Neurotoxicologyand Teratology, Vol. 12, pp. 285-292. Pergamon Press plc, 1990. Printed in the U.S.A.
0892-0362/90 $3.00 + .00
Workshop on the Qualitative and Quantitative Comparability of Human and Animal Developmental Neurotoxicity: Summary and Implications E L A I N E Z. F R A N C I S , .2 C A R O L E A. K I M M E L * A N D D. C O O P E R R E E S t 3
*Reproductive and Developmental Toxicology Branch, Office of Health and Environmental Assessment Office of Research and Development and "~Health and Environmental Review Division, Office of Toxic Substances United States Environmental Protection Agency, Washington, DC 20460 FRANCIS, E..Z., C. A. KIMMEL AND D. C. REES. Workshop on the qualitative and quantitative comparability of human and animal developmental neurotoxicity: Summary and implications. NEUROTOXICOL TERATOL 12(3) 285-292, 1990.-- The Workshop on the Qualitative and Quantitative Comparability of Human and Animal Developmental Neurotoxicity was convened by the U.S. Environmental Protection Agency (EPA) and the National Institute on Drug Abuse to address issues related to when testing should be required, what test methodologies should be required, and how the data should be interpreted and applied to the risk assessment process. The background material for Work Group discussions included presentations made at the Workshop by invited experts summarizing qualitative and quantitative human and experimental animal data on specific chemicals or classes of chemicals and EPA's proposed developmental neurotoxicity testing protocol. This overview: 1) summarizes the qualitative comparisons presented at the Workshop and attempts to make some quantitative comparisons of findings across mammalian species following exposure to developmental neurotoxicants, 2) brings the common themes that were discussed among the Work Groups together into a regulatory perspective, 3) provides a status report on EPA's developmental neurotoxicity protocol, and 4) identifies research needs in the development of test methodologies and improvement of risk assessments for developmental neurotoxicants. Developmental neurotoxicity Lead Agents of abuse Risk assessment
Phenytoin Test guideliness
Ethanol PCBs Ionizing radiation Postnatal development
THE U.S. Environmental Protection Agency (EPA) has taken the lead among the U.S. Federal Agencies in developing a protocol to assess the potential developmental neurotoxicity of agents. The first protocol for regulatory purposes specific to developmental neurotoxicity testing was proposed in 1986 by the Office of Toxic Substances (OTS) for use with glycol ethers (16). Following the receipt of public comments on this proposal, an Agency-wide guidelines committee was formed to address public comment, develop a revised protocol, and provide a forum for discussing issues related to developmental neurotoxicology. In the four years since the formation of that committee, many discussions have taken place leading to development of a testing protocol for developmental neurotoxicity that would have general applicability for testing chemical substances and pesticides and that could be modified on a case-by-case basis. In the process of developing this protocol, deliberations took place both among scientists within the Agency and with scientists
Methylmercury
from academia and the chemical industry. As these discussions progressed, it became clear that there was a need for an open forum to discuss a number of issues related to when testing should be required, what test methodologies should be required, and how the data should be interpreted and applied to the risk assessment process. Thus, the Workshop on the Qualitative and Quantitative Comparability of Human and Animal Developmental Neurotoxicity was convened to address these issues using comparisons of human and experimental animal developmental neurotoxicity data as a basis. The three-day Workshop, described by Rees et al. (10,11), provided an opportunity for scientific exchange on many of these issues. The reader is referred to other papers in these proceedings on the agents that were discussed (1, 3-6, 12, 14) at the Workshop and to the reports of the individual Work Groups (2, 9, 13, 15) for a summary of agent-specific comparisons and Work Group deliberations. This overview is intended to: 1) briefly summarize the qualitative comparisons presented at the Workshop
~The views in this paper represent those of the authors and do not necessarily reflect the views or policies of the U.S. Environmental Protection Agency. 2Requests for reprints should be addressed to Elaine Z. Francis, Ph.D.. U.S. Environmental Protection Agency (RD-689), 401 M Street, S.W., Washington, DC 20460. 3present address: RJReynolds/Nabisco, Bowman Gray Technical Center, Winston-Salem, NC 27102.
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and attempt to make some quantitative comparisons of findings across mammalian species following exposure to developmental neurotoxicants, 2) bring the common themes that were discussed among the Work Groups together into a regulatory perspective, and 3) describe the status of EPA's developmental neurotoxicity protocol and how the Workshop discussions impacted on its current design. Specific points that have been made in the manuscripts on the agents of concern and the reports of the individual Work Groups will be discussed only as they are relevant to issues addressed in this summary. Therefore, readers are strongly encouraged to refer to the rest of these proceedings for detail of the issues that were raised and discussed at the Workshop. The background material for the Work Group discussions included the presentations made at the Workshop by invited experts summarizing human and experimental animal data on specific chemicals or classes of chemicals, and EPA's proposed developmental neurotoxicity testing protocol [see Rees et al. (11) for an outline of that protocol]. The Work Groups were organized around four main topics: 1) issues related to evaluation of comparative data to provide rationale or justification for requiring testing, 2) what test methods should be required, 3) how data from such studies should be interpreted and applied to risk assessment, and 4) criteria for recommending testing. The following sections provide an overview of the Workshop proceedings addressing these specific areas, especially as they relate to implications within a regulatory setting. A section on the status of EPA's revised developmental neurotoxicity protocol also is included. The final section identifies research needs in developing test methodologies and improving risk assessments for suspect developmental neurotoxicants.
QUALITATIVE AND QUANTITATIVE COMPARABILITY OF HUMAN AND ANIMAL DEVELOPMENTAL NEUROTOXICANTS
Experts were invited to review and present, on the first day of the Workshop, the human and experimental animal data on agents known to cause developmental neurotoxicity in humans. These agents included lead, methylmercury, selected agents of abuse, phenytoin, polychlorinated biphenyls (PCBs), ethanol, and ionizing radiation. For their presentations, the experts were asked to assess the degree of qualitative and quantitative comparability between human and experimental animal data, and identify critical assumptions and areas of uncertainty required when making these qualitative and quantitative comparisons.
Qualitative Comparability The qualitative comparability of human and experimental animal data was assessed (13) and the following behavioral categories of function were selected for evaluation: motor development and function, cognitive function, motivational/arousal behavior, sensory function, and social behavior. A number of limitations associated with assessing cross-species comparability were identified. These included: 1) while some data usually were available to evaluate categories of function, often data were unavailable to make specific end point comparisons, 2) doseresponse information was limited and, therefore, it was not possible to assess comparability of results at equivalent administered dose levels or dose ranges, 3) there were differences in experimental design, such as test methodology, exposure pattern and time to testing, 4) unique underlying neurologic mechanisms for each species which may influence species sensitivity and the types of effects manifested have not been well studied. Despite the recognized limitations in making these comparisons, the degree of qualitative comparability was considered remarkable (13).
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Rees et al. (10) provide a more detailed evaluation of the qualitative and quantitative comparability between human and rodent data presented at the Workshop. Although data were presented on experimental species other than rodents, rodents were selected for comparison to humans because they are most often the experimental species in which testing is conducted in response to a regulatory action. For the category motor development and function, in general, comparable changes were observed in both humans and rodents. Specifically, alterations in motor development and motor control were observed in rodents and humans following exposure during development to methylmercury, lead, ethanol, and PCBs. Alterations in these same parameters also were observed in rodents exposed to ionizing radiation and phenytoin; however, human data were not presented for these parameters with these two agents. Effects on cognitive function were observed in both humans and rodents following exposure during development to methylmercury, lead, ethanol, PCBs, ionizing radiation, and phenytoin. In the area of sensory function, most of the agents reviewed at the Workshop have not been studied systematically. Where data were available, effects on sensory function were generally similar between humans and rodents. In general, some measure of motivational~arousal behavior was affected in rodents and humans for all of the agents discussed at the Workshop except phenytoin for which there were no human data presented on this category. This was the only functional category for which data were sufficient to indicate comparability between humans and rodents for selected agents of abuse. Finally, for social function, limited data were presented. The only agent for which there were data in both species was ethanol; following developmental exposure to ethanol, alterations in suckling behavior were observed in both rodents and humans.
Quantitative Comparability Quantitative comparability between experimental animal and human data for developmental neurotoxicants has not been well characterized. From the presentations given by the experts at the Workshop on the selected developmental neurotoxicants, several conclusions related to quantitative comparisons may be drawn. First, dose-response data are often limited; this is especially true for humans. For some agents, such as phenytoin, the administered dose in humans is known, for the most part. However, for other agents, such as ethanol or other agents of abuse, the dose that is consumed or administered is often unknown or difficult to determine accurately. Second, most of the data that are available on dose levels are based upon administered dose, although data on some internal measure of dose were available for a few agents. Third, in many cases, the most sensitive end point has not been established nor has sufficient dose-response information been gathered to permit identification of a no-observed-adverse-effectlevel (NOAEL). Therefore, it is not possible to make definitive quantitative comparisons, at this time, based upon the relationship of end points to dose although there were indications that for some of the agents discussed, cognitive function appeared to be the most sensitive category. Fourth, for a few of the agents it was possible to make comparisons based upon the administered and/or internal doses resulting in similar effect levels. Comparisons of administered doses revealed a wide range of differences across species (up to a 10,000-fold difference). On the other hand, comparisons across species using internal measurements of dose (e.g., blood or brain levels) showed a remarkable correlation (generally, a 12-fold difference). These data suggest that having internal dose values could greatly reduce the uncertainties in extrapolating dose values from experimental animals to humans and in setting allowable levels of human exposure. See Rees et al. (10) for more detailed discussion of these comparisons.
