Developmental Toxicology Evaluations—Issues with Including Neurotoxicology and Immunotoxicology Assessments in Reproductive Toxicology Studi
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《毒物学科学杂志》
The DuPont Company., Haskell Laboratory, Newark, Delaware 19714
Worldwide Safety Sciences, Pfizer Global Research and Development, Groton, Connecticut 06340
United States Food and Drug Administration, Center for Drug Evaluation Research, Office of New Drugs, Rockville, Maryland 20857
ILSI Health and Environmental Sciences Institute, Washington, DC 20005–5802
United States Environmental Protection Agency, National Center for Environmental Assessment, Washington, DC 20460–0001
Bayer CropScience LP, Stilwell, Kansas 66085–9104
The Dow Chemical Company, Toxicology & Environmental Research and Consulting, Midland, Michigan 48674
Worldwide Safety Sciences, Pfizer Global Research and Development, San Diego, CA 92064, leighann.burns@pfizer.com
ABSTRACT
Developmental and reproductive toxicology (DART) has routinely been a part of safety assessment. Attention is now focused on the effects of chemicals on the developing nervous and immune systems. This focus on developmental neurotoxicology (DNT) and developmental immunotoxicology (DIT) is based on the premise that children differ from adults in some aspects of their biology and, thus, may also differ in their responses to chemicals. This session's objective was to discuss issues common to DNT and DIT as they relate to DART protocols, including high dose selection and maternal toxicity, adequacy of pup exposure during lactation, use of a different dosing paradigm for DART versus DNT or DIT studies, and whether DIT and DNT endpoints can be incorporated into a single DART study for hazard identification purposes. Consensus was achieved on all topics except the adequacy for risk assessment purposes of the use of a limited number of endpoints for DIT and DNT, with the DNT endpoints being the primary focus of disagreement. Panelists indicated that a combination study design for hazard identification was feasible, though flexibility to meet the scientific needs of the project was emphasized. The adequacy of existing triggers for additional developmental studies was also questioned. Panelists iterated the importance of understanding pup exposure during the various life stages and the use of toxicokinetic data in designing these studies. The group agreed to consider the HESI ACSA Life Stages Task Force recommendations as a next step to address some of the issues and challenges raised during this session.
Key Words: developmental immunotoxicology; DIT; developmental neurotoxicology; DNT; developmental and reproductive toxicology; DART; study design; combination study; hazard identification; panel discussion.
INTRODUCTION
Evaluation of developmental and reproductive toxicology (DART) endpoints has been an integral part of the safety assessment process for new compounds for many years. One of the basic premises behind the testing for developmental toxicology is the understanding that children differ significantly from adults in some aspects of their basic biology and, thus, may also differ in their responses to environmental and/or pharmaceutical exposures. Considering specific organ systems, the nervous and immune systems have been identified as possibly exhibiting a unique susceptibility during development that may not be manifest if toxicological data are only acquired in adult animals (Claudio et al., 2000; Dietert et al., 2003; Hussain et al., 2005; NRC, 1993). The developing offspring may be at greater risk because chemicals may alter these organ systems with effects that are more persistent and/or more severe than those observed in adults (qualitative differences), or they may alter these systems at lower doses than in adult animals (quantitative differences).
In 1991 the U.S. Environmental Protection Agency (EPA) issued a test guideline to evaluate the effects of chemical pesticides on the developing nervous system; this developmental neurotoxicity guideline (OPPTS 870.6300) was subsequently revised in 1998. The Organization for Cooperation and Development has been engaged in the process of drafting a developmental neurotoxicology (DNT) guideline since 1995; the most recent draft version was released for international review and comment in 2003 (OECD, 2003). To address U.S. Food and Drug Administration (FDA) concerns, specific examination of DNT is performed by sponsors on a case-by-case basis for new chemical entities. The EPA is currently preparing a new test guideline for developmental immunotoxicology (DIT), while the FDA has proposed triggers for testing and suggested endpoints for the evaluation of DIT during drug development (FDA, 2002).
