1

Attachment: TCE Developmental Cardiac Toxicity Ass essment Update

Introduction

**This document is an update on the potential for car diac defects resulting from exposures to** 

**trichloroethylene (TCE), CAS No. 79-01-6.**

Background: **TCE is a volatile chemical and widely used chlorin ated solvent. It is one of the most** 

**common man-made chemicals found in the environment. TCE is frequently found in soil and ground** 

**water at Superfund sites across the country, and it s movement from contaminated ground water and soil** 

**into the indoor air of overlying buildings (i.e., v apor intrusion) is of concern.** 

**EPA completed an IRIS assessment of trichloroethyle ne (TCE) in September 2011**  **U.S. EPA (2011)**

**. After the IRIS document was finalized, some concern**  **s were raised with respect to short-term exposures**

**to TCE and one of the health effects, fetal heart d efects, identified in the IRIS assessment and on wh**  **ich**

**the inhalation reference concentration is partially based. A study by**  **Johnson et al. (2003) , which reports** 

**the results of research on TCE in drinking water, i ncluding the findings of**  **Dawson et al. (1993) , is** 

**included in a group of studies on which the referen ce values are based in the 2011 IRIS assessment, an**  **d**

**is one of several lines of evidence regarding the p otential developmental toxicity of TCE. Concerns ha**  **ve**

**been raised by stakeholders about the Johnson et al. (2003)**  **study and EPA’s use of this study for short**

**term risk evaluation. Specific issues raised includ e the need for 1) a systematic evaluation of study** 

**quality, 2) a detailed description of the study des ign (e.g., the source of concurrent controls), 3) a** 

**reexamination of the dose response for cardiac defe cts, and 4) an evaluation of the study results in l**  **ight**

**of studies that did not observe cardiac defects wit h in utero exposures. In addition, concerns have be**  **en**

**raised regarding the interpretation of the epidemio logical database for cardiac defects associated wit**  **h**

**TCE exposures.**

Purpose: **To address the identified issues and to ensure rig orous scientific review of associations** 

**between short-term exposure to TCE and fetal cardia c defects, EPA decided to update the analysis of th**  **e**

**developmental cardiac toxicity data.**

Scope: **This report covers only the fetal cardiac defects observed following gestational exposures to** 

**TCE and/or its oxidative metabolites dichloroacetic acid (DCA) and trichloroacetic acid (TCA), and** 

**does not provide an update on other developmental e ffects of TCE exposure, i.e., ocular malformations,** 

**developmental neurotoxicity, and developmental immu notoxicity. This update includes 1) identification** 

**of any new literature, 2) a systematic evaluation o f available data, 3) an evaluation of the weight of** 

**evidence for the association of TCE exposures with cardiac defects, 4) a reexamination of the dose** 

**response for cardiac defects, and 5) transparent pr esentation of the evaluation. This process is align**  **ed**

**with the NRC (2011)**  **recommendation for systematic review and weight of evidence evaluation, and** 

**presentation of information to increase transparenc y. A multi-disciplinary team of EPA scientists with** 

**expertise in developmental toxicology or biology, e pidemiology, statistics, molecular biology, risk** 

**assessment, and or TCE toxicology was assembled fro m across ORD to conduct this assessment.** 

Literature Search Update

**A systematic literature search was conducted to ide ntify all studies published subsequent to the final** 

**literature search that had been conducted by EPA du ring completion of the 2011 IRIS assessment (**  **U.S.**

**EPA, 2011 ). A total of 1686 unique citations were initially**  **identified from PubMed, Toxline, and Web**

**of Science (WoS). These citations were screened usi ng the title, abstract, and/or full text for pertin**  **ence**

**to evaluation of the developmental toxicity of TCE, TCA, and DCA exposure. The literature search** 2

**identified no new animal toxicology studies of feta l cardiac defects, one new epidemiology study that** 

**assessed the association of TCE or chlorinated solv ent exposures with cardiac defects, and two studies** 

**that provided mechanistic information relevant to a lterations of cardiac development following TCE (or** 

**metabolite) exposures.**

Study Quality Review

**For each epidemiology and toxicology study in the d evelopmental toxicity database for TCE, whether** 

**previously identified or newly identified in the up dated literature search, a detailed review of study** 

**quality was conducted.**

• Epidemiology data: **Study quality evaluation criteria and a general fo rmat for the capture of** 

**epidemiology study data and characterization have p reviously been developed in IRIS and have** 

**been presented in the** Guidelines for Developmental Toxicity Risk Assessme nt **( U.S. EPA, 1991**  **).**

**These factors include the study power, potential bi as in data collection, selection bias,** 

**measurement biases associated with exposure and out come, and consideration of potential** 

**confounding and effect modification. This format wa s used by a team epidemiologist to** 

**summarize study information and observed strengths, biases, and confounding factors for each** 

**study. An independent review of the conclusions was conducted by the IRIS Epidemiology** 

**Disciplinary Workgroup.**

•

Animal toxicology data: **Study quality evaluation criteria for in vivo, in vitro, and in ovo** 

**developmental toxicology studies were developed. Th ese criteria included considerations** 

**described in U.S. EPA (1991)**  **and focused on the adequacy of study design and do cumentation** 

**of information on the test subjects (e.g., species, strain, source, sex, age/lifestage/embryonic** 

**stage), environment (e.g., husbandry, culture mediu m), test substance (e.g., identification, purity,** 

**analytical confirmation of stability and concentrat ion), treatment (e.g., dose levels, controls,** 

**vehicle, group sizes, duration, route of administra tion), endpoints evaluated (e.g., schedule of** 

**evaluation, randomization and blinding procedures, assessment methods), and reporting (quality** 

**and completeness). An independent assessment of eac h in vivo mammalian study was conducted** 

**by at least 2 separate team members, and all team m embers evaluated study quality for 4 in vivo** 

**studies that included a detailed evaluation of card iac defects (**  **Carney et al., 2006 ;**  **Johnson et al.,**

**2003 ;**  **Fisher et al., 2001 ;**  **Dawson et al., 1993 ). Individual analyses of study strengths and** 

**limitations were collated into a summary table.**

Characterization of developmental (cardiac defect) outcomes

Epidemiology Data

**Six epidemiologic studies are available on cardiac defects and TCE exposure (**  **Ruckart et al., 2013 ;** 

**Forand et al., 2012 ;**  **Yauck et al., 2004 ;**  **Bove, 1996 ;**  **Bove et al., 1995 ;**  **Goldberg et al., 1990 ;**  **Lagakos et**

**al., 1986 ); five of the six studies were reviewed in the EPA**  **’s 2011 Trichloroethylene Toxicological**

**Review ( U.S. EPA, 2011**  **). All studies examined oral exposure except Forand et al. (2012)**  **and Yauck et** 

**al. (2004) who examined the inhalation exposure route.**  **Forand et al. (2012) is the publication of** 

**( ATSDR (2008)**  **; 2006**  **) reports referenced in U.S. EPA (2011)** 

**. Bove (1996)**  **and Bove et al. (1995)** 

**report on the same subjects and identical work. ( Ruckart et al., 2013**  **), a study published after U.S. EPA** 

**(2011) , did not draw any conclusions concerning TCE expos**  **ure and the occurrence of all cardiac defects**

**or conotruncal heart defects and, for this reason, is not discussed below.** 

**Both Forand et al. (2012)**  **and Bove (1996)**  **/ Bove et al. (1995)**  **provide evidence for an association**

**between maternal exposure to TCE or TCE and other c hlorinated solvents in drinking water and cardiac** 3

**defects. There were differences of opinions among t he epidemiology team on**  **Bove (1996) /**  **Bove et al.**

**(1995) due to the few observed TCE-exposed cardiac defect**  **cases, sparse reporting on TCE exposure**

**and cardiac defects in both publications, and the s tudy’s cross-sectional design that could not establ**  **ish**

**temporality. Two other studies with greater potenti al for biases observed elevated risk estimates betw**  **een**

**TCE exposure and cardiac defects ( Yauck et al., 2004**  **; Goldberg et al., 1990**  **) and support observations**

**in Forand et al. (2012)**  **and Bove (1996)**  **/ Bove et al. (1995)**  **. There was no evidence of an association**

**between receiving TCE–contaminated water and cardia c defects in**  **Lagakos et al. (1986) . The finding of** 

**an association between TCE exposure and cardiac def ects has coherence with the broader epidemiologic** 

**literature that observed association between matern al occupational exposure to degreasing solvents or**  **to**

**organic solvents and cardiac defects ( Gilboa et al., 2012**  **; Loffredo et al., 1991**  **; Tikkanen and Heinonen,** 

**1991 ;**  **Tikkanen and Heinonen, 1988 ). Although associations are observed in several st**  **udies, overall, the**

**studies could not establish that the association on TCE exposure and cardiac defects was causal.** 

**Studies were of different populations, living in di fferent states, and of different epidemiologic desi**  **gns.**

**Forand et al. (2012) is a small retrospective cohort study of 1,440 liv**  **e births among New York residents**

**in a TCE contaminated area via vapor intrusion. Bove (1996)**  **/ Bove et al. (1995)**  **, a cross-sectional study**

**on residents in Northern New Jersey receiving TCE i n municipal water supplies, was of 80,938** 

**singleton live-born infants and 594 singleton fetal deaths. A strength of both studies is the use of s**  **tate**

**record bases, including State Birth Defects Registr y with medically-verified outcomes that will reduce** 

**information and subject recall bias, and control fo r potential confounding factors. Both of the studie**  **s**

**observed an elevated risk estimate for major cardia c defects: a risk of 1.24 (50% confidence interval** 

**(CI): 0.75, 1.94) for >10 ppb TCE in municipal drin king water supplies compared to TCE exposure <1** 

**ppb and an estimated risk of 2.40 (95% CI: 1.00, 5. 77) compared to the rest of New York State,** 

**excluding New York City in Bove (1996)**  **/( Bove et al., 1995**  **) and Forand et al. (2012)**  **, respectively.**

**Both studies report risk estimates for specific def ects: 1.30 (50% CI: 0.88, 1.87) for ventricular sep**  **tal**

**defects and exposure to >5 ppb TCE in drinking wate r compared to no exposure**  **Bove (1996) /**  **Bove et al.**

**(1995) and 4.91 (95% CI: 1.58, 15.24) for conotruncal def**  **ect compared to no exposure ( Forand et al.,** 

**2012 ).**  **Yauck et al. (2004) , a small case-control study of 245 cases and 3,780**  **controls, living within 1.32**

