six detected IMI compounds (Σ6 IMI compounds) were found in the decreasing order; blood > testis > iWAT > kidney > brain > gWAT > mWAT > pancreas > liver. The current study provides essential data needed for effective mechanistic elucidation of compound-specific adverse outcomes associated with chronic exposures to IMI in mammalian species.

【Aim】

Several animal models of pulmonary fibrosis in rodents have been developed to search for the potential therapies for pulmonary fibrosis. The most common model is the bleomycin (BLM)-model in mice because of smaller size. Intratracheal instillation method enable administration of the test material directly to the lung. In the present study, we investigate the fibroblastic change of lung intratracheally dosed with BLM using 2 strains of mice.

【Method】

Male C57BL/6J mice (C57BL), 6-week old and Crl:CD1(ICR) mice (ICR), 9-week old were intratracheally dosed with BLM (0, 12.5 or 25 μg/body). Organ weight and histopathology of the lung were examined on 21- or 28^th day after the instillation.

【Results】

No deaths occurred in the vehicle-treated group. Moribund animals were observed on day 21 or 9 in the 12.5 or 25 μg/body treated C57BL group, on day 12 in the 12.5 μg/body treated ICR group. The survival rate at day 21 was 91.7% in the 12.5 or 25 μg/body treated C57BL group and 100 or 58.3% in the 12.5 or 25 μg/body treated ICR group. Lower body weight values were observed in the 25 μg/body treated groups of both strains compared to the vehicle groups. Higher values of absolute and relative lung weights were observed in the 12.5 and 25 μg/body treated groups of both strains compared to the vehicle groups. The lung fibroblastic lesions were observed in both strains at 21^st day after the BLM instillation. These changes were considered to be the effect of BLM instilled in the lung. In this presentation, we will report the detail results of histopathological findings in both strains.

We performed immunohistochemical analysis to develop novel method for detection of endocrine disruptors. Aminotriazole (AMT), propylthiouracil (PTU), aminoglutethimide (AGT) and other 3 chemicals were administered to SD rats for 28 days. Decreases in serum T3 and T4 in AMT and PTU groups and an increase in serum TSH in AMT, PTU and AGT groups were detected. Immunostaining in these groups revealed a decrease in T4 in the thyroid gland and an increase in TSH positive area ratio in the anterior pituitary gland, suggesting the possibility of detecting hormonal changes using pathological specimens.

The INHAND Proposal has been operational since 2005. A Global Editorial Steering Committee helps coordinate overall objectives of the project. Development of harmonized terminology for each rodent organ system or non-rodent species is the responsibility of the Organ Working Groups or Non-rodent Working Groups respectively, drawing upon experts from North America, Europe and Japan. Great progress has been made with 15 rodent organ systems published; Respiratory, Hepatobiliary, Urinary, Central/Peripheral Nervous Systems, Male Reproductive and Mammary, Zymbals, Clitoral and Preputial Glands and Hematolymphoid System in Toxicologic Pathology and the Integument and Soft Tissue, Female Reproductive System, Digestive System, Cardiovascular System, Skeletal System, Special Senses and Endocrine System in the Journal of Toxicologic Pathology as supplements and on a web site; www.goReni.org. There are 5 non-rodent working groups; non-human primate, dog, minipig, rabbit and fish. The mini-pig and dog have been published in Toxicologic Pathology (2021) and non-human primate and rabbit are scheduled for publication in the Journal of Toxicologic Pathology in 2021. The manuscript on fish will be available for review in 2021. A new group has been formed to address terminology in non-rodent ocular toxicity studies.

F-hiSIEC^TM is a human iPS cell-derived small intestinal epithelial-like cell which shows the physiological characteristics similar to those of the in vivo human small intestine such as CYP3A4 activity and expression of transporter genes. To extend the applicability of F-hiSIEC^TM to gastrointestinal (GI) toxicity assessment, several types of reference drugs were examined in this study. The results of the present study show an in vitro system using F-hiSIEC^TM can be a useful model for evaluating GI toxic agents and effects of drug candidates on differentiation of intestinal epithelium.

