- Email: [email protected]
Oral prenatal developmental toxicity study with NM-200 synthetic amorphous silica in Wistar rats
Posters / Reproductive Toxicology 32 (2011) 164–179
transport and steroid synthesis were evaluated by quantitative real time PCR (8–10 litters/dose group). Basal testosterone production ex vivo was determined by LC/MS/MS (10–12 litters/dose group). Results: Prenatal exposure to DnHP resulted in dose-dependent reductions of fetal testis mRNA levels of Scavenger receptor B-1 (SRB1), steroid acute regulatory protein (StAR), cytochrome side-chain cleavage (P450scc, Cyp11a1), 3-hydroxysteroid dehydrogenase (3-HSD), and cytochrome P450c17 (cyp17a1). Likewise, testosterone production by fetal testes was highly reduced at 125 mg/kg/day, and higher doses. There was also a decrease in the expression of Insulin-like factor-3 (Insl-3), which is essential for gubernacular development and testicular descent. Conclusion: Our results indicate that DnHP disrupts testosterone synthesis and shares target genes with DEHP. Additionally, fetal testicular testosterone production and expression of associated genes appear to be sensitive indicators of testicular response to DnHP in the fetal rat. doi:10.1016/j.reprotox.2011.06.099 Reproductive toxicology and infant immunophenotyping: Normal infant ranges for T-cell assessment Tabor Salewsky a , Tracey Warren a , Cabot Cornwall a , Paul a b a Franklin , Hideshi Tsusaki , Ryoichi Nagata , presented by Brian Bakera,∗ a b
SNBL USA Ltd., Everett, WA, USA Shin Nippon Biomedical Laboratories Ltd., Kagoshima, Japan
Reproductive toxicology is a necessary part of any drug assessment program in order to evaluate toxicological effects on mother and infant. Data is available referencing the mature immune systems which can be used as comparison for data interpretation. However, normal ranges for the developing immune system are not easily obtainable. Normal ranges are vital to the interpretation and final conclusions made for any data set. Without this baseline data conclusions derived may be flawed. Here we have compiled a set of normal infant samples to aid in the evaluation of T-lymphocytes and T-lymphocyte subsets by evaluating CD3, CD4, and CD8 cell surface markers for 82 neonates on date of births ranging from day 25 to day 35. The typical populations of CD3+CD8+ and CD3+CD4+ were assessed, as well as CD3−CD8+, CD3+CD8−, CD3−CD8−, CD3−CD4+, CD3+CD4−, CD3−CD4−. The maximum, minimum, mean, standard deviation, and confidence intervals were calculated for relative percentages. This combined information allows for an accurate assessment of changes on the developing T-lymphocyte population and confidence in conclusions with large variation.
173
approach, i.e. when studies are designed to specifically address significant concerns, either of theoretical nature or demonstrated in adult toxicity studies (when a target organ of toxicity such as the CNS undergoes significant postnatal development). At least the critical stages of development of an organ at risk should be tested. Another approach uses generalized screening tests in juvenile animals, comparable to repeat-dose studies, that may reveal toxicity not identified in adult animal studies. The rationale for using the targeted approach to assess specific concerns comes from scientific, ethical and 3R considerations, as well as business reasons. Using targeted (i.e. case-by-case) instead of general screening designs improves overall study interpretation, can avoid the assessment of irrelevant stages of development and thus the collection of potential meaningless data giving rise to warnings or delays in the approval of urgently needed paediatric medicines. In drug development, specialized and targeted reproduction toxicity studies are used, not broad multi-generation studies. It is for the same reasons that paediatric risk characterization should apply targeted, specially designed studies that deliver appropriate amount of test substance to the juvenile animal model during the critical stages of development. An ILSI expert working group concluded likewise already in 2003. Therefore, studies in juvenile animals to support paediatric development should always be performed on a case-bycase basis and applying case-by-case study design to optimize risk characterization and to minimize predictable, misleading or even inappropriate data. Such truly targeted approach is more likely to be effective in improving overall health of children as stipulated by the paediatric legislation. Only when repeat-dose studies in adult animals are not performed, the screening test approach in juvenile animals has its merit. Evidently, special investigations may need