WORKSHOP SUMMARY AND IMPLICATIONS SUPPORTFOR TESTINGREQUIREMENTSAND RISKASSESSMENTISSUES At the level of functional category (i.e., motivational/arousal behavior, sensory, motor, or cognitive function, and social behavior), there was good comparability found across species for the developmental neurotoxicants that were reviewed at this Workshop (13). In other words, if an agent produced cognitive or motor deficits in humans, corresponding deficits usually were found in experimental animals. This was particularly true at high levels of exposure. The correlation existed even though the specific end points used to assess these functions varied significantly across species. At lower levels of exposure comparability across species was more difficult to assess, possibly due to differences in specific end points assessed. When similar end points were evaluated, such as suckling behavior or sleep-wake cycles, comparable effects were observed. The degree of qualitative correlation between human and experimental data for the agents discussed at the Workshop lend strong support to the Agency's policy that experimental animals can and should be used to assess potential risk for developmental neurotoxicity in humans. The findings of this Workshop support the assumption that is made in EPA's developmental toxicity risk assessment guidelines (17) that, as for other end points of developmental toxicity, functional effects in animal studies indicate that an agent has the potential to alter development in humans. Furthermore, although the qualitative comparability of the agents assessed at this Workshop showed good correlation between human and experimental animal developmental neurotoxic effects, the assumption is made that the types of developmental effects seen in experimental animal studies may not be necessarily the same as those that may be produced in humans (as is true for the other end points of developmental toxicity). A number of issues related to interpretation of developmental neurotoxicity data and application to the risk assessment process were addressed by Workshop participants, particularly Work Group HI (•5). There was general agreement that long-term exposures were not always necessary and developmental neurotoxicity may result from as little as a single exposure. The type(s) of specific effects that may be elicited are dependent upon the dose and the developmental timing of exposure (i.e., exposure at critical or sensitive periods of development). The expression of an adverse effect may be different following a single exposure in comparison to that following longer-term exposure. In their evaluation of the data presented on specific agents at the Workshop, Work Group I (13) found that dose-response relationships exist for developmental neurotoxic effects in the species tested. Additionally, as the dose increases, different end points may be affected and timing of exposure is critical in terms of the type of effect that is elicited. Thus, the evaluation of data on the developmental neurotoxicants presented at the Workshop provides additional support for the basic principles of developmental toxicology. There was a significant amount of discussion on the issue of interpretation of developmental neurotoxicity data in the presence of other forms of developmental toxicity and/or in the presence of maternal toxicity (15). It was agreed that maternal toxicity during the period of gestation and/or lactation may confound interpretation of effects observed in offspring. The majority of the participants in Work Group III agreed that if significant effects were observed in the presence of maternal toxicity or other forms of developmental toxicity, that these effects should be presumed relevant for use in assessing potential risk to humans. Other participants in this Group raised concerns that, while these effects might be considered as developmental toxicity, they should not be considered developmental neurotoxicity necessarily. The majority opinion of this Work Group supports what is
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stated in EPA's developmental toxicity risk assessment guidelines (17), i.e., effects that are produced only at minimal maternally toxic doses should still be considered to represent developmental toxicity and should not be discounted as being secondary to maternal toxicity. Furthermore, the Work Group supported the use of a weight-of-evidence approach as is proposed in the developmental toxicity risk assessment guidelines (17) which calls for an assessment of the collective strength of all available data in order to determine whether the agent should proceed through the remainder of the risk assessment process. Only in the risk characterization phase, when hazard and dose data are compared with human exposure estimates, should a determination be made concerning the risk to humans for developmental neurotoxicity. Thus, an agent may not pose a risk at every exposure level (because of the assumption of a threshold) or in every situation (e.g., hazard may vary significantly depending on route and time of exposure). All of the conditions of the study design, levels of exposure, and other adverse effects must be taken into account in determining the potential for risk to humans. Work Group III (15) also concluded that developmental neurotoxicity data may be used in setting a reference dose (RfD) for an agent. This recommendation is consistent with the EPA's approach in its developmental toxicity risk assessment guidelines (17). However, concern was expressed by the Work Group that application of uncertainty factors to the NOAEL or LOAEL (lowest-observed-adverse-effect-level) failed to incorporate information on the slope of the dose-response curve or consider the variability in the data. Thus, efforts to improve quantitative risk assessment by encouraging the development of mathematically or physiologically based models were endorsed (see Research Needs section). EPA, in fact, is planning to encourage application of uncertainty factors to a benchmark dose derived from a doseresponse model (7) as a possible alternative to the NOAEL (or LOAEL), in its final revision of the developmental toxicity risk assessment guidelines. Use of a benchmark dose would take into account the slope of the dose-response curve and the variability in data, thus addressing concerns raised by this Work Group. Work Group III (15) also addressed interpretation of developmental neurotoxicity end points by indicating that a significant change in one parameter may be sufficient to be considered an adverse effect in certain cases, but that a statistically significant effect for a single end point in one test procedure does not necessarily reflect developmental neurotoxicity. The data must be evaluated in the context of other information (e.g., dose-response, correlation with other findings, degree of consistency in terms of other known effects of the agent) in order to provide a measure of the confidence in the data and in the interpretation of the findings. All of the known developmental neurotoxicants discussed at the Workshop produce a variety of behavioral, neuropathologic and other types of neurotoxic effects, but it is not known to what degree this sample of agents is representative of all agents. EPA's protocol contains a variety of observations and measures that generally would be expected to be highly intercorrelated. In the end, scientific judgement, which must take into account all the data available on an agent, will play a role in interpreting the toxicological significance of statistically significant findings. Transient effects and/or developmental delays were concluded to be adverse effects by participants in Work Group III (15). A number of points were made to support this conclusion, such as: 1) a transient effect may have residual persistent morphological sequelae in that some effects that appear transient may simply reflect compensation for a permanent underlying effect, 2) a transient effect may indicate that at a higher dose a permanent effect may be elicited, 3) transient effects in experimental animals may not manifest themselves as transient effects in humans, 4) in some cases, an effect may persist only in aged or stressed
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experimental animals but would appear to be transient otherwise, and 5) developmental delays may reflect a change in the sequence of maturation of organ systems. The Work Group's conclusions are consistent with EPA's approach regarding the issue of reversibility for other developmental and neurotoxic effects. The presentations on the individual agents demonstrated that when data are available on internal dose measurements rather than administered dose, this may reduce uncertainties in cross-species extrapolations (see discussion on quantitative comparisons above). Therefore, the development of data on target organ dosimetry in conjunction with end point data was supported by Workshop participants (15). STUDY DESIGNISSUESAND UPDATEON EPA'S CURRENT DEVELOPMENTALNEUROTOXICITYPROTOCOL A primary objective of the Workshop was to identify which parameters should be assessed in a developmental neurotoxicity battery. EPA's proposed protocol along with suggested modifications [Tables 3 and 4, Rees et al. (11)] had been distributed to all of the Workshop participants prior to the meeting and comments on the protocol were solicited. In addition, Work Group II was asked specifically to address the issue of test methodologies. Furthermore, each of the chemical experts was asked to address whether EPA's proposed protocol would have been sufficient to identify their particular agent as a developmental neurotoxicant. There was a consensus among the chemical experts, also supported by participants in Work Group I (13), that the proposed EPA protocol would have identified each of the agents presented at the Workshop as a potential human developmental neurotoxicant. Each agent was shown to have clear effects in rodents that would have been manifested in one or more of the testing procedures included in the protocol, especially at high levels of exposure. A concern was raised, however, that while the potential human hazard would have been identified, the risk to humans would have been underestimated for some of the agents because of species differences in the applied exposure levels (or administered doses) that produced adverse effects. For example, the mouse is a more sensitive species to methylmercury effects than is the rat which is the species used in the EPA proposed protocol, and humans are more sensitive than either rodent species. There was general agreement that assessment of developmental neurotoxicity should involve an evaluation of multiple categories of function. The converse also applies; that is, it is inappropriate to assess developmental neurotoxicity in only a single functional category, especially when little is known about the toxicity of an agent. These statements support the approach that EPA has taken in developing its proposed protocol. The protocol includes a battery of tests which evaluate the following functional categories: sensory, motivational/arousal, cognitive, and motor. While the protocol does not specify assessment of social function per se, effects on suckling, mother/infant contact, aggression, and play may be determined by cage-side observations which are called for in the protocol. Work Group I (13) concurred that in the majority of the cases presented at the Workshop, the developmental neurotoxic effects that were observed occurred at levels below those required to elicit maternal toxicity. They supported the position that the high dose used in a study should be at or just below the threshold for the production of minimal maternal toxicity. The EPA protocol states that the high dose shall "induce some overt maternal toxicity, but shall not result in a reduction in weight gain exceeding 20% during gestation and lactation" (18). This represents as minimal a toxic level as one could require in order to ensure that the agent has been adequately tested across an appropriate range of dose levels. The recommendation made at the Workshop thus supports EPA's
