EPA/690/R-22/002F | September 2022 | FINAL United States Environmental Protection Agency xvEPA Provisional Peer-Reviewed Toxicity Values for Dibenzothiophene (CASRN 132-65-0) U.S. EPA Office of Research and Development Center for Public Health and Environmental Assessment ------- A mA United States Environmental Protection »»Agency EPA 690 R-22 002F September 2022 https://www.epa.gov/pprtv Provisional Peer-Reviewed Toxicity Values for Dibenzothiophene (CASRN 132-65-0) Center for Public Health and Environmental Assessment Office of Research and Development U.S. Environmental Protection Agency Cincinnati, OH 45268 ------- AUTHORS, CONTRIBUTORS, AND REVIEWERS CHEMICAL MANAGERS Lucina E. Lizarraga, PhD Center for Public Health and Environmental Assessment, Cincinnati, OH Jeffry L. Dean II, PhD Center for Public Health and Environmental Assessment, Cincinnati, OH CONTRIBUTORS Chelsea A. Weitekamp, PhD Center for Public Health and Environmental Assessment, Research Triangle Park, NC James A. Weaver, PhD, DABT Center for Public Health and Environmental Assessment, Research Triangle Park, NC SCIENTIFIC TECHNICAL LEAD Lucina E. Lizarraga, PhD Center for Public Health and Environmental Assessment, Cincinnati, OH DRAFT DOCUMENT PREPARED BY SRC, Inc. 7502 Round Pond Road North Syracuse, NY 13212 PRIMARY INTERNAL REVIEWERS Daniel D. Petersen, MS, PhD, DABT, ATS, ERT Center for Public Health and Environmental Assessment, Cincinnati, OH Suryanarayana V. Vulimiri, BVSc, PhD, DABT Center for Public Health and Environmental Assessment, Washington, DC PRIMARY EXTERNAL REVIEWERS Eastern Research Group, Inc. 110 Hartwell Avenue Lexington, MA 02421-3136 PPRTV PROGRAM MANAGEMENT Teresa L. Shannon Center for Public Health and Environmental Assessment, Cincinnati, OH J. Phillip Kaiser, PhD, DABT Center for Public Health and Environmental Assessment, Cincinnati, OH in Dib enzothi ophene ------- Questions regarding the content of this PPRTV assessment should be directed to the U.S. EPA Office of Research and Development (ORD) Center for Public Health and Environmental Assessment (CPHEA) website at https://ecomments cpa.gov/pprtv. iv Dib enzothi ophene ------- TABLE OF CONTENTS COMMONLY USED ABBREVIATIONS AND ACRONYMS vi BACKGROUND 1 QUALITY ASSURANCE 1 DISCLAIMERS 2 QUESTIONS REGARDING PPRTVs 2 1. INTRODUCTION 3 2. REVIEW OF POTENTIALLY RELEVANT DATA (NONCANCER AND CANCER) 7 2.1. HUMAN STUDIES 11 2.1.1. Oral Exposures 11 2.1.2. Inhalation Exposures 11 2.2. ANIMAL STUDIES 11 2.2.1. Oral Exposures 11 2.2.2. Inhalation Exposures 15 2.3. OTHER DATA (SHORT-TERM TESTS, OTHER EXAMINATIONS) 15 2.3.1. Genotoxicity 15 2.3.2. Metabolism/Toxicokinetic and Supporting Animal Studies 19 3. DERIVATION 01 PROVISIONAL VALUES 23 3.1. DERIVATION OF ORAL REFERENCE DOSES 23 3 .2. DERIVATION OF INHALATION REFERENCE CONCENTRATIONS 23 3.3. SUMMARY OF NONCANCER PROVISIONAL REFERENCE VALUES 23 3.4. CANCER WEIGHT-OF-EVIDENCE DESCRIPTOR 23 3.5. DERIVATION OF PROVISIONAL CANCER RISK ESTIMATES 24 APPENDIX A. SCREENING PROVISIONAL VALUES 25 APPENDIX D. REFERENCES 55 v Dib enzothi ophene ------- COMMONLY USED ABBREVIATIONS AND ACRONYMS a2u-g alpha 2u-globulin IVF in vitro fertilization ACGIH American Conference of Governmental LC50 median lethal concentration Industrial Hygienists LD50 median lethal dose AIC Akaike's information criterion LOAEL lowest-observed-adverse-effect level ALD approximate lethal dosage MN micronuclei ALT alanine aminotransferase MNPCE micronucleated polychromatic AR androgen receptor erythrocyte AST aspartate aminotransferase MOA mode of action atm atmosphere MTD maximum tolerated dose ATSDR Agency for Toxic Substances and NAG 7V-acetyl-P-D-glucosaminidase Disease Registry NCI National Cancer Institute BMC benchmark concentration NOAEL no-observed-adverse-effect level BMCL benchmark concentration lower NTP National Toxicology Program confidence limit NZW New Zealand White (rabbit breed) BMD benchmark dose OCT ornithine carbamoyl transferase BMDL benchmark dose lower confidence limit ORD Office of Research and Development BMDS Benchmark Dose Software PBPK physiologically based pharmacokinetic BMR benchmark response PCNA proliferating cell nuclear antigen BUN blood urea nitrogen PND postnatal day BW body weight POD point of departure CA chromosomal aberration PODadj duration-adjusted POD CAS Chemical Abstracts Service QSAR quantitative structure-activity CASRN Chemical Abstracts Service registry relationship number RBC red blood cell CBI covalent binding index RDS replicative DNA synthesis CHO Chinese hamster ovary (cell line cells) RfC inhalation reference concentration CL confidence limit RfD oral reference dose CNS central nervous system RGDR regional gas dose ratio CPHEA Center for Public Health and RNA ribonucleic acid Environmental Assessment SAR structure-activity relationship CPN chronic progressive nephropathy SCE sister chromatid exchange CYP450 cytochrome P450 SD standard deviation DAF dosimetric adjustment factor SDH sorbitol dehydrogenase DEN diethylnitrosamine SE standard error DMSO dimethylsulfoxide SGOT serum glutamic oxaloacetic DNA deoxyribonucleic acid transaminase, also known as AST EPA Environmental Protection Agency SGPT serum glutamic pyruvic transaminase, ER estrogen receptor also known as ALT FDA Food and Drug Administration SSD systemic scleroderma FEVi forced expiratory volume of 1 second TCA trichloroacetic acid GD gestation day TCE trichloroethylene GDH glutamate dehydrogenase TWA time-weighted average GGT y-glutamyl transferase UF uncertainty factor GSH glutathione UFa interspecies uncertainty factor GST g 1 ii ta t h i o nc - V-1 ra ns fc ra sc UFc composite uncertainty factor Hb/g-A animal blood-gas partition coefficient UFd database uncertainty factor Hb/g-H human blood-gas partition coefficient UFh intraspecies uncertainty factor HEC human equivalent concentration UFl LOAEL-to-NOAEL uncertainty factor HED human equivalent dose UFS subchronic-to-chronic uncertainty factor i.p. intraperitoneal U.S. United States of America IRIS Integrated Risk Information System WBC white blood cell Abbreviations and acronyms not listed on this page are defined upon first use in the PPRTV document. vi Dib enzothi ophene ------- EPA/690/R-22/002F PROVISIONAL PEER-REVIEWED TOXICITY VALUES FOR DIBENZOTHIOPHENE (CASRN 132-65-0) BACKGROUND A Provisional Peer-Reviewed Toxicity Value (PPRTV) is defined as a toxicity value derived for use in the Superfund program. PPRTVs are derived after a review of the relevant scientific literature using established U.S. Environmental Protection Agency (U.S. EPA) guidance on human health toxicity value derivations. The purpose of this document is to provide support for the hazard and dose-response assessment pertaining to chronic and subchronic exposures to substances of concern, to present the major conclusions reached in the hazard identification and derivation of the PPRTVs, and to characterize the overall confidence in these conclusions and toxicity values. It is not intended to be a comprehensive treatise on the chemical or toxicological nature of this substance. Currently available PPRTV assessments can be accessed on the U.S. EPA's PPRTV website at https://www.epa.gov/pprtv. PPRTV assessments are eligible to be updated on a 5-year cycle and revised as appropriate to incorporate new data or methodologies that might