Regular articles

GROWTH AND DIFFERENTIATION OF THE FETAL MOUSE PALATE CULTURED IN VITRO : COMPARISON WITH IN VIVO PALATOGENESIS

TSUNEO KOSAZUMA
Institute of Applied Medicine, Hokkaido Radio-isotope Center, Ltd., Ishikari, Hokkaido 061-32, and Department of Hygiene Chemistry, Hoshi University, Ebara, Shinagawa, Tokyo 142, Japan

Regular articleAATEX 2(4):177-181

Abstract
Palatal primordia of day-12.5 mouse fetuses were cultivated in a chemically-defined medium by a suspension culture technique, and their growth and differentiation in vitro were compared with those of the fetal palates in vivo at the corresponding gestational stages. The maxillary explants cultivated in vitro for up to 72 hours did not grow in size and remained almost in the same size as those in day-12.5 fetuses. However, the palatal shelves elevated and grew faster towards the midline until 24 hours in culture as compared with in vivo development. In day-14.5 fetal palates in vivo, the opposing palatal shelves have contacted with each other in about 80% of cases and 73% of the palates have begun to fuse, while the corresponding rates in vitro were 19% and 6%, respectively, suggesting that in vitro palatogenesis is delayed by 48 hours in culture. At 72 hours of culture, the rate of completely closed palates was 89% against the 10% closure rate for day-15.5 fetuses in vivo. Although the comparison showed that the in vitro development of the fetal mouse palate is somewhat slower than that in vivo, the process of palate fusion in vitro was found to simulate the in vivo palatogenetic process, and suspension organ culture appears superior to the conventional static organ culture. Suspension organ culture of fetal mouse palates should be useful in the study of normal and abnormal palatogenetic processes.

Key words: palatogenesis, mouse fetus, secondary palate, in vitro, in vivo.

IN VITRO ASSASY TO PREDICT PHOTOTOXICITY OF CHEMICALS: (I) RED BLOOD CELL HEMOLYSIS ASSAY

MARIKO SUGIYAMA, HIROSHI ITAGAKI, TAKESHI HARIYA, NORIKO MURAKAMI AND SHINOBU KATO
Shiseido Safety & Analytical Research Center, 1050 Nippa-cho, Kohoku-ku, Yokohama, Japan, 223

Regular article AATEX 2(4):183-191

Abstract
Photohemolysis testing of red blood cells (RBC), a phenomenon based on photodynamic reaction of the cell membrane, was validated as a possible alternative method to predict phototoxicity of chemicals after optimization of various factors. The data from phototoxicity test results in guinea pigs for 24 chemicals (8 fragrances, 5 UV absorbers, 4 drugs, 4 antimicrobials and 3 dyes) were compared. The sensitivity, specificity, positive predictive value, negative predictive value and equivalence of the in vitro test were 67%, 73%, 60%, 79% and 73%, respectively. Four chemicals were identified as false positive compounds, and two psoralens, of which the phototoxic mechanism is different from cell membrane damage, were identified as false negative compounds in the photohemolysis test. This in vitro method is expected to be useful as a prescreening tool for evaluating phototoxic potential of chemicals, and should be an effective means of reducing the number of animals required for phototoxicity testing.

Key words: in vitro test, photohemolysis, phototoxicity.

IN VITRO ASSASY TO PREDICT PHOTOTOXICITY OF CHEMICALS: (II) YEAST GROWTH INHIBITION ASSASY AND BATTERY SYSTEM WITH PHOTOHEMOLYSIS ASSAY

MARIKO SUGIYAMA, HIROSHI ITAGAKI AND SHINOBU KATO
Shiseido Safety & Analytical Research Center, 1050 Nippa-cho, Kohoku-ku, Yokohama, Japan, 223

