Modified ESR-based photosafety test (ESR-PT) detecting singlet oxygen and free radical formation.

The electron spin resonance-based photosafety test (ESR-PT) was modified using a new parameter, photoreactivity index (PRI), to detect singlet oxygen and free radical photoproducts simultaneously. With this modification, the modified ESR-PT is expected to reduce the number of false negative results due to chemicals producing free radical photoproducts other than singlet oxygen. The assay performance of the modified ESR-PT was evaluated using 56 chemicals, including hydrophobic chemicals.

The inter-laboratory validation study of EpiSensA for predicting skin sensitization potential.

The Epidermal Sensitization Assay (EpiSensA) is a reconstructed human epidermis (RhE)-based gene expression assay for predicting the skin sensitization potential of chemicals. Since the RhE model is covered by a stratified stratum corneum, various kinds of test chemicals, including lipophilic ones and pre-/pro-haptens, can be tested with a route of exposure akin to an in vivo assay and human exposure. This article presents the results of a formally managed validation study of the EpiSensA that was carried out by three participating laboratories.

Comparison of the acceptable surface limits derived from the skin sensitization potency for workers and swab residue limits determined from the permitted daily exposure derived from the systemic effects.

Allergic contact dermatitis is a common occupational and environmental health problem and setting of health-based exposure limits (HBELs) to prevent induction of skin sensitization is strongly desired. When manufacturing pharmaceuticals in a shared facility, cleaning validation using surface residue levels (SRLs) derived from permitted daily exposures (PDEs) is conducted to avoid cross-contamination from the perspective of protecting patients; however, it is unclear whether the SRLs are sufficient to prevent induction of skin sensitization for workers as well. In this study, we compared acceptable surface limits (ASLs) derived from acceptable exposure levels (AELs) based on EC1.

Time-Course of Transcriptomic Change in the Lungs of F344 Rats Repeatedly Exposed to a Multiwalled Carbon Nanotube in a 2-Year Test.

Despite intensive toxicological studies of carbon nanotubes (CNTs) over the last two decades, only a few studies have demonstrated their pulmonary carcinogenicities in chronic animal experiments, and the underlying molecular mechanisms are still unclear. To obtain molecular insights into CNT-induced lung carcinogenicity, we performed a transcriptomic analysis using a set of lung tissues collected from rats in a 2-year study, in which lung tumors were induced by repeated intratracheal instillations of a multiwalled carbon nanotube, MWNT-7. The RNA-seq-based transcriptome identified a large number of significantly differentially expressed genes at Year 0.

Effects of inhalation of multi-walled carbon nanotube (MWCNT) on respiratory syncytial virus (RSV) infection in mice.

Multi-walled carbon nanotubes (MWCNTs), a kind of nanomaterial, are widely used in battery electrodes and composite materials, but the adverse effects associated with their accumulation in the living body have not been sufficiently investigated. MWCNTs are a fibrous material with molecules similar to asbestos fibers, and there are concerns about its effects on the respiratory system. In this study, we conducted a risk assessment by exposing mice using a previously developed nanomaterial inhalation exposure method.

Retrospective quantitative risk assessment of skin sensitization caused by an antimicrobial agent used in a consumer product: 2,3,5,6-Tetrachloro-4-(methylsulfonyl) pyridine.

Background: Serious cases of allergic contact dermatitis (ACD) caused by exposure to 3,5,6-tetrachloro-4-(methylsulfonyl)pyridine (TCMSP; CAS no. 13108-52-6) used as an antimicrobial agent for desk mats have been reported in Japan.

Objective: A quantitative risk assessment (QRA) of TCMSP contained in desk mats was performed retrospectively.

Establishment of Cell-Based Assay System for Evaluating Cytotoxic Activity Modulated by the Blockade of PD-1 and PD-L1 Interactions with a Therapeutic Antibody.

Background: Therapeutic antibodies targeting the PD-1/PD-L1 immune checkpoint are widely used in cancer therapy and are under active further development. Historically, the antitumor activity of PD-1/PD-L1 immune checkpoint inhibitors has been evaluated using and test methods; however, a simple assay method to evaluate antitumor activity accurately is needed for the efficient development of new therapeutic agents. In the present study, we attempted to establish a simple cell-based assay system to evaluate the modulating effect of PD-1/PD-L1 immune checkpoint inhibitors on cytotoxic activity.

False-negative chemicals in ESR-based photosafety test (ESR-PT) and their significance for photosafety evaluations: examples of bithionol, fenticlor and cilnidipine.