impact the toxicity values or affect the characterization of the chemical's potential for causing adverse human-health effects. Questions regarding nomination of chemicals for update can be sent to the appropriate U.S. EPA eComments Chemical Safety website at https://ecomments.epa.gov/chemicalsafetv/. QUALITY ASSURANCE This work was conducted under the U.S. EPA Quality Assurance (QA) program to ensure data are of known and acceptable quality to support their intended use. Surveillance of the work by the assessment managers and programmatic scientific leads ensured adherence to QA processes and criteria, as well as quick and effective resolution of any problems. The QA manager, assessment managers, and programmatic scientific leads have determined under the QA program that this work meets all U.S. EPA quality requirements. This PPRTV was written with guidance from the CPHEA Program Quality Assurance Project Plan (PQAPP), the QAPP titled Program Quality Assurance Project Plan (PQAPP) for the Provisional Peer-Reviewed Toxicity Values (PPRTVs) and Related Assessments/Documents (L-CPAD-0032718-QP), and the PPRTV development contractor QAPP titled Quality Assurance Project Plan—Preparation of Provisional Toxicity Value (PTV) Documents (L-CPAD-0031971-QP). As part of the QA system, a quality product review is done prior to management clearance. A Technical Systems Audit may be performed at the discretion of the QA staff. All PPRTV assessments receive internal peer review by at least two CPHEA scientists and an independent external peer review by at least three scientific experts. The reviews focus on whether all studies have been correctly selected, interpreted, and adequately described for the purposes of deriving a provisional reference value. The reviews also cover quantitative and qualitative aspects of the provisional value development and address whether uncertainties associated with the assessment have been adequately characterized. 1 Dib enzothi ophene ------- EPA/690/R-22/002F DISCLAIMERS The PPRTV document provides toxicity values and information about the adverse effects of the chemical and the evidence on which the value is based, including the strengths and limitations of the data. All users are advised to review the information provided in this document to ensure that the PPRTV used is appropriate for the types of exposures and circumstances at the site in question and the risk management decision that would be supported by the risk assessment. Other U.S. EPA programs or external parties who may choose to use PPRTVs are advised that Superfund resources will not generally be used to respond to challenges, if any, of PPRTVs used in a context outside of the Superfund program. This document has been reviewed in accordance with U.S. EPA policy and approved for publication. Mention of trade names or commercial products does not constitute endorsement or recommendation for use. QUESTIONS REGARDING PPRTVS Questions regarding the content of this PPRTV assessment should be directed to the U.S. EPA ORD CPHEA website at https://ecomments.epa.gov/pprtv. 2 Dib enzothi ophene ------- EPA/690/R-22/002F 1. INTRODUCTION Dibenzothiophene (CASRN 132-65-0) is a solid, sulfur-containing, three-ringed, heterocyclic polycyclic aromatic hydrocarbon (PAH) derivative. It is one of the organosulfur components of petroleum and coal and is used as a chemical intermediate and as an ingredient in cosmetics and pharmaceuticals (NI.M. 2021b; Blunter et al.. 201 1; Deutschmann et al.. 201 1). Dibenzothiophene is listed as an active substance in commerce on the public Toxic Substances Control Act (TSCA) inventory (U.S. EPA, 202le) and it is registered with Europe's Registration, Evaluation, Authorization, and Restriction of Chemicals (REACH) program (ECHA. 2021). Production and import volumes were not reported in U.S. EPA's Chemical Data Reporting (CDR) database (U.S. EPA. 202 la). No public information on industrial production or synthetic processes were located. The empirical formula for dibenzothiophene is C12H8S; its structure is shown in Figure 1. Experimental and estimated physicochemical properties identified for dibenzothiophene from U.S. EPA (2021c) and NLM (2021b) are presented in Table 1. When more than one experimental value was available, an experimental average is presented. Dibenzothiophene is moderately volatile from water and moist soil surfaces based on its calculated Henry's law constant; the soil adsorption coefficient indicates that dibenzothiophene will strongly sorb to soil and sediment, however, which may limit volatilization from these surfaces. Due to strong sorption and low water solubility, the potential to leach to groundwater or undergo runoff after precipitation is low. Figure 1. Dibenzothiophene (CASRN 132-65-0) Structure 3 Dib enzothi ophene ------- EPA/690/R-22/002F Table 1. Physicochemical Properties of Dibenzothiophene (CASRN 132-65-0) Property (unit) Value3 Physical state Solidb Boiling point (°C) 333 Melting point (°C) 97.0 Density (g/cm3) 1.23-1.25 (estimated) Vapor pressure (mm Hg at 25°C) 0.000205 (extrapolated)13 pH (unitless) NA pKa (unitless) NA Solubility in water (mg/L at 25°C) 1.47 (converted from PhysProp NCCT value of 7.98 x 10-6 mol/L) Log octanol/water partition coefficient (log Kow) 4.38 Henry's law constant (atm-m3/mole at 25°C) 3.38 x 10 " (calculated from vapor pressure/water solubility)0 Soil adsorption coefficient Koc (units not reported) 5,273-20,535b Atmospheric OH rate constant (cm5Vmolecule-sec at 25°C) 8.10 x 10-12b Atmospheric half-life (d) 1.3 (estimated using 12-hday; 1.5 x 106OH/cm3)d Relative vapor density (air = 1) Not applicable for solid Molecular weight (g/mol) 184.26 Flash point (°C) Not applicable for solid 'Unless otherwise noted, values are from U.S. EPA (2021c). bNLM (2021b). °U.S. EPA (2012): calculated by EPI Suite™ using a vapor pressure of 0.000205 mm Hg and a water solubility of 1.47 mg/L. dU.S. EPA (2012). EPI Suite™ = Estimation Programs Interface Suite; NA = not applicable; NCCT = National Center for Computational Toxicology; PhysProp = Physical Properties Database. A summary of available toxicity values for dibenzothiophene from U.S. EPA and other agencies/organizations is provided in Table 2. 4 Dib enzothi ophene ------- EPA/690/R-22/002F Table 2. Summary of Available Toxicity Values for Dibenzothiophene (CASRN 132-65-0) Source (parameter)ab Value (applicability) Notes Reference Noncancer IRIS NV NA U.S. EPA (202Id) HEAST NV NA U.S. EPA (2011b) DWSHA NV NA U.S. EPA (2018) ATSDR NV NA ATSDR (2021) WHO NV NA WHO (2021) CalFPA NV NA CalEPA (2021. 2020) OSHA NV NA OSHA (2021a. 2021b. 2021c) NIOSH NV NA NIOSH (2018) ACGIH NV NA ACGIH (2020) Cancer IRIS NV NA U.S. EPA (202Id) HEAST NV NA U.S. EPA (2011b) DWSHA NV NA U.S. EPA (2018) NTP NV NA NTP (2016) IARC (WOE) Group 3: not classifiable as to its carcinogenicity to humans Based on inadequate evidence for carcinogenicity in experimental animals and no available data for carcinogenicity in humans. IARC (2013) CalEPA NV NA CalEPA (2021. 