- Email: [email protected]
Negative prediction of chemical carcinogenicity using short-term effects
Abstracts / Toxicology Letters 258S (2016) S62–S324
determine transfer efficiency in order to adjust the dose volume applied to the septum ensuring the appropriate test concentration on the skin. After dose transfer, a standard DA study was performed according to OECD 428 TG. Results from 10 compounds indicated the methodology of creating and transferring DFR onto skin surfaces can be incorporated in a standard OECD 428 TG study. These data also indicated the DA from DFR was lower than that from the equivalent spray. The DA generated with this methodology is relevant for performing/refining the re-entry worker risk assessments for agrochemicals. http://dx.doi.org/10.1016/j.toxlet.2016.06.2048 P22-005 EPA’s methodology to inform TSCA premanufacturing notification decision-making: A critical analysis based on chemicals regulated by consent order R. Borotkanics ∗ , P. Locke Background: Regulatory toxicology has emerged as a necessary discipline to ensure the chemicals introduced into commerce are safe. In the United States, a number of federal laws guide regulatory toxicology, including the Toxic Substances Control Act (TSCA). The New Chemicals program is administered by US Environmental Protection Agency (EPA). This program evaluates premanufacture notifications (PMNs) of new chemicals or significant new uses of existing chemicals. PMN submitters are not required to include human health or environmental effects data, nor are they required to undertake any toxicity testing. These statutory restrictions place constraints on the EPA with respect to the basic data available to inform decisions, and the EPA has only 90 days to make regulatory decisions on PMN substances. Using publically available information about decisions on new chemicals, we sought to determine the methodology used by the TSCA New Chemicals Program, including data proffered or generated, to inform such regulatory decisions; specifically, consent order decisions. Results: The consent order regulatory decisions evaluated in this study applied analog analyses, or in silico methods to inform two-thirds of the decisions. For these decisions, no new in vivo or in vitro tests were carried out by EPA or at the direction of EPA. PMN submissions included testing data about one half of the time, which informed one third of the decisions to regulate by consent order. More broadly, test results submitted by PMN submitters were often not aligned to the health concerns identified by the EPA. Multiple human (3 or more) and environmental health concerns were associated with decisions to regulate. Discussion: Because of the way in which TSCA is written and implemented, the EPA must operate in a data poor information environment. In response, the EPA has developed alternative testing methods, which by definition include non test methods, to inform and assist in PMN regulatory decision making. This demonstrates that TSCA has resulted in the utilization of these alternative methods, demonstrating that laws can provide an opportunity for innovation and efficiencies even if explicit incentives are not designed into the law. TSCA is not the only law to promote such innovation. For instance, the European Commission required the phase out of animal tests in cosmetics. The adoption of alternative testing methods is consistent with the National Academies vision of toxicity testing in the 21st century. This study and findings are important for other regulators in the potential adoption of alternative testing methods to inform regulatory decisions going forward. http://dx.doi.org/10.1016/j.toxlet.2016.06.2049
S307
P22-006 Toxicological risk assessment of micro-organisms used as active substances in plant protection products C. Ochoa-Campuzano 1 , S. Seehase 2,∗ , R. Hauschild 2 , J. Süß 3 , W. Pfau 2 1 GAB Consulting Spain S.L.U., Av. Cortes Valencianas, No. 39, 8B, 46015 Valencia, Spain 2 GAB Consulting GmbH, Ottenbecker Damm 10, 21684 Stade, Germany 3 GAB Consulting GmbH, Heinrich-Fuchs-Str. 94, 69126 Heidelberg, Germany
