<p> Internal review charge questions - February 2017 AOP Information</p><p> AOP title: AhR activation leading to embryo toxicity  Point of contact author: Dan Villeneuve  Associated wiki page: https://aopwiki.org/aops/21</p><p>Reviewers Primary Reviewer (PR): Name: Kristie Sullivan; OECD Country/Org.: ICAPO; Email: [email protected]</p><p>Secondary reviewer 1 (SR1) Name: Sabina Halappanavar; OECD Country/Org.: Canada; Email: </p><p>Secondary reviewer 2 (SR2) Name: Gladys Ouedraogo; OECD Country/Org.: BIAC; Email:</p><p>Date review completed:</p><p>Review Section 1:</p><p>AOP identifier/Title Does the name of the AOP follow the right convention (MIE or first KE leading to AO)? Does the name of the AOP reflect its content/domain?</p><p>Reviewers' responses and comments PR: Yes, the title is according to the convention; however, there are three other AOPs which start “AhR activation” rather than “Activation of the AhR”. Is there a difference, and is there interest in consistency here? SR1: Yes the title is in accordance with the AOP convention. SR2: The title of this AOP is in line with the AOP convention: a MIE (activation of AHR) leading to an AO (early life stage mortality)</p><p>Author response: Title of the AOP was changed to “AhR activation leading to early life stage mortality” to better conform to other AhR related AOP titles. Section 2:</p><p>Authors Is it clear who the authors/developers of the AOP are? Contact information for one or more corresponding author(s) should be included.</p><p>Reviewers' responses and comments PR: Yes</p><p>SR1: Yes the authors are identified</p><p>SR2: Authors are clearly identified here. Contact for all authors is provided. Are they all corresponding authors? </p><p>Author response: No action taken.</p><p>Section 3:</p><p>Date of updating Reviewer should indicate the date stamp on the PDF snapshot under review.</p><p>Reviewers' responses and comments PR: Feb 10, 2017</p><p>SR1: The last saved document indicates Feb 10th 2017. I am using the document sent to me by email. SR2: The snapshot was created on February 10th, 2017</p><p>Author response: No action taken.</p><p>Section 4:</p><p>Abstract Does the abstract concisely describe the main content of the AOP?</p><p>Reviewers' responses and comments PR: Actually, not all of the Key Events are described in the abstract. I suggest a brief mention of the involvement of COX-2 in the AOP be added into the abstract. SR1: Yes the abstract does describe the AOP in sufficient details describing the sequential KEs leading to AO. SR2: The abstract reflects the well the whole AOP. It summarizes the sequence of events leading to the AO.</p><p>Author response: The abstract was modified to include all KEs with the addition of “This AOP can be initiated by a range of planar aromatic hydrocarbons, but is best known as the target of dioxin-like compounds (DLCs). These planar compounds are able to bind to the AhR causing heterodimerization with the aryl hydrocarbon nuclear translocator (ARNT) and interaction with dioxin-responsive elements on the DNA causing an up-regulation in dioxin responsive genes. One dioxin-responsive gene is cyclooxygenase 2 (COX-2) which has roles in development of the cardiovascular system. Up-regulation in expression of COX-2 causes alteration in cardiovascular development and function which results in reduced heart pumping efficiency, reduced blood flow, and eventual cardiac collapse and death.”</p><p>Section 5:</p><p>Molecular Initiating Event Is a MIE described? If yes, then: Is the MIE description clear and is it biologically plausible? Is the MIE described in a way that allows its use in other AOPs? Are measurement/prediction methods specified and adequately described/referenced? Is the biological context (inc. taxonomic applicability/relevance, level of biological organisation) specified and explained sufficiently? Have chemical initiators (prototypical chemicals or chemical features) been identified?</p><p>Reviewers' responses and comments PR: The MIE is extensively described; while readability may be improved by some text reorganization, that may be difficult to accomplish given that this MIE is being added to by these authors. I appreciate the bulleted text; this facilitates readability and focus on important pieces of information. While the section on measurement is detailed, it is true that there isn’t clear information about the significance of the two AHR isoforms in this section; authors should consider addressing if possible. SR1: The MIE is described. However, it seems like there is a range in terms of levels of activation depending on chemical type and species. For ease of reading, it would be good if the information can be grouped by strong activation and weak activation and not go back and forth. AHR1 and AHR2 isoforms are introduced abruptly and should be described in general early on before they are discussed It seems like Ahr2 is relevant to fish than Ahr1. Should this then be focused on Ahr2? Because there is a lot of information on Ahr1 but not relevant to fish and thus may confuse the readers. A thought to consider. May be later on there is more info on Ahr1 for fish. Stressors are identified Since the background and most of the description for this MIE comes from several species, they all should be included in the taxonomic applicability. Methods are identified. However, as mentioned earlier, it seems like AHR2 is more relevant to fish. However it is not clear if the methods recommended are capable of measuring both Ahr1 and 2 activation. In some places it is specified that the method is measuring Ahr1 activation..