Regulatory developmental neurotoxicology testing: data evaluation for risk assessment purposes

Environmental Toxicology and Pharmacology 19 (2005) 727–733

Regulatory developmental neurotoxicology testing: data evaluation for risk assessment purposes Abby A. Li∗ Exponent, 1010 14th Street, San Francisco, CA 94114, USA

Abstract The recent emphasis on children’s health has led to new requirements in developmental neurotoxicity testing and evolving new approaches to children’s risk assessment. As more regulatory DNT studies are being submitted and used for children’s risk assessment, appropriate data interpretation of developmental neurotoxicity studies is an important step to improving the scientific basis for children’s risk assessments. Preweaning motor activity testing is used to illustrate the types of issues important to appropriate data analysis and evaluation. The EPA guidelines require that motor activity be tested at PND 13, 17, 21 and 60. Total activity at each time point and activity levels at each intrasession interval must be reported. Consequently, one common method for analyzing this data is to evaluate total activity levels at each time point or activity level in each intrasession time interval as independent measurements. Review of the scientific literature indicates that data evaluation for motor activity during development should focus primarily on the overall inverted U-shaped pattern of total activity over PND 13, 17 and 21 and secondarily on the development of intrasession response decrement. The interpretation of the data for risk assessment purposes needs to be guided by our understanding of the strengths and limitations of our knowledge of the biologic basis for these tests. © 2005 Elsevier B.V. All rights reserved. Keywords: Developmental neurotoxicity; Risk assessment; Behavior; Motor activity; Development; Data analysis

1. Introduction The increased emphasis on children’s health over the past 10 years has led regulatory authorities to explicitly evaluate the risk to children from exposure to environmental chemicals. Among the many endpoints that have received more attention is developmental neurotoxicity. Recently, there has been very healthy debate and discussion of the test methods, study design and appropriate triggers for developmental neurotoxicity testing (Claudio et al., 2000; Cory-Slechta et al., 2001; Garman et al., 2001; U.S. EPA, 1999). However, there has been less consolidated discussion of data analysis and interpretation of the EPA’s developmental neurotoxicity test. Appropriate analysis of the data generated from developmental neurotoxicity (DNT) testing is a key step in improving the scientific basis for children’s risk assessment. This paper reviews the recent regulatory developments in DNT testing ∗

Tel.: +1 415 558 8802; fax: +1 415 558 8898. E-mail address: [email protected].

1382-6689/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.etap.2004.12.030

and risk assessment, and discusses approaches for analyzing and interpreting data for regulatory risk assessment decision making. 2. Recent regulatory developments in DNT testing and risk assessment There have been several regulatory developments that have impacted DNT testing and risk assessment. In 1991, the Environmental Protection Agency’s Office of Pesticide Program (EPA OPP) published the developmental neurotoxicity test guideline. These DNT guidelines were re-issued in 1998 by Office of Prevention, Pesticides and Toxic Substances Test Guideline (OPPTS) 870.6300 with minor alterations. In 2000, EPA OPP issued a Data Call-In (DCI) notice for organophosphate (OP) pesticides that included additional requirements for developmental neurotoxicity testing. These new requirements include increasing the number of rats required for pathology evaluation from six to ten; extending exposure from postnatal day 11 to postnatal day 21;