approach to selection of the high dose level. Several Work Groups raised concern regarding the interpretability of neurotoxicity data following postnatal maternal exposure to an agent. Concerns centered on the potential effects of an agent on maternal behavior, sequestering of the agent in the milk with transfer to the pup, milk production, or milk let-down and thus, potential alterations of maternal/neonatal interaction. Furthermore, it was noted that pups would be undergoing observations and testing while potentially being exposed to the agent via the milk; thus, any alterations in these measurements might be due to the pharmacologic action of the agent rather than to a neurotoxic effect. In spite of potential confounding effects, Work Group II concluded that the advantage gained in detecting developmental neurotoxicity outweighed the potential disadvantages regarding data interpretation when exposure was continued throughout the postnatal period of CNS development. Some members suggested that direct neonatal treatment be considered rather than dosing the matemal animal postpartum. However, it was noted that maternal/ neonatal interactions could just as easily be affected by the results of direct neonatal treatment. The revised EPA protocol, developed since the Workshop and discussed below, has tried to resolve some of the concerns regarding this issue. Workshop participants suggested that functional assessments should be made at different points in the life span of the animal model being used. Indeed, EPA has always used this approach. For example, the revised protocol includes assessment of motor activity at several time points prior to and around the time of weaning and, if necessary, in adulthood in order to determine a developmental profile. Similarly auditory startle habituation, as well as learning and memory, are to be evaluated around the time of weaning and, if necessary, in adulthood. Work Group II (2) focused their efforts on developing a basic screen that could be incorporated into any developmental or reproductive toxicity study. This was considered as a first tier test that should be used when there is no information available on an agent. They concluded that the basic battery should not be expected to provide detailed dose-response information or to identify a mechanism of action. Rather, the goal of this basic evaluation would be to qualitatively determine whether or not the agent is a potential developmental neurotoxicant. This Work Group recommended that a second tier test be conducted once developmental neurotoxicity has been detected (e.g., in the first tier), and that behavioral, neurochemical, and neuropathological methods should be selected for collection of dose-response and mechanistic data, based on findings in the first tier. Work Group II (2) identified some of the problems associated with trying to fit a developmental neurotoxicity test into the framework of other developmental and reproductive study designs. While it may be appropriate to incorporate neurotoxicity evaluations into a variety of protocols, there are a number of considerations to take into account. For example, there would be a lack of consistency of some important parameters, such as exposure scenarios, if in some cases testing was incorporated into a standard developmental toxicity study, in other cases a dominant lethal study, and in still other situations into a two-generation reproductive toxicity study (with a possibility of using F1 or F2 litters). This approach might slow down the progress of building a database according to a relatively consistent protocol. Perhaps a more reasonable or logistically more feasible approach would be to have a separate "basic screen" or tier 1 protocol. Work Group II considered the parameters that should be included in a basic screen (2). For the most part, the parameters were ones that were already included in EPA's protocol (e.g., assessment of morphological indices, brain weights, neuropathology following whole animal perfusion, flexibility in selection of equipment and approaches in behavioral tests). Several changes to
WORKSHOP SUMMARY AND IMPLICATIONS
TABLE 1 PROPOSED "GENERIC" DEVELOPMENTAL NEUROTOXICITY PROTOCOL FOR THE OFFICE OF PESTICIDE PROGRAMS (AUGUST 1989)
Gestation:
Sperm and/or plug positive Body weights, OBS* Begin treatment Dams weighed, OBS Pups counted, weighed, OBS Litters culled to 4 males and 4 females: neuropathology; brain weights; GFAP'~; tattoo, all pups weighed, OBS All pups weighed, OBS Day 7 Motor activity; all pups weighed, OBS Day 13 Motor activity; all pups weighed, OBS Day 17 Treatment discontinued at weaning; Day 21 motor activity; neuropathology; brain weights; GFAP; all pups weighed, OBS Learning and memory test (flexibility Days 21-24 in choice) Auditory startle response; test pups Day 22 weighed, OBS Day 60 (___2) Motor activity; learning and memory test (flexibility in choice); auditory startle response; test pups weighed, OBS End of Study Neuropathology; brain weights: GFAP
Day 0 Days 6-21 Day 6 Postnatally: Birth-Day 21 Birth Day 4
*OBS: Observations, including clinical signs and/or physical landmarks (physical landmarks include balbano-preputial separation, vaginal opening). tGFAP: Glial fibrillary acidic protein. the protocol were suggested by this Work Group, e.g., use of halbano-preputial separation as a morphological index of male sexual development instead of testes descent, eliminating the power calculation requirement for determining the number of animals to be used in the study, and eliminating the requirement for assessing glial fibrillary acidic protein (GFAP). After the Workshop in 1989, the Agency-wide guidelines committee on developmental neurotoxicity testing was requested by EPA's Office of Pesticide Programs (OPP) to prepare a "generic" developmental neurotoxicity testing protocol to include as part of a package on neurotoxicity testing of pesticides. This protocol, proposed in August 1989, is summarized in Table 1. For the most part, this protocol is similar to that described in Table 3 of Rees et al. (11) with the following exceptions: 1) use of balbano-preputial separation as a measure of male sexual maturation instead of testes descent, 2) the requirement for motor activity on day 45 was eliminated, 3) flexibility was permitted in the choice of a learning and memory test, and specific criteria that such a test should meet were identified, 4) a requirement for testing of learning and memory around the time of weaning in addition to sometime in adulthood was introduced, 5) neuropathologic evaluation and brain weights were proposed to be conducted on day 4 in addition to days 21 and adulthood, and 6) determination of GFAP values was proposed for these same three days. In its deliberations of what should be included in this proposed "generic" protocol, the Agency guidelines committee took into consideration comments that were made in association with this Workshop. The Agency received comments from over thirty individuals or organizations regarding the entire proposed adult and developmental neurotoxicity testing package. These comments were considered along with those that were prepared by OPP's Scientific Advisory Panel. In light of these comments and discussions within the Agency guidelines committee, the following changes were made to the protocol.
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A number of the public comments indicated that the developmental neurotoxicity protocol was too complex and should be restricted to those agents for which there is sufficient justification to undergo such testing. It was recommended that a simpler tier 1 type of test be developed that could be used more routinely, such as was suggested by Workshop participants as well. The Agency guidelines committee considered this suggestion and the most economical solution, in terms of time and cost, was the development of a two-tier approach within the confines of a single study design. That is, measurements are to be carried out periodically during early postnatal development, and if no statistically significant differences are observed in any measurements in any of the treated groups up to and including day 24 postnatally, the study may be terminated after the analysis of data up to that point is completed; it is presumed that the analysis could be accomplished by day 60 postnatally, the time when tier 2 testing would begin. If, however, any statistically significant differences are observed in any measurements in any of the treated groups up to and including day 24 postnatally, the study shall continue with measurements carried out into adulthood (at least day 60 postnatally). Thus, the tier 1 component is the study carded out until postnatal day 24 and the tier 2 component is an extension of tier 1 into adulthood. Whether or not the tier 2 component should be conducted would depend on the analysis of the data from the study through postnatal day 24. The advantage to this approach, as compared to doing two separate tier studies, is that if significant findings are made in the first tier component, the study does not have to repeated, but rather the animals from the same study can be continued in carrying out the remainder of the protocol. Thus, EPA's approach results in a study that will cost significantly less and use fewer animals than an approach that would use separate tier studies. Furthermore, if no significant findings are made in the first tier, it will not be necessary to carry out the second tier, thus reducing the "complexity" of the study and the cost of testing. Table 2 summarizes the main components of EPA's revised developmental neurotoxicity protocol (18). The parameters in the shaded boxes are those that would not be assessed if the study is terminated following review of data from the first tier. A number of public comments indicated that the specified duration of dosing, i.e., day 6 of gestation through day 21 postnatally, was excessive. Many of the same concerns that were identified at the Workshop were raised in the public comments. After careful consideration of all of the issues, the Agency guidelines committee revised the protocol so that the period of exposure is now from day 6 of gestation through day 10 postnatally. The rationale behind the revision was as follows: First, the Agency guidelines committee agreed that dosing should continue into the postnatal period for several reasons, including: 1) several major events that occur prenatally in the nervous system of the human are still going through critical stages postnatally in the rat, and 2) exposure to the still developing organism may occur when agents are transferred from the mother to the offspring via the milk. An alternative postnatal exposure route that has been suggested is direct dosing of neonates, but the Agency guidelines committee believes that more work is needed to develop better methods before this should be adopted. Second, although the Agency guidelines committee felt that dosing should continue into the postnatal period for the reasons identified above, it recognized that observations made during the treatment period might be influenced by concurrent pharmacologic action. Therefore, the duration of exposure in the updated protocol was modified so that it ceases on postnatal day 10. This time was chosen for cessation of exposure for the following reasons: 1) while the revised period of exposure does not cover the entire period of lactation, it should still be sufficient to detect potential
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TABLE 2 EPA'S REVISED "GENERIC" DEVELOPMENTAL NEUROTOXICITY PROTOCOL FOR THE OFFICE OF PESTICIDE PROGRAMS*
DOSING PERIOD
GD 6-PND 10"*
ROUTE
USUALLY ORAL
OBSERVATIONS
DAMS : GD 6-PND 21 PUPS: BIRTH, PND 4, 11,13 17, 21, B I W E E K L Y ~ E R E A ~ R i
6o
DEVELOPMENTAL LANDMARKS
MOTOR ACTIVITY
PND 13, 17, 21,
AUDITORY STARTLE
PND 2 2 , ~
LEARNING & MEMORY
FLEXIBILITY IN CHOICE, PND 21-24,
NEUROPA THOL OG Y
PHD 11
& BRAIN WEIGHTS
*Shaded areas indicate the parameters (Tier 2) that would not be assessed if the study is terminated following review of data from Tier 1. **GD--gestation day; PND--postnatal day.
