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Prenatal exposure to MAM forms basis for persistent neural impairment in young and adult rat offspring
Reproductive Toxicology 34 (2012) 153–154
Contents lists available at SciVerse ScienceDirect
Reproductive Toxicology journal homepage: www.elsevier.com/locate/reprotox
Free Communications III
Implementing the extended one-generation reproductive toxicity study (EOGRTS). Important points to consider Manon Beekhuijzen ∗ , Amy Zmarowski, Birgit Peter, Harry Emmen WIL Research, ‘s-Hertogenbosch, The Netherlands Aim: This abstract describes the challenges to overcome for making the newly developed EOGRTS (OECD 443) feasible. Introduction: Compared to the two-generation study (OECD 416), the EOGRTS covers more endpoints while consuming fewer animals. At weaning, selected offspring are assigned to specific subgroups (Cohorts 1-3) for further investigations, including sexual maturation, reproductive organ integrity and function, neurological and behavioural endpoints, and immune functions. These additional endpoints, the logistics and some important notes will be discussed. New parameters: The additional endpoints include extensive examinations (i.e. clinical pathology and full histopathology) for the adult animals. Labs experienced with general toxicity studies will be able to perform these measurements, however historical control data on lactating rats should be available. The reproductive part of the study (Cohort 1) is similar to the two-generation study, however it requires more flexibility as mating for the second generation is based on triggers obtained during the first generation, requiring planning-reservations for rooms and personnel and a quick review/interpretation of the data. The developmental neurotoxicity (DNT) part of the study (Cohort 2) includes neurobehavioural testing followed by detailed neurohistopathological assessment. Positive control data is required to demonstrate that testing laboratories can appropriately utilize the testing methodologies. Labs having experience with DNT studies (OECD 426) can easily incorporate these measurements, however these labs are in the minority. The developmental immunotoxicity (DIT) part of the study (Cohort 3) comprises a T-cell dependent antibody response assay (TDAR), which can also be easily incorporated in labs with immunotoxicity experience, however historical control data for 8-week-old animals is essential. Finally, there are three parameters that are unique to the EOGRTS. These include number of areolae/nipples for 13-day-old males, first day of cornified smear for females, and thyroid hormone levels for 22-day-old pups. Reference data for these parameters are typically not available. Important note: The choice of strain should be carefully considered in light of all available information (e.g. dose level selection). The guideline mentions selection of 5 pups/sex/litter. This leaves 30 reserve animals/sex/group, which can be considered excessive.
0890-6238/$ – see front matter
In addition, selection of this ratio of pups/litter can be very problematic in the case of some strains (e.g. Wistar Han). Selecting 4 pups/sex/litter is more easily attainable, which would result in sufficient animals (i.e. 10 pups/sex/group) as surplus. The forthcoming guidance document (no. 151) will additionally mention the possibility of selecting only 4 pups/sex/litter. More possibilities: To save animals, one should consider not to perform a 90-day toxicity study (OECD 408) as data obtained during the EOGRTS includes all required parameters. Vice versa, when 90-day toxicity data is already available, several parameters can be excluded from the EOGRTS. Conclusion: The EOGRTS is superior to the current twogeneration study as it obtains more information while using less animals. However, it can only be performed by an experienced and flexible laboratory having sufficient historical control data for all parameters. http://dx.doi.org/10.1016/j.reprotox.2012.05.033 Prenatal exposure to MAM forms basis for persistent neural impairment in young and adult rat offspring Didima De Groot 1,∗ , Jan Lammers 2 , Ine Waalkens 2 , Donald Fox 3 1
TNO, Zeist, The Netherlands TNO Triskelion BV, Zeist, The Netherlands 3 University of Houston, College of Optometry, Houston, TX, USA 2
An example of the Fetal Origin of Adult Health and Disease hypothesis (FOoAHaD) is the western Pacific amyotrophic lateral sclerosis (ALS) and Parkinson-dementia complex (PDC) typically appearing in populations using cycad seed kernel containing geno- and neurotoxic metabolite methylazoxymethanol (MAM), a well-known proliferation inhibitor. Researchers proposed that chemicals in the cycad seed kernels such as MAM induce a “propelling” (progressive) neurological disease. They advocate that neurodegeneration and cancer in cycling or non cycling cells such as neurons may be associated with similar pathways (Kisby and Spencer, 2011). Here, studies on neuropathology induced by prenatal exposure to MAM were re-evaluated in light of this hypothesis. Mated female rats (14 rats/dose group) were dosed ip with MAM (gestation day 13–15; doses: 0, 1.25, 2.5, 5 or 7.5 mg MAM/kg bw/day). Neuropathology survey (n = 10 litters/dose group; 1 animal/sex/litter) included: gross abnormalities, brain weight, microscopic slide reading, linear morphometry (PD22 and PD62)