728

A.A. Li / Environmental Toxicology and Pharmacology 19 (2005) 727–733

and demonstrating adequacy of exposure to pups during the postnatal period. In the OP DCI, EPA indicated that the following classes of pesticides will also receive this data callin notice in the near future: cholinesterase-inhibiting carbamates, thio- and dithiocarbamates (e.g. triallate), pyrethrin and synthetic pyrethroids, persistent organochlorines, formamidines, mectins, phoshides, organotins, dipyridyl compounds, organoarsenicals, and other pesticides with possible neurotoxic effects (U.S. EPA, 1999). Since many of the new requirements address generic concerns that are not unique to the specific mode of action for OP’s, it is expected that some of the additional requirements will be required for other pesticides. Therefore, it would behoove those planning to conduct studies according to OPPTS 870.6300 to carefully consider the additional requirements included in the OP DCI even if the test chemical is not a pesticide. The Food Quality Protection Act of 1996 (FQPA) passed by United States Congress into law has had significant regulatory impact on DNT testing and risk assessment for pesticides. FQPA requires EPA to address risks to infants and children and to publish a specific safety finding before a tolerance can be established for pesticides in food. FQPA provides for an additional 10-fold safety factor for infants and children, unless reliable data show that a different factor will be safe. EPA considers the overall weight of evidence for both the hazard and exposure assessment data and the potential for pre- and postnatal toxicity in determining whether there are any residual uncertainties (U.S. EPA, 2002). EPA has retained portions of the FQPA 10× factor in the overall risk assessment if there is concern for potential developmental neurotoxicity effects and there is no DNT test available. If a DNT study is available, EPA OPP will assess the degree of concern regarding the potential for pre- and postnatal effects. This involves examining the level of concern for sensitivity and susceptibility, and the degree of confidence in the point of departure selected for risk assessment. EPA will consider the need to retain portions of the FQPA factor if the overall database indicate that pups are more sensitive than adults qualitatively or quantitatively. Thus, an additional uncertainty factor may be considered if the most sensitive NOEL for the pup and adult are quantitatively the same, but the effect on the pup at the LOEL is judged to be more severe than in the adult. For example, persistent developmental effects with a steep dose response curve might be regarded as more adverse than an acute transient effects in adults. Therefore, the severity and specificity of functional, clinical and morphologic effects on the developing nervous system is one of the important factors considered by U.S. EPA OPP in making final risk assessment and risk management decisions for pesticides. FQPA has brought a significant new focus on evaluating children’s health risks to environmental chemicals other than just pesticides. EPA’s review of the reference dose and reference concentration processes published by the EPA’s Risk Assessment Forum specifically evaluates the FQPA factor used by EPA OPP. The EPA RfD/RfC technical panel concludes

that the FQPA factor is already accounted for in traditional UFs, which include an intraspecies and database uncertainty UF if those UFs are applied correctly. They recommend that similar to the FQPA factor, a database UF should be applied if data on specific life stages organ systems are unavailable or limited data suggest that more extensive data might decrease the point of departure used for risk assessment (U.S. EPA, 2002). The potential application of additional database UF provides incentive to conduct DNT testing proactively in order to avoid unacceptable risks assessments that could limit uses. In summary, the recent emphasis on children’s health and implementation of FQPA legislation has led to an increase in DNT testing, changes in DNT experimental design, and a new focus on application of UFs to address uncertainties in evaluating risks to children. Appropriate data analysis for developmental neurotoxicity is becoming increasingly important to improve the scientific basis for risk assessment and risk management decisions.

3. Developmental neurotoxicity test guideline requirements (OPPTS 870.6300) The EPA’s developmental neurotoxicity test guidelines (OPPTS 870.6300) require numerous endpoints to be evaluated at multiple time points (Table 1). There is clear guidance on what tests should be conducted at which time point, but less guidance on the hypothesis being tested. To some extent, this is appropriate because the guidelines should be flexible enough to allow investigators to test those hypotheses that may be the most relevant for the chemical in question. Guidelines that are too prescriptive can become rapidly outdated as the science advances. However, the hypothesis tested by the investigator should be grounded in some scientific biologic basis so that the data analysis that follows will yield results that are more useful for predicting adverse effects in humans. This point will be illustrated using motor activity as an example. In discussing the DNT endpoints, it is important to recognize that many of the tests in the developmental neurotoxicity test guidelines are apical tests that evaluate function during Table 1 EPA’s developmental neurotoxicity test guideline requirements Endpoint

Time period

Motor activity Auditory startle Learning and memory Pathology with morphometrics

PNDs 13, 17, 21, and 60 Around weaning and PND 60 Around weaning and PND 60 PNDs 11 or 21a , and PND 60

Detailed clinical observation Dams Pups a

2× During GD 6-21; 2× during LD 1-10 PNDs 4, 11, 21, 35,45 and 60

EPA guidelines require PND 11. However, with the new requirement to extend exposure to PND 21, EPA has accepted protocols that evaluate pathology at PND 21 instead of PND 11.