2020) ACGIH NV NA ACGIH (2020) aSources: ACGIH = American Conference of Governmental Industrial Hygienists; ATSDR = Agency for Toxic Substances and Disease Registry; CalEPA = California Environmental Protection Agency; DWSHA = Drinking Water Standards and Health Advisories; HEAST = Health Effects Assessment Summary Tables; IARC = International Agency for Research on Cancer; IRIS = Integrated Risk Information System; NIOSH = National Institute for Occupational Safety and Health; NTP = National Toxicology Program; OSHA = Occupational Safety and Health Administration; WHO = World Health Organization. Parameters: WOE = weight of evidence. NA = not applicable; NV = not available. Literature searches were conducted in June 2019 and updated in June 2022 for studies relevant to the derivation of provisional toxicity values for dibenzothiophene. Searches were conducted using U.S. EPA's Health and Environmental Research Online (HERO) database of scientific literature. HERO searches the following databases: PubMed, TOXLINE1 (including TSCATS1), and Web of Science. The following resources were searched outside of HERO for 'Note that this version of TOXLINE is no longer updated (https://www.nlm.nih.gov/databases/download/toxlinesubset.html'): therefore, it was not included in the literature search update from June 2022. 5 Dib enzothi ophene ------- EPA/690/R-22/002F health-related values: American Conference of Governmental Industrial Hygienists (ACGIH), Agency for Toxic Substances and Disease Registry (ATSDR), California Environmental Protection Agency (CalEPA), Defense Technical Information Center (DTIC), European Centre for Ecotoxicology and Toxicology of Chemicals (ECETOC), European Chemicals Agency (ECHA), U.S. EPA Chemical Data Access Tool (CDAT), U.S. EPA ChemView, U.S. EPA Integrated Risk Information System (IRIS), U.S. EPA Health Effects Assessment Summary Tables (HEAST), U.S. EPA Office of Water (OW), International Agency for Research on Cancer (IARC), U.S. EPA TSCATS2/TSCATS8e, U.S. EPA High Production Volume (HPV), Chemicals via International Programme on Chemical Safety (IPCS) INCHEM, Japan Existing Chemical Data Base (JECDB), Organisation for Economic Co-operation and Development (OECD) Screening Information Data Sets (SIDS), OECD International Uniform Chemical Information Database (IUCLID), OECD HPV, National Institute for Occupational Safety and Health (NIOSH), National Toxicology Program (NTP), Occupational Safety and Health Administration (OSHA), and World Health Organization (WHO). 6 Dib enzothi ophene ------- EPA/690/R-22/002F 2. REVIEW OF POTENTIALLY RELEVANT DATA (NONCANCER AND CANCER) Tables 3A and 3B provide overviews of the relevant noncancer and cancer evidence bases, respectively, for dibenzothiophene and include all repeated-dose short-term, subchronic, and chronic studies, as well as reproductive and developmental toxicity studies identified from the literature search. Principal studies used in the PPRTV assessment for derivation of provisional toxicity values are identified in bold. The phrase "statistical significance" and term "significant," used throughout the document, indicate ap-value of < 0.05 unless otherwise specified. 7 Dib enzothi ophene ------- EPA/690/R-22/002F Table 3A. Summary of Potentially Relevant Noncancer Data for Dibenzothiophene (CASRN 132-65-0) Number of Male/Female, Strain, Species, Study Type, Study Reference Category3 Duration, Reported Doses Dosimetryb Critical Effects NOAELb LOAELb (comments) Notes0 Human 1. Oral (mg/kg-day) ND 2. Inhalation (mg/m3) ND Animal 1. Oral (mg/kg-day) Short-term 6-12 IV1/6—12 F, Sprague Dawley, 0,3,10,30 Increased relative liver weight in 3 10 JECDB (201D PS, rat, unspecified oral, 28 d both males and females and NPR reduced motor activity and Reported doses: 0,3,10, increased prothrombin time in 30 mg/kg-d males at >10 mg/kg-d. Other effects occurring mostly at the highest dose (30 mg/kg-d): hepatocyte hypertrophy in males and females and changes in serum markers of liver function in males (reduced albumin protein fraction and A/G ratio); increased relative kidney weights and kidney lesions in males; increased APTT in males. 8 Dib enzothi ophene ------- EPA/690/R-22/002F Table 3A. Summary of Potentially Relevant Noncancer Data for Dibenzothiophene (CASRN 132-65-0) Category3 Number of Male/Female, Strain, Species, Study Type, Study Duration, Reported Doses Dosimetryb Critical Effects NOAELb LOAELb Reference (comments) Notes0 Subchronic to Chronic Male (number not specified), albino, rat, diet, 165 d Reported dietary concentrations: 0 (historical), 0.025, 0.05, 0.10% 0 (historical), 13, 27, 63 Increased liver weight compared with laboratory historical controls; histopathological changes in liver. NDr NDr Thomas et al. (1942) (The design and reporting limitations [including the lack of a concurrent control and information on the number of test animals] prevent the determination of effect levels for this study.) PR 2. Inhalation (mg/m3) ND aDuration categories are defined as follows: acute = exposure for <24 hours; short term = repeated exposure for >24 hours to <30 days; long-term (subchronic) = repeated exposure for >30 days or <10% life span for humans (>30 days up to approximately 90 days in typically used laboratory animal species); and chronic = repeated exposure for >10% life span for humans (>~90 days to 2 years in typically used laboratory animal species) (U.S. EPA. 2002). bDosimetry: doses are presented as ADDs (mg/kg-day) for oral noncancer effects and as HECs (in mg/m3) for inhalation noncancer effects. °Notes: NPR = not peer reviewed; PR = peer reviewed; PS = principal study. ADD = adjusted daily dose; A/G = albumin/globulin; APTT = activated partial thromboplastin time; F = female(s); HEC = human equivalent concentration; LOAEL = lowest-observed-adverse-effect level; M = male(s); ND = no data; NDr = not determined; NOAEL = no-observed-adverse-effect level. 9 Dib enzothi ophene ------- EPA/690/R-22/002F Table 3B. Summary of Potentially Relevant Cancer Data for Dibenzothiophene (CASRN 132-65-0) Category Number of Male/Female, Strain, Species, Study Type, Reported Doses, Duration Dosimetry Critical Effects Reference (comments) Notes Human 1. Oral (mg/kg-day) ND 2. Inhalation (mg/m3) ND Animal 1. Oral (mg/kg-day) ND 2. Inhalation (mg/m3) ND ND = no data. 10 Dib enzothi ophene ------- EPA/690/R-22/002F 2.1. HUMAN STUDIES 2.1.1. Oral Exposures No studies were identified. 2.1.2. Inhalation Exposures No studies were identified. 2.2. ANIMAL STUDIES 2.2.1. Oral Exposures The effects of oral exposure to dibenzothiophene in animals have been evaluated in short-term (JHCDB. 2011) and chronic (Thomas et al.. 1942) studies in rats. Short-Term Studies JECDB (2011) In an OECD Test Guideline (TG) 407 study from the Japanese literature (JECDB. 