to be included in such cases to properly address concerns, and thus a meaningful routine study design that fits all purposes does not exist. Finally, systems replacing or supplementing in vivo juvenile animal testing have not enough been considered and explored. Some concerns may best be substantiated in targeted experiments using human cells, thus avoiding investigations in juvenile animals afflicted with substantial challenges in translational data interpretation. doi:10.1016/j.reprotox.2011.06.101 Oral prenatal developmental toxicity study with NM-200 synthetic amorphous silica in Wistar rats Steffen Schneider a,∗ , Robert Landsiedel a , Wendel Wohlleben a , André Wolterbeek b , Ine Waalkens-Berendsen b , Han van de Sandt c a
BASF, Ludwigshafen, Germany TNO Triskelion BV, Zeist, The Netherlands c TNO, Zeist, The Netherlands b
doi:10.1016/j.reprotox.2011.06.100 Destination target: A vote for a true case-by-case approach to address concerns in paediatric development Georg Schmitt ∗ , Thomas Singer F. Hoffmann-La Roche Ltd., Non-Clinical Safety, CH-4070 Basel, Switzerland As emphasized by the paediatric legislation, the toxicological assessment of a new medicine may need to include juvenile animal studies. Conducting studies in juvenile, i.e. immature animals should always be considered when existing animal and human safety data are thought insufficient for the safe use in the intended paediatric age groups. The main issue with juvenile animal studies is that the true purpose of these studies has still not been resolved. The most straight forward and relevant use is the targeted
The engineering of nanomaterials offers extraordinary opportunities in various technological fields and safety assessments of new nanomaterials must complement the technological progress. Current test guidelines on developmental and reproductive toxicity are generally able to determine hazards but their specific application to nanomaterials needs to be evaluated. In this project, standard OECD test methods (OECD guidelines 414 and 416) were applied for nanomaterials hazard testing and modifications of these tests will be recommended. The prenatal developmental toxicity study (OECD 414) is complete and the results are presented here. Whereas the two-generation reproduction toxicity study (OECD 416) is ongoing and results will be presented separately. In this study, 25 female Wistar Han rats per group received Synthetic Amorphous Silica (NM-200 supplied by JRC, European Joint Research Centre in Ispra, Italy) by oral gavage at dose levels of 0, 100, 300 and
174
Posters / Reproductive Toxicology 32 (2011) 164–179
1000 mg/kg body weight/day from gestation day (GD) 6 through GD 19. The material was dispersed in 10% foetal bovine serum in water according to the standard dispersion protocol of the JRC. The nanomaterial dispersions were characterized i.a. the particle size distribution was analysed by by cryo-SEM (scanning electron microscopy after shock freezing) and AUC (in situ analytical ultracentrifugation). Food consumption and body weights of the animals were recorded regularly throughout the study. The state of health of the animals was checked each day. On gestation day 20 all females were sacrificed and assessed by gross pathology (including weight determinations of the unopened uterus and the placentas). For each dam, corpora lutea were counted and number and distribution of implants (differentiated as resorptions, live and dead fetuses) were determined. The fetuses were removed from the uterus, sexed, weighed and investigated for external findings. Half of the fetuses of each litter were examined for soft tissue findings and the remaining fetuses for skeletal (inclusive cartilage) findings. In this study we did not observe differences among the various groups for clinical observations, body weights and food consumption of the dams. No effects were observed on number of corpora lutea, implants, pre- and postimplantation losses as well as number and viability of offspring. External, soft tissue and skeletal examination revealed no test-substance-related abnormalities. Acknowledgement: The project is sponsored by CEFIC-LRI (LRIN3 project) and is monitored by Monika Maier, Evonik Degussa GmbH, Hanau, Germany on behalf of CEFIC Sector group on Synthetic Amorphous Silica (ASASP). doi:10.1016/j.reprotox.2011.06.102 Assessing sperm motility in the guinea pig, Cavia porcellus David Schreur, Elise Lewis, Alan Hoberman Charles River Laboratories, Horsham, PA, USA Fertility assessment in regulatory toxicology studies are generally performed using rats or mice. Alternate species that may more closely mimic the metabolic profile of a drug to humans or demonstrate