A Practical Approach, Second Edition=Ronald D. Ho.pdf

DEVELOPMENTAL TOXICITY TESTING — METHODOLOGY 243g. Skeletal ExaminationCurrently, the remaining 50% of the litter for rodents (intact, not decapitated) and 100% of thelitter for rabbits are processed and examined for skeletal alterations. Fetal carcasses are evisceratedand prepared for staining with alizarin red S (for ossified bone) 83 or “double stained” with alizarinred S and alcian blue (for ossified and cartilaginous bone plus other permanent cartilaginousstructures, such as the tip of the nose, external pinnae, etc.). 84,85 New guidelines require evaluationof both cartilaginous and ossified skeletal components but do not specify how (double staining isthe preferred method and strongly recommended).1) Single Staining for Ossified Bone — Two and one-half days is the minimum time requiredto hand stain rodent fetuses for skeletal evaluation. However, commercially available automaticstainers (e.g., the Sakura Teratology Processor from Sakura Finetek, USA, Inc., Torrance, CA)allow very rapid single or double staining of fetuses, with appropriate documentation for GLPcompliance.Once fetuses are eviscerated, they are immersed in 70% ethanol at least overnight in preparationfor staining. 86,87 The ethanol is drained off the next morning and replaced with 1% potassiumhydroxide (KOH) solution containing alizarin red S (25 mg/liter of solution) for 24 h. After 24 h,this is poured off and replaced with a fresh 1% KOH solution (without alizarin). 88 Six hours later,the 1% KOH is replaced with 2:2:1 solution (2 parts 70% ethanol to 2 parts glycerin to 1 partbenzyl alcohol). The next morning the 2:2:1 solution is decanted and replaced with a 1:1 solution(1 part 70% ethanol to 1 part glycerin). The skeletons can remain in this solution indefinitely forevaluation and storage.Seven to eight days is the minimum time required to process a rabbit fetus for skeletalexamination. Once the fetus has been eviscerated, it is skinned, placed in a partitioned plastic box,and either air dried overnight or placed in 70% ethanol. The following morning, the ethanol isdrained off and replaced with KOH solution containing alizarin red S as described above. Fortyeighthours later, this staining solution is decanted and replaced with a fresh 1% KOH solution.After up to 4 d, depending on the size of the fetus, the 1% KOH is replaced with the abovementioned2:2:1 solution. The next morning the 2:2:1 solution is decanted and replaced with 1:170% ethanol to glycerin for evaluation and storage.2) Double Staining for Ossified Bone and Cartilaginous Tissue — Rodents — Fetusesshould be skinned (or the skin should be separated from the underlying tissue at least) afterevisceration to allow the alcian blue to penetrate. The fetus is immersed in a 70°C water bath forapproximately 7 s (the fetus will appear to “unfold”). The epidermis is then peeled off the body,paws, and tail. This is best done by a gentle rubbing between the thumb and fingers, as the skeletonat this point is extremely fragile. The skeleton is placed in 95% ethanol overnight. The followingmorning, the ethanol is drained and replaced with alcian blue stain (15 mg alcian blue to 80 ml95% ethanol to 20 ml glacial acetic acid). This is decanted after 24 h and replaced with 95% ethanolfor 24 h. The following morning, the 95% ethanol is decanted, and the skeletons are placed inalizarin red S staining solution (25 mg/l of 1% KOH) for 24 to 48 h. The stain is drained andreplaced with 0.5% KOH for 24 h; this step may not be necessary for small specimens. After the0.5% KOH solution is decanted, it is replaced with 2:2:1 solution (70% ethanol to glycerin tobenzyl alcohol). The following morning the fetal skeletons are placed in 1:1 70% ethanol to glycerinfor evaluation and storage. 89,90Rabbits — The process is the same as for staining with alizarin red S, with the following alterations.After skinning, the fetal skeletons are placed in 95% ethanol. The following morning the ethanolis decanted and replaced with the alcian blue stain (see rodent staining for formulation). Thespecimens remain in the alcian blue for approximately 24 h and are then placed in 95% ethanol© 2006 by Taylor & Francis Group, LLC