2011). groups of 12, 6, 6, and 12 Crl:CD(SD) rats/sex received dibenzothiophene at doses of 0, 3, 10, or 30 mg/kg-day, respectively, by oral administration (additional details not available in English from the existing Japanese language report) for 28 days. At the end of exposure, six rats/sex/group were sacrificed; six rats/sex in the control and high-dose groups were followed for an additional 14 days untreated (recovery) prior to sacrifice. The animals were observed for clinical signs of toxicity, and body weight and food intake were measured once each week. Detailed clinical observations of the animals in cages, during handling, and in open field were performed weekly. During exposure week 4 and recovery week 2, the animals were subjected to functional observational battery (FOB), assessing reactivity (visual, touch, auditory, pain, proprioceptive), righting reflex, grip strength, and motor activity. Blood and urine were collected at the end of exposure and at the end of the recovery period. Hematology parameters included erythrocytes (red blood cells [RBCs] and reticulocyte counts, hemoglobin, hematocrit, mean corpuscular volume [MCV], mean corpuscular hemoglobin [MCH], mean corpuscular hemoglobin concentration [MCHC]), platelet counts, prothrombin time [PT], activated partial thromboplastin time [APTT], and white blood cells [WBCs; total and differential counts]). Serum chemistry was evaluated including total protein, albumin, globulins, albumin/globulin (A/G) ratio, aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), y-glutamyl transferase (GGT), total bilirubin, glucose, total cholesterol, triglycerides, blood urea nitrogen (BUN), creatinine, and electrolytes. Urinalysis parameters included pH, protein, glucose, ketone bodies, urobilinogen, bilirubin, occult blood, color, volume, and specific gravity. All animals received gross necropsy. The following organs were weighed in all animals: liver, kidney, spleen, heart, brain, pituitary, thymus, thyroid, adrenal, and reproductive organs (testis, epididymis, prostate, seminal vesicle, ovary, and uterus). Histopathology results were reported for the following organs: lung, cecum, ileum, pancreas, liver, kidney, testis, epididymis, prostate, and pituitary gland (other organs may have been examined as well). Both male and female rats at the high dose of 30 mg/kg-day consumed less food than controls on administration day 7 (-17 and -14% relative to controls, respectively, p < 0.01) but not at other time points (14, 21, and 28 days). Male rats receiving 30 mg/kg-day exhibited lower body weights (-7%) on Days 7 and 14, but there were no statistically significant differences in body weight at Day 21 or 28 or after the recovery period, or in body-weight gains over the full treatment or recovery periods (terminal body weights changes are displayed in Table B-l). 11 Dib enzothi ophene ------- EPA/690/R-22/002F Females exhibited no significant differences in body weight. Salivation was observed in small numbers of males and females (n = 1 or 2) at the high dose; no other clinical signs were noted. Reactivity, righting reflex, and forelimb and hindlimb grip strength were not significantly affected by exposure at any dose. At the end of exposure, statistically significant decreases in motor activity counts were observed at doses of 10 and 30 mg/kg-day in male rats (-63 and -53%, respectively; see Table B-l for more details); no significant differences were observed for females. There were no differences in motor activity between control and high-dose rats after the recovery period. No treatment-related urinalysis changes were apparent. Male rats exhibited statistically significant longer PT (23-36% at >10 mg/kg-day) and APTT (41% at 30 mg/kg-day; see Table B-l) relative to controls at the end of treatment, while females did not; there were no other effects on hematology at the end of treatment and none after recovery. Statistically significant clinical chemistry findings in males at the end of exposure were increased calcium (+5%>), increased alpha 2u-globulin (au-g) and P globulin protein fraction percentages (+14%), and decreased albumin protein fraction percentages (-6%) and A/G ratios (—13%) in males receiving 30 mg/kg-day dibenzothiophene (see Table B-l). Females receiving the highest dose had significantly higher total cholesterol than controls (+54%) (see Table B-l). After the recovery period, the only observed changes were significant decreases in blood glucose in males and decreases in calcium and increases in chloride and ai globulin protein fraction percent in females (data not shown). Although there were no significant changes in most serum hepatocellular/hepatobiliary markers (ALT, AST, ALP, GGT, and total bilirubin [data not shown]) in male and female rats, the decreases in albumin protein fraction and A/G ratio in males at 30 mg/kg-day are indicative of potential liver damage and are consistent with other liver effects observed in exposed rats (see below for more details). Dose-related, statistically significant increases in relative liver weights occurred at >10 mg/kg-day in males (11—38%) and females (10—27%) (see Table B-l). Absolute liver weight was statistically significantly increased at the low dose in females (18%) and at the high dose in males and females (29-31%); however, the changes did not follow a dose-response gradient. Female rats displayed biologically significant (>10%) increases in absolute liver weight at all doses, while male rats achieved biologically significant increases in absolute liver weight at only the low and high doses. Males, but not females, exhibited dose-related, significant increases in relative kidney weights at >10 mg/kg-day (+9% at 10 mg/kg-day and +12% at 30 mg/kg-day). Absolute kidney weights increased significantly in females at 3 mg/kg-day (+14%) but there was no dose-response correspondence. Gross necropsy findings at the end of the exposure period consisted of dark brown discoloration of the liver in six of six female rats at 30 mg/kg-day (no other female groups and no males exhibited this change). Histopathology findings in the liver (see Table B-2) consisted primarily of centrilobular hepatocyte hypertrophy (six of six high-dose animals of both sexes and one of six females at 10 mg/kg-day; graded as slight in all cases). One high-dose male rat had a finding of slight focal liver necrosis. These lesions were not observed in the control group. Males (six of six in the high-dose group, two of six in the mid-dose group, and one of six in the low-dose group) also exhibited hyaline droplets and eosinophilic bodies (all graded as slight) in the proximal tubular epithelium of the kidney (see Table B-2). The kidney changes, but not the liver changes, persisted until the end of the recovery period in some rats. No-observed-adverse-effect level (NOAEL) and lowest-observed-adverse-effect level (LOAEL) values of 3 and 10 mg/kg-day, respectively, are identified from this study based on >10% increases in relative liver weights in both sexes considered biologically significant, as well as statistically significant reductions in motor activity and increased PT in males. There is some 12 Dib enzothi ophene ------- EPA/690/R-22/002F uncertainty due to lack of a full English language report. Although >10% increases in absolute liver weights were observed at a dose of 3 mg/kg-day in rats, the changes in absolute liver weights did not follow a dose-response gradient. Further, the increase in relative liver weight in the mid- and high-dose groups is supported by increased incidence of hepatocyte lesions in male and female rats at >10 mg/kg-day (mostly hypertrophy but also possible evidence of necrosis) and significant decreases in albumin protein fraction and A/G ratio in males at 30 mg/kg-day. Biologically significant increases (>10%) in relative kidney weights were observed in males at 30 mg/kg-day and these animals also showed evidence of kidney lesions (100% incidence of hyaline droplets and eosinophilic bodies). The administered doses of 0, 3, 10, and 30 mg/kg-day correspond to human equivalent doses (HEDs) of 0, 0.75, 2.5, and 7.4 mg/kg-day for males, and 