effects following early postnatal exposure via breast milk, 2) any agent with a relatively short half-life would, theoretically, be eliminated sufficiently by the time testing begins (day 13 for motor activity) and would not significantly influence the results, and 3) while the nervous system is still undergoing some development beyond this time and effects on these events may be missed, the majority of the critical periods for CNS development have occurred by this time, most notably, proliferation of neuronal precursors in the cerebellum and hippocampus. Thus, it was felt that dosing through postnatal day 10 would maximize the detection of most developmental neurotoxic effects while minimizing the potential for neuropharmacologic influences. The public comments recommended that greater flexibility should be allowed for exposure via routes other than the oral one. The protocol has been revised to read: "The test substance or vehicle shall be administered orally. Other routes of administration may be acceptable, on a case-by-case basis, with ample justification/reasoning for this selection" (18). The Agency guidelines committee, however, recognized that conduct via other routes of exposure may necessitate modification of the protocol that may be considered too drastic and, thus, unacceptable. EPA investigators are conducting research in this area and encourage others to do so, as well, in order to address the complications that may arise in studies conducted via routes other than oral [see also Kimmel and Francis (8)]. The public recommended that additional validation was needed
before the GFAP radioimmunoassay is included as part of the battery of tests in the developmental neurotoxicity study. Although the GFAP assay has been shown to be sensitive to the neurotoxic effects of agents in both the adult and developing nervous systems, this assay has been deleted at this time, based upon some valid arguments that were provided both by the SAP and the public. However, Agency scientists will continue using this assay experimentally and other investigators are encouraged to use the assay in an effort to obtain additional validation of its use as a means to assess the neurotoxic potential of agents. In addition, if GFAP immunohistochemistry is used as a special stain in the neuropathology segment of the testing protocol and evidence of a glial response to toxicant injury is observed, application of the radioimmunoassay is encouraged in the protocol in order to provide objective, quantitative dose-response data. A number of changes have been made regarding the neuropathologic evaluation. The major ones are that the evaluation will be conducted on postnatal day 11, the day after dosing ends, rather than postnatal day 4, and will be eliminated on postnatal day 21. In addition, the protocol now includes some simple morphometric measurements that may provide indications of brain dysmorphogenesis. "'TRIGGERS" FOR REQUIRING DEVELOPMENTAL NEUROTOXICITY TESTING
Work Group IV (9) focused on the criteria considered important in determining when to require developmental neurotoxicity testing in animals (i.e., "triggers" for testing). They recommended a weight of evidence approach rather than automatic "triggers" to assist in determining which agents should undergo developmental neurotoxicity testing and to what level of testing. In addition, they recommended that when there are little or no toxicity data on agents that are expected to have widespread or high levels of exposure, data from standard developmental toxicity, reproductive toxicity, and adult neurotoxicity studies should be collected on the agent and used to determine the need for developmental neurotoxicity testing. In fact, this has been EPA's approach for testing of chemicals regulated under the Toxic Substances Control Act (TSCA) since requirements for developmental neurotoxicity testing commenced. The agents for which the test rules requiring developmental neurotoxicity were developed [reviewed in Rees e t al. (11)] were ones which were deemed as appropriate candidates for such testing, based on other toxicity information and/or structure-activity relationships. In contrast to the discussions of Work Group IV, Work Group II (2) concluded that pesticides, regulated under the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA), should be required to undergo evaluation routinely for potential developmental neurotoxicity, since they are often developed with the nervous system as the target organ. In addition, Work Group II felt that for chemicals regulated under TSCA, a basic assessment of developmental neurotoxicity should be required to accompany any developmental or reproductive toxicity study requested by EPA. Since the Workshop was held, the Agency's guidelines committee on developmental neurotoxicity testing has continued to develop criteria for deciding when developmental neurotoxicity testing should be required. Ultimately, this will be a policy decision, but there are scientific considerations that may be used to help the decision makers. The "triggers" put forth by EPA prior to the Workshop (11), the modifications made to them and the call for a weight of evidence approach rather than automatic "triggers" by Work Group IV (9), and the recommendations about testing set forth by Work Group II (2) are all valid and reasonable approaches. Initially, decisions will continue to be made on a case-by-case basis depending upon what is known about the test
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agent's biological activity, its physical/chemical properties, and its expected use and exposure patterns, taking into consideration the guidance that has been provided at this Workshop. As more and more developmental neurotoxicity studies are conducted and evaluated and as the database grows for a broader variety of agents or classes of agents, the current criteria for when to require testing may be modified to incorporate new data and considerations. RESEARCHNEEDS During discussions at the Workshop, several areas were identified where additional research may provide information to fill gaps that currently exist for developmental neurotoxicity testing and risk assessment, as outlined below. Cross-species comparisons in developmental neurotoxicology can be improved greatly by focusing attention on developmental profiles and underlying neurobiological mechanisms. Functional assessments should be made at several different points in the life span and the degree of comparability of developmental profiles across species should be assessed. Since it is important to be able to link behavioral measures to underlying neurologic mechanisms, continued improvement of animal models based on advances in the basic neurosciences is needed. Development of information on target organ dosimetry or determination of systemic dose levels should be encouraged in order to help reduce uncertainties in cross-species extrapolation in risk assessment. The developmental toxicity risk assessment guidelines (17) also encourage the use of such data, when available, to assist in the interpretation of data. Efforts to improve quantitative risk assessments including development of mathematical and physiologically based doseresponse models were encouraged at the Workshop. The developmental toxicity risk assessment guidelines (17) describe some approaches that are currently being developed. In addition, EPA is sponsoring several projects under an initiative on the development of biologically based dose-response models in an effort to generate information that could ultimately be applied in the quantitative estimation of human risk.
In many instances, the dose-response information for the agents discussed at the Workshop was limited. This was especially true for the data in humans. In order to develop better quantitative comparisons, additional data to fill these gaps are needed. The full range of critical periods of development of the nervous system in humans and experimental animals and sensitive periods for exposure to toxic agents are not well characterized. More complete information in this area will permit better study design and interpretation of findings from experimental animal studies for extrapolation to humans. SUMMARY Over the last four years, EPA has taken a vigorous approach toward developing a state-of-the-art protocol to assess chemicals and pesticides for their potential developmental neurotoxicity. During this time, the Agency has identified a number of issues related to requirements for testing, study design, interpretation and application of data from experimental animals to assess risk to humans. This Workshop brought together experts with a wide variety of backgrounds and areas of expertise to address these issues. Consensus was reached on some issues. For other issues, additional research is needed before conclusions may be drawn. The Workshop provided a forum for the education of basic researchers on the issues with which regulatory scientists must contend, and of the regulatory scientists on the neuroscience issues with which the basic researchers are grappling. It is hoped that some of the basic research that is needed to develop better animal models, design better study protocols, provide methods for target organ dosimetry, and develop physiologically based dose-response models will be conducted. It is important to continue the dialogue between regulatory scientists and basic researchers; regulatory scientists must continue to define their needs in terms of what issues should be addressed in the risk assessment process and basic researchers should be open to these needs in designing research projects, the results of which will be applicable to the regulatory setting. Such joint efforts ultimately may reduce some of the uncertainties that currently exist in extrapolation of data from experimental animals to estimates of risk in humans.
REFERENCES 1. Adams, J.; Vorhees, C. V.; Middaugh, L. D. Developmental neurotoxicity of anticonvulsants: Human and animal evidence on phenytoin. Neurotoxicol. Teratol. 12:203-214; 1990. 2. Buelke-Sam, J.; Mactutus, C. F. Workshop on the qualitative and quantitative comparability of human and animal developmental neurotoxicity, Work Group II report: Testing methods in developmental neurotoxicity for use in human risk assessment. Neurotoxicol. Teratol. 12:269-274; 1990. 3. Burbacher, T. M.; Rodier, P. M.; Weiss, B. Methylmercury developmental neurotoxicity: A comparison of effects in humans and animals. Neurotoxicol. Teratol 12:191-202; 1990. 4. Davis, J. M.; Otto, D. A.; Weil, D. E.; Grant, L. D. The comparative developmental neurotoxicity of lead in humans and animals. Neurotoxicol. Teratol. 12:215-229; 1990. 5. Driscoll, C. D.; Streissguth, A. P.; Riley, E. P. Prenatal alcohol exposure: Comparability of effects in humans and animal models. Neurotoxicol. Teratol. 12:231-237; 1990. 6. Hutchings, D. E. Issues of risk assessment: Lessons from the use and abuse of drugs during pregnancy. Neurotoxicol. Teratol. 12:183-189; 1990. 7. Kimmel, C. A. Quantitative approaches to human risk assessment for noncancer health effects. Neurotoxicology, in press; 1990. 8. Kimmel, C. A.; Francis, E. Z. Proceedings of the workshop on the acceptability and interpretation of dermal developmental toxicity studies. Fundam. Appl. Toxicol. 14:386--398; 1990. 9. Levine, T. E.; Butcher, R. E. Workshop on the qualitative and quantitative comparability of human and animal developmental neu-
10. 11. 12. 13.
14. 15.
16. 17.
rotoxicity, Work Group IV report: Triggers for developmental neurotoxicity testing. Neurotoxicol. Teratol. 12:281-284; 1990. Rees, D. C.; Francis, E. Z.; Kimmel, C. A. Qualitative and quantitative comparability of human and animal developmental neurotoxicants: A workshop summary. Neurotoxicology, in press; 1990. Rees, D. C.; Francis, E. Z.; Kimmel, C. A. Scientific and regulatory issues relevant to assessing risk for developmental neurotoxicity: An overview. Neurotoxicol. Teratol. 12:175-181; 1990. Schull, W. J.; Norton, S.; Jensh, R. P. Ionizing radiation and the developing brain. Neurotoxicol. Teratol. 12:249-260; 1990. Stanton, M. E.; Spear, L. P. Workshop on the qualitative and quantitative comparability of human and animal developmental neurotoxicity, Work Group I report: Comparability of measures of developmental neurotoxicity in humans and laboratory animals. Neurotoxicol. Teratol. 12:261-267; 1990. Tilson, H. A.; Jacobson, J. L.; Rogan, W. J. Polychlorinated biphenyls and the developing nervous system: Cross-species comparisons. Neurotoxicol. Teratol. 12:239-248; 1990. Tyl, R. W.; Sette, W.F. Workshop on the qualitative and quantitative comparability of human and animal developmental neurotoxicity, Work Group III report: Weight of evidence and quantitative evaluation of developmental neurotoxicity data. Neurotoxicol. Teratol. 12:275280; 1990. U.S. Environmental Protection Agency. Proposed test rule on triethylene glycol monomethyl, monoethyl, and monobutyl ethers. Fed. Regist. 51:17883-17893; 1986. U.S. Environmental Protection Agency. Proposed amendments to the
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guidelines for the health assessment of suspect developmental toxicants; request for comments; notice. Fed. Regist. 54:9386-9403; 1989.
