In summary, the scientific basis of the DNT test method has been subjected to an extensive history of international validation, peer review, and evaluation that is contained in the public record. Through the various collaborative efforts and workshops that have been conducted, a number of important con- clusions have been drawn. The individual test methods used in the DNT study have been found to be highly relevant for characterizing health risks of neurotoxic chemicals and to be based on solid scientific principles and expe- rience. Using exposures to known human developmental neurotoxicants, the DNT study has been shown to adequately identify the potential for adverse effects of chemical exposure on neurologic development. The intra- and interlaboratory reproducibility, reliability, and sensitivity of the DNT test method has been established, using a variety of test substances. Use of the DNT Study in Risk Assessment There is a regulatory need for DNT testing to support risk assessments in OECD member countries. Many pesticides and other chemi- cals are known to affect the adult nervous system, and there are concerns regarding the potential for DNT after early-life exposures to these substances [National Research Council (NRC) 2000]. This is particularly important because unique behaviors and activities of chil- dren place them at greater risk for increased exposure to xenobiotics by multiple routes (Brent et al. 2004; Weiss et al. 2004). The call for a more rigorous assessment of the potential for DNT has been issued by scientists from multiple and diverse sectors with an interest in public health protection. An examination of the historical and potential uses of the DNT study in risk assessment is critical to an overall evalua- tion of its value in protecting human health. Currently, the largest collection of DNT guideline studies resides with the U.S. EPA OPP, which has obtained information on DNT for specific pesticides to satisfy regu- latory mandates (Food Quality Protection Act of 1996). The U.S. EPA has furthermore engaged in an ongoing scientific analysis and discourse regarding the conduct of DNT studies, the interpretation of the data from these studies, and their regulatory impact. A review of 12 DNT studies (Makris et al. 1998) evaluated by the U.S. EPA Office of Prevention, Pesticides, and Toxic Substances (OPPTS) in support of the registration and/ or use of nine pesticides and three solvents was presented to a Scientific Advisory Panel in 1998 (U.S. EPA 1999a). For the nine pes- ticides examined, the analyses demonstrated that the DNT study includes sensitive end points that are not examined in any other test guideline, including prenatal developmen- tal, reproduction, and neurotoxicity studies (OECD 1983, 1997, 2001), thereby enhanc- ing detection of neurobehavioral and neuro- pathologic effects in offspring after exposure during sensitive periods of neurologic develop- ment. As a result, the DNT study, when pres- ent in a chemical database, is often identified as a sensitive study and an important source of quantitative and qualitative information for risk assessment. DNT data have been shown to be valuable in the selection of end points and doses for risk assessment (Makris et al. 1998; Rowland et al. 2007). As might be expected of a study that uses short-term exposures (~ 25–40 days) dur- ing development, where a single exposure dur- ing a critical period may result in developmental insult (Rice and Barone 2000; Rodier 1980, 1986, 1994), the predominant use of the DNT study in pesticide risk assessment has been for acute (single dose) reference doses (RfDs) and for short-term (1–30 days) and intermediate- term (1–6 months) nonoccupational exposures, which are especially applicable to risk assess- ments for children. Results from DNT studies have also been used in calculating a chronic RfD for lifetime exposure to a toxicant when it has been shown to be the most sensitive study in the toxicology database. A survey of the use and value of neurobe- havioral assessment in safety evaluation was also conducted by Middaugh et al. (2003). This survey included the results of multi- national studies conducted since 1990 on 174 compounds, including pharmaceutical (81%), agricultural (7%), industrial (1%), or undefined (10%) substances. Notably, the neurobehavioral screening conducted for pharmaceuticals is generally composed of developmental and behavioral assessments of second-generation (F1) offspring but does not address all of the end points assessed in a guideline DNT study. Nevertheless, this review found that F1 behavioral parameters along with other parameters defined the chronic no observed effect level (NOEL) in 17 of 113 (15%) and solely defined the NOEL in 3 of 113 (2.6%) of the studies examined. The conclusion was that F1 behav- ioral parameters sometimes improved on the standard toxicologic measures of hazard iden- tification, providing information on outcomes not addressed by other standard measures of toxicity. By early 2006, a review of regulatory and published sources revealed that approximately 114 DNT studies had been completed using either the U.S. EPA guideline or the draft OECD guideline (Table 4). The list of agents in Table 4 demonstrates the extensive history and experience regarding the conduct and interpretation of DNT studies. The outcomes of these efforts do not comprise a focused Environmental Health Perspectives • volume 117 | number 1 | January 2009 DNT: retrospective performance assessment attempt to validate the study protocol or spe- cific end points. In fact, a few of these studies did not include all the end points recom- mended by U.S. EPA or OECD guidelines. As of August 2006, approximately 75 DNT studies had been submitted to the U.S. EPA OPP in support of pesticide regis- tration. A preliminary survey of the use of DNT studies in risk assessment in OPP was conducted in March 2007 (Rowland et al. 2007). For 58 of the 75 pesticide chemicals, a DNT study had been considered in the weight-of-evidence review of the toxicology database. The DNT study was used to select end points and doses for risk assessments for eight of those chemicals. Importantly, for four of the eight DNT studies, the critical effects either included or were solely based upon off- spring behavioral and neuropathologic param- eters that are not evaluated in other guideline studies (i.e., motor activity, auditory startle habituation, learning and memory, and mor- phometric analysis). This is consistent with the conclusions of the earlier retrospective analysis (Makris et al. 1998) and provides further evidence of the sensitivity of the DNT study in identifying adverse effects in the young and the important role of DNT studies in human health risk assessments. In addition to using DNT data for regu- latory decisions, some regulatory agencies have also, on a case-by-case basis, incorpo- rated an additional database uncertainty fac- tor into their regulatory decisions because of the absence of DNT data. This approach reflects regulator views that DNT data are valuable in refining permissible exposure lev- els and that the absence of these data can increase the uncertainty about the toxicity of the chemicals (U.S. EPA 2002a, 2002b). Recent reviews have examined specific end points across multiple guideline DNT studies, to demonstrate the value of current methods in hazard characterization and explore fur- ther opportunities for methodologic refine- ment. U.S. EPA scientists have conducted cross-laboratory comparisons of methodolo- gies and results from DNT studies submitted to OPP (Crofton et al. 1991, 2004; Makris et al. 2005, 2006; Raffaele et al. 2003, 2004, 2005, 2006; Sette et al. 2004). Kaufmann and Groters (2006) reviewed the neuropathologic assessments in DNT studies, summarized practical aspects in planning neuropatho- logic assessments, and highlighted the value of morphology data in reference to both the concurrent behavioral assessments and use in assessing risk. A multisector ILSI RSI work- shop examined the evaluation and interpreta- tion of neurodevelopmental end points for human health risk assessment and addressed public health considerations, data interpreta- tion, data variability, positive control data, and statistical analysis (Crofton et al. 2008; 21