The electron spin resonance (ESR)-based photosafety test (ESR-PT) is a non-animal prediction test for photosafety evaluations that can be used even for hydrophobic chemicals; the method is based on the detection of singlet oxygen generation using ESR spectroscopy and showing high accuracy for compounds with known photosafety information. During the process of extending the application data for ESR-PT, we found three false-negative chemicals: bithionol, fenticlor and cilnidipine. These chemicals did not show the characteristic triplet signal of 4-hydroxy-2,2,6,6-tetramethyl-piperidine-1-oxyl (4-hydroxy-TEMPO), which is used as a classifier for positive or negative chemicals; instead, bithionol and fenticlor induced an apparent single peak signal with a g-value of 2.

Acceptable surface limits (ASLs) of skin sensitizers derived from the local lymph node assay (LLNA): BrdU-ELISA EC1.6 values and their relationships to known sensitization potency information.

Skin sensitization is an extremely important risk factor for occupational health and safety, and it would be desirable to set health-based exposure limits (HBELs) for the quantitative risk assessment (QRA) based on the skin sensitizing potencies of chemical. We attempted to set acceptable surface limits (ASLs) as HBELs for skin sensitizers in the workplace based on the local lymph node assay (LLNA): BrdU-ELISA EC1.6 values.

Gene expression profiles of multiple brain regions in rats differ between developmental and postpubertal exposure to valproic acid.

We have previously reported that the valproic acid (VPA)-induced disruption pattern of hippocampal adult neurogenesis differs between developmental and 28-day postpubertal exposure. In the present study, we performed brain region-specific global gene expression profiling to compare the profiles of VPA-induced neurotoxicity between developmental and postpubertal exposure. Offspring exposed to VPA at 0, 667, and 2000 parts per million (ppm) via maternal drinking water from gestational day 6 until weaning (postnatal day 21) were examined, along with male rats orally administered VPA at 0, 200, and 900 mg/kg body weight for 28 days starting at 5 weeks old.

Characterisation and validation of an in vitro transactivation assay based on the 22Rv1/MMTV_GR-KO cell line to detect human androgen receptor agonists and antagonists.

We describe the characterisation and validation of an androgen receptor (AR) transactivation assay for detection of AR agonists and antagonists using a stably transfected human prostate cancer cell line. This 22Rv1/mouse mammary tumour virus glucocorticoid knock-out cell line based AR transactivation assay was validated by criteria in Organisation for Economic Cooperation and Development Guidance Document 34 to determine if the assay performed equally well to the AR EcoScreen Assay included in Test Guideline for AR Transactivation (OECD TG 458). There was no Glucocorticoid Receptor (GR) crosstalk, and no changes in the AR DNA sequence in cells after the successful knock out of GR.

Development of a new photosafety test method based on singlet oxygen generation detected using electron spin resonance.

Photosafety evaluations of chemicals used in consumer products, such as pharmaceuticals and cosmetics, are very important. Currently, two non-animal tests for photosafety evaluations, the in vitro 3T3 neutral red uptake phototoxicity test (NRU PT) and the reactive oxygen species (ROS) assay, are used to detect photoreactive chemicals. However, these two tests are difficult to apply to hydrophobic chemicals.

Applicability of the proposed GHS subcategorization criterion for LLNA:BrdU-ELISA (OECD TG442B) to the CBA/J strain mouse.

The Globally Harmonized System of Classification and Labelling of Chemicals (GHS) is a hazard classification and communication system for providing information on the safe handling of chemicals worldwide. In this study, we evaluated the applicability of the newly proposed GHS subcategorization criterion for murine local lymph node assay:2-bromodeoxyuridine enzyme-linked immunosorbent assay (LLNA:BrdU-ELISA), Category 1A:EC1.6 ≤6%, Category 1B:EC1.

α-Sens: The improved ARE-Nrf2-based sensitization screening assay using normalized transcriptional activity.

Two non-animal test methods, KeratinoSens™ and LuSens, have been approved by the Organization of Economic Cooperation and Development (OECD) test guidelines for evaluating the sensitization potential of chemicals, and been positioned as a method for appraising key event (KE)-2, namely, the keratinocyte response component of the Adverse Outcome Pathway (AOP) in sensitization process. However, these two methods require separate cytotoxicity tests to determine the concentrations to be tested in the main test. Therefore, we developed a simple and highly accurate KE-2 test method named α-Sens that uses the dual luciferase assay system and attempted a further application of luciferase-based determination of cell viability to calculate the normalized Antioxidant response element (ARE)-mediated transcriptional activity, named normalized ARE Activity (nAA), to evaluate the sensitizing potential of chemicals.

[Transcriptomics-driven Evaluation on Liver Toxicity Using Adverse Outcome Pathways (AOP)].

Because the liver is the primary target organ for chemicals and pharmaceuticals, evaluation of these substances' liver toxicity is of critical importance. New evaluation methods without animal testing (i.e.