Numerous scientific workshops, roundtable discussions, and symposia have been held to discuss a variety of aspects of both DNT and DIT including appropriate endpoints, methodologies, and the evolving state of the science for DNT (Buelke-Sam et al., 1995; Kimmel et al., 1990; Makris et al., 1998; Mileson and Ferenc, 2001; Tilson et al., 1997) and DIT (Holsapple, 2002 ; Holsapple et al., 2005; Luster et al., 2003; Kimmel et al., 2005). It is clear that performing stand-alone studies for DNT and DIT (as proposed and/or currently envisioned) would require many more animals (80–100 litters for each study), would be very expensive and technically complicated (due to the type and variety of tests), and would be long in duration with duplication of procedures for exposures and study conduct. In fact, the general design of the current DNT testing paradigm is somewhat redundant to the standard DART protocol, because body weight, clinical observations, measures of sexual maturation, and brain weight are conducted for both studies. Even more importantly, there is a significant overlap in the developmental life stages assessed across the various study designs; in particular, each study includes exposures during gestation, to parturition, and through early postnatal life (Fig. 1). While additional animals are available from DNT designs, the incorporation of DIT endpoints into a DNT study has been deemed not generally feasible due to the size and technical complications associated with DNT protocols (Holsapple et al., 2005). However, it has been proposed that DIT endpoints be added onto existing reproductive toxicity studies where feasible (Holsapple et al., 2005), and such a design has been successfully used to evaluate maternal feed restriction as it affects immunotoxicity and reproductive toxicity endpoints in offspring (Carney et al., 2004). While the general concept of evaluating all three organ systems after developmental exposures within the same test population has been demonstrated (Chapin et al., 1997; Smialowicz et al., 2001), the potential to include specifically DNT and proposed DIT endpoints onto existing reproductive toxicity studies has only recently begun to be explored (Cooper et al., in press).
Prior workshops, symposia, and roundtable discussions have focused primarily on the state of the science of both DIT and DNT, including appropriate endpoints and methodologies to evaluate them and how they are, should, or should not yet be applied in human health risk assessment. Though the conclusion from many of these scientific gatherings has been to add endpoints onto existing studies to minimize overall animal use and maximize the data gathered from individual studies, no session has yet evaluated the actual technical feasibility and potential challenges of this approach. Therefore, the objective of this Sunset Session at the 2005 Annual Meeting of the Society of Toxicology was to provide a forum to discuss whether and how the assessment of DART, DNT, and DIT could be integrated into a single study for hazard identification purposes to conserve multiple resources, including making more efficient use of animal resources. Specific questions were posed during the session (Table 1) to guide the discussion. While the time available during the SOT session was limited, the panelists did address most of the questions.
PANEL DISCUSSION
The discussion began with the acknowledgment that a combination endpoint study has already been demonstrated as feasible (Chapin et al., 1997; Smialowicz et al., 2001). The question for consideration was whether this design could be modified to meet both EPA and FDA regulatory requirements (Question 1, Table 1). The NIEHS/EPA Juvenile Pesticide Study evaluated reproductive and developmental toxicity endpoints, neurotoxicity (including cognitive function, learning and memory), and immunotoxicity. It was noted, however, that the study was not performed under strict Good Laboratory Practices (GLP) guidelines that would be required for regulatory submissions, nor did it evaluate all of the DART, DNT, or recommended DIT endpoints under consideration. The study was technically quite challenging and required the coordinated effort of multiple laboratories, but participants felt that it provided a reasonably comprehensive screen. While the panelists recognized the technical complexity, all agreed that a combination endpoint study was important and highly desirable from an animal use and resource standpoint. A study that included all endpoints to comply with all three studies will likely be too cumbersome; however, the objectives could be satisfied in a manageable study by limiting which endpoints to retain, number of days on which tests were performed, etc. As such, the panelists agreed that the first question in Table 1 should be answered in the affirmative.
The panel noted that the scientific rigor of the stand-alone studies could be significantly improved if scientists could integrate all potential chemically mediated effects into a single study, including such endpoints as onset of sexual maturation, brain weight measurements, and maternal toxicity, rather than having to interpret data from different studies in which exposures may have been different and/or where effects may not have been observed. Examples of such integration already exist. For example, regarding crop protection chemicals, the Agricultural Chemical Safety Assessment (ACSA) Technical Committee from the International Life Sciences Institute's (ILSI) Health and Environmental Sciences Institute (HESI) was discussed during the session at the SOT meeting. As recently described by Carmichael et al. (in press), the mission of the HESI ACSA effort was to develop an international consensus across sectors (government, academia, industry and nonprofit organizations) on a scientifically credible and viable system for testing for the potential adverse effects of pesticide chemicals more efficiently, with greater accuracy and fewer artifacts, and using fewer animals. The HESI ACSA Technical Committee established three multisector, international task forces—ADME (absorption, distribution, metabolism and excretion), Life Stages, and Systemic Toxicity—with the goal being to create one coherent scheme. As described by Cooper et al. (in press), the ACSA Life Stages Task Force proposed a tiered approach, which included a novel rat DART study with enhanced endpoints addressing the potential for DNT and DIT. Some of the benefits of the proposed approach were identified as the assessment of multiple types of outcomes from the same population of animals and far fewer animals used than when separate DNT and standard DART studies are conducted.