0, 0.68, 2.2, and 6.7 mg/kg-day for females, respectively.2 Subchronic/Chrottic Studies Thomas et al. (1942) In a published, peer-re viewed study, Thomas et al. (1942) administered dibenzothiophene (purity not reported) in the diet of male albino rats (source and number not reported) aged 25- 28 days with an average body weight of 48 g at the beginning of the study. The animals received 0.25, 0.50, or 1.00% dibenzothiophene in the diet for the first 4 days of the dosing period (number of animals per dose group not reported). Because of low food intakes and decreases in body weight, the doses were decreased to 0.025, 0.050, or 0.100% dibenzothiophene for the remainder of the 165-day dosing period. Adjusted daily doses (ADDs) are estimated to be 13, 27, and 63 mg/kg-day, respectively, based on total dibenzothiophene consumption reported by the study authors and time-weighted average (TWA) body weights obtained by digitizing the growth curves provided by the study authors. Animals were housed five to a cage; other details regarding animal husbandry were not provided. Appearance and behavior were recorded by the study authors "throughout the duration of the study." Food and water were provided ad libitum; animals and food cups were weighed twice a week for the duration of the study. Experimental data for each exposure group were compared with data for age- or body-weight-matched historical control animals; the type of historical control used for each endpoint is listed below with the results for that endpoint. At study termination, animals were sacrificed, and histopathological examinations were performed. The study authors noted that they used a necropsy technique previously described by Wilson et al. (1938); the spleen, liver, adrenal glands, kidneys, testes, ovaries, and heart were weighed under this necropsy protocol. Histopathological sections of the liver, spleen, adrenal gland, heart, bladder, intestine, lung, testis, and stomach were prepared from five animals in each exposure group and stained with hematoxylin and eosin (Thomas et al.. 1942). Frozen sections of the livers from three animals in the high-dose group and all animals in the low-dose group were stained with Sudan IV. Blood was collected on Days 107 and 157 from the tails of five high-dose animals and analyzed for hemoglobin and for erythrocyte, reticulocyte, and total and differential WBC counts. Although the study authors indicated statistical significance of their findings, no information was provided regarding their statistical methods. This study was performed prior to 2Administered doses were converted to HEDs by multiplying by dosimetric adjustment factors (DAFs) of 0.250, 0.248, and 0.247 for low-, mid-, and high-dose males and 0.226, 0.223, and 0.222 for low-, mid-, and high-dose females calculated as follows: DAF = (BWa1/4 ^ BWhI/4), where BWa = animal body weight, and BWh = human body weight. Study-specific TWA animal body weights of 0.272, 0.264, and 0.259 kg for low-, mid-, and high-dose males, and 0.182, 0.174, and 0.171 kg for low-, mid-, and high-dose females were used. For humans, the reference value of 70 kg was used for body weight, as recommended by U.S. EPA (1988). 13 Dib enzothi ophene ------- EPA/690/R-22/002F the adoption of Good Laboratory Practice (GLP), and little information regarding the laboratory procedures was provided. The study authors also reported a second experiment examining the presence of dibenzothiophene metabolites in the urine of rabbits (see Table 4B in Section 2.3.2). No deaths or clinical signs of toxicity were reported during the study. Body weights throughout the course of the study were presented graphically, and mean terminal body weights were provided in numerical form for each exposure group (see Table B-3). A dose-dependent decrease in body weight was observed; however, the study authors attributed this to reduced food consumption and did not consider it a direct effect of dibenzothiophene. For the evaluation of organ weights, animals dosed with dibenzothiophene were compared with laboratory historical controls matched according to body weight. As a result, the differences from the control group approximate a change in relative (to body weight) organ weight. The only significant effects on organ weight observed were in the liver and spleen. Although statistical significance for weight changes in both the liver and spleen were noted by the study authors, neither an indication of the dose at which significance occurred nor any levels of significance were reported. Data for these organs are presented in Table B-3. Liver weights increased (7-115%) in a dose-dependent manner, with changes >10% occurring at >27 mg/kg-day. Spleen weights decreased (29-57%) in a dose-dependent manner. The decreased spleen weight may be related to the decreased food consumption, as spleen weight has been shown to decrease disproportionately to body weight when food consumption is decreased (Peters and Boyd. 1966). Gross examination revealed that the livers in the mid- and high-dose animals were large and presented a yellowish, fatty appearance. Spleens appeared normal except for a reduction in their sizes upon gross examination. Liver and kidney histopathological lesions were reported by the study authors; however, incidence was not reported, and no control group was examined. Histopathology of livers from the high-dose animals revealed extensive fatty metamorphosis of the hepatic cells, abnormal fat accumulation, and irregular vacuolation of the parenchymal cells extending throughout the lobules. Livers from high-dose animals also had some cells with indistinct borders where it appeared that adjacent cells had fused. Other liver cells had a rim of homogenous, deeply stained cytoplasm surrounding groups of vacuoles. Similar changes, but less severe, were observed in the mid-dose group. The liver effects observed in the low-dose group were described as "still less severe" than those observed at the mid-dose. There was no evidence of fibrosis or necrosis, and the Kupffer cells were unchanged. Kidneys of all exposed animals had slight-to-moderate, light brown, granular pigmentation in the epithelial cells of the proximal convoluted tubules, but there was no evidence of cell destruction. Histopathological abnormalities in other organs, including the spleen, were not observed. Hematological effects were compared to age-matched controls. There were no hematological effects observed based on the blood analyses of the high-dose animals when compared with age-matched laboratory historical controls. In addition, the study authors noted that similar blood counts were seen in previously published hematological data from untreated animals and in animals treated with the closely related compound, diphenylene oxide. The outstanding limitations in the study design and data reporting, primarily the lack of a concurrent control group or reporting on the number of test animals, prevent further interpretation of the results or the determination of NOAEL and LOAEL values. The 14 Dib enzothi ophene ------- EPA/690/R-22/002F administered doses of 0, 13, 27, and 63 mg/kg-day correspond to HEDs of 0, 2.9, 5.9, and 13 mg/kg-day, respectively.3 Reproductive/Developmental Studies No studies were identified. 2.2.2. Inhalation Exposures No studies were identified. 