Comprehensive read-across was investigated using existing large-scale chemical information as the next generation of cosmetic safety assessment without animal testing.

2,6-di-t-butyl-4-ethylphenol, which is hepatotoxic, was selected as the model compound. Comprehensive read-across was conducted, and satisfactory prediction results were obtained. On the other hand, toxicity results may differ among compounds with highly similar chemical structures, suggesting that phenotype-based read-across is necessary in addition to chemical structure-based read-across.

Photosensitization evaluation is essential for cosmetic safety assurance. We have constructed a database (DB) for substances inducing photosensitivity in humans and are developing several in vitro tests. In this study, we evaluated the photosensitization of false-negative chemicals in in vitro tests by read-across using the DB. The results showed that chemical-structure-based read-across was useful for some chemicals including 6-Methylcoumarin, and phenotype-based read-across might be required for others like carbamazepine, to enable more accurate evaluation of photosensitization.

ADRA is an alternative test for skin sensitization using cysteine and lysine derivatives (NAC and NAL) to evaluate the reactivity of chemicals with these reagents. However, since the sensitization potential is decided only by NAC/NAL depletion in this test, it is unclear whether these depletions are truly occurred by reaction with chemicals. We developed the method to identify adducts formed by NAC/NAL and chemicals, and oxidation products of NAC in reaction solutions by LC-MS analysis. We believe this method is useful for differentiating between true and false positive results in ADRA.

Read-across using existing large-scale chemical information is an essential method in the next-generation safety assessment of cosmetics without animal testing. In this study, we evaluated skin sensitization intensity in LLNA using chemical-structure-based comprehensive read-across. The results showed that most compounds (including negative ones) could be predicted to a satisfactory degree. However, read-across with gene-expression-response similarities might be required for compounds which are highly similar but have significantly different sensitization intensities.

In this study, we focused on transcriptome analysis as a technique for evaluating the cardiac toxicity potential of chemical substances. The similarity of the gene expression pattern with Terfenadine is defined as the cosine similarity. As a result, among 177 drugs, Astemizole and Prenylamine (withdrawn from market due to cardiotoxicity) were included in the top 5 drugs with similar gene expression patterns to Terfenadine. The cosine similarity also showed a good correlation with the risk classification of human cardiotoxicity.

Read-across is one of the most important methods for safety assessment of cosmetic ingredients without animal testing. However, it cannot be used when toxicity information on substance with similar chemical structures (Structure-based analogs) is lacking. Here, we report on a read-across method using chemicals with similar gene expression responses in cells (Phenotype-based analogs), focusing on developmental toxicity in this study. This time, Phenotype-based analogs of chosen targets were statistically selected from LINCS, and in most cases, their developmental toxicity tended to be similar to that of the target compounds.

Single-dose toxicity tests, genetic toxicity tests, etc. are conducted to evaluate the toxicity and safety of drugs. From the viewpoint of animal protection, the spirit of 3R has been taken in from before, and in vitro using cultured cells that have been established and differentiated into cells of various tissues. Testing is being considered.

We wondered if the particle counting analyzer could contribute to the test using cultured cells. We report the possibility of evaluating the effects of the drug from the results obtained by measuring the culture sample with the device.

In this alternative method, all 15 lipstick/lip cream raw materials were judged to be non-irritating. Eye irritation data for 32 chemical substances, all 22 raw materials distinguished to GHS categories 1, 2A and 2B were judged to be irritating. 6 raw materials including to the 10 raw materials distinguished to NC, were judged to be non-irritating, but other 4 raw materials were judged to be irritating. These results indicated a possibility of the evaluation for irritating of raw materials by using the criteria of our alternative method. In addition, we will demonstrate some results of biochemical analysis in this report.