154
Abstracts / Reproductive Toxicology 34 (2012) 153–154
and stereologic estimation (PD22) of neuron-numbers (hippocampus; cerebellum). Motor activity was performed on PD13, 17, 21 (61) (DeGroot et al., 2005a,b,c, 2006). Microencephaly implied size reduction of cerebrum but not cerebellum (7.5 and 5 mg/kg MAM groups (PD22, PD61)); no brain tumors were found or gross abnormalities in other organs. Significant dose related reduction in absolute and relative brain weight (cerebrum affected, not the cerebellum). Microscopy. No changes in layer composition and/or ectopic cells (8 brain levels) were manifest during slide reading (H&E) (PD22 and PD62) but morphometry demonstrated size reductions especially in frontal/parietal cortex and hippocampus, areas in proliferation during the time the proliferation inhibitor MAM was given to the dam (GD 13–15). In line with this, cerebellar layers appeared not affected as cerebellum develops after birth. Moreover, on PD22 a significant reduction of hippocampal neurons was observed in the 7.5 mg MAM/kg group whereas cerebellar neurons were not affected. Motor activity showed hypo-activity on PD13, hyper-activity on PD17 and 21, and differences on PD61 (DeGroot et al., 2005a,b,c, 2006). From available evidence we conclude that in our rat model study, progressive genotoxic and neurotoxic effects of MAM as suggested by others [1] are not likely to occur. Effects can be fully explained by a temporarily arrest of cell proliferation during the fetal MAM exposure window with no further effects afterwards. Although the theory on self-propelling progressive neurotoxity is interesting we were not able to support this hypothesis; under de circumstances of our rat model study effects can be fully explained by a temporarily arrest of cell proliferation during the fetal MAM exposure window with no further effects afterwards. It is however evident from these experiments that prenatal exposure to low doses of environmental chemicals can have long-lasting effects on health and disease in later life. This research was supported by the American Chemistry Council. http://dx.doi.org/10.1016/j.reprotox.2012.05.034 Alternative approaches for minipig embryo-fetal development studies Edward Marsden Ricerca Biosciences SAS, Lyon, France Undertaking embryo-fetal development studies in the minipig is demanding for economic, technical and scientific reasons so the
rabbit remains the default non-rodent species. But if the rabbit is not an appropriate model for a given test compound, the minipig is a realistic alternative for embryo-fetal development studies. Ricerca Biosciences has been looking at alternative approaches to the current study designs to ensure efficient and cost-effective running of these studies without compromising scientific validity. Early termination, at a stage when adequate morphological examinations can be performed, is routine in non-human primate embryo-fetal development studies (gestation day (GD) 100 with natural birth around GD 160). Early termination is also standard practice for minipig dose range-finding studies but only external fetal examinations are included. The results of a previous study comparing the Göttingen Minipig GD 60 and GD 110 fetuses, conducted to try and detect structural abnormalities associated with a known swine teratogen, pyrimethamine, have already been reported. Pyrimethamine-associated fetal abnormalities consistent with those described in the literature, principally limb malformations, micrognathia and cleft palate, were present amongst the GD 60 fetuses. The GD 60 minipig fetus compares closely in size and weight with a GD 29 rabbit fetus. As a consequence, the materials, methods and time required to perform the visceral and skeletal examinations are also comparable with those for the rabbit. At this stage, however, although the major period of organogenesis has been completed by GD 35, maturation and growth are obviously incomplete since the GD 60 fetus is only one third through the full fetal period. Complementary studies were therefore performed, firstly to determine the histology of the principal fetal organs at GD 60 compared with the full term fetus and, secondly, to validate a double staining technique with Alizarin red for bone and Alcian blue for cartilage of GD 60 specimens. We conclude that fetal examinations performed earlier in gestation are feasible technically, provide numerous practical and time saving advantages with respect to full term caesareans, thereby reducing study duration and associated costs. Morphological development is sufficiently advanced by GD 60 to allow the fetal external, soft tissue and skeletal examinations to be completed successfully. Staining of bone and cartilage is useful at this stage in order to enhance the skeletal examinations to better elucidate any abnormalities found in the bone. In addition, by performing full fetal examinations in dose range-finding studies with an early termination, it may be possible to include women of child bearing potential into early clinical trials according to the revised ICH M3(R2) guideline. http://dx.doi.org/10.1016/j.reprotox.2012.05.035