activity similar to man only in that species may be more appropriate. One alternate rodent species is the domestic guinea pig, Cavia porcellus. Accurate computer assisted sperm analysis (CASA) of guinea pig motility has proven difficult because of the inherent nature of the sperm in Cavia species to stack or form rouleaux of cells while developing motility. A computerized sperm analyzer cannot accurately count the number of cells in each rouleaux. Shepard reported that sperm rouleaux in the guinea pig can be dissociated into single cells by incubation in medium containing trypsin [1]. To develop a method to obtain an accurate count of motile and non-motile cells and to calculate the percentage of motile cells in a sperm sample, samples were obtained from the vas deferens and cauda epididymides of domestic guinea pigs, incubated in Ham’s F-10 medium, in Ham’s F-10 medium with 0.5% trypsin added, or 2.5% trypsin in EBSS, and images were taken of the sperm cells in the sample using a Hamilton Thorne IVOS sperm analyzer. It was determined that motile sperm from both the vas deferens and the cauda epididymis did stack into rouleaux and that incubation in medium containing trypsin did not dissociate the rouleaux of motile cells into individual motile cells sufficiently for the Hamilton Thorne IVOS to obtain an accurate count. While the use of CASA for automated determination of sperm motility could not be shown to be accurate, the Hamilton Thorne IVOS is still a useful tool for the determination of this endpoint by providing a quality video image loop of sperm motion that can be used by a trained technician to take accurate manual counts of the motile and non-motile sperm, including those formed into rouleaux, allowing calculation of the percentage of motile cells. One added benefit of
the IVOS system is providing a saved image of the sample to allow for reconstruction of the study and verification of accuracy. Reference [1] Shepard, et al. In vitro studies of Guinea pig spermatozoa in rouleaux. Biol Reprod 1974;11:470–4.
doi:10.1016/j.reprotox.2011.06.103 Dose response analysis of four monophthalates in the murine embryonic stem cell test assessed by cardiomyocyte differentiation and gene expression Sjors H.W. Schulpen a,b,∗ , Dorien van Dartel a , Jeroen Pennings a , Joshua Robinson a,c , Aldert H. Piersma a,c a
Laboratory for Health Effects Research, RIVM, Bilthoven, The Netherlands b Institute for Risk Assessment Sciences, Utrecht University, Utrecht, The Netherlands c Maastricht University, Maastricht, The Netherlands
The embryonic stem cell test (EST) is an alternative test method in which the inhibition of differentiation into beating cardiomyocytes by compound exposure is used as a determinant for developmental toxicity. Application of the test could reduce animal testing in regulatory toxicology if reliability of its predictions can be ascertained. The addition of differential gene expression parameters as an end point in EST might enhance its predictive value. Here we exposed the EST to four monophthalates. Phthalates are used as plasticizers and are known to have an effect on embryo development and on the differentiation of embryonic stem cells towards cardiomyocytes. We studied the dose dependent effects of three embryotoxic phthalates, monobutyl phthalate (MBuP), monobenzyl phthalate (MBeP) and mono-(2-ethylhexyl) phthalate (MEHP) and the non-embryotoxic monomethyl phthalate (MMP). We evaluated effects on beating cardiomyocyte formation, together with a full genome gene expression response. The concentrations of embryotoxic phthalates resulting in 50% inhibition of differentiation (ID50), based on the classical read out of the EST, was between 0.4 an 1.4 mM. In general, gene expression changes were observed already at doses below those causing morphological effects. Transcriptomics analysis showed 668 genes dose-dependently differentially expressed in all embryotoxic phthalates, as compared to the vehicle control. Cluster and pathway analysis illustrated different nuclear, apoptotic and developmental gene set responses. Excluding 66 overlapping genes shared between both the embryotoxic and non-embryotoxic phthalates, these genes gave rise to a common gene signature for embryotoxic phthalates. In addition, compound-specific gene signatures were identified indicating specificity of effects within the chemical category. It can be concluded that transcriptomics leads to a more detailed description of the EST response to compound exposure and can give additional insight into phthalate specific effects on stem cell differentiation. Such mechanistic insight provided by transcriptomics may improve EST readout significance as well as its impact on hazard and risk assessment. doi:10.1016/j.reprotox.2011.06.104