Plant protection products containing micro-organisms like bacteria, fungi or viruses as active substances are regarded as sustainable and environmentally safe tools to manage pests in agriculture. The pesticidal activity of these naturally occurring micro-organisms is mostly mediated by mechanisms highly specific to the target species, including infection, parasitism or competition for nutrients, which are usually not relevant to humans. In order to ensure a safe use for humans of microbial based plant protection products, a comprehensive toxicological assessment is required. This comprises not only the conduction of studies in vertebrates as for chemical active substances and studies addressing the infectivity and pathogenicity, but also a deep knowledge of the taxonomy, biology, physiology and the mode of action of each particular micro-organism. In addition, the critical assessment of data from the peer-reviewed literature regarding reported cases of infections or allergies as well as on the production of secondary metabolites and toxins is a valuable tool in the evaluation process. Micro-organisms used in plan protection are regulated in the EU according to Regulation (EC) No. 1107/2009 and Regulations (EU) No. 283/2013 and No. 284/2013. Although these regulations have been adapted to better meet the requirements to assess micro-organisms, discussion is ongoing on how to improve the methodology. In this work, we discuss the current testing requirements under EU regulation regarding the unique characteristics of micro-organisms used in plant protection, based on our experience in the approval of microbial strains as active substances and in the registration of microbial biopesticides in the EU. http://dx.doi.org/10.1016/j.toxlet.2016.06.2050 P22-007 Negative prediction of chemical carcinogenicity using short-term effects T. Hill III 1,∗ , M. Nelms 1 , S.W. Edwards 2 , C.E. Wood 2 1
Oak Ridge Institute for Science and Education participant at the National Health and Environmental Effects Research Laboratory, Office of Research and Development, U.S. Environmental Protection Agency (EPA), Research Triangle Park, NC 27709, USA 2 National Health and Environmental Effects Research Laboratory, Office of Research and Development, U.S. Environmental Protection Agency, Research Triangle Park, NC 27709, USA The long-term rodent bioassay has served for decades as a cornerstone for identifying and assessing chemical carcinogens. However, high resource demands make this test unfeasible for the vast majority of environmental chemicals. To help address this issue and improve methods to screen and prioritize compounds for potential carcinogenic risk, here we evaluated the negative predictivity of short-term risk factors for tumor outcomes and
S308
Abstracts / Toxicology Letters 258S (2016) S62–S324
cancer classifications of chemicals previously reviewed by the U.S. EPA. In vivo data from guideline studies were extracted from the U.S. EPA Toxicity Reference Database. Of the 147 compounds with rat subchronic and mouse/rat carcinogenicity study outcomes, 54% had a tumor outcome in at least one species. The presence of subchronic histopathologic or hormonal risk markers yielded a sensitivity (Sn) of 65%, a specificity (Sp) of 54%, and a negative predictive value (NPV) of 57% for any tumor outcome. When tested against cancer classifications requiring quantitative risk assessment (qRA), subchronic effects provided 73% Sn, 38% Sp, and 75% NPV. In comparison, bioactivity in a battery of 42 in vitro cytotoxicity assays from the U.S. EPA ToxCast library provided 57% Sn and Sp and 49% NPV for any tumor outcome and 67% Sn, 56% Sp, and 80% NPV for cancer classifications not requiring qRA. These findings indicate that short-term effects may inform prioritization of chemicals for potential cancer risk, but that integration of additional data streams are needed to improve model performance. This abstract does not necessarily represent the views or policies of the U.S. EPA. http://dx.doi.org/10.1016/j.toxlet.2016.06.2051 P22-008 Neuroprotective role of melatonin in kainic acid induced neurotoxicity: A role of neurotransmitter system and calcium V.K. Mehta ∗ , M. Bhatnagar, R. Chittora