2.3. OTHER DATA (SHORT-TERM TESTS, OTHER EXAMINATIONS) 2.3.1. Genotoxicity The available genotoxicity data for dibenzothiophene are limited and primarily indicate a lack of genotoxic activity. Dibenzothiophene was negative for mutagenicity in the Ames test involving Salmonella typhimurium strains at doses up to 5,000 (.ig/plate (JECDB. 2010a; Madill et al.. 1999; Mcfall et al.. 1984; Pelrov et al.. 1983; Dickson and Adams. 1980). with or without metabolic activation. A positive result was reported for mutation in the photoluminescent bacterium, Vibrio fisheri, in the Mutatox test (without activation), although the study authors noted that a positive response in this assay can occur without deoxyribonucleic acid (DNA) damage (Madill et al.. 1999). Studies in mammalian cells were negative for mutation in Chinese hamster ovary (CHO) cells at doses up to 100 (.ig/mL (Rasmussen et al.. 1991) and chromosomal aberrations (CAs) in Chinese hamster lung fibroblast (CHL) cells at doses up to 116 jag/m L for 24 hours with S9 or up to 1,850 |ig/L for 6 hours without activation (JHCDB. 2010b). A study in cultured rainbow trout liver RTL-W1 cells reported induction of micronucleus formation by dibenzothiophene, with an EC25 (the concentration causing 25% of the maximum effect level of the standard, 4-nitroquinoline oxide) of 10.8 mg/mL (3.2 mg/L after correction for estimated losses due to volatilization, sorption, etc.) (Brinkmann et al.. 2014). This is of uncertain relevance to mammals, however, A mat et al. (2004) observed weak DNA adduct formation at cytotoxic concentrations in HepG2 human hepatocellular carcinoma cells exposed to >50 |iM dibenzothiophene. These studies are further described in Table 4A. 3Administered doses were converted to HEDs by multiplying by DAFs of 0.225, 0.219, and 0.208 for low-, mid-, and high-dose rats calculated as follows: DAF = (BWa1/4 ^ BWh1/4), where BWa = animal body weight, and BWh= human body weight. Study-specific estimated average animal body weights of 0.179, 0.161, and 0.130 kg for low-, mid-, and high-dose rats were used. For humans, the reference value of 70 kg was used for body weight, as recommended by U.S. EPA (1988). 15 Dib enzothi ophene ------- EPA/690/R-22/002F Table 4A. Summary of Dibenzothiophene (CASRN 132-65-0) Genotoxicity Endpoint Test System Doses/ Concentrations Tested3 Results Without Activationb Results With Activationb Comments Reference Genotoxicity studies in prokaryotic organisms Reverse mutation Ames assay using Salmonella typhimurium strain TA98 treated with 10-100 |ig dibenzothiophene per plate dissolved in DMSO and incubated at 37°C for 48 h with Aroclor 1254-induced rat-liver S9 homogenate activation (S9 concentrations of 4, 10, or 20%) 100 (ig/plate ND Not mutagenic at any dose; S9 volume did not affect activity. Mcfall et al. (1984) Ames assay using S. typhimurium strains TA98, TA100, TA1535, TA1537, and TA1538 treated with an unreported quantity of dibenzothiophene dissolved in DMSO with Aroclor 1254-induced rat-liver S9 homogenate activation NR ND Not mutagenic; mutagenicity results presented as revertant ratio (number of revertants per plate/number of spontaneous revertants); dibenzothiophene reportedly had "no mutagenic response" with an average revertant ratio <2.0. Dickson and Adams (1980) Ames assay using S. typhimurium strains TA98, TA100, TA1535, and TA1537 treated with 2-500 |ig dibenzothiophene per plate dissolved in DMSO with and without Aroclor 1254-induced rat-liver S9 homogenate activation 500 (ig/plate Not mutagenic at any dose. Pel to v et al. (1983) 16 Dib enzothi ophene ------- EPA/690/R-22/002F Table 4A. Summary of Dibenzothiophene (CASRN 132-65-0) Genotoxicity Endpoint Test System Doses/ Concentrations Tested3 Results Without Activationb Results With Activationb Comments Reference Ames assay (preincubation modified plate incorporation test) using S. typhimurium strains TA98 and TA100 treated with unspecified three-log dose dilutions of dibenzothiophene in DMSO with or without S9 activation; control experiments conducted with DMSO as negative control and BaP and 4-nitroqui no 1 i nc- \ -o.xide as positive controls NR Not mutagenic at any dose. Madill et al. (1999) Ames assay using S. typhimurium strains TA98, TA100, TA1535, and TA1537 and Escherichia coli WP2uvrA treated with dibenzothiophene in DMSO at 78.1- 5,000 ng/plate with or without S9 activation; negative and positive controls included 5,000 ng/plate Not mutagenic at any dose. Precipitation and growth inhibition were seen in all strains at the higher dose levels tested (1,250-5,000 |ig/plate). JECDB (2010a) Mutation Mutatox assay in which photoluminescent bacterium Vibrio fisheri were incubated with 0.01- 5 ng/tube dibenzothiophene in methanol for 45 min with or without S9 activation; control experiments conducted with methanol as negative control and BaP and phenol as positive controls 0.38 ng/tube + In absence of activation, positive response (twofold increase in light output vs. negative control) in 5 of 10 tubes in dilution series, with 0.38 ng/tube dibenzothiophene being the lowest effective dose. In presence of activation, negative at all doses up to 5 ng/tube. Positive response in this assay can occur without DNA damage (e.g., phenol, the positive control, is nongenotoxic and noncarcinogenic). Madill et al. (1999) Genotoxicity studies in nonmammalian eukaryotic cells—in vitro Micronucleus formation Micronucleus assay conducted in rainbow trout liver RTL-W1 cells using 0.3-41 mg/L dibenzothiophene 10.8 mg/L (EC25) + ND EC25 = 10.8 mg/L for micronucleus induction (3.2 mg/L after correction for estimated losses due to volatilization, sorption, etc.). Brinktnatm et al. (2014) 17 Dib enzothi ophene ------- EPA/690/R-22/002F Table 4A. Summary of Dibenzothiophene (CASRN 132-65-0) Genotoxicity Endpoint Test System Doses/ Concentrations Tested3 Results Without Activationb Results With Activationb Comments Reference Genotoxicity studies in mammalian cells—in vitro Mutation Chinese hamster ovary (CHO-K1BH4) cells treated with 1- 100 (ig/mL dibenzothiophene with Ham's F12 medium, activated with 4% Aroclor-induced rat-liver S9 solution and incubated for 5 h; control experiments conducted with DMSO as the control and methyl methane sulfonate as a positive control 100 (ig/mL ND Not mutagenic at any dose. Rasiiius.se ti et al. (1991) CAs Cultured Chinese hamster lung fibroblast CHL/IU cells incubated with dibenzothiophene at 7.23-116 |ig/mL for 6-24 h (without S9) or 57.8-1,850 ng/mL for 6 h (with S9) 1,850 ng/mL No effect on structural or numerical aberrations at any dose. Toxicity was observed at the higher doses in the tests without S9 (86.9-116 ng/mL) but not in the tests with S9. JECDB (2010b) DNA adduct formation Cultured HepG2 human hepatocellular carcinoma cells incubated with 0.25-150 |iM dibenzothiophene (dissolved in methanol) for 24 or 48 h; control experiments conducted with culture medium alone as negative control and BaP in DMSO as positive control. 