transport and steroid synthesis were evaluated by quantitative real time PCR (8–10 litters/dose group). Basal testosterone production ex vivo was determined by LC/MS/MS (10–12 litters/dose group). Results: Prenatal exposure to DnHP resulted in dose-dependent reductions of fetal testis mRNA levels of Scavenger receptor B-1 (SRB1), steroid acute regulatory protein (StAR), cytochrome side-chain cleavage (P450scc, Cyp11a1), 3-hydroxysteroid dehydrogenase (3-HSD), and cytochrome P450c17 (cyp17a1). Likewise, testosterone production by fetal testes was highly reduced at 125 mg/kg/day, and higher doses. There was also a decrease in the expression of Insulin-like factor-3 (Insl-3), which is essential for gubernacular development and testicular descent. Conclusion: Our results indicate that DnHP disrupts testosterone synthesis and shares target genes with DEHP. Additionally, fetal testicular testosterone production and expression of associated genes appear to be sensitive indicators of testicular response to DnHP in the fetal rat. doi:10.1016/j.reprotox.2011.06.099 Reproductive toxicology and infant immunophenotyping: Normal infant ranges for T-cell assessment Tabor Salewsky a , Tracey Warren a , Cabot Cornwall a , Paul a b a Franklin , Hideshi Tsusaki , Ryoichi Nagata , presented by Brian Bakera,∗ a b
SNBL USA Ltd., Everett, WA, USA Shin Nippon Biomedical Laboratories Ltd., Kagoshima, Japan
Reproductive toxicology is a necessary part of any drug assessment program in order to evaluate toxicological effects on mother and infant. Data is available referencing the mature immune systems which can be used as comparison for data interpretation. However, normal ranges for the developing immune system are not easily obtainable. Normal ranges are vital to the interpretation and final conclusions made for any data set. Without this baseline data conclusions derived may be flawed. Here we have compiled a set of normal infant samples to aid in the evaluation of T-lymphocytes and T-lymphocyte subsets by evaluating CD3, CD4, and CD8 cell surface markers for 82 neonates on date of births ranging from day 25 to day 35. The typical populations of CD3+CD8+ and CD3+CD4+ were assessed, as well as CD3−CD8+, CD3+CD8−, CD3−CD8−, CD3−CD4+, CD3+CD4−, CD3−CD4−. The maximum, minimum, mean, standard deviation, and confidence intervals were calculated for relative percentages. This combined information allows for an accurate assessment of changes on the developing T-lymphocyte population and confidence in conclusions with large variation.
173
approach, i.e. when studies are designed to specifically address significant concerns, either of theoretical nature or demonstrated in adult toxicity studies (when a target organ of toxicity such as the CNS undergoes significant postnatal development). At least the critical stages of development of an organ at risk should be tested. Another approach uses generalized screening tests in juvenile animals, comparable to repeat-dose studies, that may reveal toxicity not identified in adult animal studies. The rationale for using the targeted approach to assess specific concerns comes from scientific, ethical and 3R considerations, as well as business reasons. Using targeted (i.e. case-by-case) instead of general screening designs improves overall study interpretation, can avoid the assessment of irrelevant stages of development and thus the collection of potential meaningless data giving rise to warnings or delays in the approval of urgently needed paediatric medicines. In drug development, specialized and targeted reproduction toxicity studies are used, not broad multi-generation studies. It is for the same reasons that paediatric risk characterization should apply targeted, specially designed studies that deliver appropriate amount of test substance to the juvenile animal model during the critical stages of development. An ILSI expert working group concluded likewise already in 2003. Therefore, studies in juvenile animals to support paediatric development should always be performed on a case-bycase basis and applying case-by-case study design to optimize risk characterization and to minimize predictable, misleading or even inappropriate data. Such truly targeted approach is more likely to be effective in improving overall health of children as stipulated by the paediatric legislation. Only when repeat-dose studies in adult animals are not performed, the screening test approach in juvenile animals has its merit. Evidently, special investigations may need to be included in such cases to properly address concerns, and thus a meaningful routine study design that fits all purposes does not exist. Finally, systems replacing or supplementing in vivo juvenile animal testing have not enough been considered and explored. Some concerns may best be substantiated in targeted experiments using human cells, thus avoiding investigations in juvenile animals afflicted with substantial challenges in translational data interpretation. doi:10.1016/j.reprotox.2011.06.101 Oral prenatal developmental toxicity study with NM-200 synthetic amorphous silica in Wistar rats Steffen Schneider a,∗ , Robert Landsiedel a , Wendel Wohlleben a , André Wolterbeek b , Ine Waalkens-Berendsen b , Han van de Sandt c a
