International Trend

Alternative to primary Draize skin irritation test using cultured human skin model: Comparison of six endpoints

Katsuyasu Morota¹, Noriyuki Morikawa¹, Shin-Ichiro Morita¹ Hajime Kojima², and Hiroaki Konishi^2
1Kyoto Research Laboratory, GUNZE Ltd., 1 Ishiburo, Inokura-shinmachi, Ayabe*city, Kyoto 623-0051, Japan
²Research Institute, Nippon Menard Cosmetic Co., Ltd., 2-7 Torimicho, Nishi-ku, Nagoya 451-0071, Japan

Correspondence: Katusyasu Morota, Kyoto Research Laboratory, GUNZE Ltd., 1 Ishiburo, Inokura-shinmachi, Ayabe-city, Kyoto 623-0051, Japan
Phone:+81-773-42-0141 Fax:+81-773-43-0362
Email:katsuyasu.morota@gunze.co.jp

Running title: Alternative to Draize skin irritation test

Original paper :AATEX 6(1):41-51,1999
Abstract
In this study, we evaluated skin irritancy of test chemicals by using six endpoints. As an endpoint, the cell viability was evaluated with 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) and neutral red (NR). In addition, lactate dehydrogenase (LDH), interleukin-1a (IL-1(alpha)), interleukin-8 (IL-8), and prostaglandin E₂ (PGE₂) releases from the cells were measured. Each assay was compared with in vivo animal testing and with one another. The results indicated the assays could be divided into 3 groups, MTT/NR, LDH/IL-1(alpha), and IL-8/PGE₂. The MTT and NR assays revealed good correlation with in vivo scores, except for croton oil; the IL-8 and PGE₂ assays could detect the irritancy of croton oil. On the whole, the LDH, IL-1(alpha), IL-8, and PGE₂ assays had poor correlation with in vivo scores.
Furthermore, time transition of skin irritancy was examined by the MTT, IL-8, and PGE₂ assays. Production of IL-8 and PGE₂ was related to cell viability as determined by the MTT assay.
This model may be reliable for predicting skin irritancy of chemicals and for studying mechanisms of irritancy by using a combination of endpoints

Key words:skin model, skin irritancy, MTT, NR, LDH, IL-1(alfa), IL-8, PGE₂

The LD50 Values of Cardiovascular Drugs at Different Developmental Stages in Chick Embryos

Hiroyuki Miyazaki, Takashi Sugiyama and Hideyo Shimada
Division of Pathophysiology, Center for Clinical Pharmacy and Clinical Sciences, School of Pharmaceutical Sciences, Kitasato University,9-1,Shirokane 5-chome,Minato-ku,Tokyo 108-8641 Japan

Correspondence: Takashi Sugiyama, Ph.D.
Division of Pathophysiology, Center for Clinical Pharmacy and Clinical Sciences, School of Pharmaceutical Sciences, Kitasato University
9-1, Shirokane 5-chome, Minato-ku, Tokyo 108-8641 Japan
Tel / Fax: 03-3446-9036
e-mail: sugiyama@platinum.pharm.kitasato-u.ac.jp

Running Title: LD50 Values of Cardiovascular Drugs in Chick Embryos

Original paper :AATEX 6(1):52-57,1999
Abstract
The LD50 values were determined for cardiovascular drugs in chick embryos at different developmental stages in order to obtain a more precise injection stage for fertile eggs of White Leghorn chickens for the prediction in rodents. First, time-course changes in the weight of fertile eggs, their air sac volume, and weight of each egg component were measured after the initiation of incubation. The weight of whole eggs decreased with incubation, while air sac volume increased. The chick embryos weight increased with decreases in albumen weight. These findings suggest that decreases in whole egg weight are due to decreases in water in the eggs. When the maximum volume of physiological saline or CMC-Na solution was injected into the air sac on different days of incubation the bulk of the vehicle did not prove to be toxic to the chick embryos. Next, several cardiovascular drugs, i.e., aloprenolol, piretanide, dipyridamol, lidocaine, propranolol, canrenoate, disopyramide and reserpine were injected into the air sac of eggs on day 2, 5, 8, or 15 of incubation. Then, the chick embryos were sacrificed on day 20 of incubation, and LD50 values were calculated. The LD50 values of these drugs increased with the developmental stages in chick embryos. The LD50 values in chick embryos on day 2 and 5 of incubation showed a fairly strong correlation to those in mice obtained from the intravenous route. Furthermore, findings obtained on day 2 of incubation were most sensitive and reliable in predicting LD50 values in rodents. In conclusion, when cardiovascular drugs were injected into the air sac of fertile eggs in the early stage of development, i.e., day 2 of incubation, LD50 values in chick embryos could be used to predict the LD50 values in rodents.