Proposal of GHS sub-categorization criteria for LLNA: BrdU-ELISA (OECD TG442B).

The Globally Harmonized System of Classification and Labelling of Chemicals (GHS) is a hazard classification and communication system for providing information on the safe handling of chemicals worldwide. While the GHS provides sub-categorization criteria for sensitizers when using the guinea pig maximization test/Buehler test (OECD TG406) and the standard radioisotopic LLNA (OECD TG429), the sub-categorization criteria for LLNA: BrdU-ELISA (OECD TG442B) are not currently provided. In this study, we re-analyzed the existing data of 32 sensitizers classified in the 1A or 1B categories of the GHS, and attempted to determine optimal criteria for GHS sub-categorization using LLNA: BrdU-ELISA.

Effects of Soluble Tumor Necrosis Factor (TNF) on Antibody-Dependent Cellular Cytotoxicity of Therapeutic anti-TNF-α Antibody.

Anti-TNF antibodies are major therapeutics for rheumatoid arthritis and have been approved for marketing in many countries. Antibody-dependent cellular cytotoxicity (ADCC) is considered to be a potential mechanism of action of anti-TNF antibodies, since some anti-TNF antibodies have been confirmed to induce cytotoxic effects on TNF-producing cells via ADCC and complement-dependent cytotoxicity (CDC) in experiments. In this study, we established a new stable effector cell line expressing human FcγRIIIa, CD16:KHYG-1, and compared the performance of this cell line with that of peripheral blood mononuclear cells (PBMCs) in ADCC assays against CHO-derived target cells expressing protease-sensitive pro-TNF.

New approach to predict photoallergic potentials of chemicals based on murine local lymph node assay.

Photoallergic dermatitis, caused by pharmaceuticals and other consumer products, is a very important issue in human health. However, S10 guidelines of the International Conference on Harmonization do not recommend the existing prediction methods for photoallergy because of their low predictability in human cases. We applied local lymph node assay (LLNA), a reliable, quantitative skin sensitization prediction test, to develop a new photoallergy prediction method.

CSAHi study-2: Validation of multi-electrode array systems (MEA60/2100) for prediction of drug-induced proarrhythmia using human iPS cell-derived cardiomyocytes: Assessment of reference compounds and comparison with non-clinical studies and clinical information.

With the aim of reconsidering ICH S7B and E14 guidelines, a new in vitro assay system has been subjected to worldwide validation to establish a better prediction platform for potential drug-induced QT prolongation and the consequent TdP in clinical practice. In Japan, CSAHi HEART team has been working on hiPS-CMs in the MEA (hiPS-CMs/MEA) under a standardized protocol and found no inter-facility or lot-to-lot variability for proarrhythmic risk assessment of 7 reference compounds. In this study, we evaluated the responses of hiPS-CMs/MEA to another 31 reference compounds associated with cardiac toxicities, and gene expression to further clarify the electrophysiological characteristics over the course of culture period.

Investigation of the early-response genes in chemical-induced renal carcinogenicity for the prediction of chemical carcinogenicity in rats.

This study was designed to identify early-response genes of chemical-induced renal carcinogenicity for the prediction of chemical carcinogenicity in rats. We conducted a 28-day repeated-dose test in male Crl:CD (SD) rats with 12 carcinogens and 10 non-carcinogens as the training dataset, and five carcinogens and five non-carcinogens as the validation dataset. Renal gene expression profiles were analyzed by using a microarray.

Effects of dose volume and delivery device on bronchoalveolar lavage parameters of intratracheally administered nano-sized TiO in rats.

The intratracheal (IT) test is useful for screening the pulmonary toxicity of inhaled materials, including nanomaterials. However, a standard procedure has not yet been authorized internationally, and the effects of different test parameters are unknown. To determine appropriate experimental conditions for the IT test, we intratracheally administered nano-sized TiO to male F344 rats at 3.

Categorization of nano-structured titanium dioxide according to physicochemical characteristics and pulmonary toxicity.

A potentially useful means of predicting the pulmonary risk posed by new forms of nano-structured titanium dioxide (nano-TiO) is to use the associations between the physicochemical properties and pulmonary toxicity of characterized forms of TiO. In the present study, we conducted intratracheal administration studies in rats to clarify the associations between the physicochemical characteristics of seven characterized forms of TiO and their acute or subacute pulmonary inflammatory toxicity. Examination of the associations between the physicochemical characteristics of the TiO and the pulmonary inflammatory responses they induced revealed (1) that differences in the crystallinity or shape of the TiO particles were not associated with the acute pulmonary inflammatory response; (2) that particle size was associated with the acute pulmonary inflammatory response; and (3) that TiO particles coated with Al(OH) induced a greater pulmonary inflammatory response than did non-coated particles.