Micro‐computed tomography and alizarin red evaluations of boric acid–induced fetal skeletal changes in Sprague‐Dawley rats

Abstract

Assessment of developmental toxicity has historically included assessment of fetal skeletal morphology after alizarin red staining. X-ray micro-computed tomography (micro-CT) produces high-resolution images of skeletal structures and was investigated as an alternative method. Groups of 5 mated Crl:CD (SD) female rats each were administered vehicle or boric acid (40 to 500 mg/kg/day) from GD 6 through 11. On GD 21, all live fetuses were weighed, euthanized, and viscera removed. Each litter was placed into a custom-made polystyrene holder and scanned in the micro-CT imaging system. Raw projection data were acquired in approximately 15 sec ( approximately 20 litters per hour) and reconstructed images at 100-micron cubic voxel dimension could be viewed as early as 20 min later. Fetuses were subsequently stained with alizarin red, and findings recorded separately for each method without knowledge of treatment group. Micro-CT evaluation of fetal rat skeletons detected essentially the same skeletal malformations, variations, and incomplete ossifications as seen by the staining method. The specific skeletal abnormalities that did not match exactly involved the smallest skeletal elements with minimal degrees of ossification (i.e., cervical ribs, hypoplastic 13(th) ribs, supernumerary ribs, the 5(th) sternebra, and numbers of caudal vertebrae), but the differences did not impact the overall conclusions. Additional measures such as femur length were easily measured by micro-CT. These results indicate that micro-CT imaging can effectively assess rat fetal skeletal structures, and for those laboratories with this resource, it may be used to significantly reduce time prior to skeletal evaluation and hazardous wastes associated with staining.