P22-009 Comprehensive assessment of reproductive toxicity of dimoxystrobin: No classification warranted S. Melching Kollmuss, C. Werner, I. Fegert ∗ Basf Se, Ludwigshafen, Germany Dimoxystrobin is a European-registered pesticidal active ingredient. Toxicologically it interferes with the iron transport in rodents, which leads to lower serum iron levels and anemia in rats. Dimoxystrobin is classified with Repr. 2, H361d (suspected of damaging the unborn child). The classification decision for R63 – taken at ECB – was based on impaired offspring body weight development, an assumed higher sensitivity of offspring vs. adult animals and heart effects seen in the 2-generation toxicity study. In a thorough re-evaluation of the data it could be shown that the impaired offspring body weight effects at the high dose of the 2-generation toxicity study only developed postnatally, when the pups are directly exposed to dimoxystrobin-treated diet. Further the cardiomegalies – seen in some 21-day old pups of the high dose – are known to be a secondary effect to anemia and were proven to be reversible. No developmental effects were seen in the rat and rabbit prenatal developmental toxicity study. In two new studies, clear NOAELs were determined in offspring and young rats, which were not lower than the adult NOAELs. Thus it was confirmed, that
ML Sukhadia University, Udaipur, India Nitric oxide (NO) plays a role in the pathophysiology of excitotoxicity. It is also possible that increase in Ca2+ overload and NO-mediated events are involved in neuronal loss during status epilepticus (SE). We investigated the effects of melatonin on NADPH-d positive hippocampal neurons after kainic acid (KA) induced SE in rats. Cytosolic Ca2+ and acetylcholine esterase (AChE) activity in all respective groups was studied. KA was administered, with a single dose of 10 mg/kg/bw. KA treated rats were given melatonin at a dose of 20 mg/kg/bw (for 21 days). On the last day of treatment, animals were transcardially perfused with 4% paraformaldehyde under deep thiopental anesthesia. Cryostat sections (20 m) were cut and stained for NADPH-d and AChE positive neurons. KA exposed animals showed significantly increased number of NO positive neurons in the dorsal and ventral blade of the dentate gyrus (DG), hilus, CA1 and CA3 area of hippocampus, with a parallel increase in intracellular free Ca2+ ion concentration. AChE activity in KA treated animals showed a significant decline in the hippocampal regions. KA +melatonin treated animal groups showed reduced number of NADPH-d positive neurons in DG, hilus, CA1, CA3 areas and a decline in cytosolic Ca2+ concentration and AChE expression, as compared to KA treated group. Our study suggests that the enhanced levels of Ca2+ and nitric oxide play an important role in kainate induced excitotoxicity. Inhibition of NO production may be another means whereby melatonin can reduce oxidative damage and seems to play important role in neuroprotection. http://dx.doi.org/10.1016/j.toxlet.2016.06.2052
- Pups and young rats were not more sensitive than adult animals - Effects on offspring body weight development occurred only after direct dimoxystrobin exposure - Reversible heart effects in offspring are not a developmental effect. Based on a comprehensive re-evaluation of existing and new data of dimoxystrobin it can be concluded that a classification for reproduction toxicity is scientifically not justified and should be reconsidered. http://dx.doi.org/10.1016/j.toxlet.2016.06.2053 P22-010 15-Day intact adult male rat assay with alpha-cypermethrin M. Kemény 1 , H.A. Marxfeld 1 , R. Buesen 1 , A. Chukwudebe 2 , B. Van Ravenzwaay 1 , I. Fegert 1,∗ 1 2
BASF SE, Ludwigshafen, Germany BASF Corporation, Research Triangle Park, USA
Objective: Cypermethrin (8 isomers) and the related betacypermethrin (4 isomers of Cypermethrin) are reported to adversely affect testes and spermatogenesis in the 15-day intact male adult rat assay via a mechanism involving reduced androgen receptor (AR) expression (Hu et al., 2011; Li et al., 2013; Liu et al., 2010). This study investigated the effects of alphacypermethrin (a-cyp; consists of 2 common cypermethrin/betacypermethrin isomers) in a comparable 15-day intact male adult rat assay. Methods: Based on results of a range-finder study and published literature, doses of 0, 2.0, 3.5, and 6.6 mg/kg bw/day a-cyp in corn oil (1 ml/kg bw) were gavaged daily to male Wistar rats (N = 15) for 15 days. Signs of toxicity were monitored daily during treatment. After cessation, sperm parameters, body