BASF, Ludwigshafen, Germany TNO Triskelion BV, Zeist, The Netherlands c TNO, Zeist, The Netherlands b
doi:10.1016/j.reprotox.2011.06.100 Destination target: A vote for a true case-by-case approach to address concerns in paediatric development Georg Schmitt ∗ , Thomas Singer F. Hoffmann-La Roche Ltd., Non-Clinical Safety, CH-4070 Basel, Switzerland As emphasized by the paediatric legislation, the toxicological assessment of a new medicine may need to include juvenile animal studies. Conducting studies in juvenile, i.e. immature animals should always be considered when existing animal and human safety data are thought insufficient for the safe use in the intended paediatric age groups. The main issue with juvenile animal studies is that the true purpose of these studies has still not been resolved. The most straight forward and relevant use is the targeted
The engineering of nanomaterials offers extraordinary opportunities in various technological fields and safety assessments of new nanomaterials must complement the technological progress. Current test guidelines on developmental and reproductive toxicity are generally able to determine hazards but their specific application to nanomaterials needs to be evaluated. In this project, standard OECD test methods (OECD guidelines 414 and 416) were applied for nanomaterials hazard testing and modifications of these tests will be recommended. The prenatal developmental toxicity study (OECD 414) is complete and the results are presented here. Whereas the two-generation reproduction toxicity study (OECD 416) is ongoing and results will be presented separately. In this study, 25 female Wistar Han rats per group received Synthetic Amorphous Silica (NM-200 supplied by JRC, European Joint Research Centre in Ispra, Italy) by oral gavage at dose levels of 0, 100, 300 and
174
Posters / Reproductive Toxicology 32 (2011) 164–179
1000 mg/kg body weight/day from gestation day (GD) 6 through GD 19. The material was dispersed in 10% foetal bovine serum in water according to the standard dispersion protocol of the JRC. The nanomaterial dispersions were characterized i.a. the particle size distribution was analysed by by cryo-SEM (scanning electron microscopy after shock freezing) and AUC (in situ analytical ultracentrifugation). Food consumption and body weights of the animals were recorded regularly throughout the study. The state of health of the animals was checked each day. On gestation day 20 all females were sacrificed and assessed by gross pathology (including weight determinations of the unopened uterus and the placentas). For each dam, corpora lutea were counted and number and distribution of implants (differentiated as resorptions, live and dead fetuses) were determined. The fetuses were removed from the uterus, sexed, weighed and investigated for external findings. Half of the fetuses of each litter were examined for soft tissue findings and the remaining fetuses for skeletal (inclusive cartilage) findings. In this study we did not observe differences among the various groups for clinical observations, body weights and food consumption of the dams. No effects were observed on number of corpora lutea, implants, pre- and postimplantation losses as well as number and viability of offspring. External, soft tissue and skeletal examination revealed no test-substance-related abnormalities. Acknowledgement: The project is sponsored by CEFIC-LRI (LRIN3 project) and is monitored by Monika Maier, Evonik Degussa GmbH, Hanau, Germany on behalf of CEFIC Sector group on Synthetic Amorphous Silica (ASASP). doi:10.1016/j.reprotox.2011.06.102 Assessing sperm motility in the guinea pig, Cavia porcellus David Schreur, Elise Lewis, Alan Hoberman Charles River Laboratories, Horsham, PA, USA Fertility assessment in regulatory toxicology studies are generally performed using rats or mice. Alternate species that may more closely mimic the metabolic profile of a drug to humans or demonstrate activity similar to man only in that species may be more appropriate. One alternate