June 19, 2019 Transmitted Electronically Document Control Manager Office of Pollution Prevention and Toxics (OPPT) US EPA 1200 Pennsylvania Avenue, NW Washington, DC 20460-0001 Dear Sir/Maddam, The Center for Environmental Health (CEH) hereby submits these comments on the proposed Toxic Substances Control Act high-priority candidates (EPA-HQ-OPPT-2019-0131- 0001). As required by the Toxic Substances Control Act (TSCA), the United States Environmental Protection Agency (USEPA) must regularly release a list of chemicals that are under review for prioritization for risk evaluation. This list consists of 20 high-priority and 20 low-priority candidates. These comments identify data on the 2019 list of the 20 high-priority candidates (Table 1) in the literature that support the notion that these chemicals may be endocrine disrupting compounds (EDCs). EDCs are a class of chemicals that can interfere with the production, action, transport, or metabolism of endogenous hormones in the body.1 For the purposes of these comments, an EDC will be considered any compound that showed an effect on an endocrine system at any dose or concentration, thus accounting for any potential dose responses that may be non-monotonic. This is supported in the literature wherein disruption of upstream signaling, even at low doses and within “normal” levels of hormone variability, adverse outcomes can be observed.2,3 These comments will describe various studies to determine whether the compound of interest could be considered an EDC. This was accomplished by searching PubMed as well as The Endocrine Disruption Exchange’s List of Potential Endocrine Disruptors for peer- reviewed articles by searching with the CAS Registry Number for each chemical; USEPA’s Computational Toxicology Dashboard was also searched. While these searches were informative, they should not be considered exhaustive. Some searches showed no data for certain compounds, and it is worth acknowledging that an absence of evidence for endocrine disruption does not mean that these compounds are not EDCs. For other compounds, the data is too abundant to condense into a short report such as this. In vitro studies are often disregarded by risk assessment and risk management practices despite being valuable and informative; these studies should be considered in risk evaluation processes, especially given USEPA’s mandate to reduce vertebrate animal testing.4 In vivo testing, while informative, is costly and may overlook more sensitive, tissue-specific or species-specific endpoints that are more easily assessed with high-throughput in vitro testing. Both in vitro and in vivo studies are essential in the risk evaluation process. In total, of the 20 high-priority candidates, 18 currently show evidence that support treating them as an endocrine disrupting compound (EDC). Risk evaluations of these compounds should review this evidence in determining whether they pose unreasonable risks to human health due to the adverse outcomes caused by endocrine disruption. For many of the 20 high-priority compounds, extensive data indicating they are endocrine disruptors is already available, such as the five phthalates.5-7 However, further data is necessary for other compounds with less publicly available data (such as p-dichlorobenzene, 1,2- dichloroethane, trans-1,2-dichloroethylene, o-dichlorobenzene, and ethylene dibromide) as studies on these compounds are limited but indicate endocrine activity. For two compounds, 1,1-dichloroethane and phthalic anhydride, no data is publicly available for whether they are or are not EDCs. Tests that should be performed to evaluate endocrine disruption can be found in the Revised Guidance Document 150 on Standardized Test Guidelines for Evaluating Chemicals for Endocrine Disruption from the Organization for Economic Cooperation and Development (OECD), and all studies should perform the tests listed in Conceptual Framework Levels 2-5.8 Table 1. Summary table for USEPA's 2019 20 High-Priority Candidates' evidence for endocrine disruption. Evidence for Endocrine Chemical CAS-RN Disruption? p-Dichlorobenzene 106-46-7 Yes 1,2-Dichloroethane 107-06-2 Yes trans-1,2-Dichloroethylene 156-60-5 Yes o-Dichlorobenzene 95-50-1 Yes 1,1,2-Trichloroethane 79-00-5 Yes 1,2-Dichloropropane 78-87-5 Yes 1,1-Dichloroethane 75-34-3 Insufficient data Dibutyl phthalate 84-74-2 Yes Butyl benzyl phthalate 85-68-7 Yes 117-81-7 Di-ethylhexyl phthalate Yes 84-69-5 Di-isobutyl phthalate Yes 84-61-7 Dicyclohexyl phthalate Yes 4,4'-(1-Methylethylidene)bis[2, 6- 79-94-7 Yes dibromophenol] 115-96-8 Tris(2-chloroethyl) phosphate Yes Triphenyl ester phosphoric acid 115-86-6 Yes Ethylene dibromide 106-93-4 Yes 106-99-0 1,3-Butadiene Yes 1,3,4,6,7,8-Hexahydro-4,6,6,7,8,8- 1222-05-5 hexamethylcyclopenta [g]-2- Yes benzopyran 50-00-0 Formaldehyde Yes 85-44-9 Phthalic anhydride Insufficient data p-Dichlorobenzene There is currently evidence within the literature to support labelling p-dichlorobenzene (PDCB) as an EDC. Most notably, an epidemiological study in adolescents in the US found that increased