In this study, we measured the chemical concentration in the yolk sac of zebrafish embryos/larvae and other sites to investigate the distribution of the drug in the body. As a result, the concentration in the yolk sac was higher than the other sites, and showed a high correlation with the drug log P (or log D) as well as the concentration of whole embryos/larvae. In addition, the area under the concentration-time curve (AUC) calculated from the concentration of CBZ in ZF embryos/larvae exposed to CBZ of 47.3 mg/L was 5,110 μg·h/g, which was 22 times higher than the human AUC.

We measured the volume in Göttingen minipigs from the CT images of 20 minipigs (11 males and 9 females), and calculated the density and sphericity. The minipigs had been restrictedly fed according to the feeding guideline. The mean ± SD for the density of the 20 pigs was 1.01 ± 0.03; 1.02 ± 0.03 in the males and 1.00 ± 0.03 in the females; no significant sex difference was seen. The mean ± SD for the sphericity of the 20 pigs was 0.606 ± 0.021; 0.593 ± 0.014 in the males and 0.620 ± 0.017 in the females. Sphericity of the females was significantly higher than that of the males（P = 0.002).

We compared two myocardial infarction models of C57BL/6J and NOD/ShiJic-ScidJcl-mice. Infarction was induced by complete ischemia of the left anterior descending artery. Cardiac function was measured by echocardiography. Infarct size was measured in Masson’s trichrome-stained specimens. In both mouse strains, EF and %FS decreased from day 1 (the first day of model preparation). EF, %FS, LVIDd, and LVIDs remained similar to those on day 7 until day 28. Myocardial fibrosis area ratios were almost equal. No differences were observed between the two mouse strains.

Diabetes mellitus (DM) is one of the serious risks in COVID-19. The virus Infects through ACE2. Increased ACE2 is reported in DM patients. To analyze the relationship between ACE2 expression and pathophysiology of DM, SDT fatty rats were fed by a normal diet or QF diet+2% cholesterol. At necropsy, lung, kidney and blood were sampled. In SDT fatty rats, infiltration of alveolar macrophage, hemorrhage and ACE2 positive cells were increased in lung. These findings were enhanced by fed QF+2% cho. These data suggested that SDT fatty rats were possible to be an animal model of the risk with COVID19.

In this study, mice were fed with a choline-deficient methionine-reduced high-fat diet (CDAHFD) for 6 and 12 weeks to confirm the development of NASH pathology. We also evaluated the effects of pemafibrate, a selective PPARα modulator, on the progression of NASH pathology.

After 6 and 12 weeks, hepatic biomarkers for NASH, and hepatic mRNA expression for lipid accumulation, inflammation, and fibrosis increased over time, revealing the development of NASH pathology.

0.1 and 0.3 mg/kg of pemafibrate administered by gavage for 12 weeks completely prevented the progression of NASH pathology.

Diphenylarsinic acid (DPAA), a pentavalent arsenic detected in the well water, induced neurological symptoms in nearby residents. DPAA could induce aberrant intracellular activation including induction/activation of antioxidant proteins, MAP kinases, and transcription factors in cultured rat cerebellar astrocytes. Here, we assessed the potential of dimercaptosuccinic acid, an antidote to lead poisoning, as a prophylactic and/or curative agent against DPAA. DMSA could inhibit DPAA-induced aberrant activation clearly, but not accelerate recovery from activated status after DPAA removal.

Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) are rare severe adverse drug reactions with skin blistering and keratinocyte death. Here Alcyomics has developed an in vitro skin model of TEN and has investigated its modulation by the TNF inhibitor, etanercept. Patients with SJS/TEN or maculopapular exanthem as well as serum and healthy volunteer skin samples were used in the study. Healthy skin biopsies exposed to TNFα (10 ng/mL), SJS/TEN (10% and 100% dilution), rash or healthy control sera (10% and 100% dilution), were cultured in the absence/presence of etanercept (1 μg/mL). A positive control (damage induced by Anakinra (10μg/mL) and a negative control (skin in media only) were used in all assays. Skin was incubated with or without sera and with or without etanercept for 3 days and graded for histopathological damage (grades I-IV). Skin sections were reported for the presence of necrosis or intra-epidermal damage. Skin cultured with TNFα showed grade II responses with vacuolisation of the epidermis in the presence and absence of etanercept. A grade III positive response was observed in response to SJS/TEN sera at 10x dilution with intra-epidermal damage and severe necrosis which was reduced to a grade II response in the presence of etanercept. The results demonstrate that the histopathological features of SJS/TEN can be induced by exposure to TNFα, SJS/TEN sera and rash sera, and inhibited by etanercept and suggest a putative ex vivo model of TEN with potential to be utilised as a nonclinical safety screen.