Regular article AATEX 2(4):193-202

Abstract
The yeast growth inhibition assay, which is based on photodynamic reactions of compounds with cells organelles or DNA, was validated as possible alternative to animal testing to predict phototoxicity of chemicals after optimization of various factors. The data for 24 chemicals (8 fragrances, 5 UV absorbers, 4 drugs, 4 antimicrobials and 3 dyes) were compared with in vivo phototoxicity test results in guinea pigs. The sensitivity, specificity, positive predictive value, negative predictive value and equivalence of the in vitro test were 89%, 80%, 73%, 92% and 81%, respectively. Three chemicals were identified as false positive compounds, and one was identified as a false negative in the yeast growth inhibition assay. Although an excellent correlation was obtained between in vivo studies and yeast growth inhibition assay, a false negative result was still observed, so the combination of the photohemolysis test, which we have already reported, and the yeast test was used as a battery system. The battery system had a sensitivity of 100%, a specificity of 67%, a positive predictive value of 64%, a negative predictive value of 100% and an equivalence of 77%. Because of the sensitivity of 100%, this battery system, which is composed of two methods based on different mechanisms, is considered to be useful as a screening tool for predicting phototoxic potential of new chemicals, as an animal test alternative.

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

AN ALTERNATIVE METHOD FOR TESTING ORAL MUCOSAL IRRITATION CAUSED BY SURFACTANTS

MAYUMI KOTANI¹, YUKIMITSU MASAMOTO¹ and MASAMI WATANABE^2
1Safety Analysis Center R&D/Mfg/Technical HQ, Sunstar Inc, 3-1 Asahi-Machi Takatsuki Oaska 569 Japan; ²Division of Radiation Biology, Department of Health Sciences, Faculty of Pharaceutical Sciences, Nagasaki University, 1-14 Bunkyo-cho Nagasaki 852, Japan

Regular article AATEX 2(4):203-214

Abstract
Surfactant induced oral mucosal irritation and desquamation were assessed by the two-dimensional in vitro test which included neutral red assay, indirect immunostaining for fibronectin and tubulin, western blot for tubulin, and scanning electron microscopic (SEM) evaluation on normal human embryo kerotinocyte monolayers. Mucosal desquamation was also assessed by the three-dimensional in vitro test which included light microscopic (LM) and SEM evaluations of a three-dimensional culture of normal human gingival keratinocytes and fibroblasts treated with surfactants. Neutral red assay, and SEM evaluation in the two-dimensional in vitro test gave an index of the oral mucosal irritation. And, indirect immunostaining in the two-dimensional in vitro test, and LM and SEM evaluations in the three-dimensional in vitro test provided an index of desquamation. Benzalkonium chloride treatment resulted in mainly inflammation, while both sodium lauryl sulfate and sodium lauroyl sarcosinate treatments caused mainly desquamation. Tween 20 and Sucrose fatty acid ester did not produce any effects.
It seems that the evaluation of intra- and extra-cellular matrix is simple in the in vitro alternative method for oral mucosal desquamation.

IN VITRO BATTERY TEST SYSTEM FOR PREDICTING EYE IRRITANCY

TOSHIKATSU HAYASHI¹, HIROSHI ITAGAKI², TOSHIO FUKUDA¹, UHEI TAMURA¹ AND SINOBU KATO^2
1Shiseido Product Research Center, ²Shiseido Safety & Analytical Research Center, 1050 Nippa-cho, Kohoku-ku, Yokohama 723, Japan

Regular article AATEX 2(4):215-223

Abstract
Based on the hypothesis that irritation is due to damage of the cellular plasma membrane and cellular proteins, we designed an in vitro battery test system for predicting eye irritancy. The system consists of 3 tests, a hemoglobin denaturation (HDR) test to evaluate the protein denaturation factor, and tests of red blood cell (RBC) Iysis and liposome Iysis to evaluate the cellular plasma membrane destruction factor. Multi-regression analysis of the data obtained yielded equations for predicting Draize eye irritation scores. A combination of HDR and liposome tests gave the highest correlation to corneal Draize score (r=0.922), while combination of HDR and RBC tests gave the best correlation to total Draize score (r=0.941). These correlations are sufficiently good that this in vitro battery test system should represent a practical alternative to the in vivo Draize test for predicting eye irritancy of chemicals.