Another example of integration regarding pharmaceuticals is the globally recognized ICH S5A guidance (ICH, 1994), which provides for a limited assessment of DNT (i.e., functional development parameters: motor, memory, auditory, startle) in the peri- and postnatal (multigeneration) study, and was acknowledged to likely provide an adequate screen for developmental neurotoxic potential of a test substance. Additionally, the 2002 FDA guidance on immunotoxicity evaluation suggests that DIT can be performed using rats available in standard reproductive studies, rather than needing a stand-alone study. Though there is no official guideline/protocol available, it was noted that EPA has consistently encouraged combination endpoint studies where it makes sense to do so. The DNT guideline (OPPTS 870.6300) specifically indicates a preference for combined testing (U.S. EPA, 1998), and this concept was reconfirmed in a Scientific Advisory Panel review of DNT studies submitted to EPA (Makris et al., 1998b). It is envisioned that, when an EPA DIT guideline is finalized, combining this evaluation with DART studies will also be encouraged. Along these lines, the panel agreed that immunotoxicity parameters assessed in a DART study would likely be sufficient to determine the immunotoxic potential of the test article in adults (Holsapple et al., 2005).
There was some discussion around whether such a combination endpoint study should be conducted routinely or only as needed [i.e., conduct based on some trigger(s)]. Triggers have been utilized in determining the need for DNT testing (based upon recommendations of Levine and Butcher, 1990) and have been proposed for the conduct of a DIT study (Holsapple et al., 2005), including the following: structure-activity relationships and other compound-specific triggers (i.e., structural analogs of known immunotoxicants), findings from animal toxicology studies and preclinical or clinical tests, the intended use of the chemical or pharmaceutical, and the potential for pediatric exposure and/or maternal exposure (i.e., maternal treatment to prevent HIV transfer) (FDA, 2002). Nevertheless, some panelists questioned whether the proposed triggers are adequate or whether sufficient data would be available early enough in product development to indicate when such a combination endpoint study is needed. However, the general consensus of the panel was that combined-endpoint studies should be encouraged for preliminary screening of new (i.e., previously untested) active ingredients, but that this approach may not be reasonable for test substances for which extensive toxicological testing has already been conducted. In that case, the rationale for conducting combined-endpoint studies should be based on a case-by-case basis depending upon specific trigger(s). It was also recently noted that these limitations could be addressed in further specialized tests within the existing testing scheme; however, such assessments may not be required in all cases, and the triggers for them in the standard two-generation DART test may be absent (Cooper et al., in press). In addition, the panelists agreed that, as neurotoxicity and immunotoxicity studies with either adults or juvenile animals become required testing in some regulatory arenas, these triggers would likely become less important, as the studies would be conducted nonetheless.
In regard to the query around the endpoints for DNT and DIT studies that have been determined to be of added value, (Question 2, Table 1), the panel did not achieve full consensus on the number or type of DART, DNT, and/or DIT endpoints that would be sufficient from a hazard identification standpoint for a combination endpoint study. This lack of consensus was primarily driven by disagreement among panelists regarding which DNT endpoints were most predictive of a potential adverse effect. It was noted that the DNT study design was conceived as a battery of tests to screen across a number of semi-independent structural and functional neurological domains (Francis et al., 1990), and it would not be logical to expect that adverse effects would be observed uniformly across all tests within a single study. Thus, because effects are often observed in some but not all tests, it was questioned how the choice could be made to eliminate specific tests and whether all currently employed tests are necessary. In order to accommodate the need to retain all currently evaluated endpoints, one suggestion was to consider conducting a second breeding of the parental generation in a two-generation reproduction study, or alternatively, using an F2 generation to assess either DNT or DART endpoints. It was reported that studies have been submitted to EPA in which serial breeding of the parental generation was conducted and DNT endpoints were successfully evaluated in the resulting offspring (F1a or F1b). Nevertheless, other panelists disagreed with the contention that certain tests cannot be eliminated, suggesting that some of the evaluations (e.g., auditory startle) have not demonstrated their value when comparing neurobehavioral effects to those observed in general toxicology studies. Still others questioned whether the functional observational battery (FOB) data from the DART or DNT studies conducted to date should be compared with the neurotoxicological findings or histopathology from general toxicology studies to help determine the most appropriate and predictive endpoints. It was indicated that some of these kinds of analyses have been conducted (Makris et al., 1998; Middaugh et al., 2003) and/or are currently ongoing at the EPA, examining the performance of the DNT study in hazard identification in comparison to parental and offspring effects observed in reproductive toxicology studies and findings in adult neurotoxicity studies.
