Dr. Yukiaki Kuroda, honorary member and former vice president of the Japanese Society for Alternatives to Animal Experiments, passed away on May 21, 2008. He was 82 years old.
Dr. Kuroda was born in Nara Prefecture on May 22, 1926. After graduating from the Faculty of Science, Kyoto University in 1950, he immediately assumed the position of research associate in the Department of Genetics, Faculty of Medicine, Osaka University. In 1957, he was appointed as an lecturer in the Department of Biology, Faculty of Science, Osaka University, and was promoted to associate professor in the same Department in 1963. In 1966, Dr. Kuroda transferred to the National Institute of Genetics as general manager, and in 1976 he was appointed as the director of the Division of Gene Expression. He was elected as professor at the same institute in 1984, and in 1990 became an honorary professor upon retirement. Also in 1990 he assumed the post of professor in charge of the Life Science Course, the School of Environmental Health, Azabu University. He served as director of the Research Institute of Biosciences at the same university from 1991 until he retired in 1997. After his retirement from the University he devoted himself to activities in research and education, while assuming such positions as honorary professor at the National Institute of Genetics, and visiting professor at Azabu University and University of Shizuoka.
Dr. Kuroda's research spanned a wide range of themes, including the establishment of cell and tissue culture techniques, analysis of gene expression in culture systems, establishment of methods for gene mutation detection, and search for environmental mutagens and mutagenesis inhibitors. …

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The human Cell Line Activation Test (h-CLAT) is an in vitro skin sensitization method based on augmentation of CD86 and CD54 expression in THP-1 cells (human monocytic leukemia cell line). In our previous Japanese inter-laboratory study, we reported that the transferability and reproducibility of the h-CLATis basically good. The aim of this study was to define the criteria for selecting appropriate THP-1 cells inthe h-CLAT. In this study, new THP-1 cell lots were obtained from three cell banks: one in America,Europe and Japan. Using these three lots plus the cell lot we had previously used and obtained from ATCCwe investigated the CD86/CD54 expression following exposure to two allergens (DNCB and Ni) and onenon-allergen (SLS). Compared with the previous ATCC lot, two new lots showed similar results. Mean-while, the third new lot showed distinctly different results in cell viability and CD86/CD54 augmentationinduced by Ni compared to the other three lots. These results showed that the variability of cellular responses in the THP-1 cells depended on the cell source. In conducting the h-CLAT, it would be importantto select appropriate THP-1 cells to predict correctly the skin sensitization potential.

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The human Cell Line Activation Test (h-CLAT) is an in vitro skin sensitization test based on the enhancement of CD86 and/or CD54 expression on THP-1 cells. The aim of this study was to examine the effect of differences of serum source on the results of h-CLAT. Three different lots of serum, obtained from three sources, were compared with the serum used in the previous Japanese ring study. With each serum, cellular proliferation in subculture, cytotoxicity, and CD86/CD54 expression on THP-1 cells were measured following exposure to two known allergens (dinitrochlorobenzene (DNCB) and nickel sulfate (Ni)) and one non-allergen (sodium lauryl sulfate (SLS)).
There was no clear difference of cellular proliferation in subculture, cytotoxicity, or CD86/54 expression among cultures in the four sera. Although the source of serum does not appear to influence the resultof h-CLAT, the validity of the test should nevertheless be confirmed when serum from a new source is introduced.

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The human Cell Line Activation Test (h-CLAT) is an in-vitro skin sensitization method based on the enhancement of CD86 and/or CD54 in THP-1 cells. Experimental conditions for h-CLAT were optimized inour previous study. This protocol defines that THP-1 cells are seeded between 0.1x10⁶ and 0.2x10⁶cells/mL, and pre-cultured for 48h or 72h before treated with a test chemical. In this study we evaluatedeffects of pre-culture conditions on the h-CLAT results minutely. We cultivated the cells on ninepre-culture conditions before exposure to allergens (dinitrochlorobenzene (DNCB) and nickel sulfate (Ni))or non-allergen (sodium lauryl sulfate (SLS)), and then measured CD86 and CD54 expressions on thesecells after the exposure. All laboratories almost correctly evaluated the skin sensitization potential of thesethree chemicals on any pre-culture conditions. However only low CD86 and CD54 RFI values induced byDNCB tend to be obtained as the final cell concentration on pre-culture became higher. For maintainingthe response of THP-1 cells to allergens and distinguishing allergens and non-allergens more clearly,THP-1 cells should be avoided being in over-growth conditions during pre-culture. Therefore a supplementary experimental condition about pre-culture for h-CLAT that final cell concentration in pre-culturemust not exceed 1.0x10⁶ cells/mL was defined.

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Recently, safety evaluation tests that do not involve animal experiments have been prosperously developing. However, the optimal evaluation materials and methods for assessing ocular irritancy have not been well investigated. In this study, we determined the optimal evaluation method for testing ocular irritation using a human cultured corneal epithelium model (corneal model). In order to assess adequate treatment conditions for the corneal model, we used cetylpyridinium chloride (CPC), which has been recognized as an irritant chemical by the Draize eye test. The irritancy elicited by multiple concentrations of CPC was evaluated by a cytotoxicity assay under nine treatment conditions and compared to the Draize score. The treatment conditions that included a 5-second exposure period followed by a 24-hour post-incubation period (hereafter called protocol "5-sec+24-h") showed a significant correlation between cytotoxicity and the Draize score. Furthermore, the dose-dependent cytotoxicity of six test chemicals was assessed by protocol "5-sec+24-h" and found to correlate with the Draize eye test results.

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