Toxicity study of ophthalmic drugs has unique characteristics of its study design and packages. The present survey was carried out in order to review toxicity of 5 intravitreal drugs those have been approved in Japan, and to compare clinical adverse effects with nonclinical toxicity. This survey is valuable for considering the toxicity study design, and suggested that there is a limitation to predict clinical adverse effects from nonclinical study results especially in retinal vascular lesions which might be attributed immunoreaction.

【Introduction】Among nanomaterials, nanosilica is a material that has been put to practical use in an extensive range of industrial fields in Japan and abroad. However, the number of test data on toxicity information is scarce, and comprehensive response to toxicity evaluation and safety is required.

【Method】In this study, we collected test data of physicochemical properties (PCPs) and harmfulness information for five types of silicon dioxide nanoparticles (SiO₂ NPs) provided by the OECD NM safety assessment test document and eNanoMapper DB and searched and scrutinized data necessary for statistical analysis using SIMCA-P. Furthermore, we analyzed the similarity of PCP values and examined the usefulness of this method for toxicity evaluation using multivariate analysis for the relationship between PCPs and hazard information.

【Result and Discussion】PCPs of about 62 items for which was collected information were classified by hierarchical clustering analysis, and their similarities were shown by Dendrogram. On the other hand, the relevance of hazard information was characterized by property items showing toxicity by OPLS (Orthogonal partial least squares). From the results obtained in this analysis, it was possible to clarify the slight differences in PCPs associated with toxicity using a vast data collection on NM's PCPs. Therefore, it is suggested that the various statistical methods used in this analysis can be useful tools for evaluating the properties of NM and be applied to toxicity evaluation methods based on the properties of NM.

The systemic dose levels of eye-drop drugs are relatively low in comparison with that of systemic route such as oral administration. We undertook risk assessment for the central nervous system (CNS) effects of eye-drop drugs, by comparing the estimated systemic dose level of eye-drop drugs with the oral dose levels of known CNS toxicants and CNS medicines (e.g., clinically used drugs of sleeping, antidepressant, antiepileptic, anti-Parkinson diseases and analgesics) of approximately 50 different modes of action (MOAs). The systemic dose levels of eye-drop drugs in human were estimated to be 0.01 to 1.0 mg/body on the assumption of 0.01% to 1% of eye-drop formulation, 0.05 mL/eye of instillation volume, administration to both eyes, 50 kg body weight, and once a day.

As the result, (1) 0.01% eye-drop drug systemic dose level was lower than the efficacy doses of all MOAs; (2) Among the MOAs investigated, the efficacy dose levels of 8 MOAs were located at the relatively low position, which was approximately x10 to x100 dose ratio against 0.01% eye-drop drug systemic dose level; (3) Even at the 1% eye-drop drug systemic dose level, it was 10- to 100-fold lower than that of approximately half of MOAs investigated.

In conclusion, our present investigation strengthens a general consideration that the risk of CNS effect in eye-drop drugs is generally low. Especially, the concentration level of 0.01% or below have less risk of CNS effects in most MOAs. This result also supports the ICH S7A guideline for mentioning unnecessity of safety pharmacology studies for eye-drop drugs.