rodent species is the domestic guinea pig, Cavia porcellus. Accurate computer assisted sperm analysis (CASA) of guinea pig motility has proven difficult because of the inherent nature of the sperm in Cavia species to stack or form rouleaux of cells while developing motility. A computerized sperm analyzer cannot accurately count the number of cells in each rouleaux. Shepard reported that sperm rouleaux in the guinea pig can be dissociated into single cells by incubation in medium containing trypsin [1]. To develop a method to obtain an accurate count of motile and non-motile cells and to calculate the percentage of motile cells in a sperm sample, samples were obtained from the vas deferens and cauda epididymides of domestic guinea pigs, incubated in Ham’s F-10 medium, in Ham’s F-10 medium with 0.5% trypsin added, or 2.5% trypsin in EBSS, and images were taken of the sperm cells in the sample using a Hamilton Thorne IVOS sperm analyzer. It was determined that motile sperm from both the vas deferens and the cauda epididymis did stack into rouleaux and that incubation in medium containing trypsin did not dissociate the rouleaux of motile cells into individual motile cells sufficiently for the Hamilton Thorne IVOS to obtain an accurate count. While the use of CASA for automated determination of sperm motility could not be shown to be accurate, the Hamilton Thorne IVOS is still a useful tool for the determination of this endpoint by providing a quality video image loop of sperm motion that can be used by a trained technician to take accurate manual counts of the motile and non-motile sperm, including those formed into rouleaux, allowing calculation of the percentage of motile cells. One added benefit of
the IVOS system is providing a saved image of the sample to allow for reconstruction of the study and verification of accuracy. Reference [1] Shepard, et al. In vitro studies of Guinea pig spermatozoa in rouleaux. Biol Reprod 1974;11:470–4.
doi:10.1016/j.reprotox.2011.06.103 Dose response analysis of four monophthalates in the murine embryonic stem cell test assessed by cardiomyocyte differentiation and gene expression Sjors H.W. Schulpen a,b,∗ , Dorien van Dartel a , Jeroen Pennings a , Joshua Robinson a,c , Aldert H. Piersma a,c a
Laboratory for Health Effects Research, RIVM, Bilthoven, The Netherlands b Institute for Risk Assessment Sciences, Utrecht University, Utrecht, The Netherlands c Maastricht University, Maastricht, The Netherlands
The embryonic stem cell test (EST) is an alternative test method in which the inhibition of differentiation into beating cardiomyocytes by compound exposure is used as a determinant for developmental toxicity. Application of the test could reduce animal testing in regulatory toxicology if reliability of its predictions can be ascertained. The addition of differential gene expression parameters as an end point in EST might enhance its predictive value. Here we exposed the EST to four monophthalates. Phthalates are used as plasticizers and are known to have an effect on embryo development and on the differentiation of embryonic stem cells towards cardiomyocytes. We studied the dose dependent effects of three embryotoxic phthalates, monobutyl phthalate (MBuP), monobenzyl phthalate (MBeP) and mono-(2-ethylhexyl) phthalate (MEHP) and the non-embryotoxic monomethyl phthalate (MMP). We evaluated effects on beating cardiomyocyte formation, together with a full genome gene expression response. The concentrations of embryotoxic phthalates resulting in 50% inhibition of differentiation (ID50), based on the classical read out of the EST, was between 0.4 an 1.4 mM. In general, gene expression changes were observed already at doses below those causing morphological effects. Transcriptomics analysis showed 668 genes dose-dependently differentially expressed in all embryotoxic phthalates, as compared to the vehicle control. Cluster and pathway analysis illustrated different nuclear, apoptotic and developmental gene set responses. Excluding 66 overlapping genes shared between both the embryotoxic and non-embryotoxic phthalates, these genes gave rise to a common gene signature for embryotoxic phthalates. In addition, compound-specific gene signatures were identified indicating specificity of effects within the chemical category. It can be concluded that transcriptomics leads to a more detailed description of the EST response to compound exposure and can give additional insight into phthalate specific effects on stem cell differentiation. Such mechanistic insight provided by transcriptomics may improve EST readout significance as well as its impact on hazard and risk assessment. doi:10.1016/j.reprotox.2011.06.104