A.A. Li / Environmental Toxicology and Pharmacology 19 (2005) 727–733

development (Rice and Barone, 2000). Effects on apical tests, such as motor activity can result from specific as well as non-specific effects on the nervous system. Results of these tests can be difficult to interpret in terms of mode of action on the developing nervous system especially at the highest dose level which is required to cause maternal or pup toxicity. The DNT guidelines were developed more than 12 years ago prior to important recent advances in our knowledge of the molecular biology of development, and in particular, of the interaction of components of intracellular genetic regulatory circuits with components of intercellular signaling pathways. As the field of developmental biology advances, new approaches to testing using endpoints that are more specific to developmental processes of the nervous system (e.g. neurogenesis, migration, differentiation, synaptogenesis, gliogenesis, myelination, and apoptosis) should gradually replace those that are less specific. In the meantime, studies must be conducted according to EPA’s DNT guidelines, and regulatory decisions must be made based on these results in spite of the limitations of our biologic understanding of the endpoints currently required. It is not the intent of this paper to defend or criticize the EPA’s DNT guidelines. Instead, the purpose of this paper is to review what is known about the biologic basis of the motor activity test as an illustration of how this information should guide data analysis and interpretation.

4. Motor activity during preweaning period EPA’s DNT guideline requires that the motor activity of rat pups be measured on PND 13, 17, 21 and 60 but provides no guidance on the biologic rationale for why several time points were selected prior to weaning. The DNT guidelines refer to the motor activity requirements in the adult neurotoxicity screening battery (OPPTS 870–6200), which focuses on adult animal testing and provides no guidance for why three early time points are required prior to weaning. The DNT guidelines specify that daily total session activity counts and intrasession subtotals must be reported for each treatment and control group. Based on this limited guidance, a common method for statistical analysis of motor activity data from DNT studies is to perform ANOVA’s on total activity counts for each day of testing or subtotal activity counts for each individual intrasession time bins. This method of statistical analysis for motor activity addresses the following questions: (1) What is the effect of the test chemical on total activity at any one time point independent of each other, and (2) What is the effect of the test chemical on activity level for each individual intrasession time bin independent of other time bins on any one day? However, these questions may not be the most relevant or appropriate questions. In determining approaches to analyze the data, the scientific literature should be consulted to determine if there is biologic basis for these and other approaches in analyzing the data.

729

5. Theoretical biologic basis for motor activity testing during development Much of the original research on the ontogeny of motor activity and habituation was conducted in the 1970s and 1980s and involved daily testing of motor activity. Campbell and his associates established that a peak in locomotor activity occurs in neonatal rats and hamsters around PND 15–20. Campbell hypothesized that the initial increase in activity results from maturation of mesencephalic arousal systems, and that the decrease in activity is a result of development of telencephalic inhibitory centers in cerebral cortex, limbic system, and basal ganglia (Campbell and Mabry, 1972; Moorcroft et al., 1971). The increase in spontaneous activity has been associated with catecholaminergic system, whereas the decrease in activity has been associated with cholinergic and serotonergic systems (Mabry and Campbell, 1973; Campbell et al., 1969). A prediction arising from this hypothesis is that animals born with more mature brains would show a peak closer to birth or none at all. Evaluation of guinea pig, rabbit and rat appears to be consistent with this hypothesis based on the assumption that brain development in the rabbit is more advanced at birth than the rat, but less so than the guinea pig (Oakley and Plotkin, 1975). Shaywitz et al. (1979) also observed a peak period of spontaneous activity in Sprague–Dawley rats between 19 and 22 days of age. The time of peak activity level depends in part upon the extent to which exploratory behavior is evaluated. The extent to which different strains of rat and/or motor activity equipment might impact this pattern of development of motor activity has not been systematically studied. Many of the theories regarding the ontogeny of motor activity reflect the level of understanding of neurotransmitters and developmental neurobiology of the 1980s. The research in this area has not made significant progress beyond the very general theories described above. In spite of these limitations in our knowledge, the fact remains that this general pattern of development of total activity levels has been replicated in several laboratories using different species and methods of measuring motor activity. Based on the research in this area, the primary focus for analysis of motor activity data is the overall pattern of total activity, whereas the predictive relevance of the development of intrasession response decrement during development is not as well understood.