**miles from at least one TCE emissions source, which used an insensitive exposure surrogate, observed a** 

**strong relative risk estimate of 6.2 (95% CI: 2.6, 14.5) for cardiac defects in infants born to mother**  **s**

**aged 38 years or older after controlling for potent ial confounding. No association for cardiac defects**  **was**

**observed among infants of mothers aged less than 38 years (RR = 0.9, 95% CI: 0.6, 1.2). The case** 

**control study by Goldberg et al. (1990)**  **used three sets of controls, two of which are subj ect to potential** 

**selection bias, and is limited in reporting. Compar ing offspring of residentially-exposed mother to in**  **fant**

**of non–exposed family, the control group believed t o have a less potential for selection bias, an** 

**unadjusted risk estimate of 2.58 (95% CI: 2.0, 3.4) was reported by**  **Bove et al. (2002)**

**. Neither**  **Forand et al. (2012) nor**  **Bove (1996) /**  **Bove et al. (1995) present exposure-response information,** 

**and the only two studies that do present exposure-r esponse information, do not observe an exposure** 

**response relationship ( Goldberg et al., 1990**  **; Lagakos et al., 1986**  **).**

**In summary, Forand et al. (2012)**  **and Bove (1996)**  **/ Bove et al. (1995)**  **provide evidence for association**

**between maternal TCE exposure and cardiac defects. A more mixed pattern of associations is seen in** 

**three other studies with greater potential for bias and confounding (**  **Yauck et al., 2004 ;**  **Goldberg et al.,**

**1990 ;**  **Lagakos et al., 1986 ); but, the association in these studies is not con**  **sidered inconsistent with**

**Forand et al. (2012) or**  **Bove (1996) /**  **Bove et al. (1995) . For the database as a whole, the epidemiologic** 

**studies are of lower statistical power due to the r arity of cardiac defects. Additionally, information**  **bias**

**of exposure in all studies may provide alternative explanations. As exposure assessment in these studi**  **es**

**is at an aggregate level, one can assume informatio n bias is non-differential. Non-differential** 

**misclassification of exposure would introduce impre cision, resulting in wider confidence intervals** 

**inhibiting the ability to detect some associations and possibly exposure-response relationships. This**  **may**

**be an explanation for the lack of statistically sig nificant findings at the 95% confidence level in**  **Bove**4

**(1996) /**  **Bove et al. (1995) . None of the studies considers maternal folic acid**  **intake, a risk factor for**

**cardiac defects. It is unclear whether maternal fol ic acid intake is a confounder of the associations**  **that**

**were noted since there is no available information to evaluate whether folic acid intake differs relat**  **ed to**

**exposure. However, both Forand et al. (2012)**  **and Bove (1996)**  **/ Bove et al. (1995)**  **adjust or examine**

**maternal risk factors, including adequate prenatal care as potential confounding factors. Observations**  **in**

**the other studies are more uncertain compared to Forand et al. (2012)**  **and Bove (1996)**  **/ Bove et al.** 

**(1995) , and may be due to alternative explanations from b**  **ias, chance, or potential confounding. Use of**

**hospital cases by Yauck et al. (2004)**  **and cases identified from cardiologist’s records b y**  **Goldberg et al.**

**(1990) may introduce possible selection bias. It is diffi**  **cult to evaluate control for potential confounding**

**in Goldberg et al. (1990)**  **due to limited reporting in the publication. The s elf-reporting of outcome in** 

**Lagakos et al. (1986) will introduce uncertainty because of potential se**  **lective reporting.**

Animal Toxicology Data

**The experimental toxicology database for the assess ment of developmental cardiac defects resulting** 

**from TCE exposures includes in ovo chicken studies, in vitro assays, and rodent studies that assessed** 

**fetal morphology following in utero exposures to TC E or its oxidative metabolites.** 

Inhalation rodent and rabbit TCE studies: **Five publications reported the conduct of studies in which** 

**TCE was administered by inhalation exposure to rats , using a prenatal developmental toxicity study** 

**design ( Carney et al., 2006**  **; Healy et al., 1982**  **; Hardin et al., 1981**  **; Dorfmueller et al., 1979**  **; Schwetz et** 

**al., 1975 ). The studies by**  **Hardin et al. (1981) also exposed rabbits to TCE, and the study by**  **Schwetz et**

**al. (1975) also exposed mice. None of these studies reported**  **cardiac defects in fetuses following in utero**

**exposures to TCE; however, of these, only the Carney et al. (2006)**  **and Schwetz et al. (1975)**  **provided**

**sufficient study detail to demonstrate that they we re conducted in accordance with good laboratory** 

**practices and examined the fetuses using methods de signed to detect abnormalities of cardiac** 

**development.**

Oral rodent TCE studies: **Six studies reported the results of oral administr ation of TCE to rodents** 

**during fetal developmental ( Johnson et al., 2003**  **; Fisher et al., 2001**  **; Narotsky and Kavlock, 1995**  **;**

**Narotsky et al., 1995 ;**  **Dawson et al., 1993 ;**  **Cosby and Dukelow, 1992 ). All studies were performed in** 

**rats, except Cosby and Dukelow (1992)**  **which used mice. In all of these rodent studies, T CE was** 

**administered by gavage, with the exception of the Dawson et al. (1993)**  **and Johnson et al. (2003)** 

**studies, in which TCE was administered via drinking water. Only the drinking water studies detected** 

**treatment-related fetal cardiac defects.**

**The gavage studies by Fisher et al. (2001)** 

**, Narotsky et al. (1995)**  **, and Narotsky and Kavlock (1995)** 

**were conducted in accordance with good laboratory p rocedures.**  **Fisher et al. (2001) used the same fetal** 

**evaluation methods as described in Johnson et al. (2003)**  **, and the first author of the Johnson et al. (2003)** 

**paper participated in the Fisher et al. (2001)**  **study, yet TCE-related cardiac defects were not de tected.** 

**The studies by Narotsky et al. (1995)**  **and Narotsky and Kavlock (1995)**  **evaluated neonatal growth and**

**viability, and examined cardiac and other soft tiss ue morphology only in pups that had died; no cardia**  **c**

**defects were reported. The study by Cosby and Dukelow (1992)**  **did not conduct a detailed assessment**

**of cardiac development.**

**The Johnson et al. (2003)**  **publication reported the results of TCE drinking w ater exposures on fetal** 

**cardiac development from a 6-year academic research program. It included data on two TCE treatment** 

**groups conducted in 1989-1991 that had previously b een published by**  **Dawson et al. (1993) , plus the** 

**data from two lower dose TCE treatment groups condu cted in 1994-1995. Cardiac malformation** 

**incidence data were compared between treated groups and combined control data from cohorts** 

**conducted concurrent to treated groups during the 6 -year research program, including controls from** 5

**studies on TCE metabolites, published in Johnson et al. (1998)**  **. Other information on the TCE studies**

**reported in Johnson et al. (2003)**  **included published communications ( Hardin et al., 2004**  **; Johnson et al.,** 

**2004 ), errata (**  **Johnson et al., 2005 )), and individual cardiac malformation findings an**  **d evaluation**

**methods provided to EPA by the study author (Johnso n, personal communications, 2009, 2014).** 

**Johnson et al. (2003) summarized the combined results from the studies t**  **hat administered TCE to**

**pregnant rats at doses of 2.5 ppb, 250 ppb, 1.5 ppm , and 1100 ppm in drinking water throughout** 

**gestation. Fetal cardiac defects, primarily valvula r and septal anomalies, were observed at ≥250 ppb.** 

**The 2011 IRIS assessment noted that there are limit ations in the**  **Johnson et al. (2003) study. The current** 

**evaluation of the Johnson et al. (2003)**  **and Dawson et al. (1993)**  **studies reaffirmed this judgment; study**

**design and reporting issues were identified. The OR D TCE team contacted the study author (P.** 

**Johnson), who provided clarification on a number of topics, i.e., a detailed description of study meth**  **ods**

**beyond what has been previously published, includin g verification that concurrent controls were** 

**conducted for each of the treated groups, informati on on fetal randomization and blinded cardiac** 

**evaluation procedures, and details of animal care a nd maintenance. As a result of discussions with the** 

**study author, an errata was submitted to** Environmental Health Perspectives **( Johnson et al., 2014**  **) to**

**update the public record regarding Johnson et al. (2003)**  **. However, some questions on that study remain**

**unresolved, i.e., the precise dates that each indiv idual control animal was on study and the detailed** 

**results of analytical chemistry testing for dose co ncentration. Additional possible sources of uncerta**  **inty**

**identified for these studies include that the resea rch was conducted over a 6-year period, combined** 

**control data were used for comparison to treated gr oups, and possible imprecision of exposure** 

**characterization due to the use of tap water in the Dawson et al. (1993)**  **study and TCE intake values that**

**were derived from water consumption measures of gro up housed animals. On the other hand, the** 

**strengths of this study include the examination of fetal hearts without knowledge of treatment (or** 

**control) group, standardized methods of fetal evalu ation, examination of the gross (in situ) and inter**  **nal**

**structure of the fetal hearts by a group of 3 senio r researchers, confirmation of cardiac anomalies by** 

**consensus agreement, and that the researchers share d individual fetal and litter cardiac abnormality d**  **ata**

**for treated groups with EPA, thereby facilitating i ndependent statistical analysis of the data.** 

Oral rodent metabolite studies: **Several studies were conducted in rats to examine t he effects of** 

**developmental exposures to the TCE oxidative metabo lites, DCA and TCA. Studies by**  **Smith et al.**

**(1992) and**  **Epstein et al. (1992) observed cardiac defects following gavage administ**  **ration of DCA**

**during pregnancy. Smith et al. (1989)**  **and Johnson et al. (1998)**  **reported cardiac defects with TCA**

**exposures administered during gestation via gavage and drinking water, respectively. However, a well** 

**conducted study by Fisher et al. (2001)**  **did not detect cardiac defects following gavage ad ministration of** 

**DCA or TCA on GD 6-15.**

In ovo avian studies: **Several studies examined cardiac development follo wing in ovo administration of** 

**TCE to chicken embryos ( Rufer et al., 2010**  **; Drake et al., 2006b**  **; Drake et al., 2006a**  **; Loeber et al.,** 

**1988 ). Abnormalities of cardiac structure and/or functi**  **on were observed in each of these studies.**

**Defects in valvuloseptal development were similar t o those that have been observed in rodents and** 

**humans, since early stages of cardiac development a re similar across species (**  **NRC, 2006 ).** 

In vitro assays: **Whole embryo culture studies that examined cardiac development were conducted by** 

**Hunter et al. (1996) using mouse embryos exposed to DCA or TCA and by**  **Mishima et al. (2006) using** 

**chicken embryos exposed to TCE. Alterations in card iac development were observed in each of these** 

**models.**

Summary of animal toxicology data: **Alterations in fetal cardiac development have been observed in** 