18. U.S. Environmental Protection Agency. Developmental Neurotoxicity Study. In draft. 1990.
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Workshop on the Qualitative and Quantitative Comparability of Human and Animal Developmental Neurotoxicity: Summary and Implications E L A I N E Z. F R A N C I S , .2 C A R O L E A. K I M M E L * A N D D. C O O P E R R E E S t 3
*Reproductive and Developmental Toxicology Branch, Office of Health and Environmental Assessment Office of Research and Development and "~Health and Environmental Review Division, Office of Toxic Substances United States Environmental Protection Agency, Washington, DC 20460 FRANCIS, E..Z., C. A. KIMMEL AND D. C. REES. Workshop on the qualitative and quantitative comparability of human and animal developmental neurotoxicity: Summary and implications. NEUROTOXICOL TERATOL 12(3) 285-292, 1990.-- The Workshop on the Qualitative and Quantitative Comparability of Human and Animal Developmental Neurotoxicity was convened by the U.S. Environmental Protection Agency (EPA) and the National Institute on Drug Abuse to address issues related to when testing should be required, what test methodologies should be required, and how the data should be interpreted and applied to the risk assessment process. The background material for Work Group discussions included presentations made at the Workshop by invited experts summarizing qualitative and quantitative human and experimental animal data on specific chemicals or classes of chemicals and EPA's proposed developmental neurotoxicity testing protocol. This overview: 1) summarizes the qualitative comparisons presented at the Workshop and attempts to make some quantitative comparisons of findings across mammalian species following exposure to developmental neurotoxicants, 2) brings the common themes that were discussed among the Work Groups together into a regulatory perspective, 3) provides a status report on EPA's developmental neurotoxicity protocol, and 4) identifies research needs in the development of test methodologies and improvement of risk assessments for developmental neurotoxicants. Developmental neurotoxicity Lead Agents of abuse Risk assessment
Phenytoin Test guideliness
Ethanol PCBs Ionizing radiation Postnatal development
THE U.S. Environmental Protection Agency (EPA) has taken the lead among the U.S. Federal Agencies in developing a protocol to assess the potential developmental neurotoxicity of agents. The first protocol for regulatory purposes specific to developmental neurotoxicity testing was proposed in 1986 by the Office of Toxic Substances (OTS) for use with glycol ethers (16). Following the receipt of public comments on this proposal, an Agency-wide guidelines committee was formed to address public comment, develop a revised protocol, and provide a forum for discussing issues related to developmental neurotoxicology. In the four years since the formation of that committee, many discussions have taken place leading to development of a testing protocol for developmental neurotoxicity that would have general applicability for testing chemical substances and pesticides and that could be modified on a case-by-case basis. In the process of developing this protocol, deliberations took place both among scientists within the Agency and with scientists
Methylmercury
from academia and the chemical industry. As these discussions progressed, it became clear that there was a need for an open forum to discuss a number of issues related to when testing should be required, what test methodologies should be required, and how the data should be interpreted and applied to the risk assessment process. Thus, the Workshop on the Qualitative and Quantitative Comparability of Human and Animal Developmental Neurotoxicity was convened to address these issues using comparisons of human and experimental animal developmental neurotoxicity data as a basis. The three-day Workshop, described by Rees et al. (10,11), provided an opportunity for scientific exchange on many of these issues. The reader is referred to other papers in these proceedings on the agents that were discussed (1, 3-6, 12, 14) at the Workshop and to the reports of the individual Work Groups (2, 9, 13, 15) for a summary of agent-specific comparisons and Work Group deliberations. This overview is intended to: 1) briefly summarize the qualitative comparisons presented at the Workshop
~The views in this paper represent those of the authors and do not necessarily reflect the views or policies of the U.S. Environmental Protection Agency. 2Requests for reprints should be addressed to Elaine Z. Francis, Ph.D.. U.S. Environmental Protection Agency (RD-689), 401 M Street, S.W., Washington, DC 20460. 3present address: RJReynolds/Nabisco, Bowman Gray Technical Center, Winston-Salem, NC 27102.
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and attempt to make some quantitative comparisons of findings across mammalian species following exposure to developmental neurotoxicants, 2) bring the common themes that were discussed among the Work Groups together into a regulatory perspective, and 3) describe the status of EPA's developmental neurotoxicity protocol and how the Workshop discussions impacted on its current design. Specific points that have been made in the manuscripts on the agents of concern and the reports of the individual Work Groups will be discussed only as they are relevant to issues addressed in this summary. Therefore, readers are strongly encouraged to refer to the rest of these proceedings for detail of the issues that were raised and discussed at the Workshop. The background material for the Work Group discussions included the presentations made at the Workshop by invited experts summarizing human and experimental animal data on specific chemicals or classes of chemicals, and EPA's proposed developmental neurotoxicity testing protocol [see Rees et al. (11) for an outline of that protocol]. The Work Groups were organized around four main topics: 1) issues related to evaluation of comparative data to provide rationale or justification for requiring testing, 2) what test methods should be required, 3) how data from such studies should be interpreted and applied to risk assessment, and 4) criteria for recommending testing. The following sections provide an overview of the Workshop proceedings addressing these specific areas, especially as they relate to implications within a regulatory setting. A section on the status of EPA's revised developmental neurotoxicity protocol also is included. The final section identifies research needs in developing test methodologies and improving risk assessments for suspect developmental neurotoxicants.
QUALITATIVE AND QUANTITATIVE COMPARABILITY OF HUMAN AND ANIMAL DEVELOPMENTAL NEUROTOXICANTS
Experts were invited to review and present, on the first day of the Workshop, the human and experimental animal data on agents known to cause developmental neurotoxicity in humans. These agents included lead, methylmercury, selected agents of abuse, phenytoin, polychlorinated biphenyls (PCBs), ethanol, and ionizing radiation. For their presentations, the experts were asked to assess the degree of qualitative and quantitative comparability between human and experimental animal data, and identify critical assumptions and areas of uncertainty required when making these qualitative and quantitative comparisons.
Qualitative Comparability The qualitative comparability of human and experimental animal data was assessed (13) and the following behavioral categories of function were selected for evaluation: motor development and function, cognitive function, motivational/arousal behavior, sensory function, and social behavior. A number of limitations associated with assessing cross-species comparability were identified. These included: 1) while some data usually were available to evaluate categories of function, often data were unavailable to make specific end point comparisons, 2) doseresponse information was limited and, therefore, it was not possible to assess comparability of results at equivalent administered dose levels or dose ranges, 3) there were differences in experimental design, such as test methodology, exposure pattern and time to testing, 4) unique underlying neurologic mechanisms for each species which may influence species sensitivity and the types of effects manifested have not been well studied. Despite the recognized limitations in making these comparisons, the degree of qualitative comparability was considered remarkable (13).
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Rees et al. (10) provide a more detailed evaluation of the qualitative and quantitative comparability between human and rodent data presented at the Workshop. Although data were presented on experimental species other than rodents, rodents were selected for comparison to humans because they are most often the experimental species in which testing is conducted in response to a regulatory action. For the category motor development and function, in general, comparable changes were observed in both humans and rodents. Specifically, alterations in motor development and motor control were observed in rodents and humans following exposure during development to methylmercury, lead, ethanol, and PCBs. Alterations in these same parameters also were observed in rodents exposed to ionizing radiation and phenytoin; however, human data were not presented for these parameters with these two agents. Effects on cognitive function were observed in both humans and rodents following exposure during development to methylmercury, lead, ethanol, PCBs, ionizing radiation, and phenytoin. In the area of sensory function, most of the agents reviewed at the Workshop have not been studied systematically. Where data were available, effects on sensory function were generally similar between humans and rodents. In general, some measure of motivational~arousal behavior was affected in rodents and humans for all of the agents discussed at the Workshop except phenytoin for which there were no human data presented on this category. This was the only functional category for which data were sufficient to indicate comparability between humans and rodents for selected agents of abuse. Finally, for social function, limited data were presented. The only agent for which there were data in both species was ethanol; following developmental exposure to ethanol, alterations in suckling behavior were observed in both rodents and humans.
Quantitative Comparability Quantitative comparability between experimental animal and human data for developmental neurotoxicants has not been well characterized. From the presentations given by the experts at the Workshop on the selected developmental neurotoxicants, several conclusions related to quantitative comparisons may be drawn. First, dose-response data are often limited; this is especially true for humans. For some agents, such as phenytoin, the administered dose in humans is known, for the most part. However, for other agents, such as ethanol or other agents of abuse, the dose that is consumed or administered is often unknown or difficult to determine accurately. Second, most of the data that are available on dose levels are based upon administered dose, although data on some internal measure of dose were available for a few agents. Third, in many cases, the most sensitive end point has not been established nor has sufficient dose-response information been gathered to permit identification of a no-observed-adverse-effectlevel (NOAEL). Therefore, it is not possible to make definitive quantitative comparisons, at this time, based upon the relationship of end points to dose although there were indications that for some of the agents discussed, cognitive function appeared to be the most sensitive category. Fourth, for a few of the agents it was possible to make comparisons based upon the administered and/or internal doses resulting in similar effect levels. Comparisons of administered doses revealed a wide range of differences across species (up to a 10,000-fold difference). On the other hand, comparisons across species using internal measurements of dose (e.g., blood or brain levels) showed a remarkable correlation (generally, a 12-fold difference). These data suggest that having internal dose values could greatly reduce the uncertainties in extrapolating dose values from experimental animals to humans and in setting allowable levels of human exposure. See Rees et al. (10) for more detailed discussion of these comparisons.