urinary levels of PDCB was associated with increased levels of thyroid stimulating hormone (TSH) and thyroglobulin (TG) in serum. This exposure was also associated with hypothyroidism.9 In female rats, PDCB increased levels of 17-beta estradiol in the serum.10 Another study performed in both rats and mice found that uterine and ovarian weight were decreased in PDCB-exposed animals. In this study, PDCB also displayed antagonistic effects, blocking estrogen signaling. It should be noted that the authors observed variability between their five studies and that different strains of mice were affected differently by PDCB, indicating that different populations may have different levels of risk associated with PDCB exposure.11 Another study in rats found that a single intraperitoneal injection of related chlorinated benzenes, o-dichlorobenzene and m- dichlorobenzene, decreased levels of T3 and T4 in the serum after just 24 hours; unfortunately, this study did not investigate did not investigate PDCB.12 Taken together, these data support PDCB as an EDC that may impact thyroid and estrogen signaling. 1,2-Dichloroethane There is currently evidence within the literature to support labelling 1,2-dichloroethane as an EDC. Only one study was found to indicate this, but its findings were detailed and compelling. Male mice were exposed for 1 week or 4 weeks to 1,2-dichloroethane via inhalation. After exposure, there was an increase in testicular weight and decrease in the diameter of the seminiferous tubules. Increases in plasma and testicular testosterone and luteinizing hormone (LH) were also increased in exposed animals. Gonadotrophic releasing hormone (GnRH) was also increased in the testes of exposed animals. Expression of genes related to testosterone production was altered at the transcript and protein levels. Histological assessment of the testes showed degeneration of the testes and immature sperm and cellular debris being released into the testicular lumen; these findings were further supported by experiments revealing induced cell death in the testes. Sperm concentration and quality were also decreased in these mice.13 While only one study was available for 1,2-dichloroethane, it was robust and informative in showing there is evidence to support the notion it is an EDC, especially as it relates to the male reproductive system. trans-1,2-Dichloroethylene There is currently evidence within the literature to support labelling trans-1,2- dichloroethylene as an EDC. This evidence comes from the USEPA’s Computational Toxicology Dashboard wherein trans-1,2-dichloroethylene showed activity in an assay that measured androgen receptor in vitro.14 Further studies, especially those with in vivo assays, are necessary to further confirm these findings; regardless, trans-1,2-dichloroethylene has endocrine disrupting potential. o-Dichlorobenzene There is currently evidence within the literature to support labelling o-dichlorobenzene (ODCB) as an EDC. The evidence, however, is minimal and further studies are needed. Described above, there is one study, of chlorinated benzenes, including ODCB, exposed rats via intraperitoneal injection and found that T3 and T4 levels in the serum were decreased 24 hours after exposure.12 This study supports that ODCB is an EDC with thyroid-related effects, but more studies are needed to support this study that is now almost 30 years old. USEPA’s Computational Toxicology Dashboard reports that ODCB is able to interfere with estrogen signaling,14 but, again, further studies are needed for measuring the endocrine disrupting potential of ODCB. 1,1,2-Trichloroethane There is currently evidence within the literature to support labelling 1,1,2- trichloroethane as an EDC. This evidence, again, comes from the USEPA’s Computational Toxicology Dashboard; 1,1,2-trichloroethane was returned as a positive hit in an in vitro assay measuring estrogen receptor agonism.14 1,1,2-Trichloroethane has endocrine disrupting potential, but further studies are necessary to confirm these findings in vivo. 1,2-Dichloropropane There is currently evidence within the literature to support labelling 1,2- dichloropropane as an EDC. The most extensive study was published 30 years ago, but it detailed exposure to 1,2-dichloropropane in rats for 1, 5, or 10 days. The researchers noted “excessive” degenerate spermatogonia as well as decreased spermatozoa in the epididymis of exposed rats. Some of the exposed animals also presented testicular degeneration as well as decreased mature sperm. Interestingly, the authors also noted increased fat storage in the adrenal glands.15 In a different study also conducted in rats, rats were exposed to 1,2- dichloropropane via inhalation for 3 weeks; female rats exposed to 1,2-dichloropropane in this study experienced more estrous cycles lasting more than 6 days, which is uncommon for rats. Researchers also observed that the exposed rats produced fewer ova per estrous cycle.16 Together, these data support that 1,2-dichloropropane is an EDC with effects that impact the male and female reproductive systems.