**rodent studies following in utero exposure to TCE a nd its oxidative metabolites, DCA and TCA). These** 6

**findings are supported by the detection of cardiac anomalies in chicken embryos exposed to TCE in ovo,** 

**and in whole embryo cultures (mouse and chicken) of TCE and/or its metabolites. In spite of the** 

**concordant evidence that TCE can be associated with cardiac defects, controversy centers on the studie**  **s**

**by Johnson et al. (2003)**  **and Dawson et al. (1993)**  **and that two other well-conducted developmental**

**toxicity studies in rats did not observe treatment- related cardiac defects following gavage or inhalat**  **ion**

**gestational exposures to TCE, i.e., in Fisher et al. (2001)**  **and Carney et al. (2006)**  **, respectively).**

**Detailed examination of the study protocols has ide ntified several differences in study design and** 

**conduct, including but not limited to differences i n route of administration that may have contributed**  **to**

**the variant study outcome. In the case of the Fisher et al. (2001)**  **study, as previously noted, care was**

**taken to follow the Johnson et al. (2003)**  **fetal evaluation procedures as closely as possible , yet a number** 

**of other differences in study design and conduct re mained. For example, the source of the animals, the** 

**route of exposure, the vehicle/control substance, f etal cardiac tissue preservation methods, and some** 

**fetal cardiac evaluation procedures were different. In conclusion, there has not been a confirmation o**  **f**

**the results of the Johnson et al. (2003)**  **and Dawson et al. (1993)**  **studies by another laboratory, but there**

**has also not been a repeat of the exact same study design that would corroborate or refute their findi**  **ngs.**

Mechanistic Information and Adverse Outcome Pathway (AOP) for Cardiac Defects

**A preliminary conceptual model of an AOP for TCE-in duced congenital heart defects was developed,** 

**based upon data identified in a systematic literatu re search as well as mechanistic data that had been** 

**discussed in the 2011 IRIS assessment U.S. EPA (2011)**  **and had provided one line of evidence**

**regarding the potential for TCE to cause cardiac de fects. Although data gaps remain, the information** 

**upon which the preliminary AOP construct is based s upports the biological plausibility that TCE** 

**exposures during development can lead to disruption of key processes in the development of cardiac** 

**valves and septa.**

**Commonly reported cardiac defects, in humans and ro dents, associated with gestational exposures to** 

**TCE and its metabolites TCA and DCA are valvulosept al defects (atrial septal defects [ASDs], muscular** 

**and membranous ventricular septal defects [VSDs]) a nd pulmonary and aortic stenosis (**  **Chiu et al.,**

**2013 ;**  **Forand et al., 2012 ;**  **Yauck et al., 2004 ;**  **Johnson et al., 2003 ;**  **Johnson et al., 1998 ;**  **Bove, 1996 ;** 

**Bove et al., 1995 ;**  **Dawson et al., 1993**

**, 1990**  **; Goldberg et al., 1990**  **; Loeber et al., 1988**  **). The period of**

**valvuloseptal morphogenesis defines a window of vul nerability to TCE and TCA in avian studies; thus** 

**an adverse outcome pathway (AOP) was explored relat ive to this dysmorphology. In normal cardiac** 

**development, valvuloseptal morphogenesis is driven by mesenchymal cells in the regions of the atrio** 

**ventricular canal (AVC) and outflow tract (OFT) reg ions. AVC cushions are formed as mesenchymal** 

**cells are derived from squamosal endothelial cells (i.e., epithelial-mesenchymal transition [EMT] with** 

**an endothelial origin [EndMT]) and invade the cardi ac jelly. These mesenchymal cells proliferate and** 

**differentiate into AV valves and membranous septum, as well as patterning the myocardium and acting** 

**to direct vascular flow. Evidence points to the ori gin of some TCE-related valvuloseptal defects throu**  **gh**

**EndMT involving the following key events in the AVC and OFT cushions (**  **Jensen et al., 2013 ;**  **Kovacic**

**et al., 2012 ;**  **von Gise and Pu, 2012 ):** 

• initiation **of EMT by signal molecules elaborated from myocard ial cells into the cardiac jelly;** 

• disassembly **of cell-cell junctions between squamosal endothelia l cells in the endocardium;** 

• delamination **by loss of polarity, cytoskeletal rearrangement, a nd breakdown of basal lamina;** 

• invasion **of cardiac jelly by newly motile mesenchymal cells;**

• proliferation **of trans-differentiated mesenchyme to ‘cellularize ’ and remodel the cardiac jelly;** 

• patterning **of the AV myocardium by flow-mediated remodeling of the looped heart;** 

• differentiation **of cardiac valves and membranous septum.**7

**Molecular initiating events may involve a cellular initiation of vascular inflammatory signals, perhap**  **s**

**through an LXR/RXR-mediated effect on cholesterol h omeostasis, vulnerability to reactive oxygen** 

**species (ROS) ( Williams et al., 2006**  **; Hassoun et al., 2005**  **; Fisher et al., 2001**  **), or disruption of the**

**downstream consequences of VEGF signaling ( Ou et al., 2003**  **). A search of the Mouse Genome**

**Informatics (MGI) database (http://www.informatics. jax.org/) for abnormalities in cardiac EMT** 

**identified mouse knockouts with phenotypes similar to those reported for avian studies with TCE,** 

**implicating the possibility of disruption of the fo llowing genetic signals and responses by TCE exposu**  **re**

**during cardiac development: the TGF-beta pathway, t he ephrin pathway, the Notch pathway, the VEGF** 

**pathway, and RXR signaling. In support of EndMT bei ng a likely target, TCE exposure in chick** 

**embryos or WECs has been associated with inhibition of cell-cell separation and mesenchymal** 

**formation ( Boyer et al., 2000**  **), alterations in mesenchymal cell migration ( Mishima et al., 2006**  **; Selmin** 

**et al., 2005 ) and alterations in endocardial proliferation patt**  **erns ( Drake et al., 2006b**  **). In ovo studies**

**have shown that TCE and TCA can alter cushion forma tion, cardiac function, and embryo survival** 

**( Drake et al., 2006a**  **), and cushion cellularity can be altered as a func tion of concentration, duration, and** 

**timing of exposure, likely mediated by the ephrin-E PH system. Endocardial disruption may have** 

**additional consequences on the developing heart, re lated to dysregulation of cellular Ca2+ fluxes and** 

**cardiac contractility ( Palbykin et al., 2011**  **; Makwana et al., 2010**  **; Caldwell et al., 2008**  **; Selmin et al.,** 

**2008 ;**  **Collier et al., 2003 ) or to alterations in cardiac hemodynamics (**  **Rufer et al., 2010 ).** 

Weight of Evidence (WOE) Analysis

**The weight of evidence for fetal cardiac defects wa s characterized according to the criteria described**  **in**

A Framework for Assessing Health Risk of Environmen tal Exposures to Children **( U.S. EPA, 2006**  **), a**

**scheme that is based upon principles of causality a ssessment developed by**  **Hill (1965) . The key** 

**components (factors) of the WOE analysis were: temp orality, strength of association, variability** 

**analysis, uncertainty analysis, qualitative dose-re sponse, experimental evidence, reproducibility** 

**(consistency), biological plausibility, alternative or multiple explanations, specificity, and coheren**  **ce.**

**Team members independently assessed the WOE, each p roviding evaluations based upon their expertise,** 

**and arrived at a group consensus of the evidence su pporting stronger and weaker weight of association** 

**for each key factor (Table 2). In summarizing and s ynthesizing the WOE, the team members considered** 

**its application to decisions regarding the potentia l hazard for cardiac defects.** 

**The following question framed the discussion: Does the weight of the evidence for the overall TCE** 

**database support the conclusion that TCE exposure a t sufficient doses during developmental is likely t**  **o**

**cause cardiac defects?**

**The majority of the team members agreed that the ov erall evidence in the TCE database supports a** 

**conclusion that TCE is likely to cause cardiac defe cts at sufficient doses when exposure occurs during**  **a**

**sensitive period of fetal development. This conclus ion was based upon the data that demonstrate or** 

**suggest a potential hazard to cardiac development, including epidemiology studies, developmental** 

**toxicology studies in rodents with TCE and its meta bolites (DCA and TCA), avian in ovo studies, in** 

**vitro assays, and mechanistic data that form the ba sis of a proposed AOP. It is, however, recognized t**  **hat**

**the “likely” descriptor reflects previously describ ed limitations within the database that increase th**  **e**

**uncertainty regarding this conclusion. The epidemio logy studies provide evidence of associations** 

**between TCE, or TCE and other chlorinated solvents, and cardiac defects, but these studies have** 

**limitations related mainly to exposure measurement error and lower statistical power due to the rarity**  **of**

**cardiac defects. The rodent developmental toxicolog y studies conducted by**  **Dawson et al. (1993)**

**, Johnson et al. (2003)**  **, and Johnson et al. (1998)**  **that have reported cardiac defects resulting from TCE** 

**(and metabolite) drinking water exposures have stud y design and reporting limitations. Additionally,** 

**two good quality (GLP) inhalation and gavage rodent studies conducted in other laboratories,**  **Carney et**

**al. (2006) and**  **Fisher et al. (2001) , respectively, have not detected cardiac defects.**  **These limitations and**8

**uncertainties were the basis of the single dissenti ng opinion of a team member regarding whether the** 

**database supports a conclusion that TCE exposures d uring development are likely to cause cardiac** 

**defects.**

Dose–Response Analysis

Suitability of Johnson et al. (2003) study for deriving a point of departure

**Given the hazard conclusion that TCE exposure durin g development is likely to cause cardiac defects,** 

**the next critical issue is the assessment of dose-r esponse. The**  **Johnson et al. (2003) study is the only** 

**available study potentially useable for dose-respon se analysis: it is conducted by a relevant route of** 

**exposure (drinking water), covers the appropriate d evelopmental window (entire period of gestation),** 

**and has multiple exposure levels. A number of the s tudy’s limitations were discussed above, and will n**  **ot**

**be repeated here. Additional issues that bear addit ional discussion relate to the robustness of the do**  **se**

**response relationship and the apparent plateau in t he level of response.** 

**There is compelling evidence of a dose-response tre nd in the**  **Johnson et al. (2003) study, from a highly** 

**significant monotonic increase with dose (P <0.001) based on a Cochran-Armitage trend test and from a** 

**significant trend even if the highest dose is dropp ed (P < 0.04).** 

**The Dawson et al. (1993)**  **study, a subset of controls plus the two highest T CE doses reported in**  **Johnson**

**et al. (2003) , used tap water as a vehicle and drinking water so**  **urce. The data from Dawson et al. (1993)** 

**were considered separately and a significant trend (P < 0.03) was still found.** 

**The TCE data appear to exhibit a plateau in respons e around 10%. Among NTP developmental** 

**toxicology studies that exhibited a significant tre nd (13 of 66 studies tested for trend), none exhibi**  **ted a**

**plateau, but that could be attributable to the narr ow dose spacing (2- to 4-fold) compared with the TC**  **E**

**study (almost 600- fold between the two highest dos es, 0.218 and 129 mg/kg-day). Four of the 13** 

**studies achieved less than 10% maximum response. Fo r most of these dose-response curves it is** 

**impossible to determine whether response is reachin g a plateau at less than 100% response. For all fou**  **r**

**having a low maximum response, there was evidence f or maternal and fetal toxicity at the high doses** 

**and often at intermediate doses.**

**( http://tools.niehs.nih.gov/ntp_tox/index.cfm?fuseac**  **tion=ntpsearch.allchemicalsforstudy&searchterm=D**

**evelopmental ).** 

**On the whole, a majority of the team members agreed that the**  **Johnson et al. (2003) is suitable for use in** 

**deriving a point of departure. The study has an app ropriate design for dose-response analysis in terms**  **of**

**route, duration, and number dose groups. Additional ly, this judgment also took into consideration the** 

**strengths and limitations of the study and uncertai nties identified in the weight of evidence analysis** 

**. Additional support was derived from the finding of**  **a robust, statistically significant dose-response**

**relationships not only for the dataset as a whole, but also for various subsets of the dataset. Althou**  **gh**

**some concern was raised regarding the plateau in th e**  **Johnson et al. (2003) response, its biological** 

**plausibility could not be ruled out based on examin ation of available historical developmental toxicit**  **y**

**datasets.**

Dose-Response Modeling of the Data from Johnson et al. (2003)