Regarding DIT endpoints, the panelists agreed with previous workshop recommendations (Holsapple et al., 2005) that a T-cell-dependent antibody response on PND42 should be conducted. However, there was some contention on what other DIT endpoints (e.g., innate immunity) should be included in a combination endpoint study, as some immune endpoints have not been fully validated (Luster et al., 2003). In this context, it is interesting to note that the centerpiece of the HESI ACSA Life Stages paradigm is an F1-extended one-generation study (Cooper et al., in press). Selected F1 generation pups in three sets are treated continuously until post-natal-day 70 (PND 70). With respect to the specific endpoints for DNT and DIT, the members of the multisector, international task force, which included several representatives from the EPA (Cooper et al., in press), recommended the following (i.e., based in part on the aforementioned analyses currently ongoing at the EPA): Set 1a for clinical pathology including, clinical chemistry (with thyroid hormones), hematology and urinalysis between PND 63 and 70 followed by gross necropsy with a determination of a number of relevant organ weights for the system under evaluation (e.g., brain, spleen); Set 1b for limited histopathology of relevant organs and DNT assessment including FOB between PND 49 and 56 and motor activity using automated apparatus between PND 56 and 63; Set 2 for DIT including an assessment of the T-cell-dependent antibody response (TDAR) between PND 63 and 70; and Set 3 for estrous cycle monitoring and any other triggered endpoints, which in regards to DIT would depend on the outcome of the TDAR. If positive responses are observed in the TDAR, then a phenotypic analysis of the lymphocytes may be conducted, and if there is no effect on the TDAR, then consideration should be given on a case-by-case basis to the conduct of a natural killer cell assay.
The panelists engaged in some limited discussion around the use of genomic markers to identify changes occurring at the molecular level before functional deficits are observed. The panel agreed that the use of such markers was an important point to consider, but that it was premature to consider genomic markers for risk assessment purposes. It was noted that the molecular markers of importance still need to be identified, and changes in such markers further correlated with functional changes.
The panel also considered the impact of maternal toxicity on hazard identification and/or risk assessment in reproductive toxicology, DNT and DIT studies (Question 3, Table 1). The group agreed that there was a need to recognize that maternal toxicity, particularly with the use of a maximum tolerated dose, has an impact on the various endpoints of DART, DNT, and DIT. As noted above, Carney et al. (2004) reached a similar conclusion for DART and immunotoxicity endpoints using feed restriction as a surrogate for maternal toxicity.
Another area of consensus among the panelists was the importance of understanding pup exposure during gestation, lactation, and juvenile life stages (Question 4, Table 1). During the discussion, the panelists agreed that pup exposure should be understood and that careful consideration should be given to determining the need to directly administer test substance to preweaning pups. This recommendation suggests that sponsors need more preliminary (e.g., range-finding) toxicokinetic data when designing definitive studies, a conclusion that is completely consistent with the international consensus from the HESI ACSA project (Carmichael et al., in press). However, it was also noted that, while it is relatively straightforward to verify exposure qualitatively, it is much more difficult to provide a robust quantitative measure of offspring exposure during gestation and lactation, and there are no generally accepted standardized procedures. The latter comments were noted to be consistent with the findings from a separate working group involved with the HESI ACSA project (Barton et al., 2005). The panelists concluded that a negative study (i.e., no compound-related effects) in the absence of exposure is not valid, and that overexposure resulting from poor high dose selection confounds the ability to determine whether effects are direct or secondary to overt toxicity. Finally, the panelists agreed that, as a general rule, compound exposure has not been characterized very well, particularly as it pertains to a study with perinatal animals. As described previously, the recommendations for a DIT framework specified that pups should be exposed throughout the various life stages (Holsapple et al., 2005).
CONCLUSIONS
When asked whether we are to a point with developmental neurotoxicology and developmental immunotoxicology that we can propose a single study design (Question 5, Table 1), the panel was in unanimous agreement regarding the potential to integrate DART, DNT, and DIT endpoints in a single study for hazard identification purposes. It was emphasized, however, that agreement on the ability to implement a combination endpoint study design should not be viewed as an endorsement of the approach as the study design for the future. All panelists agreed that flexibility to alter existing testing paradigms to meet the needs of the project was the most important message. It was stressed that studies should be designed on the basis of sound science and the profile of the chemical under evaluation. The general consensus of the panel was that combined-endpoint studies should be encouraged for preliminary screening of previously untested active ingredients, but that this approach may not be reasonable for chemicals for which extensive toxicological testing has already been conducted. In that situation, the rationale for conducting combined-endpoint studies should be considered on a case-by-case basis depending on specific triggers. Panelists also questioned the adequacy of existing triggers that would suggest that additional developmental studies, or a combined endpoint study, be conducted. The panel did not achieve consensus on whether a limited number of DART, DNT, or DIT endpoints determined useful for risk assessment (rather than for example, all endpoints currently required in DART and DNT stand-alone studies) would be sufficient for a combination endpoint study. This was primarily driven by disagreement among panelists regarding which DNT endpoints were most predictive of a potential adverse effect. The panel did agree, however, that it was important to measure and understand the exposure of pups during gestation, lactation, and juvenile life stages. Panelists further agreed upon the importance of utilizing toxicokinetic data in designing study protocols and in determining the need for direct dosing of the test substance to preweaning offspring. Finally, the group agreed to consider the recommendations from the HESI ACSA Life Stages Task Force (Cooper et al., in press) as a next step to address some of the issues and challenges raised during this session.