50 \M ± ND Weak DNA adduct formation at cytotoxic concentrations. A mat et al. (2004) aLowest effective dose for positive results, highest dose tested for negative results. b+ = positive; ± = weakly positive; - = negative. BaP = benzoic/1 pyre nc: CA = chromosomal aberration; DMSO = dimethylsulfoxide; DNA = deoxyribonucleic acid; EC25 = the concentration causing 25% of the maximum effect level of the standard, 4-nitroquinoline oxide; ND = no data. 18 Dib enzothi ophene ------- EPA/690/R-22/002F 2.3.2. Metabolism/Toxicokinetic and Supporting Animal Studies Additional studies investigating the metabolism of dibenzothiophene in rats (Jacob et al.. 1991; Vignier et al., 1985), elimination of dibenzothiophene in the urine of rabbits (Thomas et al., 1942), acute toxicity of dibenzothiophene in mice (Leigfaton. 1989), and toxicity of dibenzothiophene by weekly injection in rats (Silva et al .. 2015), as well as in vitro studies of effects of dibenzothiophene on aggregation of platelets (Chaudhurv et al .. 1988) and viability of differentiated SK-N-SH human neuroblastoma cells (Sarma et al.. 2017) are also available. See Table 4B for details of these studies. 19 Dib enzothi ophene ------- EPA/690/R-22/002F Table 4B. Other Studies Test Materials and Methods Results Conclusions Reference Supporting Animal Studies Short-term CD-I mice treated via gavage. Pilot ranse-findins studies: 4 mice/sex/dose. Pilot ranee finding studies: No treatment-related Without induction: acute LD5o of 470 mg/kg; with prior induction of MFO, acute LD5o of 335 mg/kg; preinduction of MFO potentiated the toxicity of dibenzothiophene. Leighton (1989) hematological changes seen; no treatment-related histological lesions seen in the kidney, duodenum, spleen, or heart; liver lesions included centrilobular or periacinar degeneration and necrosis. LDsu experiments: All mortality occurred within 72 h of treatment and was increased in groups with prior induction of MFO; animals were sluggish; gross lesions in mice found dead included pulmonary congestion and edema, mild to moderate hydrothorax, intestinal hemorrhage, and mottled livers; all MFO-induced mice had mild fibrinous peritonitis; histological lesions included severe centrilobular hepatic necrosis across doses in both experiments, necrosis of lymphocytes in thymic cortices at >540 mg/kg in Experiment 1 and >265 mg/kg in Experiment 2, and degenerative changes in the walls of small arteries in the lung in five mice dosed with 265-492 mg/kg (Experiment 2). single dose of 0-3,250 mg/kg or four consecutive daily doses of 0-325 mg/kg; necropsy performed 24 h after last dose; blood taken from hearts of mice and examined for hematological effects; liver, kidney, spleen, heart, lungs, thymus, and duodenum examined at necropsy. LDsu Experiment 1: 12 male mice/dose. 12 vehicle controls, 8 untreated controls; single doses of 0, 260, 374, 540, 777, 1,118, or 1,609 mg/kg; LD5o determined at 7 d; surviving mice sacrificed on day 14 for histology of liver, lung, heart, and thymus. LDsu Experiment 2: 12 mice/treatment aroiiD. 5 preinduced vehicle controls; MFO pretreatment (one i.p. injection of 3-methylcholanthrene [80 mg/kg] followed by daily i.p. injections of phenobarbital [50 mg/kg] in sterile saline for 3 d) followed 24 h later by single doses of 0, 215, 265, 325, 400, 492, 605, or 744 mg/kg. 20 Dib enzothi ophene ------- EPA/690/R-22/002F Table 4B. Other Studies Test Materials and Methods Results Conclusions Reference Injection Male Wistar rats were given weekly i.p. injections of saline (n = 5), soy oil (n = 5) or 30 mg/kg dibenzothiophene in soy oil (n = 15) for 10 wk and were examined after a latency period of 14 wk for hematology (RBC, hematocrit, WBC total and differential), serum chemistry (amylase, ALT, AST), organ weights (liver and spleen), histology (liver, spleen, lungs, and intestines), immunohistochemistry (using anti-CEA and anti-CD44 antibodies) and proteomic analysis. Body weights were recorded weekly. There were no treatment-related effects on body weight, hematology, serum chemistry, or organ weights. Histopathological examination showed effects only in the intestines, including increased incidence of cellular atypia in the mucosa and submucosa, mucosa inflammation, necrosis, and hyperplasia of lymphoid nodules in both the large and small intestines. Counts of cells positive for antibody labelling were increased threefold (either CEA or CD44), suggesting the presence of preneoplasia. Proteomic analysis identified 23 proteins showing altered levels (>1.5-fold change versus controls) in the small intestine, with functions including heat-shock response; cytoskeleton organization; antioxidant activity; cell signaling; carbohydrate, lipid and nucleotide metabolism; and protein folding. Dibenzothiophene produced dysplastic lesions in the large and small intestines of male rats treated weekly by injection of 30 mg/kg in soy oil for 10 wk and examined at 24 wk. Silva et al. (2015) Metabolism/toxico kinetic Metabolism/ Toxicokinetic Male Wistar rat (number not specified), treated with daily i.p. injections of 40 mg/kg dibenzothiophene for 3 d, 3-methylcholanthrene for 3 d, 500 mg/kg Aroclor 1254 for 5 d, or twice daily i.p. injections of 40 mg/kg phenobarbital for 4 d, then starved for 24 h after final injection; liver microsomes isolated; in vitro oxidation assay performed using dibenzothiophene (0.02-0.50 mM) and rat liver microsomal suspension (10 |iL). Dibenzothiophene metabolic pathway determined to be S-oxidation with metabolites of dibenzothiophene-5-oxide (primary) and dibenzothiophene -5 -dioxide (secondary); Aroclor 1254, 3-methylcholanthrene, and phenobarbital increased rate of formation of sulfoxide, but dibenzothiophene pretreatment had no effect; carbon monoxide inhibited sulfoxidation. Dibenzothiophene metabolite, dibenzothiophene-5-oxide, was further oxidized to dibenzothiophene-5-dioxide; CYP450 monooxygenases most likely involved in the metabolism. Vienier et al. (1985) 21 Dib enzothi ophene ------- EPA/690/R-22/002F Table 4B. Other Studies Test Materials and Methods Results Conclusions Reference Male Wistar rat (number not specified), treated with i.p. injections of 40 mg 5,6-benzoflavone/kg for 3 d, 200 mg Aroclor/kg once, or 80 mg phenobarbital/kg in 0.9% NaCl over 3 d and sacrificed 24 h after last dose; microsomes from four animals per group were incubated with 50 (imol/L dibenzothiophene for 20 min at 37°C and analyzed; solvent-only controls. Metabolic products were sulfoxide (main product) and sulfone; no pretreatments affected sulfoxide formation, but pretreatments with phenobarbital and Aroclor increased sulfone formation. Dibenzothiophene metabolites (using rat microsomes) were sulfoxide and sulfone, controlled by different enzymes; only the one responsible for sulfone formation can be induced by CYP450 inducers such as phenobarbital. Jacob et al. (1991) One rabbit (sex and strain not specified) given an emulsion of 2 g dibenzothiophene in water administered via stomach tube; urine collected (time not specified) and analyzed. Main excretion product was mono-hydroxy- diphenylene sulfone. Dibenzothiophene oxidized to mono-hydro xy-diphenylene sulfone in the rabbit. Thomas et al. (1942) Mode of action/mechanistic In vitro Aggregation of platelets from blood collected from male Sprague Dawley rats was measured photometrically after incubation for 2 min with 1. 