Hajime Kojima

The Epi2SensA method is a method similar to the validated EpiSensA assay for assessing the skin sensitization potential of chemicals. The Epi2SensA protocol includes adaptation (changes to exposure conditions and the controls) for using an alternative reconstructed human epidermis (RhE) model, the EpiDerm™ model. The interlaboratory validation study evaluated the reliability and predictive capacity of Epi2SensA according to OECD Performance Standards. Four laboratories (Mattek, Now Part of Sartorius, Eurofins Munich, Burleson Research Technologies, Inc., and Food and Drug Safety Center) conducted blinded testing of 20 coded reference substances representing various chemical categories and sensitization potencies. Statistical analysis using modified acceptance criteria (a 60% cell viability threshold) and a modified prediction model (requiring at least two positive gene markers) demonstrated substantially improved performance compared to the original EpiSensA criteria. The between-laboratory reproducibility (BLR) was 85%, the average within-laboratory reproducibility (WLR) was 83.3%, and the average predictivity parameters were 88.1% for sensitivity, 88.9% for specificity, and 88.3% for accuracy. Epi2SensA achieved performance metrics comparable to the validated reference method (EpiSensA), supporting regulatory acceptance of the Epi2SensA assay using the EpiDerm™ model (Mattek Corporation, Now Part of Sartorius, Ashland, MA, USA) as an alternative RhE source for OECD TG 442D skin sensitization testing.

In a previous study, a novel human corneal-like epithelium model utilizing an immortalized human corneal epithelial cell line (iHCE-NY1) was developed as an alternative to animal models to identify chemicals not classified under the United Nations Globally Harmonized System of Classification and Labeling of Chemicals (GHS) and was evaluated following the criteria of Test Guideline 492 of the Organization for Economic Co-operation and Development (OECD). In the present study, our aim was to establish an eye irritation test protocol using the iHCE-NY1 model to classify liquid chemicals under the GHS ocular hazard categories: no effect, no classification (No Cat.), Category 2 (Cat. 2) reversible effects, and Category 1 (Cat. 1) irreversible eye damage. The protocol involved exposing the iHCE-NY1 model to 31 liquid test chemicals for 5 min, followed by observation at post-incubation periods (PIPs) to assess recovery. Classification was based on cell viability, and histopathological findings on PIP days 7, 14, and 21. The outcomes were compared with an established database of classifications. All Cat. 1 liquid chemicals, 62.5% of No Cat., and 63.2% of Cat. 2 were correctly categorized. This study demonstrates that the iHCE-NY1 model can not only distinguish No Cat. test liquid chemicals but also differentiate between Cat. 2 and Cat. 1 liquid chemicals.

Detecting the toxic effects of chemicals on reproduction and development without using mammalian animal models is crucial in the exploitation of pharmaceuticals for human use. Zebrafish are a promising animal model for investigating pharmacological effects and toxicity during vertebrate development. Several studies have suggested the use of zebrafish embryos for the assessment of malformations or embryo-fetal lethality (MEFL). However, a reproducible protocol as a standard for the zebrafish MEFL test method that fulfills global requests has not been established based on the International Council of Harmonisation (ICH) S5 (R3) guidelines. To establish such a toxicity test method, we developed a new and easy protocol to detect MEFL caused by chemicals, especially those with teratogenic potential, using fertilized zebrafish eggs (embryos) within 5 days of development. Our toxicity test trials using the same protocol in two to four different laboratories corroborated the high inter-laboratory reproducibility. Our test method enabled the detection of 18 out of 22 test compounds that induced rat MEFL. Thus, the prediction rate of our zebrafish test method for MEFL was almost 82% compared with that of rat MEFL. Collectively, our study proposes the establishment of an easy and reproducible protocol for the zebrafish MEFL test method for reproductive and developmental toxicity that meets ICH guideline S5 (R3), which can be further considered in combination with information from other sources for regulatory use.

昨今，創薬開発においても作用機構をもとにした安全性評価の重要性が謳われ，国際機関からもヒト由来‍細‍胞や組織を用いた動物実験によらない代替試験法（以下，代替法と記す）を利用した安全性評価の要‍望が増えている．ただし，動物実験で捉えられる多様な現象を，従来開発されてきたような，ひとつの代替法で網羅できないことは明らかであり，そもそも動物実験データとの比較に留まりヒトの毒性を評価す‍る‍こ‍とを想定していない場合もある．そこで，創薬開発にNew Approach Method（NAM）の活用への期待が高まっている．本稿では，NAMの事例として，生殖毒性試験の代替法および人体摸倣システム（Microphysiological system：MPS）による安全性評価の行政的な受け入れに関する昨今の状況をまとめた．