NOTES
The views expressed in this document are those of the authors and do not necessarily reflect the views or policies of the U.S. Environmental Protection Agency or the U.S. Food and Drug Administration. As well, no official support or endorsement by these agencies is intended or should be inferred.
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Worldwide Safety Sciences, Pfizer Global Research and Development, Groton, Connecticut 06340
United States Food and Drug Administration, Center for Drug Evaluation Research, Office of New Drugs, Rockville, Maryland 20857
ILSI Health and Environmental Sciences Institute, Washington, DC 20005–5802
United States Environmental Protection Agency, National Center for Environmental Assessment, Washington, DC 20460–0001
Bayer CropScience LP, Stilwell, Kansas 66085–9104
The Dow Chemical Company, Toxicology & Environmental Research and Consulting, Midland, Michigan 48674
Worldwide Safety Sciences, Pfizer Global Research and Development, San Diego, CA 92064, leighann.burns@pfizer.com
ABSTRACT
Developmental and reproductive toxicology (DART) has routinely been a part of safety assessment. Attention is now focused on the effects of chemicals on the developing nervous and immune systems. This focus on developmental neurotoxicology (DNT) and developmental immunotoxicology (DIT) is based on the premise that children differ from adults in some aspects of their biology and, thus, may also differ in their responses to chemicals. This session's objective was to discuss issues common to DNT and DIT as they relate to DART protocols, including high dose selection and maternal toxicity, adequacy of pup exposure during lactation, use of a different dosing paradigm for DART versus DNT or DIT studies, and whether DIT and DNT endpoints can be incorporated into a single DART study for hazard identification purposes. Consensus was achieved on all topics except the adequacy for risk assessment purposes of the use of a limited number of endpoints for DIT and DNT, with the DNT endpoints being the primary focus of disagreement. Panelists indicated that a combination study design for hazard identification was feasible, though flexibility to meet the scientific needs of the project was emphasized. The adequacy of existing triggers for additional developmental studies was also questioned. Panelists iterated the importance of understanding pup exposure during the various life stages and the use of toxicokinetic data in designing these studies. The group agreed to consider the HESI ACSA Life Stages Task Force recommendations as a next step to address some of the issues and challenges raised during this session.
Key Words: developmental immunotoxicology; DIT; developmental neurotoxicology; DNT; developmental and reproductive toxicology; DART; study design; combination study; hazard identification; panel discussion.
INTRODUCTION
Evaluation of developmental and reproductive toxicology (DART) endpoints has been an integral part of the safety assessment process for new compounds for many years. One of the basic premises behind the testing for developmental toxicology is the understanding that children differ significantly from adults in some aspects of their basic biology and, thus, may also differ in their responses to environmental and/or pharmaceutical exposures. Considering specific organ systems, the nervous and immune systems have been identified as possibly exhibiting a unique susceptibility during development that may not be manifest if toxicological data are only acquired in adult animals (Claudio et al., 2000; Dietert et al., 2003; Hussain et al., 2005; NRC, 1993). The developing offspring may be at greater risk because chemicals may alter these organ systems with effects that are more persistent and/or more severe than those observed in adults (qualitative differences), or they may alter these systems at lower doses than in adult animals (quantitative differences).
In 1991 the U.S. Environmental Protection Agency (EPA) issued a test guideline to evaluate the effects of chemical pesticides on the developing nervous system; this developmental neurotoxicity guideline (OPPTS 870.6300) was subsequently revised in 1998. The Organization for Cooperation and Development has been engaged in the process of drafting a developmental neurotoxicology (DNT) guideline since 1995; the most recent draft version was released for international review and comment in 2003 (OECD, 2003). To address U.S. Food and Drug Administration (FDA) concerns, specific examination of DNT is performed by sponsors on a case-by-case basis for new chemical entities. The EPA is currently preparing a new test guideline for developmental immunotoxicology (DIT), while the FDA has proposed triggers for testing and suggested endpoints for the evaluation of DIT during drug development (FDA, 2002).
Numerous scientific workshops, roundtable discussions, and symposia have been held to discuss a variety of aspects of both DNT and DIT including appropriate endpoints, methodologies, and the evolving state of the science for DNT (Buelke-Sam et al., 1995; Kimmel et al., 1990; Makris et al., 1998; Mileson and Ferenc, 2001; Tilson et al., 1997) and DIT (Holsapple, 2002 ; Holsapple et al., 2005; Luster et al., 2003; Kimmel et al., 2005). It is clear that performing stand-alone studies for DNT and DIT (as proposed and/or currently envisioned) would require many more animals (80–100 litters for each study), would be very expensive and technically complicated (due to the type and variety of tests), and would be long in duration with duplication of procedures for exposures and study conduct. In fact, the general design of the current DNT testing paradigm is somewhat redundant to the standard DART protocol, because body weight, clinical observations, measures of sexual maturation, and brain weight are conducted for both studies. Even more importantly, there is a significant overlap in the developmental life stages assessed across the various study designs; in particular, each study includes exposures during gestation, to parturition, and through early postnatal life (Fig. 1). While additional animals are available from DNT designs, the incorporation of DIT endpoints into a DNT study has been deemed not generally feasible due to the size and technical complications associated with DNT protocols (Holsapple et al., 2005). However, it has been proposed that DIT endpoints be added onto existing reproductive toxicity studies where feasible (Holsapple et al., 2005), and such a design has been successfully used to evaluate maternal feed restriction as it affects immunotoxicity and reproductive toxicity endpoints in offspring (Carney et al., 2004). While the general concept of evaluating all three organ systems after developmental exposures within the same test population has been demonstrated (Chapin et al., 1997; Smialowicz et al., 2001), the potential to include specifically DNT and proposed DIT endpoints onto existing reproductive toxicity studies has only recently begun to be explored (Cooper et al., in press).
