Research Article Pharmacokinetics, Dose-Range, and Mutagenicity Studies of Methylphenidate Hydrochloride in B6C3F1 Mice Mugimane G. Manjanatha, 1 * Sharon D. Shelton, 1 Vasily N. Dobrovolsky, 1 Joseph G. Shaddock, 1 Lynda G. McGarrity, 1 Daniel R. Doerge, 2 Nathan W.Twaddle, 2 Chien-Ju Lin, 3 James J. Chen, 3 Donald R. Mattison, 4 and Suzanne M. Morris 1 1 Division of Genetic and Reproductive Toxicology, National Center for Toxicological Research (NCTR), Food and Drug Administration (FDA), Jefferson, Arkansas 2 Division of Biochemical Toxicology, NCTR, FDA, Jefferson, Arkansas 3 Division of Personalized Medicine and Nutrition, NCTR, FDA, Jefferson, Arkansas 4 NIH, NICHD, For the Best Pharmaceuticals for Children’s Act, Center for Research for Mothers and Children, Bethesda, Maryland Methylphenidate hydrochloride (MPH) is one of the most frequently prescribed pediatric drugs for the treatment of attention deficit hyperactivity dis- order. In a recent study, increased hepatic adeno- mas were observed in B6C3F1 mice treated with MPH in their diet. To evaluate the reactive metab- olite, ritalinic acid (RA) of MPH and its mode of action in mice, we conducted extensive investiga- tions on the pharmacokinetics (PK) and genotoxic- ity of the drug in B6C3F1 mice. For the PK study, male B6C3F1 mice were gavaged once with 3 mg/kg body weight (BW) of MPH and groups of mice were sacrificed at various time points (0.25–24 hr) for serum analysis of MPH and RA concentrations. Groups of male B6C3F1 mice were fed diets containing 0, 250, 500, 1,000, 2,000, or 4,000 ppm of MPH for 28 days to determine the appropriate doses for 24-week transgenic mutation studies. Also, the micronu- cleus frequencies (MN-RETs and MN-NCEs), and the lymphocyte Hprt mutants were determined in peripheral blood and splenic lymphocytes, respec- tively. Mice fed 4,000 ppm of MPH lost signifi- cant BW compared to control mice (P < 0.01). There was a significant increase in the average liver weights whereas kidneys, seminal vesicle, testes, thymus, and urinary bladder weights of mice fed higher doses of MPH were significantly lower than the control group (P  0.05). There was no significant increase in either the Hprt mu- tant frequency or the micronucleus frequency in the treated animals. These results indicated that although MPH induced liver hypertrophy in mice, no genotoxicity was observed. Environ. Mol. Mutagen. 49:585–593, 2008. Published 2008 Wiley-Liss, Inc. y Key words: methylphenidate hydrochloride; ritalinic acid; Hprt mutation; micronucleus assay The views presented in this article do not necessarily reflect those of the FDA or NICHD. The experiments described here were part of the scien- tific response developed by a working group of HHS staff that was con- vened as part of the NICHD BPCA 2002 responsibility to evaluate the findings of El-Zein et al. [2005] on the genotoxicity of methylphenidate in pediatric patients. The working group members represented NICHD (Donald R. Mattison), NIEHS (Mike Shelby and Kristine Witt), FDA/ CDER (David Jacobson-Kram), NIMH (Ben Vitiello), and FDA/NCTR (Bill Slikker and Suzanne Morris). Grant sponsors: Interagency Agreement between the Food and Drug Administration/National Center for Toxicological Research and the National Institute for Child Health and Human Development. *Correspondence to: Mugimane G. Manjanatha, Division of Genetic and Reproductive Toxicology, National Center for Toxicological Research (NCTR), Food and Drug Administration (FDA), 3900 NCTR Rd., Jefferson, AR, USA. E-mail: mugimane.manjanatha@fda.hhs.gov Received 4 December 2007; provisionally accepted 9 April 2008; and in final form 11 April 2008 DOI 10.1002/em.20407 Published online 10 July 2008 in Wiley InterScience (www.interscience. wiley.com). Published 2008 Wiley-Liss, Inc. y This article is a US Government work and, as such, is in the public domain in the United States of America. Environmental and Molecular Mutagenesis 49:585^593 (2008)