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Toxicity Potential of Absorbed-Retained Ethylene Oxide Residues in Culture Dishes on Embryo Development in Vitro^1,2,

National Zoological Park, Smithsonian Institution, Washington, DC 20008 and National Institutes of Health, Bethesda, MD 20205

Abstract

Four hundred eight-cell mouse embryos were cultured in vitro in either polystyrene (plastic) dishes (Exp. 1) or watch glasses (Exp. 2) to analyze the toxicity potential of absorbed-retained ethylene oxide (EtO). Culture dishes were gas-sterilized with Anprolene® equipment and allowed to aerate (21 C) for varying durations. Post-sterilization EtO residues, as determined by weight measurement, were eluted from polystyrene dishes at an exponential rate. After 1 wk of aeration, .625 mg EtO was retained/g of polystyrene product. To determine the effect of EtO residue on in vitro culture, embryos were collected from superovulated donor mice (C57BL/6N), pooled in a modified Dulbecco's phosphate buffered saline (PB1) medium and then randomly allotted to dishes subjected to various aeration durations. Embryos were cultured in Whitten's medium +3 mg/ml bovine serum albumin (BSA) under a humidified 5% CO₂ in air atmosphere at 37 C. Gross assessments of embryo development were made using standard morphology and quality grading systems and a fluorescein diacetate staining assay. In Exp. 1, embryo development was retarded at the 8- to 16-cell stage when <12 h aeration of polystyrene dishes was permitted. Aeration for 24 and 36 h resulted in suboptimal embryo development with fewer (P<.01) blastocysts and greater degeneration rates at 48 h of in vitro culture compared with the control (manufacturer-packaged dishes, no EtO) and 1-wk aeration treatment groups. In Exp. 2, no difference (P>.05) was observed among treatment groups in embryo development or quality through 24 h of culture in glassware. However, at 48 h a greater (P<.01) percentage of blastocysts, fewer (P<.05) degenerate embryos and improved (P<.01) embryo quality were detected in the manufacturer-packaged plastic dishes (control) compared with the other treated glassware groups. These results demonstrate that sterilization of polystyrene culture dishes with EtO gas requires careful attention to aeration duration to avoid potentially toxic effects on embryo development. When EtO is used to gas-sterilize plastic culture-ware an aeration interval greater than 36 h is required to sustain optimal embryo development in vitro.

¹ This project was supported, in part, by Friends of the National Zoo (FONZ). Special acknowledgement is extended to Dr. S. R. Andersen and Thomas Benjamin of H. W. Andersen Products for their sincere interest and donation of Anprolene® materials.

² Names of commercial manufacturers and trade names are provided for identification only and inclusion does not imply endorsement by the National Institutes of Health, U.S. Public Health Serivce, U.S. Department of Health and Human Services or the National Zoological Park.

³ Dept. of Animal Health, National Zoological Park.

⁴ From whom reprints should be requested.

⁵ Veterinary Resources Brarich, National Institutes of Health.