determine transfer efficiency in order to adjust the dose volume applied to the septum ensuring the appropriate test concentration on the skin. After dose transfer, a standard DA study was performed according to OECD 428 TG. Results from 10 compounds indicated the methodology of creating and transferring DFR onto skin surfaces can be incorporated in a standard OECD 428 TG study. These data also indicated the DA from DFR was lower than that from the equivalent spray. The DA generated with this methodology is relevant for performing/refining the re-entry worker risk assessments for agrochemicals. http://dx.doi.org/10.1016/j.toxlet.2016.06.2048 P22-005 EPA’s methodology to inform TSCA premanufacturing notification decision-making: A critical analysis based on chemicals regulated by consent order R. Borotkanics ∗ , P. Locke Background: Regulatory toxicology has emerged as a necessary discipline to ensure the chemicals introduced into commerce are safe. In the United States, a number of federal laws guide regulatory toxicology, including the Toxic Substances Control Act (TSCA). The New Chemicals program is administered by US Environmental Protection Agency (EPA). This program evaluates premanufacture notifications (PMNs) of new chemicals or significant new uses of existing chemicals. PMN submitters are not required to include human health or environmental effects data, nor are they required to undertake any toxicity testing. These statutory restrictions place constraints on the EPA with respect to the basic data available to inform decisions, and the EPA has only 90 days to make regulatory decisions on PMN substances. Using publically available information about decisions on new chemicals, we sought to determine the methodology used by the TSCA New Chemicals Program, including data proffered or generated, to inform such regulatory decisions; specifically, consent order decisions. Results: The consent order regulatory decisions evaluated in this study applied analog analyses, or in silico methods to inform two-thirds of the decisions. For these decisions, no new in vivo or in vitro tests were carried out by EPA or at the direction of EPA. PMN submissions included testing data about one half of the time, which informed one third of the decisions to regulate by consent order. More broadly, test results submitted by PMN submitters were often not aligned to the health concerns identified by the EPA. Multiple human (3 or more) and environmental health concerns were associated with decisions to regulate. Discussion: Because of the way in which TSCA is written and implemented, the EPA must operate in a data poor information environment. In response, the EPA has developed alternative testing methods, which by definition include non test methods, to inform and assist in PMN regulatory decision making. This demonstrates that TSCA has resulted in the utilization of these alternative methods, demonstrating that laws can provide an opportunity for innovation and efficiencies even if explicit incentives are not designed into the law. TSCA is not the only law to promote such innovation. For instance, the European Commission required the phase out of animal tests in cosmetics. The adoption of alternative testing methods is consistent with the National Academies vision of toxicity testing in the 21st century. This study and findings are important for other regulators in the potential adoption of alternative testing methods to inform regulatory decisions going forward. http://dx.doi.org/10.1016/j.toxlet.2016.06.2049
S307
P22-006 Toxicological risk assessment of micro-organisms used as active substances in plant protection products C. Ochoa-Campuzano 1 , S. Seehase 2,∗ , R. Hauschild 2 , J. Süß 3 , W. Pfau 2 1 GAB Consulting Spain S.L.U., Av. Cortes Valencianas, No. 39, 8B, 46015 Valencia, Spain 2 GAB Consulting GmbH, Ottenbecker Damm 10, 21684 Stade, Germany 3 GAB Consulting GmbH, Heinrich-Fuchs-Str. 94, 69126 Heidelberg, Germany