WORKSHOP SUMMARY AND IMPLICATIONS SUPPORTFOR TESTINGREQUIREMENTSAND RISKASSESSMENTISSUES At the level of functional category (i.e., motivational/arousal behavior, sensory, motor, or cognitive function, and social behavior), there was good comparability found across species for the developmental neurotoxicants that were reviewed at this Workshop (13). In other words, if an agent produced cognitive or motor deficits in humans, corresponding deficits usually were found in experimental animals. This was particularly true at high levels of exposure. The correlation existed even though the specific end points used to assess these functions varied significantly across species. At lower levels of exposure comparability across species was more difficult to assess, possibly due to differences in specific end points assessed. When similar end points were evaluated, such as suckling behavior or sleep-wake cycles, comparable effects were observed. The degree of qualitative correlation between human and experimental data for the agents discussed at the Workshop lend strong support to the Agency's policy that experimental animals can and should be used to assess potential risk for developmental neurotoxicity in humans. The findings of this Workshop support the assumption that is made in EPA's developmental toxicity risk assessment guidelines (17) that, as for other end points of developmental toxicity, functional effects in animal studies indicate that an agent has the potential to alter development in humans. Furthermore, although the qualitative comparability of the agents assessed at this Workshop showed good correlation between human and experimental animal developmental neurotoxic effects, the assumption is made that the types of developmental effects seen in experimental animal studies may not be necessarily the same as those that may be produced in humans (as is true for the other end points of developmental toxicity). A number of issues related to interpretation of developmental neurotoxicity data and application to the risk assessment process were addressed by Workshop participants, particularly Work Group HI (•5). There was general agreement that long-term exposures were not always necessary and developmental neurotoxicity may result from as little as a single exposure. The type(s) of specific effects that may be elicited are dependent upon the dose and the developmental timing of exposure (i.e., exposure at critical or sensitive periods of development). The expression of an adverse effect may be different following a single exposure in comparison to that following longer-term exposure. In their evaluation of the data presented on specific agents at the Workshop, Work Group I (13) found that dose-response relationships exist for developmental neurotoxic effects in the species tested. Additionally, as the dose increases, different end points may be affected and timing of exposure is critical in terms of the type of effect that is elicited. Thus, the evaluation of data on the developmental neurotoxicants presented at the Workshop provides additional support for the basic principles of developmental toxicology. There was a significant amount of discussion on the issue of interpretation of developmental neurotoxicity data in the presence of other forms of developmental toxicity and/or in the presence of maternal toxicity (15). It was agreed that maternal toxicity during the period of gestation and/or lactation may confound interpretation of effects observed in offspring. The majority of the participants in Work Group III agreed that if significant effects were observed in the presence of maternal toxicity or other forms of developmental toxicity, that these effects should be presumed relevant for use in assessing potential risk to humans. Other participants in this Group raised concerns that, while these effects might be considered as developmental toxicity, they should not be considered developmental neurotoxicity necessarily. The majority opinion of this Work Group supports what is
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stated in EPA's developmental toxicity risk assessment guidelines (17), i.e., effects that are produced only at minimal maternally toxic doses should still be considered to represent developmental toxicity and should not be discounted as being secondary to maternal toxicity. Furthermore, the Work Group supported the use of a weight-of-evidence approach as is proposed in the developmental toxicity risk assessment guidelines (17) which calls for an assessment of the collective strength of all available data in order to determine whether the agent should proceed through the remainder of the risk assessment process. Only in the risk characterization phase, when hazard and dose data are compared with human exposure estimates, should a determination be made concerning the risk to humans for developmental neurotoxicity. Thus, an agent may not pose a risk at every exposure level (because of the assumption of a threshold) or in every situation (e.g., hazard may vary significantly depending on route and time of exposure). All of the conditions of the study design, levels of exposure, and other adverse effects must be taken into account in determining the potential for risk to humans. Work Group III (15) also concluded that developmental neurotoxicity data may be used in setting a reference dose (RfD) for an agent. This recommendation is consistent with the EPA's approach in its developmental toxicity risk assessment guidelines (17). However, concern was expressed by the Work Group that application of uncertainty factors to the NOAEL or LOAEL (lowest-observed-adverse-effect-level) failed to incorporate information on the slope of the dose-response curve or consider the variability in the data. Thus, efforts to improve quantitative risk assessment by encouraging the development of mathematically or physiologically based models were endorsed (see Research Needs section). EPA, in fact, is planning to encourage application of uncertainty factors to a benchmark dose derived from a doseresponse model (7) as a possible alternative to the NOAEL (or LOAEL), in its final revision of the developmental toxicity risk assessment guidelines. Use of a benchmark dose would take into account the slope of the dose-response curve and the variability in data, thus addressing concerns raised by this Work Group. Work Group III (15) also addressed interpretation of developmental neurotoxicity end points by indicating that a significant change in one parameter may be sufficient to be considered an adverse effect in certain cases, but that a statistically significant effect for a single end point in one test procedure does not necessarily reflect developmental neurotoxicity. The data must be evaluated in the context of other information (e.g., dose-response, correlation with other findings, degree of consistency in terms of other known effects of the agent) in order to provide a measure of the confidence in the data and in the interpretation of the findings. All of the known developmental neurotoxicants discussed at the Workshop produce a variety of behavioral, neuropathologic and other types of neurotoxic effects, but it is not known to what degree this sample of agents is representative of all agents. EPA's protocol contains a variety of observations and measures that generally would be expected to be highly intercorrelated. In the end, scientific judgement, which must take into account all the data available on an agent, will play a role in interpreting the toxicological significance of statistically significant findings. Transient effects and/or developmental delays were concluded to be adverse effects by participants in Work Group III (15). A number of points were made to support this conclusion, such as: 1) a transient effect may have residual persistent morphological sequelae in that some effects that appear transient may simply reflect compensation for a permanent underlying effect, 2) a transient effect may indicate that at a higher dose a permanent effect may be elicited, 3) transient effects in experimental animals may not manifest themselves as transient effects in humans, 4) in some cases, an effect may persist only in aged or stressed
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experimental animals but would appear to be transient otherwise, and 5) developmental delays may reflect a change in the sequence of maturation of organ systems. The Work Group's conclusions are consistent with EPA's approach regarding the issue of reversibility for other developmental and neurotoxic effects. The presentations on the individual agents demonstrated that when data are available on internal dose measurements rather than administered dose, this may reduce uncertainties in cross-species extrapolations (see discussion on quantitative comparisons above). Therefore, the development of data on target organ dosimetry in conjunction with end point data was supported by Workshop participants (15). STUDY DESIGNISSUESAND UPDATEON EPA'S CURRENT DEVELOPMENTALNEUROTOXICITYPROTOCOL A primary objective of the Workshop was to identify which parameters should be assessed in a developmental neurotoxicity battery. EPA's proposed protocol along with suggested modifications [Tables 3 and 4, Rees et al. (11)] had been distributed to all of the Workshop participants prior to the meeting and comments on the protocol were solicited. In addition, Work Group II was asked specifically to address the issue of test methodologies. Furthermore, each of the chemical experts was asked to address whether EPA's proposed protocol would have been sufficient to identify their particular agent as a developmental neurotoxicant. There was a consensus among the chemical experts, also supported by participants in Work Group I (13), that the proposed EPA protocol would have identified each of the agents presented at the Workshop as a potential human developmental neurotoxicant. Each agent was shown to have clear effects in rodents that would have been manifested in one or more of the testing procedures included in the protocol, especially at high levels of exposure. A concern was raised, however, that while the potential human hazard would have been identified, the risk to humans would have been underestimated for some of the agents because of species differences in the applied exposure levels (or administered doses) that produced adverse effects. For example, the mouse is a more sensitive species to methylmercury effects than is the rat which is the species used in the EPA proposed protocol, and humans are more sensitive than either rodent species. There was general agreement that assessment of developmental neurotoxicity should involve an evaluation of multiple categories of function. The converse also applies; that is, it is inappropriate to assess developmental neurotoxicity in only a single functional category, especially when little is known about the toxicity of an agent. These statements support the approach that EPA has taken in developing its proposed protocol. The protocol includes a battery of tests which evaluate the following functional categories: sensory, motivational/arousal, cognitive, and motor. While the protocol does not specify assessment of social function per se, effects on suckling, mother/infant contact, aggression, and play may be determined by cage-side observations which are called for in the protocol. Work Group I (13) concurred that in the majority of the cases presented at the Workshop, the developmental neurotoxic effects that were observed occurred at levels below those required to elicit maternal toxicity. They supported the position that the high dose used in a study should be at or just below the threshold for the production of minimal maternal toxicity. The EPA protocol states that the high dose shall "induce some overt maternal toxicity, but shall not result in a reduction in weight gain exceeding 20% during gestation and lactation" (18). This represents as minimal a toxic level as one could require in order to ensure that the agent has been adequately tested across an appropriate range of dose levels. The recommendation made at the Workshop thus supports EPA's