**The 2011 TCE assessment applied the nested logistic model to the**  **Johnson et al. (2003) data and found** 

**a BMDL 01**  **of 0.02 mg/kg-day. The benchmark response (BMR) of 0.01 (1%) extra risk was justified by** 

**the severity of the effect. The data were re-analyz ed using this and other BMDS models to evaluate** 

**uncertainty related to model selection and modeling assumptions (**  **http://www.epa.gov/ncea/bmds/ ).** 

**Dose-response modeling of the data from Johnson et al. (2003)**  **identified several sources of uncertainty**

**related to modeling assumptions, presented as quest ions here:** 9

**(1) Does the data have a plateau at less than 100% response? The evidence is equivocal and does not** 

**permit a clear answer. A model with a plateau is pl ausible, but does not alter the general** 

**conclusion and results. [Note: BMDS has only one di chotomous model with a plateau that can be** 

**estimated rather than being fixed at 100%].**

**(2) Is it better to drop or retain the high dose? F or the 2011 TCE assessment (**  **U.S. EPA, 2011 ), the** 

**high dose was dropped on the strength of an examina tion of residuals at the low doses for the** 

**nested model. The decision to drop the high dose is confirmed in this re-examination, using non** 

**nested dichotomous models. Dropping the high dose l eads to higher model goodness of fit and** 

**better fit in the region of the BMD 01**  **and BMD 05** 

**. (3) Are there sufficient data in the low-dose regio**  **n and near the BMD 01**  **to permit reliable inference**

**about the dose-response curve shape (which influenc es the BMD and BMDL)? BMD inference** 

**at the 1% extra-risk level is highly uncertain, bec ause BMD and BMDL values vary by several** 

**orders of magnitude depending on the modeling assum ptions. This is attributed to the apparent** 

**(lack of) steepness of the response curve at low do ses and the lack of additional doses that would** 

**be required to fully specify curve shape in the low dose part of the curve. The data and models** 

**allow more reliable inference for BMD 05**  **and BMD 10** 

**.** Uncertainty in the Point of Departure

**There is substantial model- and parameter uncertain ty at the 1% level of extra risk, although 1% is th**  **e**

**appropriate BMR based on severity of the effect. Th ese uncertainties can be attributed primarily to** 

**having too few data points in the low-dose range, w here more data would be required to adequately** 

**characterize the dose-response shape. Uncertainty d ecreases for higher BMR levels (5% and 10% extra** 

**risk), although 10% exceeds the range of the data f or some models. Thus, a “science-policy” choice may** 

**have to be made about a risk level and/or a POD, ac knowledging the uncertainty involved. Some options** 

**for arriving at a POD include the following:**

• **Use the BMDL 01**  **(0.0207 mg/kg-day) for the nested log-logistic mod el selected in the 2011 TCE** 

**assessment (constrained and without the high dose g roup) because it provides a compromise** 

**value from the range of BMDLs derived from the vari ety of models examined and because** 

**EPA’s science policy practices put forward in the a ssessment still apply.** 

• **Use the 5% BMR for the nested log-logistic model (B MDL**  **05 is 0.108 mg/kg-day) and divide the** 

**resulting BMDL by an adjustment factor to extrapola te to a lower dose that is potentially** 

**protective for a smaller BMR.**

• **Use a model-averaged BMDL 01**  **or BMDL 05**  **for dichotomous models (using data after Rao-Scott**

**transformation to adjust for intra-litter correlati on) with the high dose dropped to achieve better** 

**fit in the low-dose range (BMD 01**  **0.0809 mg/kg-day and BMDL 01**  **0.0225 mg/kg-day, BMD 05**  **0.282 mg/kg-day and BMDL 05**  **0.178 mg/kg-day). (Note that this option yields re sults similar to** 

**that of the modeling approach used in the 2011 TCE assessment.)** 

• **Use the LOAEL/NOAEL approach, although there is als o uncertainty about defining the point of** 

**departure (POD) for this approach. Specifically, ei ther the second highest dose (0.218 mg/kg** 

**day) or the next lower dose (0.048 mg/kg-day) could be defended biologically as a LOAEL** 

**because the apparent extra risk values calculated f rom the observed responses of 2.9% and 2.5%,** 

**respectively, exceed 1%, the level identified as a suitable BMR because of the outcome.** 

**In summary, additional dose-response analyses were performed to characterize the uncertainty in the** 

**point of departure (POD). Alternative PODs were der ived based on use of alternative models, alternativ**  **e**

**benchmark response levels, or alternative procedure s (such as a LOAEL/NOAEL approach), each with** 

**different strengths and limitations. These alternat ives were within about an order of magnitude of the** 

**POD derived in the 2011 TCE assessment.**

**Given the numerous uncertainties in the dose-respon se analysis derived from both the nature of the dat**  **a**

**and from constraints inherent in BMD modeling progr ams and procedures, the majority of team** 10

**members considered the confidence in the point of d eparture based on (**  **Johnson et al., 2003 ) to be** 

**between “low” and “medium.” Overall, however, the t eam members concluded that the point of** 

**departure derived in the 2011 TCE assessment, which used an approach consistent with standard U.S.** 

**EPA dose-response practices, remained a reasonable choice.** 

Summary/Conclusions

**This updated analysis of the potential for exposure to TCE during critical windows of development to** 

**result in cardiac defects was conducted as an indep endent review by expert multidisciplinary EPA** 

**scientists from ORD/NCEA, ORD/NHEERL, and NCCT. Thi s review resulted in: 1) an updated** 

**characterization of uncertainties in the TCE databa se for cardiac defects, 2) documentation of data an**  **d**

**weight of evidence evaluation, 3) an extended chara cterization of uncertainties in the dose-response** 

**modeling, and 4) an expanded consideration of the m echanistic database that may support future** 

**research to develop an adverse outcome pathway for cardiac defects resulting from TCE exposures.** 

**Key conclusions of the team include:**

• **Six epidemiological studies are available on total or specific cardiac defects and TCE exposure;** 

**five of the six studies were reviewed in the EPA 20 11 Trichloroethylene Toxicological Review** 

**( U.S. EPA, 2011**  **). One study did not report on total cardiac defect s and was not included in the** 

**review. All studies have limited sensitivity for de tecting an association between exposure to TCE** 

**and other chlorinated solvents and cardiac defects in offspring. One study did not observe an** 

**association, likely a result of lower sensitivity a nd statistical power. Two studies provide** 

**evidence for an association between maternal exposu re to TCE or TCE and other chlorinated** 

**solvents in drinking water and cardiac defects. Two other studies with greater potential for biases** 

**observed elevated risk estimates between TCE exposu re and cardiac defects in offspring, and** 

**provide supportive evidence.**

• **Alterations in fetal cardiac development have been observed in two rodent studies following in** 

**utero exposure to TCE and in four studies (of three laboratories) following exposure to its** 

**oxidative metabolites, DCA and TCA). These findings are supported by the detection of cardiac** 

**anomalies in three studies (conducted in two labora tories) that exposed chick embryos to TCE in** 

**ovo, and in two whole embryo culture studies (mouse and chicken), conducted in two** 

**laboratories, of TCE and/or its metabolites.**

• **An evaluation of the Johnson et al. (2003)**  **and Dawson et al. (1993)**  **study (and published errata)**

**identified and characterized both strengths and lim itations in the study design and reporting.** 

**Strengths of this study include the examination of fetal hearts without knowledge of treatment** 

**(or control) group, standardized methods of fetal e valuation, examination of the gross (in situ)** 

**and internal structure of the fetal hearts by a gro up of 3 senior researchers, confirmation of** 

**cardiac anomalies by consensus agreement, and that the researchers shared individual fetal and** 

**litter cardiac abnormality data for treated groups with EPA, thereby facilitating independent** 

**statistical analysis of the data. Limitations inclu de reporting deficits and study design issues** 

**(e.g., the study was conducted over a period of 6 y ears, combined control data were used for** 

**comparison to treated groups, and concerns about po ssible imprecision in exposure** 

**characterization)**

• **Two other high quality rodent developmental toxicit y studies did not identify cardiac fetal** 

**defects following exposure to TCE by gavage or inha lation ((**  **Fisher et al., 2001 ) and (**  **Carney et**

**al., 2006 ), respectively). It is not clear why there is a di**  **fference in outcomes between the**

**Johnson et al. (2003) study versus the Fisher and Carney studies, which**  **did not detect cardiac**

**defects. There were differences in study design and conduct, in spite of efforts by**  **Fisher et al.**

**(2001) to minimize such differences, but the team cannot**  **determine from available data the**

**reason that outcomes differed across these studies.**11

• **A preliminary conceptual model of an Adverse Outcom e Pathway (AOP) for TCE-induced** 

**congenital heart defects supports the biological pl ausibility that TCE exposures during** 

**development can lead to disruption of key processes in the development of cardiac valves and** 

**septa. The stage of development for such defects is conserved across species, hence supporting** 

**relevance of the chicken embryo in ovo and mouse an d chicken whole embryo findings.** 

• **A weight of evidence evaluation of the TCE database based on the criteria described in**  A

Framework for Assessing Health Risk of Environmenta l Exposures to Children **( U.S. EPA, 2006**  **)**

**supports a conclusion that TCE is likely to cause c ardiac defects at sufficient doses when** 

**exposure occurs during a sensitive period of fetal development.** 

• **The weight of evidence for developmental cardiac to xicity based on the**  Guidelines for

Developmental Toxicity Risk Assessment **( U.S. EPA, 1991**  **) categorized as “Sufficient**

**Experimental Animal Evidence” and “Limited Human Da ta.”** 

• **The Johnson et al. (2003)**  **study data show a statistically-significant trend with dose.** 

**An expanded dose-response analysis of the Johnson et al. (2003)**  **data using additional models**

**illuminates some of the model and parameter uncerta inty in the derivation of a BMDL to use as a** 

**point of departure; however, the range of results s upports the findings of the 2011 TCE** 

**assessment, which used an approach consistent with EPA science practices.** 

• **A number of different approaches could yield points of departure (PODs) for a 1% extra risk** 

