Prediction of Human Skin Irritancy Using a Cultured Human Skin Model: Comparison of Chemical Application Procedures and Development of a Novel Chemical Application Procedure Using the Vitrolife-Skin^TM Model

Noriyuki Morikawa¹, Katsuyasu Morota¹, Shin-Ichiro Morita¹, Hajime Kojima², Satoru Nakata² and Hiroaki Konishi²
1 GUNZE Ltd., 2 Nippon Menard Cosmetic Co., Ltd.

Corresponding author: Noriyuki Morikawa,
Division of Research & Development, GUNZE Ltd.
1 Ishiburo, Inokura-shinmachi, Ayabe, Kyoto 623-8512, Japan
Phone: +81-773-42-0141, Fax: +81-773-42-0036, E-mail: noriyuki.morikawa@gunze.co.jp

Original paper :AATEX 9(1):1-10
Abstract
When cultured skin models are used to evaluate skin irritancy, it is important that appropriate procedures for chemical application are utilized. In this study, we used several test substances and evaluated their skin irritancy by employing in vitro examinations, that is, evaluations of time scores for 50% cytotoxicity (ET50 values) and concentration scores for 50% cytotoxicity (EC50 values), and compared them to in vivo examination, that is, human patch test scores. There are great differences between chemical application procedures to predict skin irritancy. Therefore, to prevent both false negative and false positive results, we have developed a novel chemical application procedure, which is termed the post-incubation (PI) method. The PI method to evaluate EC50 values showed better correlation compared with other methods. In conclusion, considering the characteristics of these chemical application procedures, it is possible to confidently predict skin irritancy. It is also expected that cultured skin models will be very useful for purposes of predicting in vivo skin irritancy of chemicals and for studying mechanistic aspects of such irritancy.

Key words: cultured skin model, skin irritancy, EC50, ET50, human patch test

Kinetic Analysis of Cell Killing Effect Induced by Busulfan in Chinese Hamster Cells

Mariko Fukumoto, Takashi Sugiyama and Akira Ogamo
Kitasato University

Correspondence: Mariko Fukumoto
Center for Clinical Pharmacy and Clinical Sciences, School of Pharmaceutical Sciences, Kitasato University, 5-9-1, Shirokane, Minato-ku, Tokyo 108-8641, Japan
Tel: 03-5791-6248, Fax 03-3442-1946, E-mail: fukumotom@pharm.kitasato-u.ac.jp

Original paper :AATEX 9(1):11-16
Abstract
High dosage busulfan (1 mg/kg orally every 6 hours x16 doses) is an important component of many of the myeloablative protocols for hematopoietic stem cell transplantation (HSCT) in both adults and children. At an orally given dose, there is considerable variability in the systemic exposure of busulfan, typically expressed as the area under the drug concentration-time curve (AUC) or average concentration at steady state (Css = AUC/dose interval). Several investigators have identified relationships between busulfan AUC (or Css) and clinical outcome in patients undergoing HSCT. Generally, the risk of hepatic veno-occlusive disease of the liver (VOD), the severest toxicity of busulfan, is increased with busulfan AUC>5400ug h/L. In Europe and America, dose adjustment based on measured AUC is common.
However, analysis of cell killing action of busulfan has not yet been presented on a kinetic basis. In this report, we have attempted to make a more kinetically based analysis of the cell killing effect of busulfan, and to determine which type of cell killing action of busulfan shows. We assessed the busulfan concentration necessary for 90% cell kill (IC90) with various exposure times and the degradation rate constants under the culture conditions used. Busulfan was slowly eliminated during incubation, with a half-life of 19.3h and residual busulfan amounted to 40.75% of the initial level after a 24-h incubation at 37 oC. When IC90fs and exposure times were plotted on a log scale, a linear relationship with a slope of ?1 was seen for busulfan during shorter than 100 h. We analyzed cell kill kinetics of busulfan and showed the concentration-time product(C x T) dependence of cell killing action of the drug by colony-forming inhibition assays using Chinese hamster V79 cells. These results suggested that the AUC of busulfan is an important determinant of graft rejection and regimen-related toxicity in HSCT patients and indicated dose modification based on measured AUC is necessary for these patients in Japan.