Plant protection products containing micro-organisms like bacteria, fungi or viruses as active substances are regarded as sustainable and environmentally safe tools to manage pests in agriculture. The pesticidal activity of these naturally occurring micro-organisms is mostly mediated by mechanisms highly specific to the target species, including infection, parasitism or competition for nutrients, which are usually not relevant to humans. In order to ensure a safe use for humans of microbial based plant protection products, a comprehensive toxicological assessment is required. This comprises not only the conduction of studies in vertebrates as for chemical active substances and studies addressing the infectivity and pathogenicity, but also a deep knowledge of the taxonomy, biology, physiology and the mode of action of each particular micro-organism. In addition, the critical assessment of data from the peer-reviewed literature regarding reported cases of infections or allergies as well as on the production of secondary metabolites and toxins is a valuable tool in the evaluation process. Micro-organisms used in plan protection are regulated in the EU according to Regulation (EC) No. 1107/2009 and Regulations (EU) No. 283/2013 and No. 284/2013. Although these regulations have been adapted to better meet the requirements to assess micro-organisms, discussion is ongoing on how to improve the methodology. In this work, we discuss the current testing requirements under EU regulation regarding the unique characteristics of micro-organisms used in plant protection, based on our experience in the approval of microbial strains as active substances and in the registration of microbial biopesticides in the EU. http://dx.doi.org/10.1016/j.toxlet.2016.06.2050 P22-007 Negative prediction of chemical carcinogenicity using short-term effects T. Hill III 1,∗ , M. Nelms 1 , S.W. Edwards 2 , C.E. Wood 2 1
Oak Ridge Institute for Science and Education participant at the National Health and Environmental Effects Research Laboratory, Office of Research and Development, U.S. Environmental Protection Agency (EPA), Research Triangle Park, NC 27709, USA 2 National Health and Environmental Effects Research Laboratory, Office of Research and Development, U.S. Environmental Protection Agency, Research Triangle Park, NC 27709, USA The long-term rodent bioassay has served for decades as a cornerstone for identifying and assessing chemical carcinogens. However, high resource demands make this test unfeasible for the vast majority of environmental chemicals. To help address this issue and improve methods to screen and prioritize compounds for potential carcinogenic risk, here we evaluated the negative predictivity of short-term risk factors for tumor outcomes and
S308
Abstracts / Toxicology Letters 258S (2016) S62–S324
cancer classifications of chemicals previously reviewed by the U.S. EPA. In vivo data from guideline studies were extracted from the U.S. EPA Toxicity Reference Database. Of the 147 compounds with rat subchronic and mouse/rat carcinogenicity study outcomes, 54% had a tumor outcome in at least one species. The presence of subchronic histopathologic or hormonal risk markers yielded a sensitivity (Sn) of 65%, a specificity (Sp) of 54%, and a negative predictive value (NPV) of 57% for any tumor outcome. When tested against cancer classifications requiring quantitative risk assessment (qRA), subchronic effects provided 73% Sn, 38% Sp, and 75% NPV. In comparison, bioactivity in a battery of 42 in vitro cytotoxicity assays from the U.S. EPA ToxCast library provided 57% Sn and Sp and 49% NPV for any tumor outcome and 67% Sn, 56% Sp, and 80% NPV for cancer classifications not requiring qRA. These findings indicate that short-term effects may inform prioritization of chemicals for potential cancer risk, but that integration of additional data streams are needed to improve model performance. This abstract does not necessarily represent the views or policies of the U.S. EPA. http://dx.doi.org/10.1016/j.toxlet.2016.06.2051 P22-008 Neuroprotective role of melatonin in kainic acid induced neurotoxicity: A role of neurotransmitter system and calcium V.K. Mehta ∗ , M. Bhatnagar, R. Chittora