**within about an order of magnitude of the BMDL valu e used in the 2011 IRIS assessment. (The** 

**BMDL in the 2011 IRIS assessment was 0.021 mg/kg-da y using a target BMR of 1%.)** 

• **Based on these conclusions, the hazard and dose-res ponse database for developmental cardiac** 

**toxicity is categorized as “Sufficient Experimental Animal Evidence” and “Limited Human** 

**Data” in accordance with the** Guidelines for Developmental Toxicity Risk Assessme nt **( U.S. EPA,** 

**1991 ).** 

**The database was considered by the majority of team members to be adequate in support of the** 

**categorization of the health-related database for h azard and dose-response (**  **U.S. EPA, 1991 ), with the** 

**determination that there was “Sufficient Experiment al Animal Evidence” and “Limited Human Data”** 

**for developmental cardiac toxicity. According to th e Guidelines, this category “includes data from** 

**experimental animal studies and/or limited human da ta that provide convincing evidence for the** 

**scientific community to judge that a potential for developmental toxicity exists.” The minimum evidenc**  **e**

**that would be necessary to determine whether there is or is not sufficient evidence of developmental** 

**toxicity is the existence of appropriate, well-cond ucted animal study(ies). The overall TCE database m**  **et**

**this criterion, although limitations and uncertaint ies in the primary study used in dose response (**  **Johnson**

**et al., 2003 ) are acknowledged. Those limitations and uncertain**  **ties were the basis of the only dissenting**

**opinion (i.e., of one team member) regarding whethe r the database supports a conclusion that TCE** 

**exposures during development are likely to cause ca rdiac defects.** 

**The team had a range of views as to their confidenc e in the conclusion regarding hazard for cardiac** 

**defects – with three out of nine scientists express ing an opinion concluding the confidence should be** 

**medium to high, and six of nine concluding confiden ce should be “low” or “medium.” These ratings** 

**were influenced by whether the primary focus was on the uncertainties and limitations of the**  **Johnson et**

**al. (2003) study or whether it was on the weight of evidence**  **consideration of the entire database. The**

**2011 IRIS TCE assessment did not assess the confide nce in the hazard for cardiac defects** 

**independently, but judged the confidence in overall developmental toxicity to be “medium-to-high”. It** 

**also stated that the overall weight of evidence sup ported an effect of TCE on cardiac development,** 

**although recognizing that the Johnson et al. (2003)**  **study has “important limitations”.**12

**Overall, taking into account the study’s design, it s strengths and limitations, and uncertainties in t**  **he**

**weight of evidence, a majority of the team members agreed that the**  **Johnson et al. (2003) study was** 

**suitable for use in deriving a point of departure. However, confidence of team members in the dose** 

**response evaluation of the cardiac defect data from the**  **Johnson et al. (2003) study was characterized as** 

**between “low” and “medium” (with 7 of 11 team membe rs rating confidence as “low” and four team** 

**members rating confidence as “low to medium”). None theless, the team members concluded that the** 

**point of departure derived in the 2011 TCE assessme nt, which used an approach consistent with** 

**standard U.S. EPA dose response practices, remained a reasonable choice. The IRIS TCE assessment** 

**( U.S. EPA, 2011**  **) indicated “moderate” confidence in the candidate reference values for developmental** 

**cardiac effects (pp. 5-96 and 5-100) and “high” con fidence for the non-cancer reference values based** 

**upon multiple effects which included the developmen tal cardiac defects (p. 6-43). The majority of the** 

**team agreed that the results of the present analysi s are consistent with the dose-response conclusions**  **of**

**the 2011 IRIS TCE assessment.**

Future Research: **Future research should address the recommendations of the**  **NRC (2006) . Three** 

**general areas might be explored: 1) In vivo studies should be conducted to evaluate the lowest-observe**  **d**

**adverse-effect levels and mode of action for TCE-in duced developmental function and morphology,** 

**gain information on metabolic activation in the avi an model for the evaluation of interspecies** 

**differences and determine the most appropriate spec ies for human modeling, evaluate tissue-specific** 

**concentrations of TCE and its metabolites. Advanced imaging techniques (e.g., ultrasound, micro-CT)** 

**could be utilized. 2) Research is needed to explore linkages between proposed adverse-outcome** 

**pathways and TCE, TCA, and DCA exposure in mammals. Research should be conducted to** 

**characterize susceptible populations, including the influence of genetic polymorphisms or maternal age** 

**as risk factors for TCE-induced adverse development al effects. 3) Additional research is needed to bet**  **ter**

**characterize human exposures and outcomes. An epide miology research program should include the** 

**examination of TCE and other solvents in large case -control studies of congenital malformations that** 

**include cardiac defects, such as the Center for Dis ease Control’s National Birth Defects Prevention** 

**Study ( http://nbdps.org/**  **).**

Team Members:

• **Susan Makris (Team lead), Andrew Hotchkiss, Xabier Arzuaga, Susan Euling, Christina Powers,** 