</p><p>SR2: The MIE, activation of AHR is well described –a transcription factor with multiple target genes-, references are provided as well as ways to measure this event. This MIE is already used in other AOPs. Biological plausibility and biological context are well described. Stressors are provided, TCDD being one of the most potent activator of AhR. PAHs are a class of AhR activators.</p><p>Author response: In an attempt to improve readability, the title “The molecular mechanism of activation of gene expression by AHR1” was changed to “….AHR”. Further, the title “Roles of isoforms in Birds:” was moved to supersede all discussion of AhRs in birds. Therefore, all information is now taxa and AHR isoform independent up until the title for AhR of birds. Following this title, entries are taxa dependent and described under the heading “Roles of isoforms in birds”, “Roles of isoforms in fishes”, and “Roles of isoforms in amphibians and reptiles”.</p><p>SR1 Since the background and most of the description for this MIE comes from several species, they all should be included in the taxonomic applicability. White sturgeon (Acipenser transmontanus) and Atlantic salmon (Salmo salar) were added to taxonomic applicability for the AOP based on in vitro AhR activation studies with these species. Evidence is listed as moderate. White sturgeon, Atlantic salmon, lake sturgeon (Acipenser fulvescens), red seabream (Pagrus major), African clawed frog (Xenopus laevis), American alligator (Alligator mississippiensis), rainbow trout (Oncorhynchus mykiss), and salamander (Ambystoma mexicanum) were added to the MIE taxonomic applicability based on in vitro AhR activation studies with these species. Evidence is listed as strong.</p><p>SR2 Methods are identified. However, as mentioned earlier, it seems like AHR2 is more relevant to fish. However it is not clear if the methods recommended are capable of measuring both Ahr1 and 2 activation. In some places it is specified that the method is measuring Ahr1 activation.. The methods were clarified to be applicable to all AhRs by the addition of: “For demonstrative purposes, a luciferase reporter gene assay used to measure AHR1-mediated transactivation for avian species is described here. However, comparable assays are utilized for investigating both AHR1s and AHR2s of all taxa.” All AhRs that have been investigated by use of the LRG assay was clarified and listed as: “Luciferase reporter gene (LRG) assay The described luciferase reporter gene (LRG) assays have been used to investigate activation of AhRs of:  Humans (Homo sapiens) (Abnet et al 1999)  Species of birds, namely chicken (Gallus gallus), ring-necked pheasant (Phasianus colchicus), Japanese quail (Coturnix japonica), and common tern (Sterna hirundo) (Farmahin et al 2012; Manning et al 2013), Mutant AhR1s with ligand binding domains resembling those of at least 86 avian species have also been investigated (Farmahin et al 2013). AhR2s of birds have only been investigated in black-footed albatross (Phoebastria nigripes) and common cormorant (Phalacrocorax carbo) (Yasio et al 2007).  American alligator (Alligator mississippiensis) is the only reptile for which AhR activation has been investigated (Oka et al 2016), AhR1A, AhR1B, and AhR2 of American alligator were assayed (Oka et al 2016).  AhR1 of two amphibians have been investigated, namely African clawed frog (Xenopus laevis) and salamander (Ambystoma mexicanum) (Lavine et al 2005; Shoots et al 2015; Ohi et al 2003),  AhR1s and AhR2s of several species of fish have been investigated, namely Atlantic salmon (Salmo salar), Atlantic tomcod (Microgadus tomcod), white sturgeon (Acipenser transmontanus), rainbow trout (Onchorhynchys mykiss), red seabream (Pagrus major), lake sturgeon (Acipenser fulvescens), and zebrafish (Danio rerio) (Andreasen et al 2002; Abnet et al 1999; Bak et al 2013; Doering et al 2014; 2015; Evans et al 2005; Hansson & Hahn 2008; Karchner et al 1999; Tanguay et al 1999; Wirgin et al 2011).”</p><p>Key Events Are the KE descriptions clear on how the events work and are they biologically plausible? Are the KEs described in a way that allows their reuse in other AOPs? Are measurement methods specified and adequately described/referenced? Is the biological context (inc. taxonomic applicability/relevance, level of biological organisation) specified and explained sufficiently? </p><p>Reviewers' responses and comments PR: In general the Key Events are well-described and informative. I agree with the other reviewers that specific KEs could be improved by the suggestions below. Regarding ARNT, in the paragraph that starts “This mechanism…”; the sentence describes killifish AHR1 and AHR2 and zebrafish AHR2 and ARNT2; is that correct? Regarding COX-2, the measurement description could be more informative (species, assay system, etc.). Regarding altered cardiovascular development/function, the authors should describe how these malformations have been measured—there is no section listed here. SR1: Again information regarding the isoforms of ARNT should come before being discussed