Contents lists available at SciVerse ScienceDirect
Reproductive Toxicology journal homepage: www.elsevier.com/locate/reprotox
Free Communications III
Implementing the extended one-generation reproductive toxicity study (EOGRTS). Important points to consider Manon Beekhuijzen ∗ , Amy Zmarowski, Birgit Peter, Harry Emmen WIL Research, ‘s-Hertogenbosch, The Netherlands Aim: This abstract describes the challenges to overcome for making the newly developed EOGRTS (OECD 443) feasible. Introduction: Compared to the two-generation study (OECD 416), the EOGRTS covers more endpoints while consuming fewer animals. At weaning, selected offspring are assigned to specific subgroups (Cohorts 1-3) for further investigations, including sexual maturation, reproductive organ integrity and function, neurological and behavioural endpoints, and immune functions. These additional endpoints, the logistics and some important notes will be discussed. New parameters: The additional endpoints include extensive examinations (i.e. clinical pathology and full histopathology) for the adult animals. Labs experienced with general toxicity studies will be able to perform these measurements, however historical control data on lactating rats should be available. The reproductive part of the study (Cohort 1) is similar to the two-generation study, however it requires more flexibility as mating for the second generation is based on triggers obtained during the first generation, requiring planning-reservations for rooms and personnel and a quick review/interpretation of the data. The developmental neurotoxicity (DNT) part of the study (Cohort 2) includes neurobehavioural testing followed by detailed neurohistopathological assessment. Positive control data is required to demonstrate that testing laboratories can appropriately utilize the testing methodologies. Labs having experience with DNT studies (OECD 426) can easily incorporate these measurements, however these labs are in the minority. The developmental immunotoxicity (DIT) part of the study (Cohort 3) comprises a T-cell dependent antibody response assay (TDAR), which can also be easily incorporated in labs with immunotoxicity experience, however historical control data for 8-week-old animals is essential. Finally, there are three parameters that are unique to the EOGRTS. These include number of areolae/nipples for 13-day-old males, first day of cornified smear for females, and thyroid hormone levels for 22-day-old pups. Reference data for these parameters are typically not available. Important note: The choice of strain should be carefully considered in light of all available information (e.g. dose level selection). The guideline mentions selection of 5 pups/sex/litter. This leaves 30 reserve animals/sex/group, which can be considered excessive.
0890-6238/$ – see front matter
In addition, selection of this ratio of pups/litter can be very problematic in the case of some strains (e.g. Wistar Han). Selecting 4 pups/sex/litter is more easily attainable, which would result in sufficient animals (i.e. 10 pups/sex/group) as surplus. The forthcoming guidance document (no. 151) will additionally mention the possibility of selecting only 4 pups/sex/litter. More possibilities: To save animals, one should consider not to perform a 90-day toxicity study (OECD 408) as data obtained during the EOGRTS includes all required parameters. Vice versa, when 90-day toxicity data is already available, several parameters can be excluded from the EOGRTS. Conclusion: The EOGRTS is superior to the current twogeneration study as it obtains more information while using less animals. However, it can only be performed by an experienced and flexible laboratory having sufficient historical control data for all parameters. http://dx.doi.org/10.1016/j.reprotox.2012.05.033 Prenatal exposure to MAM forms basis for persistent neural impairment in young and adult rat offspring Didima De Groot 1,∗ , Jan Lammers 2 , Ine Waalkens 2 , Donald Fox 3 1
TNO, Zeist, The Netherlands TNO Triskelion BV, Zeist, The Netherlands 3 University of Houston, College of Optometry, Houston, TX, USA 2
An example of the Fetal Origin of Adult Health and Disease hypothesis (FOoAHaD) is the western Pacific amyotrophic lateral sclerosis (ALS) and Parkinson-dementia complex (PDC) typically appearing in populations using cycad seed kernel containing geno- and neurotoxic metabolite methylazoxymethanol (MAM), a well-known proliferation inhibitor. Researchers proposed that chemicals in the cycad seed kernels such as MAM induce a “propelling” (progressive) neurological disease. They advocate that neurodegeneration and cancer in cycling or non cycling cells such as neurons may be associated with similar pathways (Kisby and Spencer, 2011). Here, studies on neuropathology induced by prenatal exposure to MAM were re-evaluated in light of this hypothesis. Mated female rats (14 rats/dose group) were dosed ip with MAM (gestation day 13–15; doses: 0, 1.25, 2.5, 5 or 7.5 mg MAM/kg bw/day). Neuropathology survey (n = 10 litters/dose group; 1 animal/sex/litter) included: gross abnormalities, brain weight, microscopic slide reading, linear morphometry (PD22 and PD62)