6. Evaluation of EPA DNT test data based on published literature Laboratories developing test capabilities for DNT testing are encouraged to compare data variability and method sensitivity with published reports of untreated and positive control data (Crofton et al., 2004). Ruppert et al. (1985a,b) and Buelke-Sam et al. (1985) systematically evaluated the use of motor activity in developmental testing. These studies provided much of the experimental basis for including motor activity testing in the EPA DNT guidelines. However, there

730

A.A. Li / Environmental Toxicology and Pharmacology 19 (2005) 727–733

are important differences in experimental design that must be considered when comparing data from these papers with results of EPA DNT guideline studies. Ruppert et al. (1985a) reported that Long-Evan rat pups tested once a day from PND 13 to 21 exhibit a developmental profile of activity level that is an inverted U-shaped function with a peak in activity between PND 14 and 16. In contrast, when three separate groups of pups were tested naively on PND 15 only, PND 18 only, or PND 21 only, the developmental profile of activity level was flat. Ruppert et al. (1985a) also reported that pups repeatedly tested every third day did not exhibit peak in activity at PND 15. Thus, the frequency and timing of repeated testing is an important component in the development of the inverted U-shaped curve for total activity levels. Based on this data, the patterns of activity reported by Ruppert et al. (1985a,b) following repeated daily testing should not be regarded as the “gold standard” for EPA DNT guideline studies that test activity every 3–4 days. Other investigators were able to obtain an inverted U-shaped curve with intermittent testing every 5 days starting at PND 10 in a 23-h stabilimeter test or a 5-min nose-poke test (Mabry and Campbell, 1973; Ba and Seri, 1995). However, these data also have limitations as standards for comparison because these test paradigms are not typical of activity measurements used for EPA DNT testing. These two studies evaluated activity of individual pups from an unreported number of litters. The litter and not the pup should be considered the experimental unit for developmental toxicity evaluations. A well-conducted collaborative behavioral teratology study provides published untreated and positive control data from several laboratories (Buelke-Sam et al., 1985). Motor activity in a Figure-8 maze was evaluated on PND 21 and 60 for 1 h and on PND 100 and 120 for 23 h (Adams et al., 1985; Buelke-Sam et al., 1985). When only the first hour of activity is evaluated over all the time periods, an inverted Ushaped curve is observed with activity levels at PND 60 and 100 higher than PND 21 and 120 (Buelke-Sam et al., 1985). However, the use of these data as a standard for comparison is limited by the fact that the inter-laboratory collaborative study did not test animals at PND 13 and 17. Thus, there are important differences in experimental design and conditions of testing that can lead to differences in behavior that need to be considered when comparing test guidelines data with the published literature. Although the general expectation might be to see an inverted U-shaped function between PND 13 and 21 with peak activity levels at PND 17, other developmental profiles (e.g. relatively flat, monotonically increasing) may be equally valid depending on the strain of animal, activity device, and other experimental conditions. Comparisons of variability of data and sensitivity to known positive controls also need to take into account differences in experimental design and conditions of testing. The mean coefficient of variation (averaged across sex, 2 studies) was 38 and 20% for PND 21 and 60, respectively, for the National Center for Toxicological Research (NCTR) laboratory

responsible for training all other participating laboratories in the collaborative research study (Buelke-Sam et al., 1985). These means were reported to be generally representative of the other five participating laboratories although actual values were not reported. All laboratories were trained by NCTR to follow standard operating procedures specifying the strain and source of rat, number and type of activity device, housing conditions, time of day of testing, season of testing, replicate design, etc (Adams et al., 1985). These CV’s are useful to compare against provided the specific conditions of the testing procedures are taken into account. They do not apply to PND 13 and 17. Historical control data from laboratories conducting EPA DNT guideline studies consistently demonstrate that motor activity is much more variable at PND 13 and 17 than at later ages (Barnett et al., 2002; Raffaele et al., 2003). Mean CV’s for 14 laboratories was approximately 50–140% at PND 13; 40–100% at PND 17; 20–60% at PND 21 and 18–30% at PND 60 (Raffaele et al., 2003; data based on inspection of graphs). It is possible that some of this variability could be reduced by improving environmental experimental conditions (sound, temperature, walking surface, light, type of activity monitoring device, time of day, length of session). However, since the variability at PND 21 and 60 is comparable to that observed under the ideal conditions of the collaborative study, the variability at PND 13 and 17 may also be due to normal variability of motor activity during a period of especially rapid development of motor function. In addition, animals with the same birth date can be different in age by 23-h which can make a difference to motor development especially in young pups. Finally, the EPA DNT test requires at least 80 litters of pups with representation from all litters required. Consequently, motor activity testing for one time point must occur in different sessions over several hours during the day as well as across multiple days of testing depending on when the pups were born. All these factors are important variables that have not been systematically studied in well-controlled studies. In summary, the reproducibility, variability, and detection sensitivity of motor activity testing according to EPA guidelines requirements has not been well-characterized. To a large extent, the laboratories currently conducting DNT studies will generate the data that will increase our understanding of how reproducible, variable and sensitive these endpoints are. Important differences in methodology need to be considered when comparing EPA DNT data with published literature of untreated and positive control data.