in the context of KE. Since different isoforms are involved, the methods should clarify if the method is specific to one or the other form of an isoform or measures all of them. The information on COX2 is scarce compared to other key events. Does this mean that this KE is not as well recognized as others and that it may not be essential? The information on the level of biological organization is missing. How is cardiovascular development and function is measured. The table gives a range of endpoints to be measured – are they all equally footed? Do all these endpoints reflective of end mortality? SR2: The keys events of this AOP are clearly described: the dimerization of AhR with ARNT, increase of COX2 expression, altered cardiovascular development. Regarding COX2, it would be informative to have further information on the biological organization: localization in organisms, function and how it is regulated. This may help better understand the sequence between COX2 increased expression and impaired cardiovascular development.</p><p>Author response: PR: Regarding ARNT, in the paragraph that starts “This mechanism…”; the sentence describes killifish AHR1 and AHR2 and zebrafish AHR2 and ARNT2; is that correct? This statement was written by another author as part of another AOP and therefore we are unsure about this statement. PR: Regarding COX-2, the measurement description could be more informative (species, assay system, etc.). Specifics of measurement have been updated to include: “Transcript abundance of COX-2 has been measured in whole embryos of fishes (Dong et al 2010; Teraoka et al 2008; 2014) and embryonic hepatic and cardiac tissue of birds (Fujisawa et al 2014).” PR: Regarding altered cardiovascular development/function, the authors should describe how these malformations have been measured—there is no section listed here. This section was added as: “Altered cardiovascular development/function can be measured in numerous ways: 1) As blood flow in the mesencephalic vein by use of time-lapse recording using a digital video camera (Teraoka et al 2008; 2014). Blood flow is measured as the number of red blood cells passing the mesencephalic vein per second (Teraoka et al 2008; 2014). This method is described in detail by Teraoka et al (2002). However, some studies have assessed blood flow through visualized scoring techniques by use of a microscope as (1) same rate as control, (2) slower rate than control, or (3) no flow (Henry et al 1997). 2) As heart area, pericardial edema area, or yolk sac edema area quantified with area analysis by use of a microscope linked digital camera and conventional image software (Dong et al 2010; Teraoka et al 2008; 2014; Yamauchi et al 2006). Images at the same magnification are used to obtain the area measured as number of pixels (Teraoka et al 2008; 2014). This method can use either live individuals or histologic samples. This method is described in detail by Teraoka et al (2003). 3) As basic physical measurements such as heart weight, heart aspect ratio (horizontal length versus vertical length), heart weight to body weight ratio (Fujisawa et al 2014). 4) As incidence of malformation measured as percent occurrence among individuals (Buckler et al 2015; Dong et al 2010; Park et al 2014; Yamauchi et al 2006). This method is described in detail by Dong et al (2010). 5) As heartbeat rate measured by direct observation by use of a microscope (Park et al 2014). This method is described in detail by Park et al (2014).” SR1: Again information regarding the isoforms of ARNT should come before being discussed in the context of KE. Since different isoforms are involved, the methods should clarify if the method is specific to one or the other form of an isoform or measures all of them. In order to clarify the ARNT page several changes were made: 1) How this Key Event works was modified under two headings A) “Structure and Function of ARNT” which gives background information and lists isoforms of ARNT and their physiological roles. And an addition, B) “Interaction with AHR” which lists isoforms specific potential for heterodimerization with the AHR” that states:  Both ARNT1s and ARNT2s are able to heterodimerize with AhR and interact with dioxin-responsive elements on the DNA in in vitro systems (Hirose et al 1996; Lee et al 2007; Lee et al 2011; Prasch et al 2004).  Selective knockdown of ARNTs in zebrafish (Danio rerio) demonstrates that ARNT1s, but not ARNT2s, are required for activation of the AhR in vivo (Prasch et al 2004; 2006).  In limited investigations ARNT3 has not been demonstrated to interact with the AHR either in vivo or in vitro (Jain et al 1998). These two sections in the How this Key Event Works are followed by text from other authors as part of other AOPs. 2)How it is Measured or Detected was clarified by addition of “AhR/ARNT heterodimerization can be measured in several ways:” and “2) Species-specific differences in dimerization and differences in dimerization between ARNT isoform and AhR isoform combinations have been assessed through luciferase reporter gene (LRG) assays utilizing COS-7 cells transfected with expression constructs of AhR and ARNT isoforms of mammals, birds, and fishes (Abnet et al 1999; Bak et al 2013; Doering et al 2014; 2015; Hansson & Hahn 2008; Hirose et al 1996; Karchner et al 1999; Lee et al 2007; Lee et al 2011; Prasch et al 2004; Wirgin et al 2011). However, this method is indirect as it also includes binding of a ligand to the AhR, and interaction of the AhR/ARNT heterodimer with dioxin-responsive elements on the DNA.” 3) Evidence Supporting Taxonomic Applicability was clarified with addition of “Taxonomic Applicability of Heterodimerization of ARNT isoforms with AhR isoforms:</p><p> In mouse (Mus mus) and chicken (Gallus gallus) both the ARNT1 and ARNT2 were capable of heterdimerizing with AHR and interacting with dioxin-responsive elements on the DNA in vitro (Hirose et al 1996; Lee et al 2007; Lee et al 2011; Prasch et al 2004). However, no studies have yet confirmed involvement of both ARNT1 and ARNT2 in vivo.  