Prior workshops, symposia, and roundtable discussions have focused primarily on the state of the science of both DIT and DNT, including appropriate endpoints and methodologies to evaluate them and how they are, should, or should not yet be applied in human health risk assessment. Though the conclusion from many of these scientific gatherings has been to add endpoints onto existing studies to minimize overall animal use and maximize the data gathered from individual studies, no session has yet evaluated the actual technical feasibility and potential challenges of this approach. Therefore, the objective of this Sunset Session at the 2005 Annual Meeting of the Society of Toxicology was to provide a forum to discuss whether and how the assessment of DART, DNT, and DIT could be integrated into a single study for hazard identification purposes to conserve multiple resources, including making more efficient use of animal resources. Specific questions were posed during the session (Table 1) to guide the discussion. While the time available during the SOT session was limited, the panelists did address most of the questions.
PANEL DISCUSSION
The discussion began with the acknowledgment that a combination endpoint study has already been demonstrated as feasible (Chapin et al., 1997; Smialowicz et al., 2001). The question for consideration was whether this design could be modified to meet both EPA and FDA regulatory requirements (Question 1, Table 1). The NIEHS/EPA Juvenile Pesticide Study evaluated reproductive and developmental toxicity endpoints, neurotoxicity (including cognitive function, learning and memory), and immunotoxicity. It was noted, however, that the study was not performed under strict Good Laboratory Practices (GLP) guidelines that would be required for regulatory submissions, nor did it evaluate all of the DART, DNT, or recommended DIT endpoints under consideration. The study was technically quite challenging and required the coordinated effort of multiple laboratories, but participants felt that it provided a reasonably comprehensive screen. While the panelists recognized the technical complexity, all agreed that a combination endpoint study was important and highly desirable from an animal use and resource standpoint. A study that included all endpoints to comply with all three studies will likely be too cumbersome; however, the objectives could be satisfied in a manageable study by limiting which endpoints to retain, number of days on which tests were performed, etc. As such, the panelists agreed that the first question in Table 1 should be answered in the affirmative.
The panel noted that the scientific rigor of the stand-alone studies could be significantly improved if scientists could integrate all potential chemically mediated effects into a single study, including such endpoints as onset of sexual maturation, brain weight measurements, and maternal toxicity, rather than having to interpret data from different studies in which exposures may have been different and/or where effects may not have been observed. Examples of such integration already exist. For example, regarding crop protection chemicals, the Agricultural Chemical Safety Assessment (ACSA) Technical Committee from the International Life Sciences Institute's (ILSI) Health and Environmental Sciences Institute (HESI) was discussed during the session at the SOT meeting. As recently described by Carmichael et al. (in press), the mission of the HESI ACSA effort was to develop an international consensus across sectors (government, academia, industry and nonprofit organizations) on a scientifically credible and viable system for testing for the potential adverse effects of pesticide chemicals more efficiently, with greater accuracy and fewer artifacts, and using fewer animals. The HESI ACSA Technical Committee established three multisector, international task forces—ADME (absorption, distribution, metabolism and excretion), Life Stages, and Systemic Toxicity—with the goal being to create one coherent scheme. As described by Cooper et al. (in press), the ACSA Life Stages Task Force proposed a tiered approach, which included a novel rat DART study with enhanced endpoints addressing the potential for DNT and DIT. Some of the benefits of the proposed approach were identified as the assessment of multiple types of outcomes from the same population of animals and far fewer animals used than when separate DNT and standard DART studies are conducted.
