Comparison of embryotoxicity of ESBO and phthalate esters using an in vitro battery system

Epoxidized soy bean oil (ESBO) and phthalate esters have been used as a plasticizer in polyvinyl chloride products. In this study, the embryotoxicity of ESBO and phthalate esters, namely, diethyl hexyl phthalate (DEHP), butylbenzyl phthalate (BBP) and dibutyl phthalate (DBP) was evaluated using short-term in vitro battery system, such as the whole embryo, midbrain and limb bud culture systems. Whole embryos at gestation day 9.5 were cultured for 48 h and the morphological scoring was measured. The cytotoxic effect and differentiation for mid-brain (MB) and limb bud (LB) cell were assessed by 50% inhibition concentration (IC 50) with neutral red uptake and hematoxylin-stained foci (MB) or Alcian Blue staining (LB), respectively. In the whole embryo culture assay, ESBO (83, 250 and 750 μg/ml) exerted no toxic effect on growth and development of the embryo, whereas phthalate esters (1, 10, 100 μg/ml for DEHP, 10, 100, 1000 μg/ml for BBP and DBP) inhibited growth and development dose dependently. In mid-brain and limb bud culture, the IC 50 of differentiation and cytotoxicity in BBP was 412.24 and 231. 76 μg/ml for mid-brain, and 40.13 and 182.38 μg/ml for limb bud, respectively. The IC 50 of differentiation and cytotoxicity in DBP was 27.47 and 44.53 μg/ml for mid-brain, and 21.21 and 25.54 μg/ml for limb bud cells, respectively. The lower IC 50 in both cells was obtained from DBP when compared to BBP. From these results, limb bud cells responded more sensitively to BBP and DBP than mid-brain cells. The IC 50 of limb bud cell differentiation and cytotoxicity in DBP is 1.9 and 7.1 less than that of BBP. However, any alteration in cytotoxicity and differentiation was observed with ESBO treatment. These studies suggested that ESBO is not embryotoxic; however, DEHP, BBP and DBP exhibit embryotoxic potential at high concentration.