In zebrafish, all adverse effects of DLCs so far examined in vivo are mediated solely by ARNT1 based on knockdown studies, although ARNT2 is capable of heterodimerizing with AHR2 and interacting with dioxin-responsive elements on the DNA in vitro (Prasch et al 2004; Prasch et al 2006). In addition to AHRs of zebrafish, AHRs of Atlantic salmon (Salmo salar), Atlantic tomcod (Microgadus tomcod), mummichog, rainbow trout, and red seabream (Pagrus major) have been demonstrated to heterodimerize with ARNT1 in vitro (Abnet et al 1999; Bak et al 2013; Hansson & Hahn 2008; Karchner et al 1999; Wirgin et al 2011), while AHRs of white sturgeon (Acipenser transmontanus), and lake sturgeon (Acipenser fulvescens) have been demonstrated to heterodimerize with ARNT2 in vitro (Doering et al 2014b; 2015b; Prasch et al 2004; 2006). “ SR1: The information on COX2 is scarce compared to other key events. Does this mean that this KE is not as well recognized as others and that it may not be essential? The information on the level of biological organization is missing. SR2: Regarding COX2, it would be informative to have further information on the biological organization: localization in organisms, function and how it is regulated. It is true that less is known about COX-2 relative to AhR and ARNT. An increase in COX- 2 is essential for altered cardiovascular development and function as described in the essentially calls as “Knockdown of COX-2 and selective antagonists of COX-2 prevent TCDD induced alteration in cardiovascular development and function (Dong et al 2010; Teraoka et al 2008; 2014). COX-2 inducers that are not agonists of the AhR cause altered cardiovascular development and function that is consistent with activation of the AhR (Huang et al 2007). Knockdown of and selective antagonists of thromboxane A synthase 1 (CYP5A), which is down-stream of COX-2 in the prostaglandin synthesis pathway, prevents TCDD induced alteration in cardiovascular development and function (Teraoka et al 2008). Exposure to the substrate for COX-2, arachidonic acid, causes an up-regulation in COX-2 and altered cardiovascular development and function that is consistent with exposure to TCDD (Dong et al 2010).” However, despite essentiality the precise mechanism(s) by which COX-2 causes cardiovascular defects is not currently clear despite numerous studies demonstrating roles of COX-2 in proper cardiovascular development. The COX-2 KE page was improved through addition of a figure under How This Key Event Works. Also, significantly more details were added to the page and the page was better organized as: How This Key Event Works: COX Pathway: https://aopwiki.org/system/dragonfly/production/2017/05/08/7vmvnr8r73_COX_pathway. pdf  Prostaglandin-endoperoxide synthase (PTGS; KEGG ID E.C. 1.14.99.1) is an enzyme that has two catalytic sites.  Cyclooxygenase site (COX) catalyzes conversion of arachidonic acid into endoperoxide prostaglandin G2 (Simmons et al 2004).  Peroxidase active site converts PGG2 to PGH2 (KEGG reactions 1599, 1590). PGH2 is a precursor for synthesis of other prostaglandins (PGEs, PGFs), prostacyclin, and thromboxanes (Simmons et al 2004; Botting & Botting 2011).  There are two isoforms, COX-1 and COX-2  COX-2 is inducible by certain chemical exposures, inflammation, during discrete stages of gamete maturation, and more (Green et al 2012).  However, COX biology is complex and important details of the pathway remain unknown (Grosser 2006). COX Cardiovascular Roles:  Prostaglandins which are catalyzed by COX and have roles in cellular homeostasis and in promoting inflammatory responses (Chien et al 2015; Smith et al 2000; Tilley et al 2001; Vane et al 1994).  Significant evidence suggests a link between COX-2 mediated inflammatory responses and progression of alterations in cardiovascular development and function in murine models, humans, and zebrafish (Danio rerio) (Delgado et al 2004; Gullestad & Aukrust 2005; Hocherl et al 2002; Huang et al 2007; Wong et al 1998 ).  However, the precise mechanism by which prostaglandins produce alterations in cardiovascular development have not been clearly elucidated (Hocherl & Dreher 2002). Evidence Supporting Taxonomic Applicability: COX-2 Structure and Function:  There is a high level of conservation of COX-2, as well as its function, especially across vertebrates (Havird et al 2008; 2015), indicating that numerous vertebrate taxa might be susceptible to up-regulation in COX-2.  Typically, teleost fish genomes contain more than one COX-2 gene, likely a result of genome duplication after divergence of teleosts from tetrapods (Ishikawa et al 2007; Havird et al 2015). In zebrafish there are two isoforms, COX-2a and COX-2b (Teraoka et al 2014).  