Makris et al. Fenner-Crisp et al. 2005; Holson et al. 2008; Raffaele et al. 2008; Tyl et al. 2008). These various review efforts and result- ing publications provide transparent decision criteria for the analysis and interpretation of DNT test results, in accordance with the prin- ciples described in GD34 (OECD 2005a). Additionally, they demonstrate test method reliability, reproducibility, and relevance, which is attributable in part to the high level of standardization of the test methods that are recommended in the test guideline. Future Activities Although the overall performance of the DNT test method and its ability to detect effects of concern from a regulatory perspec- tive have been well established, the recent increase in the number of regulatory DNT studies being conducted has refocused atten- tion on this test method. Although some argue that specific tests are insensitive (e.g., assessment of cognitive and sensory dysfunc- tion), others suggest that the tests are overly sensitive and have a high rate of false positives (U.S. EPA 1995; Claudio et al. 1999, 2000; Cory-Slechta et al. 2001; U.S. EPA 2006). Diverse groups have advocated increased testing for DNT (Andersen et al. 2000; Grandjean and Landrigan 2006; NRC 1992, 1993; Nelson 1986; Office of Technology Assessment 1990; Stein et al. 2002; Vorhees 1986). There have also been calls to include evaluations of end points not currently assessed, such as social behavior (Cory-Slechta et al. 2001), pharmacokinetics and neuro- chemistry (Andersen et al. 2000; Dorman et al. 2001), and changes during senescence (Cory-Slechta et al. 2001). In addition, there have been criticisms of the complexity of the study, accompanied by calls for deleting some test components from the protocol (Li 2005) or using screening approaches that incorpo- rate DNT testing into other testing proto- cols (Cooper et al. 2006; Ladics et al. 2005). Critics also claim that variability of some end points (e.g., motor activity, morpho- metrics) is too great to be useful (Chemical Manufacturers Association 1987; Nolen 1985; York et al. 2004) and that this in vivo test is not necessary to detect DNT (Balls and Combes 2005). These diverse opinions do not invalidate the DNT study but rather highlight the need for ongoing scientifically based evaluation of this test method and the incorporation of appropriate revisions as sci- entific knowledge advances and as experience with the DNT study warrants. Table 4. Examples of chemicals and other stressors tested using the U.S. EPA DNT guideline or Dichlorvos (2) Dicrotophos Dietary restriction Dimethoate Disulfoton Emamectin Epidermal growth factor s-Ethyldipropylthiocarbamate Ethoprophos Ethylbenzene Etofenprox Fenamidone Fenamiphos Fentin hydroxide Fipronil Flubendiamide Flufenacet Glufosinate ammonium Glyphosate trimesium GN1180 (MN rgp120/HIV-1) Hydrogen sulfide Imidacloprid Iminodiproprionitrile Indoxacarb Isopropanol Isoxaflutole Lead nitrate Lindane Malathion Maternal separation Methamidaphos p-Methane-3,8-diol Methimazole (6) Methyl bromide Methyl parathion Methylazoxymethanol (2) Methylmercury n-Methylneodecanamide Molinate Naled Nelfinavir Nitrous oxide Octamethylcyclotetrasiloxane Perchlorate Phorate (2) Prochloraz Profenofos Propylthiouracil (2) Pymetrozine Pyrasulfotole Spirodiclofen Prothioconazole Styrene Tebuconazole Terbufos Tetrachlorvinphos Thiamethoxam Thiocloprid Tetrabromobisphenol A Thiram Triallate Tribufos Trichlorfon 1,1,1-Trichloroethane Trichloroethylene Triethylene glycol monomethyl ether Trimethyltin Ziram A number of efforts are currently under way to review data from existing DNT stud- ies, identify ways to refine the DNT test, and, if possible, reduce the number of animals used. It has been proposed that a reduction in animal use can be achieved by applying certain statistical approaches to the behav- ioral analysis (Chiarotti and Puopolo 2000; Puopolo 2004). Reviews of historical and positive control data have demonstrated the need for more standardized reporting requirements (Crofton et al. 2004, 2008). Further retrospective reviews of control data have identified differences among laborato- ries in data quality and variability, suggest- ing methods to decrease variability (Crofton et al. 1991, 2004; Raffaele et al. 2003, 2004; Sette et al. 2004). Conversely, a review of various neuropathology assessments (e.g., brain weight, standard histopathology, and morphometric assessments) identified low variability for these measures (Crofton et al. 2001), concluding that no one postmortem measure is more sensitive, with each provid- ing important data (Raffaele et al. 2005). The outcome of this continuing effort will allow better data interpretation, help refine requirements for future testing, and guide new methods development. In addition to the goal of refinement of the current approach to DNT testing, there is another and more pressing driver of change in the science arena of DNT. Currently, thousands of chemicals lack even simple, basic toxicologic data (e.g., high-production- volume chemicals, pesticide inert ingredi- ents, antimicrobial pesticides) but have a high potential for human exposure (NRC 1984). Assessing potential neurotoxicologic effects for these chemicals is a major challenge confront- ing the chemical industry, international and national regulatory agencies, and associated stakeholders (Dix et al. 2007; Kavlock et al. 2008; NRC 2007). New tools and methods are required to move toward a more sustain- able risk assessment paradigm for these types of chemicals. Although the current DNT guidelines generate useful data for risk assess- ment purposes, this in vivo test is costly and time-consuming and uses a relatively large number of naive animals when conducted as a stand-alone study (compared with incorpo- rating the DNT testing into other protocols, e.g., as proposed in Cooper et al. 2006 and U.S. EPA 2002b). A pressing goal of future research is to develop a validated true first-tier screening paradigm (e.g., a high-throughput in vitro screening battery) that can rapidly screen large numbers of chemicals for their potential to cause DNT (Coecke et al. 2007; Lein et al. 2007; NRC 2007; U.S. EPA 2006). Coupled with development of deci- sion frameworks (e.g., Combes et al. 2003), data from these high-throughput screens may ovlume 117 | number 1 | January 2009 • Environmental Health Perspectives OECD TG 426.a Abamectin Acephate Acetamiprid Acibenzolar-s-methyl Acrylamide AE-0172747 Aldicarb Alitame Amicarbazone Atorvastatin Azinphos methyl BAS 510F BAS 670H Bifenthrin Carbaryl Carbofuran Chlorfenapyr Chlorite, sodium Chlorpyrifos CI-943 Cigarette smoke Clodinafop propargyl Clothianidin Coumaphos Cyclohexanemethanol λ-Cyhalothrin β-Cyfluthrin Cymoxanil ϑ-Cypermethrin p,p-DDT Deltamethrin Diazepam Diazinon Decamethylcyclopentasiloxane DEET (N,N-diethyl-meta-toluamide) 22 aNumbers indicate the number of studies conducted for that chemical.