Under the Japanese Water Supply Act, the water quality standard has been established for the safe and secure of drinking water. Water Authorities are required to inspect the 51 standard items, and water supplying must fulfill the required conditions pursuant to the provision of “Drinking Water Quality Standards”. Twenty-six substances not yet fully assessed for their risks are listed in “Complementary Items”, not-legally binding with target values. In addition, 47 substances are regarded as “Items for Further Study” that require further study and a grip of detection level. For instance, the typical disinfection by-product, total trihalomethane is included in the 51 standard items, whereas BDCAA, a halogenated acetic acid among the disinfection by-products, is listed in “Items for Further Study”, because the risk assessment had been only provisional, and its target value had never been established at the last revision of the Act. Therefore, we conducted the hazard assessment to derive its health-based guidance value. Carcinogenetic potential of BDCAA was reported in rat and mouse 2-year drinking water studies as reliable information. The benchmark dose analysis was adopted for incidences of all neoplastic endpoints in rats and mice. The most appropriate BMDL10 of 2.83 mg/kg/day was obtained for male rat malignant mesothelioma in all organs.

Because BDCAA is considered to be a genotoxic carcinogen, the Virtually Safe Dose at 10−5 risk level was calculated as 2.83 × 10−4 mg/kg. This will be useful to establish the safety standard of BDCAA in drinking water.

The Drinking Water Quality Standards for lifetime exposure of contaminants have been established for 51 items under the Japanese Water Supply Act. In addition, non-legally binding target values are also notified for “Complementary Items” and “Items for Further Study” that can be detected in drinking water or water sources. Vinyl acetate is used in materials of drinking water apparatus directory contact to drinking water and listed in “Items for Further Study.” The target value of vinyl acetate in drinking water has not been established; therefore, we conducted the hazard assessment of vinyl acetate to derive a health-based guidance value. Carcinogenetic potential was observed in rat and mouse 2-year drinking water studies. We conducted the benchmark dose analysis for incidences of squamous cell carcinoma and/or papilloma in rats and mice. The most appropriate BMDL₁₀ of 231 mg/kg/day was obtained for male rat squamous cell carcinoma in oral cavity. The reference values were derived as both a genotoxic carcinogen and a non-genotoxic carcinogen because its genotoxicity was inconclusive. The Tolerable Daily Intake (TDI) was calculated as 0.231 mg/kg/day with an Uncertainty Factor of 1000, and the Virtually Safe Dose (VSD) at 10^-5 risk level was calculated as 0.0231 mg/kg/day. The above TDI and VSD values will be useful to establish the target value of vinyl acetate in drinking water. ACKNOWLEGMENT: This study was supported by a Health and Labour Sciences Research Grant (19LA1005) from the Ministry of Health, Labour and Welfare, Japan.

The setting of health-based exposure limit (HBEL) for chemicals is important for avoidance of cross-contamination and for occupational safety. In this study, we tried to set Acceptable Surface Limit (ASL) and Occupational Exposure Band (OEB) for skin sensitizers based on EC1.6 derived from LLNA:BrdU-ELISA. Adjustment factors for extrapolating EC1.6 to human risk were set then quantitative ASL and OEB were proposed. Chemicals with ASL less than 1 mg/100 cm² were consistent with GHS 1A chemicals, suggesting our method enables setting HBEL corresponding to the international hazard classification.

Pfizer’s Drug Safety Group has been systematically evaluating drug targets for safety risks in the form of Target Knowledge Reviews (TKRs), requiring time-consuming, manual curation of target data. To allocate more time for risk assessment, we have developed Automated Target Knowledge Reviews (AutoTKR) application. AutoTKR automates information curation including orthologs/similar targets cross-species, target tissue expression, genetics, and compounds, and formats for into a report. This application opens up more time for deeper dives into target knowledge and greater insights.

Combined oral repeated-dose and reproduction/developmental toxicity screening tests were conducted in SD rats for 3-methylpentane, isooctane, and isononane, whose toxicological information was not available for human health effects. As the results, effects on the liver (weight increase, hypertrophy) and male kidney (α2u-globulin nephropathy, tubular regeneration), and no notable reproductive/developmental toxicity were observed in all compounds. This information will be useful to fill in the data gaps when evaluating other acyclic branched saturated hydrocarbons lacking toxicity information.