Keywords: busulfan, Chinese hamster V79 cells, colony formation assay, cell cycle phase-nonspecific agents, AUC

Development of an Antigenicity Study Consisting of in vitro Priming in Mouse Peritoneal Exudate Cells and in vitro Anaphylaxis Test in Rat Mast Cells

Tomoaki Inoue¹ and Ikuo Horii²
1 Nippon Roche Research Center, 2 Pfizer Inc.

Correspondence: Ikuo Horii
Drug Safety Evaluation, Pfizer Global Research & Development, Nagoya Lab., Pfizer Inc.
5-2, Taketoyo, Aichi, 470-2393, Japan.
Tel: 0569-74-4762, Fax: 0569-74-4450, E-mail: Ikuo.Horii@japan.pfizer.com

Original paper :AATEX 9(1):17-28
Abstract
To develop an antigenicity study with exposure to test compounds in in vitro systems, we investigated suitable experimental conditions for the in vitro exposure. In the antigenicity evaluation system, mouse peritoneal exudate cells (PECs) were exposed to test compounds in in vitro priming culture, the primed PECs were intraperitoneally injected into nontreat mice, and the sera were isolated after an immunizing period. The antigen-specific IgE in the sera was measured by in vitro anaphylaxis test which rat mast cells were exposed to the mouse sera with following exposure to the test compounds (antigens). Histamine levels in the supernatant was measured to evaluate the degranulation of the mast cells. Optimum conditions for the in vitro anaphylaxis test were as follows. The mast cell number: 1-3×10⁴cells/test mast cells, dilution of the test sera: 10:1, incubation of the mast cells with the test sera: 4 hr, concentrations of antigens: 100ug/ml for macromolecular antigens or maximum non-cytotoxic concentrations for low molecular weight antigens, incubation with the antigens: 45 min. By using low molecular weight reference antigens such as penicillin G and nafamostat mesilate, low but positive reactions were observed in the antigenicity evaluation system consisting of in vitro priming culture and in vitro anaphylaxis test. From these results, this antigenicity testing system would help reductions of the number of animals, required amount of test compounds, and the experimental period, and make it possible to evaluate antigenicity in early stages of drug discovery.

Keywords: antigenicity, in vitro, priming culture, anaphylaxis, peritoneal cells

A Strategic Approach for Predicting Phototoxicity of Cosmetic Ingredients

Mariko Sugiyama, Masaaki Mori, Masatoshi Hoya, Morihiko Hirota and Hiroshi Itagaki
Shiseido Safety and Analytical Research Center

Correspondence: Mariko Sugiyama
Shiseido Safety and Analytical Research Center
2-2-1 Hayabuchi, Tsuzuki-ku, Yokohama-shi 224-8558, Japan
Tel: 045-590-6062, Fax: 045-590-6094, E-mail: mariko.sugiyama@to.shiseido.co.jp

Original paper :AATEX 9(1):29-39
Abstract
Many in vitro methods to predict phototoxicity, based on various mechanisms, have been developed and reported. We have evaluated 3 types of in vitro methods and used this battery of assays to assess the phototoxicity of cosmetic ingredients. The in vitro methods used were photohemolysis testing of red blood cells (RBC), a technique to predict damage to cellular membranes, yeast growth inhibition assay, and the 3T3 mouse fibroblast neutral red uptake phototoxicity test (3T3 NRU PT), a candidate for the OECD guideline procedure to estimate phototoxic effects on cell organelles.
In comparison with in vivo data in guinea pigs, the yeast growth inhibition assay was the most highly correlated method. No false negatives were observed in the battery system of yeast growth inhibition assay and RBC photohemolysis assay. Because only a few chemicals gave positive reactions in the RBC photohemolysis or 3T3 NRU PT assay, it was suggested that a combination of methods based on different mechanisms would be needed for the safety assessment of phototoxicity of chemicals. Our results indicated that the battery system of the yeast growth inhibition assay and RBC photohemolysis assay is the most effective tool for assessing the phototoxicity of cosmetic ingredients. It is also important to select methods compatible with the physicochemical properties of the chemicals to be tested.

Key words: phototoxicity, in vitro test, battery system, yeast growth inhibition assay, photohemolysis