In invertebrates, COX is found in most crustaceans, the majority of molluscs, but only in specific lineages within Cnidaria and Annelida. COX genes are not found in Hemichordata, Echinodermata, or Platyhelminthes. Insecta COX genes lack in homology, but might function as COX enzymes based on structural analyses (Havird et al 2015).</p><p>Adverse Outcome Is an AO described? If yes, then: Is the AO description clear and is it biologically plausible? Is the AO described in a way that allows its use in other AOPs? Are measurement methods specified and adequately described/referenced? Is the biological context (inc. taxonomic applicability/relevance, level of biological organisation) specified and explained sufficiently? Has the regulatory relevance of the AO been described? </p><p>Reviewers' responses and comments PR: The AO is called “Increase, Mortality” but at the individual level, wouldn’t the outcome just be “mortality”? Is there another modifier work which could be considered? I would think an “increase” in mortality would only happen at the population level. Also, there are two AOs in the AOP Diagram which aren’t described (decrease, population trajectory and community composition/food web alteration). Why is this the case? I also found that there is another event (KE351: https://aopwiki.org/events/351) which is named “Increased, mortality” and is used in several other AOPs. Is there a reason for two separate AOs here? And finally, while an extensive description of “mortality” and how it is detected may not be necessary, some basic descriptive text is warranted. SR1: AO has very little information and as such it goes back to the comment I made for the previous KE. SR2: This section needs more information: little is said on the AO, how does it works, how is it detected?</p><p>Author response: PR: I also found that there is another event (KE351: https://aopwiki.org/events/351) which is named “Increased, mortality” and is used in several other AOPs. Is there a reason for two separate AOs here? KE350 has been changed to KE351 at the population level. Also, KE442 has been added (decreased, population trajectory). Corresponding KERs have also been added for these KEs. PR: Also, there are two AOs in the AOP Diagram which aren’t described (decrease, population trajectory and community composition/food web alteration). Why is this the case? A new AOP Diagram has been uploaded which conforms to the modified KEs. PR, SR1, SR2: And finally, while an extensive description of “mortality” and how it is detected may not be necessary, some basic descriptive text is warranted. The AO page for increased mortality (KE351) was completed with applicable text in all sections as: How This Key Event Works Increased mortality refers to an increase in the number of individuals dying in an experimental replicate group or in a population over a specific period of time. How It Is Measured or Detected Mortality is typically measured by observation. Lack of any heart beat, gill movement, and body movement are typical signs of death used in the evaluation of mortality of animals. Evidence Supporting Taxonomic Applicability All living things are susceptible to mortality. Regulatory Examples Using This Adverse Outcome Increased mortality is one of the most common regulatory assessment endpoints, along with reduced growth and reduced reproduction.</p><p>Section 6:</p><p>Key Event Relationships Are the KERs well described and in a way that allows their use in other AOPs? Are the KERs biologically plausible and is there sufficient evidence presented? Is the level of empirical support adequately described in accordance with the OECD AOP Handbook? Are inconsistencies, uncertainties and level of confidence adequately described? Is the quantitative understanding of the KER described?" [refer to Tables 2 & 3 in the handbook]</p><p>Reviewers' responses and comments PR: Regarding the quantitative understanding of the KERs, the authors summarize the literature but do not describe the nature of the quantitative information. Can some of these data, particularly for the Altered Cardiovascular development/ function leads to increase in mortality KER, be described in more detail? SR1: In the KER dimerisation leading to COX2 expression, knockdown studies state that dioxin responsive genes are not upregulated. Was COX2 measured in these experiments? The essentiality of COX2 is quite questionable as not much information is provided regarding the same. Is it possible that multiple dioxin responsive genes are implicated in this KE and not just COX2? Similarly KER between COX2 expression and consequent altered cardiovascular function is not solid. KER with AO is also weak. Essentiality of COX is not strong as stated in the document. SR2: The KERs are discussed in light of their biological plausibility, the biological context, including the empirical support. References are provided. The KER between increase COX2 gene expression and cardiovascular development impairment seems rather weak (current rating is moderate): it has been evidenced only in zebrafish and the authors state that “the precise role that COX2 plays in altered cardiovascular development/function has not been investigated”. In terms of empirical support, the arguments put forward are “pumping efficiency of the hart, measured blood cell perfusion” and “severe hemorrage”. Can this be related to impairment of the endothelial network similar to what is described in AOP150? There may be room for cross-fertilization between this AOP and AOP150.