Since the outbreak of the new coronavirus infection in 2020, we have been forced to change the way of working. Telework is a typical example. It is suitable for tasks that are well-defined. The Japanese employment system does not clearly define the scope of responsibility, so simply replacing work processes is not enough to manage the business. The SOP document in GLP test facilities clearly states who has to do what. Therefore, if we proceed with the application of telework based on SOPs, we can expect a smooth implementation. By considering it, we need to find some clues for the adoption of telework in Japan.

Positive list system for utensils, container and packaging (UCP) was enforced in June, 2020 in Japan. The toxicity profiles of the designated substances should be evaluated for at least genotoxicity, and the assessment is ongoing by an Integrated Approach, reviewing experimental genotoxicity data combined with (Quantitative) Structure Activity Relationship ((Q)SAR).

4,4'-Oxybis(benzenesulfonohydrazide) is a listed substance for UCP and known to be an additive of polyvinyl chloride. There are a number of evidences which lead 4,4'-oxybis(benzenesulfonohydrazide) was genotoxic in vitro and classified as a genotoxic substance in Industrial Safety and Health Act in Japan. In our literature review, positive results were obtained in two independent Ames tests (TG471), an unscheduled DNA synthesis (UDS) test in hepatocyte primary culture, and an in vitro chromosome aberration test (TG473). Moreover, (Q)SAR analysis supported genotoxicity in vitro. On the other hand, one negative result was obtained for mammalian in vivo micronucleus test (TG474). No toxicity data were available on its carcinogenicity. To confirm in vivo mutagenicity, we conducted the transgenic rodent gene mutation assay (TG488). After the transgenic Muta™ mice were administered 4,4'-oxybis(benzenesulfonohydrazide) orally for 28 days, genomic DNA in liver and glandular stomach, a major metabolizing organ and a primary exposed area via gavage route, were examined. Negative results were obtained in both liver and glandular stomach. We conclude that 4,4'-oxybis(benzenesulfonohydrazide) is not genotoxic in vivo.

In cases where chemical-specific toxicity data are absent or limited, the threshold of toxicological concern (TTC) offers an alternative to assess human exposure below which “there would be no appreciable risk to human health.” The application of TTC to non-cancer systemic endpoints has been pursued for decades using a chemical classification and Point of Departure (POD). This study presents a new POD dataset of oral subacute/subchronic toxicity studies in rats for 656 industrial chemicals retrieved from the Hazard Evaluation Support System (HESS) Integrated Platform, which contains hundreds of reliable repeated-dose toxicity test data of industrial chemicals under the Chemical Substances of Control Law in Japan. The HESS TTC dataset was found to have less duplication with substances in other reported TTC datasets. Each chemical was classified into a Cramer Class, with 68%, 3%, and 29% of these 656 chemicals distributed in Classes III, II, and I, respectively. For each Cramer Class, a provisional Tolerable Daily Intake (TDI) was derived from the 5th percentile of the lognormal distribution of PODs. The TDIs were 1.9 and 30 μg/kg bw/day for Class III and I, respectively. The TDI for Cramer Class II could not be determined due to insufficient sample size. This work complements previous studies of the TTC approach and increases the confidence of the thresholds for non-cancer endpoints by including unique chemical structures. This new TTC dataset is publicly available and can be merged with existing databases to improve the TTC approach.

In order to ensure the reliability of SEND data, Japan Society of Quality Assurance (JSQA) analyzed the flow of SEND data creation, identified the risks in ensuring reliability, and proposed the points to consider and countermeasures for preparing SEND data based on the ISO9001 by International Organization for Standardization.

In the current presentation, we would like to discuss the points that should be particularly focused on to ensure the reliability for the SEND data based on the results of the questionnaire in each member company of JSQA GLP Division.