154
Abstracts / Reproductive Toxicology 34 (2012) 153–154
and stereologic estimation (PD22) of neuron-numbers (hippocampus; cerebellum). Motor activity was performed on PD13, 17, 21 (61) (DeGroot et al., 2005a,b,c, 2006). Microencephaly implied size reduction of cerebrum but not cerebellum (7.5 and 5 mg/kg MAM groups (PD22, PD61)); no brain tumors were found or gross abnormalities in other organs. Significant dose related reduction in absolute and relative brain weight (cerebrum affected, not the cerebellum). Microscopy. No changes in layer composition and/or ectopic cells (8 brain levels) were manifest during slide reading (H&E) (PD22 and PD62) but morphometry demonstrated size reductions especially in frontal/parietal cortex and hippocampus, areas in proliferation during the time the proliferation inhibitor MAM was given to the dam (GD 13–15). In line with this, cerebellar layers appeared not affected as cerebellum develops after birth. Moreover, on PD22 a significant reduction of hippocampal neurons was observed in the 7.5 mg MAM/kg group whereas cerebellar neurons were not affected. Motor activity showed hypo-activity on PD13, hyper-activity on PD17 and 21, and differences on PD61 (DeGroot et al., 2005a,b,c, 2006). From available evidence we conclude that in our rat model study, progressive genotoxic and neurotoxic effects of MAM as suggested by others [1] are not likely to occur. Effects can be fully explained by a temporarily arrest of cell proliferation during the fetal MAM exposure window with no further effects afterwards. Although the theory on self-propelling progressive neurotoxity is interesting we were not able to support this hypothesis; under de circumstances of our rat model study effects can be fully explained by a temporarily arrest of cell proliferation during the fetal MAM exposure window with no further effects afterwards. It is however evident from these experiments that prenatal exposure to low doses of environmental chemicals can have long-lasting effects on health and disease in later life. This research was supported by the American Chemistry Council. http://dx.doi.org/10.1016/j.reprotox.2012.05.034 Alternative approaches for minipig embryo-fetal development studies Edward Marsden Ricerca Biosciences SAS, Lyon, France Undertaking embryo-fetal development studies in the minipig is demanding for economic, technical and scientific reasons so the
rabbit remains the default non-rodent species. But if the rabbit is not an appropriate model for a given test compound, the minipig is a realistic alternative for embryo-fetal development studies. Ricerca Biosciences has been looking at alternative approaches to the current study designs to ensure efficient and cost-effective running of these studies without compromising scientific validity. Early termination, at a stage when adequate morphological examinations can be performed, is routine in non-human primate embryo-fetal development studies (gestation day (GD) 100 with natural birth around GD 160). Early termination is also standard practice for minipig dose range-finding studies but only external fetal examinations are included. The results of a previous study comparing the Göttingen Minipig GD 60 and GD 110 fetuses, conducted to try and detect structural abnormalities associated with a known swine teratogen, pyrimethamine, have already been reported. Pyrimethamine-associated fetal abnormalities consistent with those described in the literature, principally limb malformations, micrognathia and cleft palate, were present amongst the GD 60 fetuses. The GD 60 minipig fetus compares closely in size and weight with a GD 29 rabbit fetus. As a consequence, the materials, methods and time required to perform the visceral and skeletal examinations are also comparable with those for the rabbit. At this stage, however, although the major period of organogenesis has been completed by GD 35, maturation and growth are obviously incomplete since the GD 60 fetus is only one third through the full fetal period. Complementary studies were therefore performed, firstly to determine the histology of the principal fetal organs at GD 60 compared with the full term fetus and, secondly, to validate a double staining technique with Alizarin red for bone and Alcian blue for cartilage of GD 60 specimens. We conclude that fetal examinations performed earlier in gestation are feasible technically, provide numerous practical and time saving advantages with respect to full term caesareans, thereby reducing study duration and associated costs. Morphological development is sufficiently advanced by GD 60 to allow the fetal external, soft tissue and skeletal examinations to be completed successfully. Staining of bone and cartilage is useful at this stage in order to enhance the skeletal examinations to better elucidate any abnormalities found in the bone. In addition, by performing full fetal examinations in dose range-finding studies with an early termination, it may be possible to include women of child bearing potential into early clinical trials according to the revised ICH M3(R2) guideline. http://dx.doi.org/10.1016/j.reprotox.2012.05.035