P22-009 Comprehensive assessment of reproductive toxicity of dimoxystrobin: No classification warranted S. Melching Kollmuss, C. Werner, I. Fegert ∗ Basf Se, Ludwigshafen, Germany Dimoxystrobin is a European-registered pesticidal active ingredient. Toxicologically it interferes with the iron transport in rodents, which leads to lower serum iron levels and anemia in rats. Dimoxystrobin is classified with Repr. 2, H361d (suspected of damaging the unborn child). The classification decision for R63 – taken at ECB – was based on impaired offspring body weight development, an assumed higher sensitivity of offspring vs. adult animals and heart effects seen in the 2-generation toxicity study. In a thorough re-evaluation of the data it could be shown that the impaired offspring body weight effects at the high dose of the 2-generation toxicity study only developed postnatally, when the pups are directly exposed to dimoxystrobin-treated diet. Further the cardiomegalies – seen in some 21-day old pups of the high dose – are known to be a secondary effect to anemia and were proven to be reversible. No developmental effects were seen in the rat and rabbit prenatal developmental toxicity study. In two new studies, clear NOAELs were determined in offspring and young rats, which were not lower than the adult NOAELs. Thus it was confirmed, that
ML Sukhadia University, Udaipur, India Nitric oxide (NO) plays a role in the pathophysiology of excitotoxicity. It is also possible that increase in Ca2+ overload and NO-mediated events are involved in neuronal loss during status epilepticus (SE). We investigated the effects of melatonin on NADPH-d positive hippocampal neurons after kainic acid (KA) induced SE in rats. Cytosolic Ca2+ and acetylcholine esterase (AChE) activity in all respective groups was studied. KA was administered, with a single dose of 10 mg/kg/bw. KA treated rats were given melatonin at a dose of 20 mg/kg/bw (for 21 days). On the last day of treatment, animals were transcardially perfused with 4% paraformaldehyde under deep thiopental anesthesia. Cryostat sections (20 m) were cut and stained for NADPH-d and AChE positive neurons. KA exposed animals showed significantly increased number of NO positive neurons in the dorsal and ventral blade of the dentate gyrus (DG), hilus, CA1 and CA3 area of hippocampus, with a parallel increase in intracellular free Ca2+ ion concentration. AChE activity in KA treated animals showed a significant decline in the hippocampal regions. KA +melatonin treated animal groups showed reduced number of NADPH-d positive neurons in DG, hilus, CA1, CA3 areas and a decline in cytosolic Ca2+ concentration and AChE expression, as compared to KA treated group. Our study suggests that the enhanced levels of Ca2+ and nitric oxide play an important role in kainate induced excitotoxicity. Inhibition of NO production may be another means whereby melatonin can reduce oxidative damage and seems to play important role in neuroprotection. http://dx.doi.org/10.1016/j.toxlet.2016.06.2052
- Pups and young rats were not more sensitive than adult animals - Effects on offspring body weight development occurred only after direct dimoxystrobin exposure - Reversible heart effects in offspring are not a developmental effect. Based on a comprehensive re-evaluation of existing and new data of dimoxystrobin it can be concluded that a classification for reproduction toxicity is scientifically not justified and should be reconsidered. http://dx.doi.org/10.1016/j.toxlet.2016.06.2053 P22-010 15-Day intact adult male rat assay with alpha-cypermethrin M. Kemény 1 , H.A. Marxfeld 1 , R. Buesen 1 , A. Chukwudebe 2 , B. Van Ravenzwaay 1 , I. Fegert 1,∗ 1 2
BASF SE, Ludwigshafen, Germany BASF Corporation, Research Triangle Park, USA
Objective: Cypermethrin (8 isomers) and the related betacypermethrin (4 isomers of Cypermethrin) are reported to adversely affect testes and spermatogenesis in the 15-day intact male adult rat assay via a mechanism involving reduced androgen receptor (AR) expression (Hu et al., 2011; Li et al., 2013; Liu et al., 2010). This study investigated the effects of alphacypermethrin (a-cyp; consists of 2 common cypermethrin/betacypermethrin isomers) in a comparable 15-day intact male adult rat assay. Methods: Based on results of a range-finder study and published literature, doses of 0, 2.0, 3.5, and 6.6 mg/kg bw/day a-cyp in corn oil (1 ml/kg bw) were gavaged daily to male Wistar rats (N = 15) for 15 days. Signs of toxicity were monitored daily during treatment. After cessation, sperm parameters, body