Regular articles

DEVELOPMENTAL TOXICITY OF BUTYLATED HYDROXYTOLUENE USING RAT AND HUMAN EMBRYONIC CELL ASSAYS

T. TSUCHIYA¹, N. MIYATA², S. KAMIYA², Y. IKARASHI¹, A. NAKAMURA¹ and A. TAKAHASHI^3
1Division of Medical Devices, ²Division of Orgunic Chemistry and ³Division of Xenobiotic Metabolism and Disposition, National Institute of Health Sciences, Tokyo, Japan

Regular article :AATEX3(1):1-8

Abstract
Butylated hydroxytoluene (BHT, 3, 5-ditert-butyl-4-hydroxytoluene) and 4-hydroxy-4-methyl-2,6-di-t-butylcyclohexa-2,5-dienone (BHTOH) were assayed using cell culture methods for assessing potential teratogenicity. In the rat embryonic cell differentiation assay, BHT and BHTOH showed similar inhibitory effects on the differentiation of both midbrain (MB) and limb bud (LB) cells. From the dose-response curve, the concentrations of BHT and BHTOH that inhibited the production of differentiated foci by 50%) IC50) in MB ceils were 254 and 245 ƒÊM, respectively. The inhibitory action of BHT on the human embryonic palatal mesenchymal (HEPM) cell growth was increased in the presence of S-9 mix prepared using livers from untreated f344 rats and from those treated with phenobarbital and 5,6-benzoflavone (PB-BF). In the HEPM cell growth assay, hamster and mouse PB-BF-induced S-9 were also effective in causing the metabolic activation of BHT. In vivo/in vitro methods for determining teratogenicity were investigated using a rat embryonic cell differentiation assay. BHT was orally administered to pregnant rats at a dose of 1000 mg/kg on day 11 of gestation. Embryonic MB and LB cells were then prepared from day-12 embryos, and cultured. The differentiated foci slightly reduced to 92-94% of the control values in MB and LB cultures. It was assumed that BHT up to 1000 mg/kg as a single oral dose was not harmful to the rat embryos during organogenesis under our experimental conditions.

VALIDATION OF POSTIMPLANTATION RODENT WHOLE EMBRYO CULTURE FOR IN VITRO TERATOGENICITY TESTING

KIYOHITO NAKASHIMA
Department of Chemistry Asahi University School of Dentistry, 1851-1 Hozumi, Hozumi-cho, Motosu-gun, Gifu 501-02, Japan

Regular article :AATEX3(1):9-16

Abstract
Teratogenicity data obtained from rat and mouse whole embryo culture (WEC) concerning the agents compiled in the Smith list were collected from the literature. Following analysis of these data, each technique for an in vitro teratogenicity testing was validated for its ability to evaluate teratogenic potential.
The rodent WEC techniques were variable between investigators with regard to culture medium, exogenous metabolizing source, and endpoint measurements. Of 34 agents tested in cultured rat embryos, two showed variable results. The remaining 32 agents had accuracy values of 90.0, 84.4, and 66.7% when the teratogenic potentials in vitro were compared with those available from rats, mammals, and humans in vivo, respectively. Of 15 known mammalian teratogens in vivo tested in cultured mouse embryos, 14 were positive and one was variable.
The results indicated that the rat WEC assay has poorer predictive value of teratogenic potential in vivo in mammals or humans than in rats. Also, the validity of the results obtained by the mouse WEC assay is unknown because agents known to be nonteratogenic in mammals in vivo have not been tested. Standardization of rodent WEC techniques is critically important before further validation efforts are made.

CARDIOVASCULAR MALFORMATIONS INDUCED BY CAFFEINE AND PHENOBARBITAL IN CHICK EMBRYOS

TAKASHI KOBAYASHI, ATSUYUKI NISHIDA, AKANE KUROKAWA, and FUMIO ARIYUKI
Safety Research Laboratory, Tanabe Seiyaku Co., Ltd., 3-16-89 Kashima, Yodogawa-ku, Osaka 532, Japan

Regular article :AATEX3(1):17-27

Abstract
The usage of chick embryos for studying the mechanism of cardiovascular malformations caused by chemicals was studied. Caffeine (CA), well known as a teratogen for the cardiovascular system of chick embryos, was administered at the dose of 3.0 mg per egg to chick embryos from two different breeds between incubation day 2 (ID2, H-H stage 14) and 5 (H-H stage 27). The eggs were incubated until ID12 to examine the cardiovascular malformation. Treatment at H-H stage 19 (ID3) was highly lethal and treatment at this stage and at stages 23-24 (ID4) induced a high degree of cardiovascular malformation in the embryos from both breeds. The embryotoxicity caused by CA in the two different breeds similarly varied with the developmental stage of treatment. Phenobarbital (PB), also a teratogen, was administered to chick embryos during H-H stages 22-24 at the doses of 1.13, 2.25 and 4.5 mg/egg to compare its embryotoxicity with that caused by treatment with CA. PB at the dose of 2.25 mg/egg or more was highly lethal and induced cardiovascular malformation. Administration of 13 jug of adenosine (AD) before CA and PB treatment reduced the lethality of PB but the effects of CA and teratogenicity of PB were not changed. However, in cultured cardiac cells at H-H stages 22-24 the frequency of spontaneous cell beating increased by treatment with 5 and 10 ƒÊM CA and decreased by that with 20 ƒÊM CA and those effects were reduced by co-administration of 0.5 ƒÊM AD. PB at the concentration of 6 ƒÊM or more reduced the frequency of cell beating and its effect was not changed by co-administration of 0.5 ƒÊM AD. Th responses to CA and PB treatment in whole chick embryos and cultured cardiac ceils were different. We believe that chick embryos can produce reproducible data when the developmental stage is precisely judged and the interactions of some chemicals on whole embryos and selected tissue or cells can be easily examined, so that they are very convenient to explore the mechanism of teratogenicity of chemicals.

NEUTRAL RED ASSAY USING NORMAL RABBIT CORNEAL EPITHELIAL CELLS GROWN IN SERUM-FREE MEDIUM AS AN ALTERNATIVE TO THE DRAIZE IRRITATION TEST

HISASHI TORISHIMA¹, RYOHEI YAMAMOTO¹ and MASAMI WATANABE^2
1Biochemical Department, Technical Research Laboratory, Kurabo Industries Ltd., 14-5 Shimokida, Neyagwa, Osaka 572, Japan; ²School of Pharmaceutical Science, Nagasaki University, 1-14 Bunkyo-Machi, Nagasaki 852, Japan

Regular article :AATEX3(1):29-36

Abstract
Neutral red assay using normal rabbit corneal epithelial (NRCE) cells were examined as an in vitro alternative to the Draize ocular irritation test. The cells were cultured in serum-free medium to reduce interference with serum components, especially serum proteins.
It was confirmed that the amount of neutral red incorporated into the cells was proportional to the number of viable cells, and the incorporation of the dye was affected by the concentration of calcium ion in the medium. Seventeen detergents were compared for the NR50 values obtained by this method to Draize ocular irritation test scores. The correlation coefficient between the NR50 value and the Draize rank was -0.694, and that between the NR50 and the DS20, a Draize score equal to 20 units from a possible total of 110, was 0.644.
By using neutral red assay, the sensitivity of chemicals was compared NRCE cells with established cells (SIRC) grown in serum-containing medium. NRCE cells cultured in serum-free medium were more sensitive to the cytotoxicity of chemicals than SIRC cells in serum containing medium. The higher sensitivity of the NRCE cells was mainly due to the use of serum-free medium, and also due to the higher sensitivity of normal cells to irritants tested.