**Jennifer Jinot, John Fox, Karen Hogan, Cheryl Siege l Scott, Jamie Strong, Weihsueh Chiu** 

**(NCEA, ORD)**

• **Barbara Abbott, Sid Hunter, Michael Narotsky (NHEER L, ORD)** 

• **Thomas Knudsen (NCCT, ORD)**13Table 1. WOE Evaluation of the Potential for Development Exposures to TCE to Result in Cardiac DefectsKey Factora,bType ofevidence toconsiderDataEvidence for strongerweight of associationEvidence for weakerweight of associationCommentsor Null EvidenceTemporality**Timing of exposures and** **response Tox Studies in various species in** **which TCE (or metabolites DCA or TCA) were** **administered during a sensitive period of in utero cardiac** **development resulted in morphological and/or** **functional alterations.**•**Drinking water administration of TCE to rats** **on GD 1-22 resulted in a statistically significant** **treatment-related increase in the incidence of cardiac** **defects ( Johnson et al., 2003** **; Dawson et al., 1993** **).**•**Drinking water administration of TCA (the** **TCE oxidative metabolite) to rats on GD 1-22 resulted in a** **statistically significant treatment-related increase in** **the incidence of cardiac defects (** **Johnson et al., 1998 )** **. Gavage administration of** **TCE metabolites (DCA and TCA) on GD 6-15 (** **Smith et Some in vivo or in vitro studies** **rodent studies in which TCE (or metabolites DCA or TCA) was** **administered during a sensitive period of in utero cardiac** **development resulted in no morphological alterations.** •**Gavage administration of TCE or metabolites (DCA and TCA)** **to rats on GD 6-15 did not result in treatment-related** **cardiac defects ( Fisher et al.,** **2001 ).** •**Inhalation exposures of TCE to rats on GD 6-20 (** **Carney et al., 2006** **) or to rats and mice on GD 6-15 (** **Schwetz et al., 1975 ) did** **not result in treatment-related cardiac defects.** 14Table 1. WOE Evaluation of the Potential for Development Exposures to TCE to Result in Cardiac DefectsKey Factora,bType ofevidence toconsiderDataEvidence for strongerweight of associationEvidence for weakerweight of associationCommentsor Null Evidence**al., 1992 ,** **1989 ) or of DCA** **during discrete windows of time within GD 6-15 (** **Epstein et al., 1992** **) resulted in treatment-related increases in** **the incidences of cardiac defects.** •**Avian in ovo studies that administered TCE or TCA** **during the period of valvuloseptal morphogenesis** **(e.g., HH 15-20) resulted in altered cardiac morphology** **and/or function ( Rufer et al.,** **2010 ;** **Drake et al., 2006a ;** **Loeber et al., 1988 ).** •**A study of DCA exposure to zebra fish (** **Hassoun et al., 2005** **) demonstrated evidence of a disruption in cardiac** **development (pericardial edema and altered heart rate).** •**Mouse whole embryo culture studies of DCA and TCA** **administered at the period of 3-6 somites detected cardiac** **defects ( Hunter et al., 1996** **); a chicken whole embryo** **culture study of TCE administered at HH 13-14** 15Table 1. WOE Evaluation of the Potential for Development Exposures to TCE to Result in Cardiac DefectsKey Factora,bType ofevidence toconsiderDataEvidence for strongerweight of associationEvidence for weakerweight of associationCommentsor Null Evidence**detected alterations in AV cushion (** **Mishima et al., 2006** **).**•**Avian atrioventricular canal cell culture (HH 16) study** **found evidence of inhibited endothelial cell separation** **and early events of mesenchymal cell formation** **in the heart following TCE exposures (** **Boyer et al., 2000** **). Exposure** **occurs before outcomes onset** **Epi**•**Four cohort or case-control studies consider temporality** **( Ruckart et al., 2013** **; Forand** **et al., 2012 ;** **Yauck et al., 2004** **; Goldberg et al., 1990** **). Three studies observe an** **association between the TCE exposure surrogate and major** **cardiac defects ( Forand et al.,** **2012 ;** **Yauck et al., 2004 ;** **Goldberg et al., 1990 ). An** **association with conotruncal defects, specifically, observed** **in Forand et al. (2012)** **.**•**Temporality was not considered in** **Bove (1996) /** **Bove et al. (1995)** **, Goldberg et al. (1990)** **, or** **Lagakos et al. (1986) .** •**The small numbers of conotruncal heart** **defects in Ruckart et** **al. (2013) precluded** **any analysis of this endpoint and TCE** **exposure.**Strength ofassociation**Study quality, including study** **strengths and Tox** •**For Johnson et al. (2003)** **, Dawson et al. (1993)** **, and Johnson et al. (1998)** **, all of**•**For Johnson et al. (2003)** **major study quality limitations** **include the use of data pooled**•**Some studies that reported no cardiac** **defects following**16Table 1. WOE Evaluation of the Potential for Development Exposures to TCE to Result in Cardiac DefectsKey Factora,bType ofevidence toconsiderDataEvidence for strongerweight of associationEvidence for weakerweight of associationCommentsor Null Evidence**limitations which detected cardiac** **malformations, study quality strengths include randomized** **assignment to test group, detailed description of fetal** **cardiac dissection and evaluation procedures,** **evaluation of fetal hearts without knowledge of** **treatment group, and confirmation of all cardiac** **defects by consensus of 3 experts. Statistical analysis** **of data from this study was appropriately conducted by** **EPA statisticians using individual fetal and litter data** **that were provided by the study author.** •**The power of detection in the Johnson et al. (2003)** **study was enhanced by the use of** **historical controls that did not demonstrate a temporal** **shift in cardiac defects. A significant dose related trend** **in cardiac defects was observed even without large** **group sizes.**•**A strong association of from separate study cohorts** **conducted over an approximately 6-year period,** **the use of tap water as the vehicle for some of control and** **treated groups (as reported by Dawson et al. (1993)** **with no characterization of possible** **contaminants and incomplete reporting of study methods and** **results.**•**While Dawson et al. (1993)** **indicated that levels of TCE in dose formulations were tested** **by gas chromatography, the analytical findings were not** **reported. Johnson et al. (2003)** **did not report whether dose formulations were analyzed.** **Further, levels of TCE were not assessed in the vehicle control** **water; therefore, it is plausible that TCE contaminated the** **water and that doses were actually higher than measured.** •**The Dawson et al. (1993)** **and Johnson et al. (2003)** **studies estimated doses based on the** **average water consumption. This method does not provide** **TCE gestational exposures (** **Narotsky and Kavlock, 1995** **; Narotsky et al., 1995** **; Healy et al., 1982** **; Hardin et al., 1981** **) or avian in ovo studies** **( Bross et al., 1983** **; Elovaara et al., 1979** **) did not indicate that** **detailed evaluation of fetal hearts was** **conducted.**•**A rat whole embryo culture study of TCE** **administered at the period of 4-7 somites** **detected no cardiac defects in a study by** **( Saillenfait et al.,** **1995 ); however, the** **study methods indicate that there was** **no evaluation of the embryonic heart.** 17Table 1. WOE Evaluation of the Potential for Development Exposures to TCE to Result in Cardiac DefectsKey Factora,bType ofevidence toconsiderDataEvidence for strongerweight of associationEvidence for weakerweight of associationCommentsor Null Evidence**exposure to response was observed at high dose levels** **in multiple studies that identified cardiac defects. In** **Johnson et al. (2003) there** **was a highly significant positive trend for cardiac** **defects.**•**Potential confounding factors exist in studies that did not** **identify cardiac defects (e.g., different routes of exposure,** **the use of different rodent strains or suppliers across** **studies, and the use of soybean oil as a vehicle in** **Fisher et al. (2001) .** **precise information to calculate TCE dose because variability in** **drinking water consumption among dams is not** **characterized.**•**The dose selection for Johnson** **et al. (2003) resulted in a** **NOAEL that is approximately 700-fold lower than the next** **highest dose.**•**Some studies that did not identify treatment-related** **cardiac defects following developmental exposures to** **TCE, e.g., Carney et al. (2006)** **, Fisher et al. (2001)** **, and Schwetz et al. (1975)** **, were well-conducted and adequately** **reported GLP and/or guideline studies with no substantive** **limitations identified.**•**One study Fisher et al. (2001)** **attempted to replicate the methods used in the** **Johnson et al. (2003)** **study, utilizing the same fetal cardiac dissection** **and evaluation techniques, and including one of the** **Johnson et al. (2003)** **study authors in the assessment team, yet found no** 18Table 1. WOE Evaluation of the Potential for Development Exposures to TCE to Result in Cardiac DefectsKey Factora,bType ofevidence toconsiderDataEvidence for strongerweight of associationEvidence for weakerweight of associationCommentsor Null Evidence**treatment-related cardiac defects.** **Magnitude of the effect** **measure Epi** •**Increased risk estimates between all or major cardiac** **defects ranged from 1.24 (95% CI: 0.75, 1.94) to 2.40** **(95% CI: 1.27, 3.62) observed in 3 studies (** **Forand et al., 2012** **; Bove, 1996** **; Bove et al., 1995** **; Goldberg et** **al., 1990 ). Stronger** **associations, observed with the TCE exposure surrogate** **for conotruncal defects and ventricular septal defects than** **for major cardiac defects, a broader category (** **Forand et al., 2012** **; Bove, 1996** **; Bove** **et al., 1995 ). A fourth study** **observed an increased risk estimate of 6.2 (95% CI: 2.6,** **14.5) for cardiac defects in infants of mothers aged >38** **years and maternal residence within 1.32 miles from at** **least one TCE emissions source (** **Yauck et al., 2004 ).** •**No association in Yauck et al.** **(2004) in mothers <38 years of** **age and maternal residence within 1.32 miles from at least** **one TCE emissions source nor in** **Lagakos et al. (1986) , which** **does not observe an association with cardiac defects.** **Alternative reasons such as lower statistical power may** **explain these observations.**•Variabilityanalysis**Sources of within- and** **Tox**•**Johnson et al. (2003) test** **subject source, husbandry, and randomization** •**The Johnson et al. (2003)** **study reported data from several** **cohorts of animals, which were**•**Based upon the toxicokinetic profile** **of TCE ( U.S. EPA,** 19Table 1. WOE Evaluation of the Potential for Development Exposures to TCE to Result in Cardiac DefectsKey Factora,bType ofevidence toconsiderDataEvidence for strongerweight of associationEvidence for weakerweight of associationCommentsor Null Evidence**cross-study variability that** **contribute to uncertainty** **procedures were consistent across all cohorts, i.e.,** **including Dawson et al.** **(1993) and metabolite studies** **Johnson et al. (2003) . Fetal** **cardiac evaluation methodology, which included** **evaluation without knowledge of treatment** **group and confirmation of all cardiac anomalies by 3 expert** **scientists, was also consistently applied across** **cohorts and studies from the UAZ laboratory. This had** **the result of reducing intra and inter-study variability in** **the assessment.**•**Johnson et al. (2003) reported** **that cardiac defect incidences were consistent across all** **control cohorts (55 litters over approximately 6 years).** **An EPA review of the available control data did not** **observe unusual heterogeneity in prevalence** **of malformations.**•**Studies that reported cardiac defects following** **on study over a period of approximately 6 years. The** **data included control cohorts, some of which were concurrent** **and some that were non concurrent to the TCE-treated** **groups ( Johnson et al., 2014** **, 2005** **). Data that definitively link the individual control litter** **response data with each particular cohort are no longer** **available for independent examination.** •**Different study outcomes were observed in studies that had** **many similarities in study design and conduct, i.e.,** **Dawson et al. (1993) and** **Johnson et al. (2003) identified** **exposure related cardiac defects while** **Fisher et al. (2001) did** **not. In the Fisher et al. (2001)** **study, care was taken to ensure that the same cardiac evaluation** **methods were used as in the Dawson et al. (1993)** **and Johnson et al. (2003)** **studies, including fetal evaluation with** **knowledge of treatment group, and one of the study authors of** **2011 ), it is considered** **unlikely that toxicokinetic factors** **contributed significantly to** **differences in response across study** **protocols.**20Table 1. WOE Evaluation of the Potential for Development Exposures to TCE to Result in Cardiac DefectsKey Factora,bType ofevidence toconsiderDataEvidence for strongerweight of associationEvidence for weakerweight of associationCommentsor Null Evidence**administration of metabolites (DCA and TCA) used** **randomized assignment of maternal animals to test** **group, thus reducing intra study variability.** •**Although Dawson et al.** **(1993) and** **Johnson et al. (2003)** **identified cardiac defects following exposures** **to TCE during development, Fisher et al. (2001)** **, Carney et** **al. (2006) , and** **Schwetz et al. (1975)** **did not find treatment related cardiac abnormalities.** **This may be the result of differences in the study** **design and assessment methods. This includes such** **aspects as animal strain, age, source, exposure route and** **vehicle, duration of exposure, and cardiac evaluation** **methods. Johnson et al. (2003)** **participated in the fetal examination.** •**The use of soy bean oil in the Fisher et al. (2001)** **study vs. water vehicle and control for** **Johnson et al. (2003) and** **Dawson et al. (1993) studies.** •**The Johnson et al. (2003)** **and Dawson et al. (1993)** **studies did not calculate variability in TCE** **dose by measuring individual dam water consumption.** **Sources of within- and** **cross-study variability that** **contribute to Epi** •**NE (not considered in Hill analysis)** •**NE (not considered in Hill analysis)** •**Studies examined different populations,** **exposure levels, gradients, and media.** **Additionally, different sets of** 21Table 1. WOE Evaluation of the Potential for Development Exposures to TCE to Result in Cardiac DefectsKey Factora,bType ofevidence toconsiderDataEvidence for strongerweight of associationEvidence for weakerweight of associationCommentsor Null Evidence**uncertainty strengths and** **uncertainties in this set of studies would** **contribute to observed cross-study** **variability.**Uncertaintyanalysis**Missing information or** **data gaps, within and** **across studies Tox** •**For the studies conducted by the UAZ laboratory (** **Johnson et al., 2003** **; Johnson et al.,** **1998 ;** **Dawson et al., 1993 )** **that identified cardiac defects following exposures to TCE,** **DCA, or TCA, detailed descriptions of evaluation** **methods for assessment of cardiovascular effects were** **provided.**•**Individual fetal and litter cardiac findings data, as well** **as detailed information on study conduct and fetal** **evaluation methods, were provided to the EPA for** **( Dawson et al., 1993** **) and (** **Johnson et al., 2003 ).** •**The publications for studies conducted by the UAZ** **laboratory that identified cardiac defects following** **exposures to TCE, DCA, or TCA (** **Johnson et al., 2003 ;** **Johnson et al., 1998 ;** **Dawson et al., 1993** **) did not report essential study details, and** **generally did not include summaries of maternal data or** **fetal data for endpoints other than cardiac defects.** •**For well-conducted studies that did not detect cardiac defects** **following developmental exposures to TCE or** **metabolites ( Carney et al.,** **2006 ;** **Fisher et al., 2001 )** **adequate descriptions of study methodology and summary data** **for maternal and fetal findings were reported.** •**Mechanistic data for alterations**•22Table 1. WOE Evaluation of the Potential for Development Exposures to TCE to Result in Cardiac DefectsKey Factora,bType ofevidence toconsiderDataEvidence for strongerweight of associationEvidence for weakerweight of associationCommentsor Null Evidence**in cardiac development are limited and do not identify** **initiating events for the putative AOP.** **Missing information or** **data gaps, within and** **across studies Epi** •**NE (not considered in Hill analysis)** •**NE (not considered in Hill analysis)** •Qualitativedose-response**Association between** **exposure/dose and degree of** **effect Tox** •**Alterations in cardiac development were observed** **in multiple studies at high dose levels following TCE,** **DCA, or TCA exposures (** **Johnson et al., 2003 ;** **Johnson et al., 1998 ;** **Dawson et al., 1993** **; Smith et al.,** **1992 ,** **1989 ).** •**The incidence of cardiovascular effects** **increased as a function of dose in** **Johnson et al. (2003) .** •**An association between exposure to TCE (or DCA or** **TCA) and alterations in cardiac development was** **reported in various animal models, i.e., LE and SD rats,** •**The dose response for cardiac defects identified by** **Johnson et al. (2003)** **could only be fit to a model with elimination of the** **high dose data from the analysis. The lowest dose tested** **had a zero response for cardiac defects, below the historical** **control incidence. The doses tested were spaced over several** **orders of magnitude, with wide gaps.