</p><p>Author response: PR: Regarding the quantitative understanding of the KERs, the authors summarize the literature but do not describe the nature of the quantitative information. Can some of these data, particularly for the Altered Cardiovascular development/ function leads to increase in mortality KER, be described in more detail? Although significant quantitative information has been generated linking altered cardiovascular development/function and mortality, no studies have directly correlated these endpoints and quantitative relationships cannot be accurately generated without the raw data. In order to better represent this, quantitative understanding has been changed to weak or moderate for each KER. However, significant quantitative information is available for the indirect link between activation of the AhR and mortality. This KER has been added to the AOP with a quantitative understanding of Strong and the KER page has been completely populated with applicable text and quantitative models as: How Does This Key Event Relationship Work  This Key Event Relationship describes the indirect link between the Molecular Initiating Event (activation of the AhR) and the Adverse Outcome (increased mortality).  Despite decades of research into the molecular initiating event (i.e., binding of chemicals to the AhR) and resulting adverse outcomes (i.e. mortality), less is known about the cascade of key events that link activation of the AhR to the adverse outcome (Doering et al 2016).  Current evidence supports a cyclooxygenase-2 (COX-2) mediated mechanism linking activation of the AhR to early life stage mortality (Dong et al 2010; Teraoka et al 2008; 2014).  However, hundreds to thousands of different genes are regulated, either directly or indirectly, by activation of the AhR, which presents major uncertainties in the precise pathway of key events or whether perturbation to multiple pathways is the cause of mortality (Brinkmann et al 2016; Doering et al 2016; Huang et al 2014; Li et al 2013; Whitehead et al 2010).  Despite these uncertainties in the AOP, considerable research has investigated the indirect relationship between activation of the AhR and increased mortality among different chemicals, species, and taxa (Doering et al 2013). Biological Plausibility The AhR has key functions in critical physiological and developmental processes, including regulation of the cell cycle, cellular proliferation and differentiation, and cell-to- cell communications, angiogenesis, regulation of the immune system, neuronal processes, metabolism, development of the heart and other organ systems, and detoxification (Duncan et al., 1998; Hahn et al 2002; Lahvis and Bradfield, 1998; Emmons et al., 1999). Chemicals that bind to and activate the AhR are known to cause early life stage mortality in vertebrates (Van den Berg et al 1998).</p><p>Empirical Support for Linkage Mammals:  AhR deficient strains of mice (Mus musculus) are unaffected by exposure to agonists of the AhR (Fernandez-Salguero et al 1996).  Strains of mice that express AhRs with lesser affinity for agonists are more tolerant to adverse effects of exposure relative to strains of mice that express AhRs with greater affinity for agonists (Bisson et al 2009; Ema et al 1993). Birds:  Species of birds that are sensitive to adverse effects of exposure to agonists of the AhR express AhR1s with greater affinity for agonists relative to AhR1s expressed by species of birds that are tolerant to adverse effects of exposure to agonists (Karchner et al 2006). Fish:  Knockdown of the AhR2 prevents mortality following exposure to agonist of the AhR in fishes (Clark et al 2010; Hanno et al 2010; Prasch et al 2003; Van Tiem & Di Giulio 2011). Amphibians:  AhR1s of amphibians studied to date are insensitive to activation by dioxin-like compounds in vitro, while amphibians studies to date are extremely tolerant to adverse effects of exposure to dioxin-like compounds in vivo (Jung et al 1997; Lavine et al 2005; Shoots et al 2015). Invertebrates:  Chemicals that activate the AhR of vertebrates are not known to bind AhRs of invertebrates and increased mortality in invertebrates has never been observed as a result of exposure to these agonists (Hahn 2002; Hahn et al 1994). Uncertainties or Inconsistencies Uncertainites:  Only limited AhR activation information is currently available for fishes.  