Keywords: ; cardiovascular drugs, chick embryo, developmental stage, lethal toxicity

Effects of Ca Channel Blockers under Different Lighting Conditions in the Chick-embryonic Heart

Hiroyuki Miyazaki, Takashi Sugiyama and Hideyo Shimada
Division of Pathophysiology, Center for Clinical Pharmacy and Clinical Sciences, School of Pharmaceutical Sciences, Kitasato University,9-1,Shirokane 5-chome,Minato-ku,Tokyo 108-8641 Japan

Correspondence: Takashi Sugiyama, Ph.D.
Division of Pathophysiology, Center for Clinical Pharmacy and Clinical Sciences, School of Pharmaceutical Sciences, Kitasato University
9-1, Shirokane 5-chome, Minato-ku, Tokyo 108-8641 Japan
Tel / Fax: 03-3446-9036
e-mail: sugiyama@platinum.pharm.kitasato-u.ac.jp

Running Title: Effects of Ca Channel Blockers in Chick-embryonic Heat

Original paper :AATEX 6(1):58-64,1999
Abstract
The pharmacological effects of Ca channel blockers on electrocardiogram (ECG) in the chick embryonic-heart were investigated under different lighting conditions. The fertile eggs of White Leghorn chickens were incubated in dark conditions and used on day 16 of incubation. Three different types of Ca channel blocker, nicardipine, diltiazem and verapamil were injected into the air sac of the fertile eggs under light conditions (450 Lux) or dark conditions (12 Lux). The bipolar lead of ECG patterns was recorded using ECG systems. Heart rates (HRs) were calculated from R-R intervals. Under light conditions, 3 kinds of Ca channel blockers caused a decrease in HRs in a dose-dependent manner and also arrhythmia was provoked. These phenomena were observed more clearly under dark conditions. Furthermore, these enhancements of the drug effects under dark conditions were also observed by concomitant injection of 15-50ƒÊg/egg of melatonin even under light conditions. In conclusion, these results suggest that chick embryos are very useful models for investigating the chronopharmacological effects and for evaluating drug interaction of cardiovascular drugs.

Keywords: Ca channel blocker, chick embryo, electrocardiogram, chronopharmacology, melatonin

Cause of low embryogenesis-promoting ability of bovine and swine sera for 9.5-day rat embryo in culture

Miyuki Katoh, Reiko Kimura and Ryujiro Shoji
Department of Embryology, Institute for Developmental Research, Aichi Human Service Center, 713-8 Kamiya-cho, Kasugai, Aichi 480-0392, Japan.

Correspondence: Miyuki Katoh, Department of Embryology, Institute for Developmental Research, Aichi Human Service Center, 713-8 Kamiya-cho, Kasugai, Aichi 480-0392, Japan
Tel: 0568-88-0811, Fax: 0568-88-0829

Running Title: Embryogenesis-promoting ability of bovine and swine sera in rat embryo

Original paper :AATEX 6(1):65-71,1999
Abstract
Were bovine and swine sera to have the same embryogenesis-promoting ability as rat serum, they might be able to replace rat serum as a medium for whole rat embryo culture, given their availability at any butchery. When 9.5-day rat embryos were cultured in 50% and 100% of bovine and swine sera, respectively, the embryogenesis-promoting abilities were compared with those of rat serum. Development of rat embryos cultured in 50% and 100% of these sera was significantly poorer than that of embryos cultured in 50% rat sera. Therefore, these sera probably have low embryogenesis-promoting ability for 9.5-day rat embryos in culture. Furthermore, the bovine and swine sera contained a 180 kDa serum component in place of the 190 kDa component in rat serum, and DEAE-eluate in these sera also contained most of the 180 kDa serum component. In addition, development of 9.5-day rat embryos that were cultured in serum-free medium, containing rat transferrin, supplemented with DEAE-eluate in these sera, also was poorer than that of embryos cultured in medium with rat serum. The difference in presence of the defined serum components seemed to reflect the limited embryogenesis-promoting abilities in these sera

Keywords: whole rat-embryo culture, culture medium, domestic-animal’s sera, rodent sera

Utilization of Chick Embryonic Electrocardio-grams to Detect the Proarrhythmic Actions by Antiarrhythmic Drugs

Takashi Sugiyama, Hiroyuki Miyazaki and Hideyo Shimada
Division of Pathophysiology, Center for Clinical Pharmacy and Clinical Sciences, School of Pharmaceutical Sciences, Kitasato University,@9-1, Shirokane 5-chome, Minato-ku, Tokyo 108-8641, Japan

Correspondence: Takashi Sugiyama, Ph.D.
Division of Pathophysiology, Center for Clinical Pharmacy and Clinical Sciences, School of Pharmaceutical Sciences, Kitasato University, 9-1, Shirokane 5-chome, Minato-ku, Tokyo 108-8641, Japan
Tel and Fax; +81-03-3446-9036
E mail; sugiyamat@platinum.pharm.kitasato-u.ac.jp

Running title: Proarrhythmic actions by antiarrhythmic drugs in chick embryos

Original paper :AATEX 6(1):72-78,1999
Abstract

We have studied the pharmacological and toxicological effects of cardiovascular drugs on chick embryos using electrocardiograms in order to develop alternatives to the use of mammals. Recently, proarrhythmic actions induced by antiarrhythmic drugs have been widely studied. In this study, we investigated the pharmacological effects of antiarrhythmic drugs, Ia(procainamide), Ib(lidocaine), Ic(flecainide), II(propranolol) and IV(verapamil)types, as classified by Vaughan Williams. When a single injection of drugs was performed into the air sac of fertile eggs of White Leghorns on the 16th day of incubation, all drugs decreased HR in a dose-dependent manner. Also, when higher dosages of drugs were injected, these drugs induced various kinds of arrhythmias, i.e., S-A or A-V blocks, as shown in mammals. The intervals of PQ, QRS and QT waves varied depending on the drugs used. However, the ECG waves exhibited the same changes seen in mammals with a few exceptions. Our ECG recording system using chick embryos may be applicable for screening tests for proarrhythmic actions of antiarrhythmic drugs.

Key words: Chick embryo, alternative animal, antiarrhythmic drug, electrocardiogram, proarrhythmic action