** •**Carney et al. (2006) was the** **only other study in the database that evaluated developmental** **effects of TCE over multiple dose levels. In that study, no** **fetal toxicity and minimal maternal toxicity was reported.** •**TCE doses tested in Dawson et al. (1993)** **and Johnson et al.** **(2003) (drinking** **water): 2.5 ppb, 250 ppb, 1.5 ppm, or 1100** **ppm (0, 0.00045, 0.048, 0.218, or** **129 mg/kg-day)**•**TCE doses tested in Fisher et al. (2001)** **(gavage): 500 mg/kg day** •**TCE doses tested in Carney et al. (2006)** **(inhalation): 50, 150, or 600 ppm (268.5,** **805.5, or 3222 mg/m 3** **)**23Table 1. WOE Evaluation of the Potential for Development Exposures to TCE to Result in Cardiac DefectsKey Factora,bType ofevidence toconsiderDataEvidence for strongerweight of associationEvidence for weakerweight of associationCommentsor Null Evidence**CD-1 mice, chicken embryos, and zebra fish (** **Drake et al., 2006b** **; Drake et al., 2006a** **; Williams et al., 2006** **; Hassoun et al., 2005** **; Johnson** **et al., 2003 ;** **Dawson et al., 1993** **; Smith et al., 1992** **, 1989** **).**•**A BMDL for Johnson et al.** **(2003) was derived by EPA** **statisticians from individual cardiac defect data provided** **to EPA. Litter contribution to the outcome of interest was** **incorporated in the analysis. A significant dose-response** **trend was identified, whether or not the high dose value** **was included in the analysis. Exposure** **response gradient:** **Association between** **exposure/dose and degree of** **effect Epi** •**NE**•**Goldberg et al. (1990) and** **Lagakos et al. (1986) examined** **exposure-response; none observed.** •Experimental**Hypothesis Tox**•**A study by ( Epstein et al.,** •**Studies in rodents that**•**Studies that**24Table 1. WOE Evaluation of the Potential for Development Exposures to TCE to Result in Cardiac DefectsKey Factora,bType ofevidence toconsiderDataEvidence for strongerweight of associationEvidence for weakerweight of associationCommentsor Null Evidenceevidence**testing: manipulation of** **exposure scenario with** **resulting alterations in** **response 1992** **) administered the metabolite DCA to rats on** **varied days of gestation and identified critical windows of** **exposure for eliciting cardiac developmental defects.** •**No statistically significant increases in congenital heart** **defects were observed in groups of rats that were** **exposed to TCE prior to pregnancy only (** **Dawson et al., 1993** **).**•**( Drake et al., 2006b** **) demonstrated that cardiac** **defects did not occur in chick embryos exposed to TCE and** **TCA during the period of cardiac specification** **(approximately GD 6 in rats) rather than the period of** **valvuloseptal morphogenesis. administered TCE via drinking** **water detected an increase in fetuses with cardiac defects** **( Johnson et al., 2003** **; Dawson** **et al., 1993 ); studies that** **administered TCE via other routes (gavage and inhalation)** **were negative for this response(** **Carney et al., 2006 ;** **Fisher et al., 2001 ;** **Schwetz et al., 1975** **).**•**In a whole embryo culture (WEC) study of DCA and TCA** **( Hunter et al., 1996** **), that identified cardiac defects, the** **acid nature of DCA and TCA may have impacted** **dysmorphogenesis. manipulated the** **gestational exposure period were not** **conducted with TCE. Association not** **observed once exposure ceases** **Epi**•**NE**•**No differences between observed and expected numbers** **of cardiac defect cases once wells were closed in** **contaminated area ( Goldberg et** **al., 1990 ).** •Reproducibility**Reproducibility: Tox**•**Studies that administered**•**Studies conducted in other**•**Studies that did not**25Table 1. WOE Evaluation of the Potential for Development Exposures to TCE to Result in Cardiac DefectsKey Factora,bType ofevidence toconsiderDataEvidence for strongerweight of associationEvidence for weakerweight of associationCommentsor Null Evidence[Consistency]**Corroboration across studies,** **labs, routes of exposure,** **species, etc. TCE in drinking water to rats** **on GD 1-22 were conducted over a period of** **approximately 6 years by researchers at the same** **academic facility (UAZ, Tucson) used the same** **cardiac evaluation methods and identified treatment and** **dose-related cardiac malformations (** **Johnson et al., 2003** **; Johnson et al.,** **1998 ;** **Dawson et al., 1993 ). A** **preliminary screening study that utilized intrauterine** **administration of TCE also detected cardiac defects** **( Dawson et al., 1990** **). The types of cardiac** **malformations observed were similar across study cohorts** **and treatment groups throughout the duration of the** **research program.**•**Studies on TCE metabolites (TCA and TCA) conducted in** **other laboratories ( Epstein et** **al., 1992 ;** **Smith et al., 1992 ,** **1989 ) identified cardiac** **defects similar to those laboratories than UAZ and that** **administered TCE by gavage or inhalation (** **Carney et al., 2006 ;** **Fisher et al., 2001 ;** **Schwetz et al., 1975** **) did not identify statistically significant increases** **in cardiac defects. Fisher et al.** **(2001) used the same cardiac** **evaluation methods as the UAZ lab.** **identify cardiac defects with TCE** **and/or metabolite exposures (** **Carney et al., 2006** **; Fisher et al.,** **2001 ;** **Schwetz et al., 1975** **) did not replicate all aspects of the** **Johnson et al. (2003) study, even though** **Fisher et al. (2001) used the same cardiac** **evaluation techniques as (** **Johnson et al., 2003** **) and Dawson et** **al. (1993) , and** **therefore provide only limited evidence** **of lack of reproducibility.** 26Table 1. WOE Evaluation of the Potential for Development Exposures to TCE to Result in Cardiac DefectsKey Factora,bType ofevidence toconsiderDataEvidence for strongerweight of associationEvidence for weakerweight of associationCommentsor Null Evidence**observed in the UAZ studies.**•**Cardiac septal anomalies were observed in avian in ovo** **studies ( Rufer et al., 2010** **; Drake et al., 2006a** **), and in WEC assays (** **Mishima et al., 2006** **; Hunter et al., 1996** **) with TCE and/or metabolite** **exposures. Zebrafish studies also demonstrated evidence** **of alterations in cardiac development (** **Williams et al., 2006** **; Hassoun et al., 2005** **). Consistency:** **Association observed in** **different populations,** **places, time and circumstances.** **Epi**•**Association between cardiac defects and TCE exposure** **surrogate observed in four studies. These studies were** **of different populations living in different state (NY,** **NJ) and covered slightly different time period (1983-** **2000, 1985-1988) ( Forand et** **al., 2012 ;** **Bove, 1996 ;** **Bove et al., 1995** **). Two other studies of weaker designs** **were of different populations and carried out in two** **different locations in the United States, and provide** **supporting evidence ( Yauck** •**Lagakos et al. (1986) compared** **a pregnancy receiving contaminated residential well** **water to a pregnancy not receiving residential water from** **contaminated wells and does not observed an association** **between cardiac defects and contaminated drinking water.** •27Table 1. WOE Evaluation of the Potential for Development Exposures to TCE to Result in Cardiac DefectsKey Factora,bType ofevidence toconsiderDataEvidence for strongerweight of associationEvidence for weakerweight of associationCommentsor Null Evidence**et al., 2004 ;** **Goldberg et al., 1990** **).**Biologicalplausibility**Observed outcome can be** **attributed to toxic insult** **given the known science** **Tox**•**Avian in ovo studies and atrioventricular cell culture** **studies support the biological plausibility of effects of TCE** **on cardiac development, given that early chick heart** **development is similar to mammalian (including** **human), particularly regarding the role of the** **cardiac cushion in septation (** **NRC, 2006 ).** •**Preliminary exploration of a possible adverse outcome** **pathway (AOP) has resulted in a reasonable conceptual** **model for TCE-induced congenital heart defects. In** **this construct, the vulnerable period is defined by** **endocardial morphogenesis. Endothelial–mesenchyme** **transition is disrupted in the area of the atrioventricular** **canal, leading to septal defects. Possible genetic** **contributions to abnormal cardiac development include** •**A definitive AOP for TCE induced cardiac defects,** **including a putative initiating event, has not yet been** **characterized. Additional mechanistic data are needed to** **support the hypothesized AOP.**•**There are insufficient mechanistic data to characterize** **additional potential MOAs other than that hypothesized in** **the AOP.**•**It is possible that multiple modes of** **action are involved in alterations to cardiac** **development.**28Table 1. WOE Evaluation of the Potential for Development Exposures to TCE to Result in Cardiac DefectsKey Factora,bType ofevidence toconsiderDataEvidence for strongerweight of associationEvidence for weakerweight of associationCommentsor Null Evidence**disruption of TGF-beta pathway, endrin pathway,** **Notch pathway, VEGF pathway, and RXR signaling.** **At a cellular level, epithelial mesenchymal transition may** **be affected in the endocardium, at the tissue** **level, there is altered cellularity of the endocardial** **cushion, and secondary effects such as dysregulation** **of cellular Ca 2+** **fluxes may result in additional impacts** **on the developing heart. Observed** **association plausible given** **the known science** **Epi**•**NE**•**NE**•**In vitro and in vivo animal studies report** **cardiac defects with TCE and TCE** **metabolite exposure.**Alternative ormultipleexplanations**Other possible explanations for** **observed outcome after** **the exposure of interest** **Tox**•**Given the presumed contribution of both** **environmental exposures and genetic predisposition in** **human congenital heart disease (Richards and Garg,** **2010), it is possible that the test subjects used in the** •**There is a possibility that cardiac defects detected in the** **Dawson et al. (1993) study** **were associated in part with the use of tap water as a control** **vehicle (i.e., possible presence of contaminants).** •29Table 1. WOE Evaluation of the Potential for Development Exposures to TCE to Result in Cardiac DefectsKey Factora,bType ofevidence toconsiderDataEvidence for strongerweight of associationEvidence for weakerweight of associationCommentsor Null Evidence**Johnson et al. (2003) study** **and others conducted in that laboratory may have been** **particularly susceptible to alterations in cardiac** **development.**•**Other contributing factors or confounding factors were not** **specifically identified in the evaluated in-vivo studies.** •**It is possible that the absence of treatment-related cardiac** **defects in well-conducted TCE studies (** **Carney et al., 2006** **; Fisher et al., 2001** **) or metabolite studies (** **Fisher et al., 2001** **) was due to confounding variables such** **as differences in strain/source of animal model, route of** **exposure, toxicokinetics, vehicle [e.g., soybean oil in** **Fisher et al. (2001) ], or** **differences in cardiac evaluation methods.** •**It is unlikely that the cardiac defects observed by** **Johnson et al. (2003)** **were an artifact of the evaluation procedures** **used, since a study by Fisher** 30Table 1. WOE Evaluation of the Potential for Development Exposures to TCE to Result in Cardiac DefectsKey Factora,bType ofevidence toconsiderDataEvidence for strongerweight of associationEvidence for weakerweight of associationCommentsor Null Evidence**et al. (2001) , using the same** **fetal cardiac evaluation procedures, did not identify** **an association between TCE exposure and the incidence of** **cardiac defects. Other possible** **explanations for observed** **outcome after the exposure of** **interest (not considered in** **Hill analysis) Epi** •**Potential maternal risk factors were adjusted in** **statistical analysis in Forand** **et al. (2012) and** **Yauck et al. (2004)** **or were not found in statistical analyses to** **influence observed association by +15% (** **Bove, 1996** **; Bove et al., 1995** **).**•**Potential for confounding from another exposure given the poor** **exposure definition in Yauck et** **al. (2004) . The positive** **association in Goldberg et al.** **(1990) may result from likely** **selection biases in controls.**•Specificity**Single cause and effect** **relationship resulting from** **exposure to test substance** **Tox**•**Cardiac defects in rats appear to be attributable to direct** **chemical exposure to TCE or metabolites (DCA or TCA)** **and are unlikely to be the result of secondary effect of** **maternal toxicity. Johnson et** **al. (2003) reported that TCE** **exposure via drinking water to pregnant rats did not result** **in maternal toxicity. Carney** **et al. (2006) reported minimal** **decreases in body weight gain in dams, with no adverse fetal** •**Studies conducted in other laboratories than UAZ and that** **administered TCE by gavage or inhalation (** **Carney et al., 2006 ;** **Fisher et al., 2001 ;** **Schwetz et al., 1975** **) did not identify cardiac defects.** **Fisher et al. (2001)** **used the same cardiac evaluation methods as the UAZ** **lab.**•**The cardiac defects detected in the** **Dawson et al. (1993) study** **may have been related to the use of tap water as a vehicle** •31Table 1. WOE Evaluation of the Potential for Development Exposures to TCE to Result in Cardiac DefectsKey Factora,bType ofevidence toconsiderDataEvidence for strongerweight of associationEvidence for weakerweight of associationCommentsor Null Evidence**outcomes. In fetuses, there was no indication of TCE** **related fetal weight deficits, external or skeletal** **anomalies, or of soft tissue alterations other than cardiac** **defects in Johnson et al.** **(2003) , nor in any other** **study.**•**The majority of the cardiac malformations following** **TCE exposures to rats (** **Johnson et al., 2003 ;** **Dawson et al., 1993 ) or** **chicks ( Rufer et al., 2010** **; Drake et al., 2006a** **) during sensitive periods of cardiac** **development were ventricular septal defects, valve defects,** **or outflow tract abnormalities. Mechanistic** **data suggest a common etiology (disruption of the** **cardiac cushion formation) for the observed cardiac** **defects Boyer et al. (2000)** **. (i.e., possible contaminants).** **Single cause and effect** **relationship Epi** •**NE**•**Specificity not a critical compared to other Hill aspects** **since outcomes may have several risk factors. Maternal** •32Table 1. WOE Evaluation of the Potential for Development Exposures to TCE to Result in Cardiac DefectsKey Factora,bType ofevidence toconsiderDataEvidence for strongerweight of associationEvidence for weakerweight of associationCommentsor Null Evidence**resulting from exposure to test** **substance risk factors, specifically** **chemical risk factors, associated with cardiac defects** **in infants have not been well studied.** Coherence**Summary: Extent to which** **data are similar in outcome and** **exposure across database** **Tox**•**Multiple studies were conducted at UAZ (** **Johnson et al., 2003** **; Johnson et al.,** **1998 ;** **Dawson et al., 1993 ),** **in which rats were administered TCE or** **metabolites DCA or TCA in drinking water on GD 1-22** **and for which study design and cardiac evaluation** **methodologies were consistent. The outcomes of** **these studies (detection of cardiac defects, particularly** **septal defects, valve abnormalities, and outflow** **tract anomalies) are consistent across these** **studies. Additionally, these outcomes are supported by** **the results of avian in ovo and in vitro studies, studies with** **TCE metabolites (DCA and TCA) in rodents, in vitro** **whole embryo culture studies,**•**Developmental toxicity studies with TCE that were conducted** **in other laboratories ( Carney et** **al., 2006 ;** **Fisher et al., 2001 ;** **Schwetz et al., 1975 )** **administered TCE to rats of other strains or sources, using** **different routes of exposure (inhalation or gavage),** **administered on different days of gestation (i.e., not including** **GD 1-6) than the UAZ studies and did not identify cardiac** **defects. No other study in the TCE database reported cardiac** **defects at the low dose levels reported by** **Johnson et al. (2003)** **.**•33Table 1. WOE Evaluation of the Potential for Development Exposures to TCE to Result in Cardiac DefectsKey Factora,bType ofevidence toconsiderDataEvidence for strongerweight of associationEvidence for weakerweight of associationCommentsor Null Evidence**and mechanistic data. Cause and** **effect interpretation** **should not conflict with the** **generally known facts of** **the natural history and** **biology of the disease** **Epi**•**Associations in epidemiologic studies of** **cardiac defects and maternal occupational exposure to** **degreasing solvents or to organic solvents (** **Gilboa et al., 2012** **; Loffredo et al.,** **1991 ;** **Tikkanen and Heinonen, 1991** **; Tikkanen** **and Heinonen, 1988 ).** •**NE**•**NE = No relevant evidence. HH = Hamburger-Hamilton stages of chick development** **( Hamburger and Hamilton, 1951** **). Tox = Animal toxicology studies; Epi = Epidemiologi** **cal studies Key Factor references:** **a U.S. EPA (2006)** **b Hill (1965)** 34