Introduction: Animal tests of cosmetic ingredients and products have been banned in the EU since 2013. However, in Japan, the application of new quasi-drugs requires the generation of 24-hour data on primary and cumulative skin irritation by animal testing. Such data are unreliable because an ingredient predicted as nonirritating after short exposure (4 hours), based on the Organization for Economic Co-operation and Development (OECD) test guidelines (TG)404, may cause irritation after a longer application period in human skin irritation tests. With insufficient data to draw conclusions about the irritation potential of an ingredient, there remains a high probability of skin irritation occurrence after extended exposure to the ingredient. Materials and Methods: This study assessed whether the skin irritation caused by quasi-drugs and cosmetic products can be evaluated in a step-by-step manner. Results: A workflow was developed considering several key steps such as the component characteristics based on physicochemical properties or the ingredient category based on existing information from animal tests and human patch test results, and its utility was assessed using the reconstructed human epidermis (RhE) test (OECD TG439), animal testing, the human patch test, and the human cumulative skin irritation test. Conclusion: The RhE test and the aforementioned human skin tests can be employed to evaluate test substances that cause weak or nonskin irritation categorized as “harmless ingredients”—thereby avoiding animal testing.

The Amino acid Derivative Reactivity Assay (ADRA) is a convenient and effective in chemico test method for assessing covalent binding of test chemicals with protein‐derived nucleophilic reagents as a means of predicting skin sensitization potential. Although the original molar‐concentration approach to ADRA testing was not suitable for testing multiconstituent substances of an unknown composition, a weight‐concentration approach that is suitable for such substances was developed, which also led to the realization that test chemical solutions prepared to molar concentrations higher than the original 1 mM would reduce false negative results as well as enhance predictive capacity. The present study determined an optimal molar‐concentration that achieves even higher predictive capacity than the original ADRA. Eight chemicals that were false negatives when tested with 1 mM test chemical solutions were retested with test chemical solutions between 2 and 5 mM, which showed 4 mM to be the optimal molar‐concentration for ADRA testing. When 82 chemicals used in the original development were retested with 4 mM test chemical solutions, false negative results were reduced by four. When an additional 85 chemicals used to evaluate the weight‐concentration approach to ADRA were retested, the results essentially replicated those obtained with 0.5 mg/ml test chemical solutions and gave 10 fewer false negatives than original ADRA with 1 mM solutions. A comparison of these results for 136 chemicals showed that ADRA testing with 4 mM solutions achieved a four percentage point improvement in accuracy over original ADRA and a two percentage point improvement over DPRA testing.

This paper reviews the usefulness, current status, and potential of primary human hepatocytes (PHHs) in three-dimensional (3D) cultures, also known as spheroids, in the field of pharmacokinetics (PK). Predicting PK and toxicity means pharmaceutical research can be conducted more efficiently. Various in vitro test systems using human hepatocytes have been proposed as tools to detect hepatic toxicity at an early stage in the drug development process. However, such evaluation requires long-term, low-level exposure to the test compound, and conventional screening systems such as PHHs in planar (2D) culture, in which the cells can only survive for a few days, are unsuitable for this purpose. In contrast, spheroids consisting of PHH are reported to retain the functional characteristics of human liver for at least 35 days. Here, we introduce a fundamental PK and toxicity assessment model of PHH spheroids and describe their applications for assessing species-specific metabolism, enzyme induction, and toxicity, focusing on our own work in these areas. The studies outlined in this paper may provide important information for pharmaceutical companies to reduce termination of development of drug candidates.

Introduction: In the last decade, a variety of in vitro eye irritation test (EIT) methods have been developed and validated as an alternative to animal testing to assess the ocular toxicity of chemicals. Among these in vitro test methods, that using reconstructed human corneal epithelium (RhCE) is considered to be most useful, but RhCE is expensive and cannot be purchased at any time in Japan. Thus, we undertook this study to establish a method for in house fabrication of RhCE using the immortalized human corneal epithelial cell lines. Materials and Methods: An RhCE fabricated from the immortalized human corneal epithelial cell line (iHCE-NY1) was evaluated in accordance with the performance standards of Test Guideline 492 issued by the Organization for Economic Cooperation and Development (OECD). Results: Histological analysis of the iHCE-NY1 model showed a complete corneal epithelium containing three corneal epithelial layers and the corneal epithelial marker, Mucin-16, was expressed in the appropriate regions. Prediction of eye irritation potential of the 30 reference chemicals based on performance standards using the iHCE-NY1 model correlated well with in vivo test results for eye irritation potential, with a sensitivity of 100%, a specificity of 60.0%, and an accuracy of 80.0%. Discussion and Conclusion: This development successfully established a simple and inexpensive method for in house fabrication of RhCE at laboratories, thereby providing a promising alternative to animal testing for assessing eye irritation potential using the iHCE-NY1 model.