7. Approaches to data analysis of preweaning motor activity The approach used by Ruppert et al. (1983, 1985b) in evaluating the toxic effects of metals on the development of motor activity provides an example of one approach to analyze data. Acute doses of cadmium (4 mg/kg), triethyltin (4, 5, 6 mg/kg), and trimethyltin (6 mg/kg) injected into pups at

A.A. Li / Environmental Toxicology and Pharmacology 19 (2005) 727–733

731

PND 5 caused increases in total activity producing an obvious alteration in the normal inverted U-shaped curve. Total activity at each age was analyzed by a repeated measures ANOVA using sex and dose as between-animal factors; age and interactions of sex and dose with age were within-animal factors. In addition to total activity, both within-session habituation (the decrease in activity during each test session) and between-session habituation (the decrease in activity over successive days of testing) were examined. Activity counts for each intrasession intervals for each day of testing was analyzed by a repeated measures ANOVA using sex and dose as between-animal factors; age and intrasession interval and interactions with these variables were used as within-animal factors. Ruppert et al. (1985b) distinguished between changes in intrasession data that resulted in a lack of habituation (or response decrement) from a parallel shift in the habituation curve to the right. At all dosages of metals, which produced alterations in motor activity, preweaning growth was also reduced. Ruppert et al. (1985b) evaluated the possibility that undernutrition in the postnatal period caused by alteration in suckling could be the cause of the behavioral changes. Important elements of the approach to data analysis are that statistical analysis (e.g. repeated measures analysis) designed to evaluate the effect on motor activity in the same animals over the different test dates was used; the graphed data was evaluated to understand the overall pattern of effect on intrasession activity, rather than merely relying on statistical significance; and the impact of general toxicity effects on the functional changes was considered. If this inverted U-shaped pattern can be reliably demonstrated for the specific equipment and strain of rat, then this overall pattern can be utilized as a theoretical framework for interpreting data even though the biologic basis for this test might be limited. Using this framework, a significant increase in activity at PND 21 with no intrasession response decrement is easier to regard as an adverse effect if control animals consistently exhibit a decrease in activity at this time point. It is more difficult to interpret a small statistically significant decrease in activity at PND 21 (compared to controls) as a developmental effect if a normal peak in activity is observed at PND 17 and normal activity is noted at PND 60. Likewise, it is more difficult to determine if small statistically significant increase or decrease in activity at PND 13 is a meaningful adverse effect on development if the overall inverted U-shaped pattern in activity is observed and no changes are observed at other timepoints. Evaluation of historical control data along with evaluation of other functional and morphologic effects will become important in evaluating the biological significance of these types of effects.