Table 2. Comparison of TCE Prenatal Developmental T oxicity Study Methods

Dawson et

al. (1993) Johnson et

al. (2003) Fisher et

al. (2001) Carney

et al.

(2006)

Study Description/Objective

**GLP; guideline [G], guideline-type**

**[GT], or research [R] protocol R R R GLP, G** 

Test Subjects

**Species Rat Rat Rat Rat**

**Strain SD SD SD SD**

**Source (company) Harlan Harlan CRL CRL**

**Source (location Indianapoli**

**s, IN Indianapoli** 

**s, IN Raleigh,** 

**NC Portage,** 

**MI**

**Dates of study conduct 1989-1990 1989-1995 NR NR**

**Day of mating confirmation (GD**

**0 or GD 1) NR NR GD 0 GD 0** 

**Day of cesarean section GD 22 GD 22 GD 21 GD 21**

Treatment

**Test Substance TCE TCE TCE TCE**

**Source Aldrich Aldrich Aldrich Dow**

**Purity (%) NR NR NR 99%**

**Negative control (vehicle) Tap water Distilled**

**water Soybean** 

**oil Ambien** 

**t air**

**Positive control N N RA N**

**No. of treated groups 2 4 1 3**

**Group size (litters/group) 9-15 9-12 19-25 27**

**Random assignment of test subjects**

**to groups Y Y Y Y** 35

Table 2. Comparison of TCE Prenatal Developmental T oxicity Study Methods

Dawson et

al. (1993) Johnson et

al. (2003) Fisher et

al. (2001) Carney

et al.

(2006)

**Dose period (duration, gestation**

**only groups) GD 1-22 GD 1-22 GD 6-15 GD 6-** 

**20**

**Daily dosing schedule Ad libitum Ad libitum 1x/day 6** 

**hrs/day,**

**7**

**days/wk**

**Route of administration DW DW Gavage Inhalati**

**on a**

Maternal evaluation

**In-life data (BW, FC, WC, and/or**

**clinobs) Y Y Y Y** 

**Postmortem data (necropsy, organ**

**wts, pathology, and/or CL) Y Y Y Y** 

Fetal evaluation

**Implantations and resorptions (early**

**and late) Y Y Y Y** 

**Fetal weight, length, sex Y Y Y Y**

**External fetal exam Y Y Y Y**

**Percent fetuses (litters) evaluated**

**for external findings 100 (100) 100 (100) 100 (100) 100** 

**(100)**

**Visceral examination Y Y Y Y**

**Percent fetuses (litters) evaluated**

**for visceral findings 100 (100) 100 (100) 100 (100) 50 (100)** 

**Fresh dissection (in situ organ**

**examination) Y (heart) Y (heart) Y (heart) Y** 

**(viscera**

**)**

**Wilson exam (Bouins fixation,**

**free-hand sections) N N N Y** 

**(head)**36

Table 2. Comparison of TCE Prenatal Developmental T oxicity Study Methods

Dawson et

al. (1993) Johnson et

al. (2003) Fisher et

al. (2001) Carney

et al.

(2006)

**Fetal cardiac examination**

**methods Y Y Y Y** 

**Fresh dissection and**

**evaluation UAZ** 

**method UAZ** 

**method UAZ** 

**method Staples** 

**exam**

**Free-hand section of**

**decalcified fetuses N N N N** 

**Preservation GLA flush**

**&**

**immersion GLA flush** 

**&**

**immersion formalin** 

**immersio**

**n NR** 

**Confirmation of findings Y b Y b NR NR**

**Skeletal examination NR NR NR Y**

**Percent fetuses (litters) evaluated**

**for skeletal findings NR NR NR 50 (100)** 

**Bone development NR NR NR Y**

**Cartilage development NR NR NR Y**

**Random selection of fetuses for**

**visceral or skeletal evaluation NA NA NA NA** 

**Assessment of fetuses without**

**knowledge of treatment group Y Y Y Y** 

Footnotes:

**This table only includes mammalian studies with pre natal TCE exposures and an** 

**evaluation of fetal morphology.**

**NR = not reported; NA = not applicable; Y = yes, N = No; DW = drinking water; GD =** 

**gestation day; RA = retanoic acid; GLA = gluterald ehyde** 

**Test subject strain: SD = Sprague-Dawley**

**Test subject source: CRL = Charles River Laboratori es; Harlan = Harlan Laboratories** 37

Table 2. Comparison of TCE Prenatal Developmental T oxicity Study Methods

Dawson et

al. (1993) Johnson et

al. (2003) Fisher et

al. (2001) Carney

et al.

(2006)

**Group sizes are range of actual group size (i.e., n o. of dams) on study; numbers in** 

**parentheses () indicate target group size.**

**a = Whole-body exposure, dynamic air flow, analyti cal chamber concentrations.** 

**b = Unanimous agreement of cardiac diagnoses by st udy investigators: a pathologist, a** 

**pediatric cardiologist, and a veterinarian.**

**Cardiac evaluation references: Staples exam: ( Stuckhardt and Poppe, 1984**  **; Staples,** 

**1974 ); University of AZ exam: (**  **Johnson et al., 2003 ;**  **Dawson et al., 1993 )** 

References

**ATSDR (Agency for Toxic Substances and Disease Regi stry). (2006). Health consultation: Endicott area** 

**investigation: Health statistics review: Cancer and birth outcome analysis, Endicott area, town of** 

**Union, Broome County, New York. Atlanta, GA: U.S. D epartment of Health and Humans** 

**Services.**

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