Only limited AhR activation information and mortality information is currently available for reptiles and amphibians. Inconsistencies:  There are no currently known inconsistencies between AhR activation and increased mortality among vertebrates. Quantitative Understanding of the Linkage Mammals: A quantitative model has been developed linking in silico activation of the AhR with acute lethality (measured as dose to cause 50 % lethality; LD50) among 7 species of mammals with an R2 of 0.99 (Wang et al 2013). The model is described in detail by Wang et al (2013). The model is described as: If steric (LJ12-6) < 0 then Log (LD50) = 13.273Log(NOQ) + 5.167Log(-Steric(PLP))- 0.157Log(-steric(LJ12-6))-1.799Log(-(H-bond))-24.625 If steric (LJ12-6) > 0 then Log (LD50) = 13.273Log(NOQ) + 5.167Log(-Steric(PLP)) +0.157Log(-steric(LJ12-6))-1.799Log(-(H-bond))-24.625 Birds: A quantitative model has been developed linking in vitro activation of the AhR1 in transfected COS-7 cells (measured as concentration to cause 50 % effect; EC50) with early life stage mortality (measured as dose to cause 50 % lethality; LD50) among three species of birds across 3 chemicals with an R2 of 0.81 (Farmahin et al 2012). This model has been demonstrated to be applicable across all species of birds and for all dioxin-like compounds (Farmahin et al 2012; 2013; Manning et al 2012). The model is described in detail by Farmahin et al (2012). The model is described as: LD50 = 0.7276*(EC50)+0.2248 Fishes: Limited information is currently available across fishes. However, a quantitative model has been developed linking in vitro activation of the AhR2 alpha in transfected COS-7 cells (meaured as concentration to cause 50 % effect; EC50) with early life stage mortality (measured as dose to cause 50 % lethality; LD50) for rainbow trout (Oncorhynchus mykiss) across 6 chemicals with an R2 of 0.81 (Abnet et al 1999). The model is described in detail by Abnet et al (1999). The model is described as: LD50 = 1.57*(EC50)-0.2418 Amphibians and reptiles: No quantitative models are currently available for amphibians or reptiles. Evidence Supporting Taxonomic Applicability This KER is believed to be applicable to all vertebrates based on mortality as a result of exposure to known agonists of the AhR (Buckler et al 2015; Cohen-Barnhouse et al 2011; Elonen et al 1998; Johnson et al 1998; Jung et al 1997; Kopf & Walker 2009; Park et al 2014; Tillitt et al 2016; Toomey et al 2001; Walker et al 1991; Wang et al 2013; Yamauchi et al 2006; Zabel et al 1995).</p><p>SR1: In the KER dimerisation leading to COX2 expression, knockdown studies state that dioxin responsive genes are not upregulated. Was COX2 measured in these experiments? The essentiality of COX2 is quite questionable as not much information is provided regarding the same. Is it possible that multiple dioxin responsive genes are implicated in this KE and not just COX2? Similarly KER between COX2 expression and consequent altered cardiovascular function is not solid. KER with AO is also weak. 1) Quantitative understanding for “activation, AhR leads to dimerization, AHR/ARNT” has been changed to “Weak” as few studies specifically measure AHR/ARNT dimerization quantitatively. Added to the Quantitative Understanding of Linkage section to read “However, no studies specifically investigate AHR/ARNT dimerization quantitatively despite considerable indirect quantitative information.” 2) Empirical Support for linkage has been clarified by addition of “However, expressions of COX-2 have not yet been investigated following targeted knockdown of either AhR or ARNT1 preventing dimerization and interaction with DREs.” 3) COX-2 to Cardiovascular effects was clarified with empirical support as:</p><p>COX-2 Cardiovascular Development Roles:</p><p> Transgenic mice (Mus musculus) that over express COX-2 display altered cardiac remodeling that results in cardiomyocyte hypertrophy (Streicher et al 2010). However, significantly impaired cardiac function was not observed in the strain of transgenic mice investigated in this study (Streicher et al 2010).  Embryos of zebrafish (Danio rerio) exposed to the COX-2 inducers, aristolochic acid and doxorubicin, develop hypertrophy of the heart, disorganization of cardiomyocytes, and loss of endocardium (Huang et al 2007). These effects result in reduced function of the heart and eventually cause death in zebrafish (Huang et al 2007).  COX-2 has also been demonstrated to have roles in cardiovascular development in humans, chicken (Gallus gallus), and Japanese medaka (Oryzias latipes) (Fujisawa et al 2014; Gullestad & Aukrust 2005; Hocherl et al 2002; Huang et al 2007; Wong et al 1998).</p><p>Section 7:</p><p>Overall Assessment of the AOP Is the domain of applicability of the AOP defined appropriately? Is the level of support for essentiality of the KEs adequately described and assessed? Has the degree of quantitative understanding of KERs been assessed properly? Has consideration been given to the overall weight of evidence for the AOP? Are the calls on Overall WoE and Quantitative Understanding supported?</p><p>Reviewers' responses and comments PR: From the evidence provided in the KE Essentiality section, I find the evidence for a role for COX2 in the cardiotoxicity described to be compelling; I agree that there is uncertainty in the precise mechanistic steps leading from COX2 increase to altered CVD, however for the essentiality assessment specifically, the call by the authors (of strong) follows the Handbook guidance. Inconsistencies and uncertainties are described by the authors. SR1: Already addressed above. For most part the details provided support the essentiality except in the case of COX2 and downstream of COX2. SR2: The authors provide the applicability domain of this AOP; discuss the essentiality of the KEs, the quantitative features of the KERs and a weight of evidence of this AOP. Generally the argument support WoE. There is a weakness in the essentiality of the role of COX2 increased expression in cardiovascular