of response decrement has also been evaluated. Shaywitz et al. (1979) hypothesized that the decrease in total activity was related to the animal’s ability to begin to “habituate”. It is not the intent of this paper to review the complex literature on habituation, but there are three important cautions in interpretation of the intrasession activity level that should be discussed. First, indiscriminate use of terminology can lead to inappropriate assumptions of mechanisms of behavior. File (1981) suggested that the terms “response decrement” and “response increment” should be used to describe observable progressive decreases and increases in response amplitude, respectively. The terms “habituation” and “sensitization” should be used to denote one of several possible hypothetical mechanisms underlying progressive response decrement or increment. The reason this caution is important is that effects on intrasession activity are sometimes assumed to be effects on habituation, and hence, on a simple form of learning and memory. These conclusions stretch the science beyond our current understanding of motor activity intrasession activity. Second, it is much more difficult to use motor activity to study “habituation” because of the lack of control over both the stimulus and the response. Those who attempt to study habituation to spatial novelty try to develop more sophisticated methods of evaluating exploration (File, 1981; Ross, 2001; Cerbone and Sadile, 1994). More progress has been made using other systems, such as auditory startle because of the improved ability to control the stimulus and response and because of a better understanding of the neuroanatomical and pharmacological pathways involved in auditory startle (Davis, 1980; Davis et al., 1982a,b; Pilz and Schnitzler, 1996). Some might argue that it is not important to understand the biologic basis of a behavioral test as long as the behavior is reliable and sensitive in detecting effects of chemicals. Indeed, the pattern of response decrement within a session has been reliably established in adult rats across different laboratories (Crofton et al., 1991). However, this is not the case for pups. This leads to the third point which is that the normal variation in intrasession motor activity patterns during development under conditions of periodic (not daily) testing on PND 13, 17, and 21 is not sufficiently understood. Based on these considerations, data evaluation for motor activity during development should focus primarily on the overall inverted U-shaped pattern of activity and secondarily on significant changes to the overall pattern of intrasession response. The practice of statistically analyzing changes in activity for each individual intrasession time intervals (time bins) independent of evaluating the overall pattern of behavior can yield uninterpretable statistically significant changes that should not be used for risk assessment.

8. Evaluation of intrasession activity levels

9. General risk assessment considerations for evaluating ontogeny of motor activity

Although the primary emphasis in evaluating the ontogeny of motor activity has been on the inverted U-shaped pattern for the total activity, the development of intrasession pattern

The ultimate purpose of conducting DNT tests is to evaluate risks to the developing human child. Although humans

732

A.A. Li / Environmental Toxicology and Pharmacology 19 (2005) 727–733

would not be expected to exhibit the ontogenic pattern of total activity seen in rats, the possible relevance of this developmental behavior to humans is that effects might indicate possible alterations in neurochemical or neuroanatomical processes important to human brain development. A concern is that changes in behavior could reflect a specific effect on delay in development that may lead to increased vulnerability later in life. The challenge in interpreting this data is that the specific biological process and pathways underlying this ontogenic pattern is not understood, making it difficult to develop testable hypothesis. In addition, tests such as motor activity are apical tests that reflect the integrated output of sensory, motor, and associative processes of the nervous system (Crofton, 1991). Changes in motor activity can also result from non-specific effects on the nervous system resulting from abnormal maternal behavior, undernutrition, growth retardation and other more general effects on development. Therefore, it becomes important for changes in motor activity to be evaluated in association with other effects on the DNT and other toxicity studies. EPA’s guidelines for neurotoxicity risk assessment (U.S. EPA, 1998) provides the following important guidance on evaluating motor activity and functional observational battery that is directly applicable to evaluation of behavioral endpoints in the DNT studies: “Motor activity measurements are typically used with other tests (e.g., FOB) to help detect neurotoxic effects. Agent-induced changes in motor activity associated with other overt signs of toxicity (e.g., loss of body weight, systemic toxicity) or occurring in non-dose-related fashion are of less concern than changes that are dose dependent, are related to structural or other functional changes in the nervous system, or occur in the absence of life-threatening toxicity.” “If unrelated measures in the FOB are affected, or the effects are unrelated to dose, the results may not be considered evidence of a neurotoxic effect. If several neurological signs are affected, but only at the high dose and in conjunction with other overt signs of toxicity, including systemic toxicity, large decreases in body weight, decreases in body temperature, or debilitation, there is less persuasive evidence of a direct neurotoxic effect. In cases where several related measures in a battery of tests are affected and the effects appear to be dose dependent, the data are considered to be evidence of a neurotoxic effect, especially in the absence of systemic toxicity. The risk assessor should be aware of the potential for a number of false positive statistical findings in these studies because of the large number of endpoints customarily included in the FOB.” The EPA requires that the highest dose level must cause toxicity to either the dams or pups. These high doses can cause effects on behavioral endpoints that result from non-