Another example of integration regarding pharmaceuticals is the globally recognized ICH S5A guidance (ICH, 1994), which provides for a limited assessment of DNT (i.e., functional development parameters: motor, memory, auditory, startle) in the peri- and postnatal (multigeneration) study, and was acknowledged to likely provide an adequate screen for developmental neurotoxic potential of a test substance. Additionally, the 2002 FDA guidance on immunotoxicity evaluation suggests that DIT can be performed using rats available in standard reproductive studies, rather than needing a stand-alone study. Though there is no official guideline/protocol available, it was noted that EPA has consistently encouraged combination endpoint studies where it makes sense to do so. The DNT guideline (OPPTS 870.6300) specifically indicates a preference for combined testing (U.S. EPA, 1998), and this concept was reconfirmed in a Scientific Advisory Panel review of DNT studies submitted to EPA (Makris et al., 1998b). It is envisioned that, when an EPA DIT guideline is finalized, combining this evaluation with DART studies will also be encouraged. Along these lines, the panel agreed that immunotoxicity parameters assessed in a DART study would likely be sufficient to determine the immunotoxic potential of the test article in adults (Holsapple et al., 2005).
There was some discussion around whether such a combination endpoint study should be conducted routinely or only as needed [i.e., conduct based on some trigger(s)]. Triggers have been utilized in determining the need for DNT testing (based upon recommendations of Levine and Butcher, 1990) and have been proposed for the conduct of a DIT study (Holsapple et al., 2005), including the following: structure-activity relationships and other compound-specific triggers (i.e., structural analogs of known immunotoxicants), findings from animal toxicology studies and preclinical or clinical tests, the intended use of the chemical or pharmaceutical, and the potential for pediatric exposure and/or maternal exposure (i.e., maternal treatment to prevent HIV transfer) (FDA, 2002). Nevertheless, some panelists questioned whether the proposed triggers are adequate or whether sufficient data would be available early enough in product development to indicate when such a combination endpoint study is needed. However, the general consensus of the panel was that combined-endpoint studies should be encouraged for preliminary screening of new (i.e., previously untested) active ingredients, but that this approach may not be reasonable for test substances for which extensive toxicological testing has already been conducted. In that case, the rationale for conducting combined-endpoint studies should be based on a case-by-case basis depending upon specific trigger(s). It was also recently noted that these limitations could be addressed in further specialized tests within the existing testing scheme; however, such assessments may not be required in all cases, and the triggers for them in the standard two-generation DART test may be absent (Cooper et al., in press). In addition, the panelists agreed that, as neurotoxicity and immunotoxicity studies with either adults or juvenile animals become required testing in some regulatory arenas, these triggers would likely become less important, as the studies would be conducted nonetheless.
In regard to the query around the endpoints for DNT and DIT studies that have been determined to be of added value, (Question 2, Table 1), the panel did not achieve full consensus on the number or type of DART, DNT, and/or DIT endpoints that would be sufficient from a hazard identification standpoint for a combination endpoint study. This lack of consensus was primarily driven by disagreement among panelists regarding which DNT endpoints were most predictive of a potential adverse effect. It was noted that the DNT study design was conceived as a battery of tests to screen across a number of semi-independent structural and functional neurological domains (Francis et al., 1990), and it would not be logical to expect that adverse effects would be observed uniformly across all tests within a single study. Thus, because effects are often observed in some but not all tests, it was questioned how the choice could be made to eliminate specific tests and whether all currently employed tests are necessary. In order to accommodate the need to retain all currently evaluated endpoints, one suggestion was to consider conducting a second breeding of the parental generation in a two-generation reproduction study, or alternatively, using an F2 generation to assess either DNT or DART endpoints. It was reported that studies have been submitted to EPA in which serial breeding of the parental generation was conducted and DNT endpoints were successfully evaluated in the resulting offspring (F1a or F1b). Nevertheless, other panelists disagreed with the contention that certain tests cannot be eliminated, suggesting that some of the evaluations (e.g., auditory startle) have not demonstrated their value when comparing neurobehavioral effects to those observed in general toxicology studies. Still others questioned whether the functional observational battery (FOB) data from the DART or DNT studies conducted to date should be compared with the neurotoxicological findings or histopathology from general toxicology studies to help determine the most appropriate and predictive endpoints. It was indicated that some of these kinds of analyses have been conducted (Makris et al., 1998; Middaugh et al., 2003) and/or are currently ongoing at the EPA, examining the performance of the DNT study in hazard identification in comparison to parental and offspring effects observed in reproductive toxicology studies and findings in adult neurotoxicity studies.
Regarding DIT endpoints, the panelists agreed with previous workshop recommendations (Holsapple et al., 2005) that a T-cell-dependent antibody response on PND42 should be conducted. However, there was some contention on what other DIT endpoints (e.g., innate immunity) should be included in a combination endpoint study, as some immune endpoints have not been fully validated (Luster et al., 2003). In this context, it is interesting to note that the centerpiece of the HESI ACSA Life Stages paradigm is an F1-extended one-generation study (Cooper et al., in press). Selected F1 generation pups in three sets are treated continuously until post-natal-day 70 (PND 70). With respect to the specific endpoints for DNT and DIT, the members of the multisector, international task force, which included several representatives from the EPA (Cooper et al., in press), recommended the following (i.e., based in part on the aforementioned analyses currently ongoing at the EPA): Set 1a for clinical pathology including, clinical chemistry (with thyroid hormones), hematology and urinalysis between PND 63 and 70 followed by gross necropsy with a determination of a number of relevant organ weights for the system under evaluation (e.g., brain, spleen); Set 1b for limited histopathology of relevant organs and DNT assessment including FOB between PND 49 and 56 and motor activity using automated apparatus between PND 56 and 63; Set 2 for DIT including an assessment of the T-cell-dependent antibody response (TDAR) between PND 63 and 70; and Set 3 for estrous cycle monitoring and any other triggered endpoints, which in regards to DIT would depend on the outcome of the TDAR. If positive responses are observed in the TDAR, then a phenotypic analysis of the lymphocytes may be conducted, and if there is no effect on the TDAR, then consideration should be given on a case-by-case basis to the conduct of a natural killer cell assay.