development impairment. Can the author provide more information to support the essentiality of COX2? </p><p>Author response: Additional text has been added to the COX-2 pages as described in prior responses. Additionally, a new page for activation of the AhR leads to increased mortality has been added to the page. Although there are uncertainties in the precise KEs linking activation of the AhR with increased mortality, there is considerable evidence bridging the MIE and AO, and therefore knowing the precise series of KEs linking the MIE to AO might not be necessary beyond current evidence. This has been stated on the main AOP page under uncertainties as: “Despite these uncertainties, the strong, quantitative link between activation of the AhR and early life stage mortality means that elucidating the precise series of key events is less critical. Therefore, the evidence suggesting COX-2 as a primary mechanism might be all that is necessary, although multiple mechanisms acting together is the most likely true mechanism.”</p><p>Section 8:</p><p>Potential application of the AOP (optional): Is any context provided as regards the reason for development or the intended use?</p><p>Reviewers' responses and comments PR: This has not been provided.</p><p>SR1: The reasons for development or intended use is not provided but may be implied.</p><p>SR2: The authors did not address potential application of this AOP.</p><p>Author response: Potential applications was added as “There are great differences in sensitivity to agonists of the AhR among species and among taxa and great differences in potency among agonists of the AhR. Therefore, this AOP has utility towards the mechanistic understanding of adverse effects of agonists of the AhR with regard to cross-chemical, cross-species, and cross-taxa extrapolations. This is particularly true regarding ongoing development of a quantitative AOP.”</p><p>General Observations and Conclusions of the Reviewer</p><p>Reviewers' responses and comments PR: I agree with the other two reviewers that after revisions this AOP will be ready for external review. In addition to reviewer 1 and 2’s suggested revisions, please consider:  Describing quantitative information for KERs in more detail where possible  Using KE 351 rather than KE 350 (for the AO) and providing more information on this KE  Providing more information on the measurement of COX2 and altered cardiovascular development/function  Minor revision of the abstract SR1: This is one of the detailed AOP I have reviewed so far and the AOP itself is very well written. Some issues remain as already discussed above. The Cox2 and downstream of COX2 events are not convincing and need another consideration. SR2: The authors have done a fair amount of work in gathering information to build this AOP. The multiple targets of AhR and its isoforms make this exercise quite complex. I recommend considering this AOP for external review after the following revisions: - Identify contact person(s) - Provide further information on the biological organization of COX2: localization in organisms, function and how it is regulated - Provide more information on essentiality of COX2 in impairment of cardiovascular development - Cross fertilization with AOP150 if deemed appropriate by the authors</p><p>Author response: PR: Describing quantitative information for KERs in more detail where possible Additional quantitative information was added where possible as described in prior responses. This was mostly done for a new KER describing the indirect relationship between the MIE and AO. PR: Using KE 351 rather than KE 350 (for the AO) and providing more information on this KE KE 351 is now part of this AOP as described in a prior response. PR: Providing more information on the measurement of COX2 and altered cardiovascular development/function Significant details on measurement of these KEs has been added as described in prior responses. PR: Minor revision of the abstract All KEs are now discussed in the abstract as described in prior responses. SR1: The Cox2 and downstream of COX2 events are not convincing and need another consideration. As discussed in prior comments, additional details of COX-2 have been included, however there is limited amounts of information on this topic. However, with the addition of the indirect KER between the MIE and AO, the precise series of KEs is less critical and a COX-2/cardiovascular mechanism is proposed as a probable, primary mediator with uncertainties being acknowledged. SR2: Identify contact person(s) Contacts are included. SR2: Provide further information on the biological organization of COX2: localization in organisms, function and how it is regulated All available information on COX-2 has now been included as described in prior responses. However, significant information is currently unavailable. SR2: Provide more information on essentiality of COX2 in impairment of cardiovascular development All available information on COX-2 has now been included as described in prior responses. However, significant information is currently unavailable. SR2: Cross fertilization with AOP150 if deemed appropriate by the authors Cross fertilization is included as appropriate. However, the authors on AOP 150 propose very different KEs which we believe to be weak candidates for KEs leading to mortality.</p>