specific effects on the nervous system. These effects need to be protected against, but should not be incorrectly regarded as specific developmental neurotoxic effects. Since the risk assessment process will already protect children from high dose toxicity causing general pup or maternal toxicity, it would be advantageous to conduct DNT studies at doses just below that which causes frank toxicity to either the dam or the pup. This will improve our ability to evaluate the significance of behavioral effects as potential specific effects on the nervous system. EPA’s new requirement to extend exposure throughout the preweaning period to PND 21 introduces an additional confounder to interpretation of preweaning motor activity data. This new requirement was added because of concern that significant rat brain development occurs from PND 11 to 21. Dosing of pregnant rats and offspring only up to postnatal day 10 could miss exposure during critical periods in nervous system development. However, exposure to the chemical will occur during the period when ontogeny of spontaneous activity is evaluated (PND 13, 17, 21). It will be difficult, if not impossible, to determine if a change on PND 17 only, for example, is an acute reversible effect of the chemical on motor activity or a developmental delay. Both effects are important to protect children against, and the risk assessment process allows for this. However, the latter interpretation could be considered a much more severe effect that has a much higher level of concern and can result in more severe labeling or other risk management decisions. The ability to distinguish between chemicals that cause more severe developmental effects from those of lesser concern makes a difference to how regulatory priorities and resources are focused effectively. From this perspective, sensitive methods and experimental designs that more specifically and directly evaluate neuronal development are much more preferable and should eventually replace less specific approaches.

10. Conclusion In conclusion, the example of motor activity illustrates the importance of using the biological basis of the specific test to guide the approaches used for data analysis and interpretation. These considerations apply to other endpoints on the DNT study. Automated equipment for auditory startle and learning and memory tests can measure multiple parameters. There are numerous brain morphometric measurements and detailed clinical observations that can be made. There are many possible questions that can be asked in analyzing data and many data points that can be statistically analyzed. Knowledge of the scientific literature supporting these approaches should be used to guide interpretation of the data. Understanding the differences between the actual testing requirements and the conditions used in the scientific literature is necessary so that the limitations or uncertainties in interpreting effects are taken into consideration. Our current

A.A. Li / Environmental Toxicology and Pharmacology 19 (2005) 727–733

scientific understanding should be used to identify those approaches that are most relevant, reliable and predictable in identifying patterns of effects that are biologically meaningful to humans.

References Adams, J., Buelke-Sam, J., Kimmel, C.A., Nelson, C.J., Reiter, L.W., Sobotka, T., Tilson, H.A., Nelson, B.K., 1985. Collaborative behavioral teratology study: protocol design and testing procedures. Neurobehav. Toxicol. Teratol. 7, 579–586. Ba, A., Seri, B.V., 1995. Psychomotor functions in developing rats: ontogenetic approach to structure-function relationships. Neurosci. Biobehav. Rev. 19, 413–425. Barnett Jr., J.A., Giknis, M.L.A., Clifford, C.B., 2002. Postnatal growth, development and behavioral/functional evaluation in Crl:CD® (SD)IGS BR Rats. Charles River Laboratories. Buelke-Sam, J., Kimmel, C.A., Adams, J., Nelson, C.J., Vorhees, C.V., Wright, D.C., Omer, V.St., Korol, B.A., Butcher, R.E., Geyer, M.A., Holson, J.F., Kutscher, C.L., Wayner, M.J., 1985. Collaborative behavioral teratology study: results. Neurobehav. Toxicol. Teratol. 7, 591–624. Campbell, B.A., Lytle, L.D., Fibiger, H.C., 1969. Ontogeny of adrenergic arousal and cholinergic inhibitory mechanisms in the rat. Science 166, 635–637. Campbell, B.A., Mabry, P.D., 1972. Ontogeny of behavioral arousal: a comparative study. J. Comp. Phys. Psych. 67, 15–22. Cerbone, A., Sadile, A.G., 1994. Behavioral habituation to spatial novelty: interference and noninterference studies. Neurosci. Biobehav. Rev 18, 497–518. Claudio, L., Kwa, W.C., Russell, A.L., Wallinga, D., 2000. Testing methods for developmental neurotoxicity of environmental chemicals. Tox. Appl. Pharm. 164, 1–14. Cory-Slechta, D.A., Crofton, K.M., Foran, J.A., Ross, J.F., Sheets, L.P., Weiss, B., Mileson, B., 2001. Methods to identify and characterize developmental neurotoxicity for human health risk assessment. I: Behavioral effects. Crofton, K.M., Howard, J.L., Moser, V.C., Gill, M.W., Reiter, L.W., Tilson, H.A., McPhail, R.C., 1991. Interlaboratory comparison of motor activity experiments: implication for neurotoxicology assessments. Neurotox. Teratol. 13, 599–609. Crofton, K.M., Makris, S.L., Sette, W.F., Mendez, E., Raffaele, K.C., 2004. A qualitative retrospective analysis of positive control data in developmental neurotoxicity studies. Neurotoxicol. Teratol. 26, 342–352. Davis, M., 1980. Neurochemical modulation of sensory-motor reactivity: acoustic and tactile startle reflexes. Neurosci. Biobehav. Rev. 4, 241–263. Davis, M., Gendelman, D.S., Tischler, M.D., Gendelman, P.M., 1982a. A primary acoustic startle circuit: lesion and stimulation studies. J. Neurosci. 2, 791–805.

