Dose-Response for Retinoic Acid-Induced Forelimb Malformations and Cleft Palate: A Comparison of Computerized Image Analysis and Visual Inspection Jerry L. Campbell Jr., 1 Mary Alice Smith, 1 * Jeffrey W. Fisher 2 and D. Alan Warren 2 1 Department of Environmental Health Science, University of Georgia, Athens, Georgia 2 Air Force Research Laboratory, Human Effectiveness Directorate, Wright-Patterson Air Force Base, Dayton, Ohio BACKGROUND: The objectives of this study were to (1) compare two techniques (computerized image analysis and visual morphological evaluation) for the assessment of fetal forelimb malformations and (2) increase the robustness of the dose-response curve for forelimb and cleft palate malformations resulting from all-trans retinoic acid (RA) exposure in GD 11 mice. METHODS: Pregnant CD-1 mice were administered a single oral dose of all-trans RA (0, 2.5, 10, 30, 60, or 100 mg/kg) on GD 11. GD 18 fetuses were examined for malformations using visual morphological scoring and computerized image analysis. RESULTS: Dose-dependent changes occurred in the size and shape of the humerus, radius, and ulna based on both assessment methodologies. The most sensitive indicators for the lowest effect level (10 mg/kg) on forelimbs were roundness, a shape measurement determined by image analysis, and visual morphological scoring. For all other bone measurements (proximal and distal width, area, length, and perimeter), the lowest effect level was 30 mg/kg. The maximum effect for limb defects and total malformed fetuses was seen at 60 mg/ kg and higher. Incidence of cleft palate increased over the entire range of administered doses reaching a maximum of 74% (100 mg/kg). CONCLUSIONS: Overall, results indicate that computerized image analysis was no more sensitive in detecting changes in the humerus, radius, and ulna than gross visual examination. Dose-response modeling of developmental endpoints yielded comparable benchmark dose levels for long bones and cleft palate that ranged from 0.24 to 7.6 mg/kg all-trans RA. Birth Defects Res B 71:289–295, 2004. r 2004 Wiley-Liss, Inc. Key words: retinoic acid; limb malformation; cleft palate; mouse; computerized image analysis; benchmark dose INTRODUCTION all-trans Retinoic acid (RA), an endogenous metabolite of vitamin A, is required for normal pattern formation during embryogenesis. However, abnormally high con- centrations in both experimental animals (Kochhar et al., 1996) and humans (Rosa, 1983; Lammer et al., 1985) result in fetal malformations. The teratogenic effects of retinoids on humans were demonstrated in the 1980s when Accutanes (13-cis retinoic acid, 13-cis RA), a metabolite of all-trans RA, was marketed for use as a treatment for acne. When pregnant women took Accu- tanes, their fetuses were at increased risk for adverse effects including malformations of the limbs, ears, heart, brain, and thymus, and reduction in IQ (Rosa, 1983; Lammer et al., 1985; Rizzo et al., 1991). While the use of 13-cis RA as a drug is now restricted in pregnancy, research related to the potential therapeutic use of retinoids continues in the treatment of acute promyelo- cytic leukemia (Grimwade and Lo Coco, 2002), the treatment of acne (Shalita, 2001), and AIDS-related Kaposi’s sarcoma (Cattelan et al., 2002). Excessive amounts of all-trans RA can affect almost any organ system, depending upon the stage of devel- opment in which exposure occurs (Kochhar, 1967; Shenefelt, 1972; Kamm et al., 1984). In mouse embryos, all-trans RA has been shown to produce severe limb defects and craniofacial malformations when treatment occurs between gestation days (GD) 11 and 14.5 (Kwasigroch and Kochhar, 1980; Kochhar et al., 1996; Hansen et al., 2001). A single 100 mg/kg dose of all-trans RA to GD 11 mice has been shown to result in 92% (Creech Kraft et al., 1989) and 100% (Soprano et al., 1994; Kochhar et al., 1996) malformed fetuses. At 10 mg/kg, all- trans RA exposure resulted in 23–39% of the fetuses being malformed (Soprano et al., 1994; Kochhar et al., 1996). No fetal limb malformations are seen when a dose of 1 mg/ kg all-trans RA or less is administered to pregnant mice Published online in Wiley InterScience (www.interscience.wiley.com) DOI: 10.1002/bdrb.20015 Grant sponsor: National Research Council; Grant sponsor: AFOSR Summer Research Program; Grant sponsor: Hatch Funds. Jef frey W. Fisher’s current address: Department of Environmental Health Science, University of Georgia, Athens, GA 30602-2102. D. Alan Warren’s current address: Academic Program Director, Environmental Health Science, University of South Carolina-Beaufort, Beaufort, SC 29902. *Correspondence to: M.A. Smith, Ph.D., Department of Environmental Health Science, University of Georgia, 206 Environmental Health Science Bldg, Athens, GA 30602-2102. E-mail: masmith@uga.edu Received 25 March 2004; Accepted 3 June 2004 Birth Defects Research (Part B) 71:289–295 (2004) & 2004 Wiley-Liss, Inc.