approach to selection of the high dose level. Several Work Groups raised concern regarding the interpretability of neurotoxicity data following postnatal maternal exposure to an agent. Concerns centered on the potential effects of an agent on maternal behavior, sequestering of the agent in the milk with transfer to the pup, milk production, or milk let-down and thus, potential alterations of maternal/neonatal interaction. Furthermore, it was noted that pups would be undergoing observations and testing while potentially being exposed to the agent via the milk; thus, any alterations in these measurements might be due to the pharmacologic action of the agent rather than to a neurotoxic effect. In spite of potential confounding effects, Work Group II concluded that the advantage gained in detecting developmental neurotoxicity outweighed the potential disadvantages regarding data interpretation when exposure was continued throughout the postnatal period of CNS development. Some members suggested that direct neonatal treatment be considered rather than dosing the matemal animal postpartum. However, it was noted that maternal/ neonatal interactions could just as easily be affected by the results of direct neonatal treatment. The revised EPA protocol, developed since the Workshop and discussed below, has tried to resolve some of the concerns regarding this issue. Workshop participants suggested that functional assessments should be made at different points in the life span of the animal model being used. Indeed, EPA has always used this approach. For example, the revised protocol includes assessment of motor activity at several time points prior to and around the time of weaning and, if necessary, in adulthood in order to determine a developmental profile. Similarly auditory startle habituation, as well as learning and memory, are to be evaluated around the time of weaning and, if necessary, in adulthood. Work Group II (2) focused their efforts on developing a basic screen that could be incorporated into any developmental or reproductive toxicity study. This was considered as a first tier test that should be used when there is no information available on an agent. They concluded that the basic battery should not be expected to provide detailed dose-response information or to identify a mechanism of action. Rather, the goal of this basic evaluation would be to qualitatively determine whether or not the agent is a potential developmental neurotoxicant. This Work Group recommended that a second tier test be conducted once developmental neurotoxicity has been detected (e.g., in the first tier), and that behavioral, neurochemical, and neuropathological methods should be selected for collection of dose-response and mechanistic data, based on findings in the first tier. Work Group II (2) identified some of the problems associated with trying to fit a developmental neurotoxicity test into the framework of other developmental and reproductive study designs. While it may be appropriate to incorporate neurotoxicity evaluations into a variety of protocols, there are a number of considerations to take into account. For example, there would be a lack of consistency of some important parameters, such as exposure scenarios, if in some cases testing was incorporated into a standard developmental toxicity study, in other cases a dominant lethal study, and in still other situations into a two-generation reproductive toxicity study (with a possibility of using F1 or F2 litters). This approach might slow down the progress of building a database according to a relatively consistent protocol. Perhaps a more reasonable or logistically more feasible approach would be to have a separate "basic screen" or tier 1 protocol. Work Group II considered the parameters that should be included in a basic screen (2). For the most part, the parameters were ones that were already included in EPA's protocol (e.g., assessment of morphological indices, brain weights, neuropathology following whole animal perfusion, flexibility in selection of equipment and approaches in behavioral tests). Several changes to
WORKSHOP SUMMARY AND IMPLICATIONS
TABLE 1 PROPOSED "GENERIC" DEVELOPMENTAL NEUROTOXICITY PROTOCOL FOR THE OFFICE OF PESTICIDE PROGRAMS (AUGUST 1989)
Gestation:
Sperm and/or plug positive Body weights, OBS* Begin treatment Dams weighed, OBS Pups counted, weighed, OBS Litters culled to 4 males and 4 females: neuropathology; brain weights; GFAP'~; tattoo, all pups weighed, OBS All pups weighed, OBS Day 7 Motor activity; all pups weighed, OBS Day 13 Motor activity; all pups weighed, OBS Day 17 Treatment discontinued at weaning; Day 21 motor activity; neuropathology; brain weights; GFAP; all pups weighed, OBS Learning and memory test (flexibility Days 21-24 in choice) Auditory startle response; test pups Day 22 weighed, OBS Day 60 (___2) Motor activity; learning and memory test (flexibility in choice); auditory startle response; test pups weighed, OBS End of Study Neuropathology; brain weights: GFAP
Day 0 Days 6-21 Day 6 Postnatally: Birth-Day 21 Birth Day 4
*OBS: Observations, including clinical signs and/or physical landmarks (physical landmarks include balbano-preputial separation, vaginal opening). tGFAP: Glial fibrillary acidic protein. the protocol were suggested by this Work Group, e.g., use of halbano-preputial separation as a morphological index of male sexual development instead of testes descent, eliminating the power calculation requirement for determining the number of animals to be used in the study, and eliminating the requirement for assessing glial fibrillary acidic protein (GFAP). After the Workshop in 1989, the Agency-wide guidelines committee on developmental neurotoxicity testing was requested by EPA's Office of Pesticide Programs (OPP) to prepare a "generic" developmental neurotoxicity testing protocol to include as part of a package on neurotoxicity testing of pesticides. This protocol, proposed in August 1989, is summarized in Table 1. For the most part, this protocol is similar to that described in Table 3 of Rees et al. (11) with the following exceptions: 1) use of balbano-preputial separation as a measure of male sexual maturation instead of testes descent, 2) the requirement for motor activity on day 45 was eliminated, 3) flexibility was permitted in the choice of a learning and memory test, and specific criteria that such a test should meet were identified, 4) a requirement for testing of learning and memory around the time of weaning in addition to sometime in adulthood was introduced, 5) neuropathologic evaluation and brain weights were proposed to be conducted on day 4 in addition to days 21 and adulthood, and 6) determination of GFAP values was proposed for these same three days. In its deliberations of what should be included in this proposed "generic" protocol, the Agency guidelines committee took into consideration comments that were made in association with this Workshop. The Agency received comments from over thirty individuals or organizations regarding the entire proposed adult and developmental neurotoxicity testing package. These comments were considered along with those that were prepared by OPP's Scientific Advisory Panel. In light of these comments and discussions within the Agency guidelines committee, the following changes were made to the protocol.
289
A number of the public comments indicated that the developmental neurotoxicity protocol was too complex and should be restricted to those agents for which there is sufficient justification to undergo such testing. It was recommended that a simpler tier 1 type of test be developed that could be used more routinely, such as was suggested by Workshop participants as well. The Agency guidelines committee considered this suggestion and the most economical solution, in terms of time and cost, was the development of a two-tier approach within the confines of a single study design. That is, measurements are to be carried out periodically during early postnatal development, and if no statistically significant differences are observed in any measurements in any of the treated groups up to and including day 24 postnatally, the study may be terminated after the analysis of data up to that point is completed; it is presumed that the analysis could be accomplished by day 60 postnatally, the time when tier 2 testing would begin. If, however, any statistically significant differences are observed in any measurements in any of the treated groups up to and including day 24 postnatally, the study shall continue with measurements carried out into adulthood (at least day 60 postnatally). Thus, the tier 1 component is the study carded out until postnatal day 24 and the tier 2 component is an extension of tier 1 into adulthood. Whether or not the tier 2 component should be conducted would depend on the analysis of the data from the study through postnatal day 24. The advantage to this approach, as compared to doing two separate tier studies, is that if significant findings are made in the first tier component, the study does not have to repeated, but rather the animals from the same study can be continued in carrying out the remainder of the protocol. Thus, EPA's approach results in a study that will cost significantly less and use fewer animals than an approach that would use separate tier studies. Furthermore, if no significant findings are made in the first tier, it will not be necessary to carry out the second tier, thus reducing the "complexity" of the study and the cost of testing. Table 2 summarizes the main components of EPA's revised developmental neurotoxicity protocol (18). The parameters in the shaded boxes are those that would not be assessed if the study is terminated following review of data from the first tier. A number of public comments indicated that the specified duration of dosing, i.e., day 6 of gestation through day 21 postnatally, was excessive. Many of the same concerns that were identified at the Workshop were raised in the public comments. After careful consideration of all of the issues, the Agency guidelines committee revised the protocol so that the period of exposure is now from day 6 of gestation through day 10 postnatally. The rationale behind the revision was as follows: First, the Agency guidelines committee agreed that dosing should continue into the postnatal period for several reasons, including: 1) several major events that occur prenatally in the nervous system of the human are still going through critical stages postnatally in the rat, and 2) exposure to the still developing organism may occur when agents are transferred from the mother to the offspring via the milk. An alternative postnatal exposure route that has been suggested is direct dosing of neonates, but the Agency guidelines committee believes that more work is needed to develop better methods before this should be adopted. Second, although the Agency guidelines committee felt that dosing should continue into the postnatal period for the reasons identified above, it recognized that observations made during the treatment period might be influenced by concurrent pharmacologic action. Therefore, the duration of exposure in the updated protocol was modified so that it ceases on postnatal day 10. This time was chosen for cessation of exposure for the following reasons: 1) while the revised period of exposure does not cover the entire period of lactation, it should still be sufficient to detect potential
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TABLE 2 EPA'S REVISED "GENERIC" DEVELOPMENTAL NEUROTOXICITY PROTOCOL FOR THE OFFICE OF PESTICIDE PROGRAMS*
DOSING PERIOD
GD 6-PND 10"*
ROUTE
USUALLY ORAL
OBSERVATIONS
DAMS : GD 6-PND 21 PUPS: BIRTH, PND 4, 11,13 17, 21, B I W E E K L Y ~ E R E A ~ R i
6o
DEVELOPMENTAL LANDMARKS
MOTOR ACTIVITY
PND 13, 17, 21,
AUDITORY STARTLE
PND 2 2 , ~
LEARNING & MEMORY
FLEXIBILITY IN CHOICE, PND 21-24,
NEUROPA THOL OG Y
PHD 11
& BRAIN WEIGHTS
*Shaded areas indicate the parameters (Tier 2) that would not be assessed if the study is terminated following review of data from Tier 1. **GD--gestation day; PND--postnatal day.