The panelists engaged in some limited discussion around the use of genomic markers to identify changes occurring at the molecular level before functional deficits are observed. The panel agreed that the use of such markers was an important point to consider, but that it was premature to consider genomic markers for risk assessment purposes. It was noted that the molecular markers of importance still need to be identified, and changes in such markers further correlated with functional changes.
The panel also considered the impact of maternal toxicity on hazard identification and/or risk assessment in reproductive toxicology, DNT and DIT studies (Question 3, Table 1). The group agreed that there was a need to recognize that maternal toxicity, particularly with the use of a maximum tolerated dose, has an impact on the various endpoints of DART, DNT, and DIT. As noted above, Carney et al. (2004) reached a similar conclusion for DART and immunotoxicity endpoints using feed restriction as a surrogate for maternal toxicity.
Another area of consensus among the panelists was the importance of understanding pup exposure during gestation, lactation, and juvenile life stages (Question 4, Table 1). During the discussion, the panelists agreed that pup exposure should be understood and that careful consideration should be given to determining the need to directly administer test substance to preweaning pups. This recommendation suggests that sponsors need more preliminary (e.g., range-finding) toxicokinetic data when designing definitive studies, a conclusion that is completely consistent with the international consensus from the HESI ACSA project (Carmichael et al., in press). However, it was also noted that, while it is relatively straightforward to verify exposure qualitatively, it is much more difficult to provide a robust quantitative measure of offspring exposure during gestation and lactation, and there are no generally accepted standardized procedures. The latter comments were noted to be consistent with the findings from a separate working group involved with the HESI ACSA project (Barton et al., 2005). The panelists concluded that a negative study (i.e., no compound-related effects) in the absence of exposure is not valid, and that overexposure resulting from poor high dose selection confounds the ability to determine whether effects are direct or secondary to overt toxicity. Finally, the panelists agreed that, as a general rule, compound exposure has not been characterized very well, particularly as it pertains to a study with perinatal animals. As described previously, the recommendations for a DIT framework specified that pups should be exposed throughout the various life stages (Holsapple et al., 2005).
CONCLUSIONS
When asked whether we are to a point with developmental neurotoxicology and developmental immunotoxicology that we can propose a single study design (Question 5, Table 1), the panel was in unanimous agreement regarding the potential to integrate DART, DNT, and DIT endpoints in a single study for hazard identification purposes. It was emphasized, however, that agreement on the ability to implement a combination endpoint study design should not be viewed as an endorsement of the approach as the study design for the future. All panelists agreed that flexibility to alter existing testing paradigms to meet the needs of the project was the most important message. It was stressed that studies should be designed on the basis of sound science and the profile of the chemical under evaluation. The general consensus of the panel was that combined-endpoint studies should be encouraged for preliminary screening of previously untested active ingredients, but that this approach may not be reasonable for chemicals for which extensive toxicological testing has already been conducted. In that situation, the rationale for conducting combined-endpoint studies should be considered on a case-by-case basis depending on specific triggers. Panelists also questioned the adequacy of existing triggers that would suggest that additional developmental studies, or a combined endpoint study, be conducted. The panel did not achieve consensus on whether a limited number of DART, DNT, or DIT endpoints determined useful for risk assessment (rather than for example, all endpoints currently required in DART and DNT stand-alone studies) would be sufficient for a combination endpoint study. This was primarily driven by disagreement among panelists regarding which DNT endpoints were most predictive of a potential adverse effect. The panel did agree, however, that it was important to measure and understand the exposure of pups during gestation, lactation, and juvenile life stages. Panelists further agreed upon the importance of utilizing toxicokinetic data in designing study protocols and in determining the need for direct dosing of the test substance to preweaning offspring. Finally, the group agreed to consider the recommendations from the HESI ACSA Life Stages Task Force (Cooper et al., in press) as a next step to address some of the issues and challenges raised during this session.
NOTES
The views expressed in this document are those of the authors and do not necessarily reflect the views or policies of the U.S. Environmental Protection Agency or the U.S. Food and Drug Administration. As well, no official support or endorsement by these agencies is intended or should be inferred.
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