was distributed for comment in 1998, and significant technical issues identified by this review (e.g., the optimal duration of treat- ment, direct dosing of preweaning rodents, and conduct of morphometric evaluations) were further discussed at an expert consul- tation meeting in 2000 (OECD 2003). A revised draft was subsequently circulated for review, and comments from OECD member countries were addressed at a 2005 expert consultation meeting (OECD 2005b). The final version of TG 426 was adopted by the OECD Council in 2007 (OECD 2007). In the context of toxicologic screening and testing to support human health risk assessment and chemical regulatory activities, the DNT study fills an information require- ment that is not satisfied by other OECD test guidelines. Notably, it is the only test guide- line that includes functional, behavioral, and anatomical evaluations of the nervous system at multiple time points, in test subjects that were exposed to test substance during critical pre- and early postnatal periods of nervous system development. This test method has been used extensively in the past two decades on a wide variety of chemicals (Table 3). Scientific Basis of DNT Guideline The test methods recommended in the DNT guideline have been extensively reviewed and evaluated over the last 25 years. This has included the conduct of a number of meetings and collaborative studies involving experts from academic, industry, regulatory, and pub- lic interest groups. Pivotal influences and key events in the history of the development of the DNT guideline (Table 2) include both research on test methods development and efforts to characterize and document the sen- sitivity, reliability, and performance of the test methods, including a number of intra- laboratory collaborative efforts. In the 1970s, a series of studies were conducted in which rats were developmentally exposed to a vari- ety of xenobiotics and subsequently tested during postnatal development using a bat- tery of neurobehavioral tests (Butcher and Vorhees 1979; Vorhees et al. 1979). Other laboratories used behavioral and histologic batteries, focusing on sensory and motor function, in adult rodents exposed to a wide variety of neurotoxicants (Pryor et al. 1983; Tilson et al. 1979). A large body of research has provided an immense database on the ability of the functional observational bat- tery to detect and characterize the effects of drugs and environmental chemicals in adult and developing animal models (Gad 1982; Irwin 1968; Moser et al. 1988). This early work was followed by wide-ranging efforts to characterize the specificity of these test methods and the impact of both organismal and experimental factors (e.g., noise, spe- cies, strain, gender, test history) (Gerber and O’Shaughnessy 1986; Levine and Butcher 1990; MacPhail et al. 1989; Spencer et al. 1993). Ultimately, the result of more than 30 years of work in this area is a consensus opinion of neurotoxicologists that proper use and interpretation of the data derived from these test methods provide unique insight into the impact of xenobiotics on the developing and adult nervous system [Cory-Slechta et al. 2001; International Programme on Chemical Safety (IPCS) 2001; Tyl et al. 2008]. The development of test methods in neurotoxicology also includes a long history of efforts to characterize the interlaboratory reliability and sensitivity of the test meth- ods now included in the DNT study design. An article comparing a learning and reten- tion method among three laboratories (Butcher et al. 1979) was followed by the CBTS (Buelke-Sam et al. 1985; Kimmel and Buelke-Sam 1985) and the “Williamsburg Workshop” on qualitative and quantitative comparability of human and animal DNT (Kimmel et al. 1990). These efforts addressed various aspects of DNT study design and conduct, providing a sound scientific basis for the test method and its use in hazard evaluation. Since the publication of the U.S. EPA DNT guideline (U.S. EPA 1991), a continued scientific effort has reviewed and updated methodologies, for neurotoxicol- ogy in general and for developmental neuro- toxicology in particular. Examples of such reviews include the IPCS collaborative study on neurobehavioral screening methodolo- gies (MacPhail et al. 1997), an International Life Sciences Institute (ILSI) Risk Science Institute (RSI) workshop on Developmental Neurotoxicity and Risk Assessment (Mileson and Ferenc 2001), a collaborative study on neurobehavioral screening in 11 Japanese laboratories (Okazaki et al. 2003), and a Behavioral Test Methods Workshop (Slikker et al. 2005). Descriptions of each of these efforts and their contributions to the scien- tific basis for DNT testing follow. The CBTS. Several of the test pro- cedures developed in early behavioral teratol- ogy studies underwent validation in a large interlaboratory effort, the CBTS. This proj- ect characterized the performance of a stan- dardized neurodevelopmental test battery in six different laboratories after in utero and lactational exposure to two known neuro- toxicants, methylmercury and amphetamine. The study examined the intra- and inter- laboratory reliability and sensitivity of several behavioral test methods and the effects of a number of other litter- and gender-related variables. The peer-reviewed publications that resulted from the CBTS included descriptions of the background and overview (Kimmel Environmental Health Perspectives • volume 117 | number 1 | January 2009 DNT: retrospective performance assessment and Buelke-Sam 1985), protocol and test procedures (Adams et al. 1985b), data entry and test systems (Adams et al. 1985c), pre- liminary research (Adams et al. 1985a), sta- tistical approach (Nelson et al. 1985), results (Buelke-Sam et al. 1985), and implications, current applications, and future directions (Kimmel et al. 1985). Additionally, the results of a workshop held to review the CBTS data were published (Butcher and Nelson 1985; Geyer and Reiter 1985; Kutscher and Nelson 1985; Sobotka and Vorhees 1985; Tilson and Wright 1985). In a corollary study, many end points in the CBTS study were compared within one laboratory to an additional set of behavioral end points, named the Cincinnati Test protocol, using both methylmercury and d -amphetamine (Vorhees et al. 1985a, 1985b). The conclusion was that the effects of methylmercury were detected by both the CBTS end points and some of the additional end points in the Cincinnati Test protocol (Vorhees 1985c). The CBTS showed that rep- licability of data among laboratories using a standardized protocol was excellent and that both positive effects (e.g., with methyl- mercury exposure) and the lack of effects (e.g., after low-level amphetamine exposure) were replicable. The CBTS also demonstrated that the DNT test procedures were sufficiently sensitive; no more than a 5–20% change from control values was required to detect an effect. T h e E u r o p e a n I n t e r l a b o r a t o r y Collaborative Study. In the 1980s, the European Interlaboratory Study Group on Behavioural Teratology conducted a study of behavioral test methods (Alder et al. 1986; Elsner 1986; Elsner et al. 1986; Schreiner et al. 1986; Suter and Schon 1986). Three labora- tories, one each from industry, academia, and government, tested animals perinatally exposed to methylmercury. The results indicated that behavioral tests were more sensitive than repro- ductive end points and that automated proce- dures and measures aimed at specific functional capacities were more sensitive than nonspecific behavioral measures (Elsner et al. 1986, 1988). The Williamsburg Workshop. In 1989, the U.S. EPA held a workshop on the Qualitative and Quantitative Comparability of Human and Animal Developmental Neurotoxicity Table 3. Number of chemicals studied using the U.S. EPA DNT guideline or draft OECD DNT guideline. Chemical class Industrial chemicals Miscellaneous agentsa Pharmaceuticals Pesticides Positive control chemicals Solvents No. of studies 8 4 3 73 15 7 aFood additives, cigarette smoke, dietary restriction, and maternal separation. 19