effects following early postnatal exposure via breast milk, 2) any agent with a relatively short half-life would, theoretically, be eliminated sufficiently by the time testing begins (day 13 for motor activity) and would not significantly influence the results, and 3) while the nervous system is still undergoing some development beyond this time and effects on these events may be missed, the majority of the critical periods for CNS development have occurred by this time, most notably, proliferation of neuronal precursors in the cerebellum and hippocampus. Thus, it was felt that dosing through postnatal day 10 would maximize the detection of most developmental neurotoxic effects while minimizing the potential for neuropharmacologic influences. The public comments recommended that greater flexibility should be allowed for exposure via routes other than the oral one. The protocol has been revised to read: "The test substance or vehicle shall be administered orally. Other routes of administration may be acceptable, on a case-by-case basis, with ample justification/reasoning for this selection" (18). The Agency guidelines committee, however, recognized that conduct via other routes of exposure may necessitate modification of the protocol that may be considered too drastic and, thus, unacceptable. EPA investigators are conducting research in this area and encourage others to do so, as well, in order to address the complications that may arise in studies conducted via routes other than oral [see also Kimmel and Francis (8)]. The public recommended that additional validation was needed
before the GFAP radioimmunoassay is included as part of the battery of tests in the developmental neurotoxicity study. Although the GFAP assay has been shown to be sensitive to the neurotoxic effects of agents in both the adult and developing nervous systems, this assay has been deleted at this time, based upon some valid arguments that were provided both by the SAP and the public. However, Agency scientists will continue using this assay experimentally and other investigators are encouraged to use the assay in an effort to obtain additional validation of its use as a means to assess the neurotoxic potential of agents. In addition, if GFAP immunohistochemistry is used as a special stain in the neuropathology segment of the testing protocol and evidence of a glial response to toxicant injury is observed, application of the radioimmunoassay is encouraged in the protocol in order to provide objective, quantitative dose-response data. A number of changes have been made regarding the neuropathologic evaluation. The major ones are that the evaluation will be conducted on postnatal day 11, the day after dosing ends, rather than postnatal day 4, and will be eliminated on postnatal day 21. In addition, the protocol now includes some simple morphometric measurements that may provide indications of brain dysmorphogenesis. "'TRIGGERS" FOR REQUIRING DEVELOPMENTAL NEUROTOXICITY TESTING
Work Group IV (9) focused on the criteria considered important in determining when to require developmental neurotoxicity testing in animals (i.e., "triggers" for testing). They recommended a weight of evidence approach rather than automatic "triggers" to assist in determining which agents should undergo developmental neurotoxicity testing and to what level of testing. In addition, they recommended that when there are little or no toxicity data on agents that are expected to have widespread or high levels of exposure, data from standard developmental toxicity, reproductive toxicity, and adult neurotoxicity studies should be collected on the agent and used to determine the need for developmental neurotoxicity testing. In fact, this has been EPA's approach for testing of chemicals regulated under the Toxic Substances Control Act (TSCA) since requirements for developmental neurotoxicity testing commenced. The agents for which the test rules requiring developmental neurotoxicity were developed [reviewed in Rees e t al. (11)] were ones which were deemed as appropriate candidates for such testing, based on other toxicity information and/or structure-activity relationships. In contrast to the discussions of Work Group IV, Work Group II (2) concluded that pesticides, regulated under the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA), should be required to undergo evaluation routinely for potential developmental neurotoxicity, since they are often developed with the nervous system as the target organ. In addition, Work Group II felt that for chemicals regulated under TSCA, a basic assessment of developmental neurotoxicity should be required to accompany any developmental or reproductive toxicity study requested by EPA. Since the Workshop was held, the Agency's guidelines committee on developmental neurotoxicity testing has continued to develop criteria for deciding when developmental neurotoxicity testing should be required. Ultimately, this will be a policy decision, but there are scientific considerations that may be used to help the decision makers. The "triggers" put forth by EPA prior to the Workshop (11), the modifications made to them and the call for a weight of evidence approach rather than automatic "triggers" by Work Group IV (9), and the recommendations about testing set forth by Work Group II (2) are all valid and reasonable approaches. Initially, decisions will continue to be made on a case-by-case basis depending upon what is known about the test
WORKSHOP SUMMARY AND IMPLICATIONS
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agent's biological activity, its physical/chemical properties, and its expected use and exposure patterns, taking into consideration the guidance that has been provided at this Workshop. As more and more developmental neurotoxicity studies are conducted and evaluated and as the database grows for a broader variety of agents or classes of agents, the current criteria for when to require testing may be modified to incorporate new data and considerations. RESEARCHNEEDS During discussions at the Workshop, several areas were identified where additional research may provide information to fill gaps that currently exist for developmental neurotoxicity testing and risk assessment, as outlined below. Cross-species comparisons in developmental neurotoxicology can be improved greatly by focusing attention on developmental profiles and underlying neurobiological mechanisms. Functional assessments should be made at several different points in the life span and the degree of comparability of developmental profiles across species should be assessed. Since it is important to be able to link behavioral measures to underlying neurologic mechanisms, continued improvement of animal models based on advances in the basic neurosciences is needed. Development of information on target organ dosimetry or determination of systemic dose levels should be encouraged in order to help reduce uncertainties in cross-species extrapolation in risk assessment. The developmental toxicity risk assessment guidelines (17) also encourage the use of such data, when available, to assist in the interpretation of data. Efforts to improve quantitative risk assessments including development of mathematical and physiologically based doseresponse models were encouraged at the Workshop. The developmental toxicity risk assessment guidelines (17) describe some approaches that are currently being developed. In addition, EPA is sponsoring several projects under an initiative on the development of biologically based dose-response models in an effort to generate information that could ultimately be applied in the quantitative estimation of human risk.
In many instances, the dose-response information for the agents discussed at the Workshop was limited. This was especially true for the data in humans. In order to develop better quantitative comparisons, additional data to fill these gaps are needed. The full range of critical periods of development of the nervous system in humans and experimental animals and sensitive periods for exposure to toxic agents are not well characterized. More complete information in this area will permit better study design and interpretation of findings from experimental animal studies for extrapolation to humans. SUMMARY Over the last four years, EPA has taken a vigorous approach toward developing a state-of-the-art protocol to assess chemicals and pesticides for their potential developmental neurotoxicity. During this time, the Agency has identified a number of issues related to requirements for testing, study design, interpretation and application of data from experimental animals to assess risk to humans. This Workshop brought together experts with a wide variety of backgrounds and areas of expertise to address these issues. Consensus was reached on some issues. For other issues, additional research is needed before conclusions may be drawn. The Workshop provided a forum for the education of basic researchers on the issues with which regulatory scientists must contend, and of the regulatory scientists on the neuroscience issues with which the basic researchers are grappling. It is hoped that some of the basic research that is needed to develop better animal models, design better study protocols, provide methods for target organ dosimetry, and develop physiologically based dose-response models will be conducted. It is important to continue the dialogue between regulatory scientists and basic researchers; regulatory scientists must continue to define their needs in terms of what issues should be addressed in the risk assessment process and basic researchers should be open to these needs in designing research projects, the results of which will be applicable to the regulatory setting. Such joint efforts ultimately may reduce some of the uncertainties that currently exist in extrapolation of data from experimental animals to estimates of risk in humans.
REFERENCES 1. Adams, J.; Vorhees, C. V.; Middaugh, L. D. Developmental neurotoxicity of anticonvulsants: Human and animal evidence on phenytoin. Neurotoxicol. Teratol. 12:203-214; 1990. 2. Buelke-Sam, J.; Mactutus, C. F. Workshop on the qualitative and quantitative comparability of human and animal developmental neurotoxicity, Work Group II report: Testing methods in developmental neurotoxicity for use in human risk assessment. Neurotoxicol. Teratol. 12:269-274; 1990. 3. Burbacher, T. M.; Rodier, P. M.; Weiss, B. Methylmercury developmental neurotoxicity: A comparison of effects in humans and animals. Neurotoxicol. Teratol 12:191-202; 1990. 4. Davis, J. M.; Otto, D. A.; Weil, D. E.; Grant, L. D. The comparative developmental neurotoxicity of lead in humans and animals. Neurotoxicol. Teratol. 12:215-229; 1990. 5. Driscoll, C. D.; Streissguth, A. P.; Riley, E. P. Prenatal alcohol exposure: Comparability of effects in humans and animal models. Neurotoxicol. Teratol. 12:231-237; 1990. 6. Hutchings, D. E. Issues of risk assessment: Lessons from the use and abuse of drugs during pregnancy. Neurotoxicol. Teratol. 12:183-189; 1990. 7. Kimmel, C. A. Quantitative approaches to human risk assessment for noncancer health effects. Neurotoxicology, in press; 1990. 8. Kimmel, C. A.; Francis, E. Z. Proceedings of the workshop on the acceptability and interpretation of dermal developmental toxicity studies. Fundam. Appl. Toxicol. 14:386--398; 1990. 9. Levine, T. E.; Butcher, R. E. Workshop on the qualitative and quantitative comparability of human and animal developmental neu-
10. 11. 12. 13.
14. 15.
16. 17.
rotoxicity, Work Group IV report: Triggers for developmental neurotoxicity testing. Neurotoxicol. Teratol. 12:281-284; 1990. Rees, D. C.; Francis, E. Z.; Kimmel, C. A. Qualitative and quantitative comparability of human and animal developmental neurotoxicants: A workshop summary. Neurotoxicology, in press; 1990. Rees, D. C.; Francis, E. Z.; Kimmel, C. A. Scientific and regulatory issues relevant to assessing risk for developmental neurotoxicity: An overview. Neurotoxicol. Teratol. 12:175-181; 1990. Schull, W. J.; Norton, S.; Jensh, R. P. Ionizing radiation and the developing brain. Neurotoxicol. Teratol. 12:249-260; 1990. Stanton, M. E.; Spear, L. P. Workshop on the qualitative and quantitative comparability of human and animal developmental neurotoxicity, Work Group I report: Comparability of measures of developmental neurotoxicity in humans and laboratory animals. Neurotoxicol. Teratol. 12:261-267; 1990. Tilson, H. A.; Jacobson, J. L.; Rogan, W. J. Polychlorinated biphenyls and the developing nervous system: Cross-species comparisons. Neurotoxicol. Teratol. 12:239-248; 1990. Tyl, R. W.; Sette, W.F. Workshop on the qualitative and quantitative comparability of human and animal developmental neurotoxicity, Work Group III report: Weight of evidence and quantitative evaluation of developmental neurotoxicity data. Neurotoxicol. Teratol. 12:275280; 1990. U.S. Environmental Protection Agency. Proposed test rule on triethylene glycol monomethyl, monoethyl, and monobutyl ethers. Fed. Regist. 51:17883-17893; 1986. U.S. Environmental Protection Agency. Proposed amendments to the
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guidelines for the health assessment of suspect developmental toxicants; request for comments; notice. Fed. Regist. 54:9386-9403; 1989.
18. U.S. Environmental Protection Agency. Developmental Neurotoxicity Study. In draft. 1990.