733

Davis, M., Parisi, T., Gendelman, D., Tischler, M., Kehne, J.H., 1982b. Habituation and sensititization of startle reflexes elicited electrically from the brainstem. Science 218, 688–689. File, S.E., 1981. Pharmacological manipulations of responses to novelty and their habituation. In: Cooper, S.J. (Ed.), Theory in Psychopharmacology. Academic Press. Garman, R.H., Fix, A.S., Jortner, B.S., Jensen, K.F., Hardisty, J.F., Claudio, L., Ferenc, S., 2001. Methods to identify and characterize developmental neurotoxicity for human health risk assessment. II: Neuropathology. Mabry, P.D., Campbell, B.A., 1973. Serotonergic inhibition of catecholamine-induced behavioral arousal. Brain Res. 49, 381–391. Moorcroft, W.H., Lytle, L.D., Campbell, B.A., 1971. Ontogeny of starvation-induced behavioral arousal in the rat. J. Comp. Phys. Psych. 75, 59–67. Oakley, D.A., Plotkin, H.C., 1975. Ontogeny of spontaneous locomotor activity in rabbit, rat, and guinea pig. J. Comp. Phys. Psych. 89, 267–273. Pilz, P.K.D., Schnitzler, H.-U., 1996. Habituation and sensitization of the acoustic startle response in rats: amplitude, threshold and latency measures. Neurobiol. Learn. Mem. 66, 67–79. Raffaele, K.C., Sette, W.F., Makris, S.L., Moser, V.S., Crofton, K.M., 2003. Poster Presented at the 2003 Meeting of Society of Toxicology. Motor activity in developmental neurotoxicity testing: a crosslaboratory comparison of control data. Rice, D., Barone, S., 2000. Critical periods of vulnerability for the developing nervous system: evidence from humans and animal models. Env. Health Persp. 108, 511–533. Ross, J.F., 2001. Tier 1 neurological assessment in regulated animal-safety studies. In: Massaro, E.J. (Ed.), Handbook of Neurotoxicology, vol. 2. Ruppert, P.H., Dean, K.F., Reiter, L.W., 1983. Comparative developmental toxicity of triethyltin using split-litter and whole-litter dosing. J. Tox. Env. Health 12, 73–87. Ruppert, P.H., Dean, K.F., Reiter, L.W., 1985a. Development of locomotor activity of rat pups in figure-eight mazes. Dev. Psychobiol. 18, 247–260. Ruppert, P.H., Dean, K.F., Reiter, L.S., 1985b. Development of locomotor activity of rat pups exposed to heavy metals. Tox. Appl. Pharm. 78, 69–77. Shaywitz, B.A., Gordon, J.W., Klopper, J.H., Zelterman, D.A., Irvine, J., 1979. Ontogenesis of spontaneous activity and habituation of activity in the rat pup. Dev. Psychobiol. 12, 359–367. U.S. EPA, 1998. Guidelines for neurotoxicity risk assessment. Fed Reg 63 (93): 26926–26954 (published May 14, 1998). U.S. EPA, 1999. Data call-in notice for organophosphate chemicals, September 10, 1999. U.S. EPA, 2002a. Determination of the appropriate FQPA safety factor(s) in tolerance assessment. http://www.epa.gov/oppfead1/trac/ science/#10-fold (published February 28, 2002). U.S. EPA, 2002b. A review of the reference dose and reference concentration processes. Prepared for the Risk Assessment Forum U.S. EPA. EPA/630/P-02/002F.