3. or 5 |iL of dibenzothiophene in DMSO or DMSO alone. Tests were also conducted with addition of thrombin or ADP to stimulate platelet aggregation, mobilization of internal calcium stores within platelets, and uptake of extracellular calcium by platelets. Platelet aggregation was significantly reduced in a dose-related manner by dibenzothiophene relative to controls. Dibenzothiophene also reduced aggregation stimulated by thrombin or ADP and reduced extracellular calcium uptake by platelets. Dibenzothiophene may inhibit platelet aggregation by bringing about alterations in the platelet plasma membrane. Chaudhurv et al. (1988) Differentiated SK-N-SH human neuroblastoma cells were cultured for 24 h with 5-100 |iIVI dibenzothiophene and assessed for viability and production of ROS relative to unexposed controls. Neuroblastoma cell viability was reduced relative to controls at all test concentration, with LCio = 5.07 nM and LC20 = 47.26 ^M. Significant increases in ROS were seen at >10 ^M. Dibenzothiophene induces neuronal cell damage by a mechanism that involves generation of oxidative stress. Sarma et al. (2017) ADP = adenosine diphosphate; ALT = alanine aminotransferase; AST = aspartate aminotransferase; CYP450 = cytochrome P450; DMSO = dimethyl sulfoxide; i.p. = intraperitoneal; MFO = mixed function oxidase; NaCl = sodium chloride; RBC = red blood cell; ROS = reactive oxygen species; WBC = white blood cell. 22 Dib enzothi ophene ------- EPA/690/R-22/002F 3. DERIVATION OF PROVISIONAL VALUES 3.1. DERIVATION OF ORAL REFERENCE DOSES The database of repeat-dose oral studies for dibenzothiophene is limited to a non-peer-revievved, 28-day study in Japanese with only tables and figures in English (JHCDB. 2011) and a peer-reviewed, 165-day study from 1942 that relied on historical laboratory control groups instead of a concurrent control (Thomas et al.. 1942). Due to the shortcomings of these studies, reference doses (RfDs) cannot be confidently derived here. However, the studies provide sufficient data to develop a screening subchronic provisional reference dose (p-RfD) value (see Appendix A). 3.2. DERIVATION OF INHALATION REFERENCE CONCENTRATIONS No subchronic or chronic provisional reference concentration (p-RfC) can be derived because no inhalation studies on exposure to dibenzothiophene were identified. The feasibility of using an analogue approach was attempted for the derivation of screening-level p-RfC values via read-across but no candidate analogues with inhalation toxicity values were identified (see Appendix A). 3.3. SUMMARY OF NONCANCER PROVISIONAL REFERENCE VALUES Table 5 presents a summary of the noncancer provisional references values. Table 5. Summary of Noncancer Risk Estimates for Dibenzothiophene (CASRN 132-65-0) Toxicity Type (units) Species/ Sex Critical Effect p-Reference Value POD Method POD (HED/HEC) UFc Principal Study Screening subchronic p-RfD (mg/kg-d) (see Appendix A) Rat/F Increased hepatocyte hypertrophy 3 x 1(T3 BMDL io 1.04 300 JHCDB (20ID Chronic p-RfD (mg/kg-d) NDr Subchronic p-RfC (mg/m3) NDr Chronic p-RfC (mg/m3) NDr BMD = benchmark dose; BMDL = 95% benchmark dose lower confidence limit on the BMD (subscripts denote BMR: i.e., 10 = dose associated with 10% extra risk); HEC = human equivalent concentration; HED = human equivalent dose; NDr = not determined; POD = point of departure; p-RfC = provisional reference concentration; p-RfD = provisional reference dose; UFC = composite uncertainty factor. 3.4. CANCER WEIGHT-OF-EVIDENCE DESCRIPTOR No human or animal data were located on the carcinogenicity of dibenzothiophene by oral or inhalation exposure. One available injection study observed dysplastic lesions in the intestines of rats treated for 10 weeks, suggesting that dibenzothiophene may have some carcinogenic potential (Silva et al.. 2015). Genotoxicity studies were largely negative, including 23 Dib enzothi ophene ------- EPA/690/R-22/002F multiple Ames tests for mutation in bacteria and assays for mutation and CAs in mammalian cells (JHCDB. 2010a. b; Madill et al.. 1999; Rasmussen et al.. 1991; Mcfall et al.. 1984; Pelrov et al.. 1983; Dickson and Adams, 1980). Under the U.S. EPA Cancer Guidelines (U.S. EPA, 2005), there is "Inadequate Information to Assess Carcinogenic PotentiaT of dibenzothiophene by oral or inhalation exposure (see Table 6). Table 6. Cancer WOE Descriptor for Dibenzothiophene (CASRN 132-65-0) Possible WOE Descriptor Designation Route of Entry (oral, inhalation, or both) Comments "Carcinogenic to Humans" NS NA No human carcinogenicity data are available. "Likely to Be Carcinogenic to Humans" NS NA No adequate animal cancer bioassays or human cancer data are available. "Suggestive Evidence of Carcinogenic Potential" NS NA No adequate animal cancer bioassays or human cancer data are available. "Inadequate Information to Assess Carcinogenic Potential" Selected Both Selected due to the lack of adequate data on carcinogenicity. One injection study provided limited evidence of carcinogenic potential. "Not Likely to Be Carcinogenic to Humans" NS NA No evidence of noncarcinogenicity is available. NA = not applicable; NS = not selected; WOE = weight-of-evidence. 3.5. DERIVATION OF PROVISIONAL CANCER RISK ESTIMATES The absence of suitable data precludes the development of cancer risk estimates for dibenzothiophene (see Table 7). Table 7. Summary of Cancer Risk Estimates for Dibenzothiophene (CASRN 132-65-0) Toxicity Type Species/Sex Tumor Type Cancer Value Principal Study p-OSF (mg/kg-d) 1 NDr p-IUR (mg/m3) 1 NDr NDr = not determined; p-IUR = provisional inhalation unit risk; p-OSF = provisional oral slope factor. 24 Dib enzothi ophene ------- EPA/690/R-22/002F APPENDIX A. SCREENING PROVISIONAL VALUES Due to the lack of evidence described in the main Provisional Peer-Reviewed Toxicity Value (PPRTV) document, it is inappropriate to derive provisional toxicity values for dibenzothiophene. However, some information is available for this chemical, which although insufficient to support derivation of a provisional toxicity value under current guidelines, may be of limited use to risk assessors. In such cases, the Center for Public Health and Environmental Assessment (CPHEA) summarizes available information in an appendix and develops a "screening value." Appendices receive the same level of internal and external scientific peer review as the provisional reference values to ensure their appropriateness within the limitations detailed in the document. Users of screening toxicity values in an appendix to a PPRTV assessment should understand that there could be more uncertainty associated with deriving an appendix screening toxicity value than for a value presented in the body of the assessment. Questions or concerns about the appropriate use of screening values should be directed to the CPHEA. A screening subchronic provisional reference doses (p-RfD) was derived for dibenzothiophene as described in the section below. For inhalation, an alternative analogue approach was evaluated (see APPLICATION OF AN ALTERNATIVE ANALOGUE APPROACH (METHODS) below), but suitable analogues were not identified and a screening value was not derived. DERIVATION OF SCREENING PROVISIONAL REFERENCE DOSES As discussed in the main body of the report, the available repeat-dose oral studies for dibenzothiophene include only JECDB (2011) and Thomas et al. (19421 both of which have limitations precluding their use in deriving provisional toxicity values. In order to account for the uncertainty associated with basing a toxicity assessment on these studies, the assessment is considered a screening-level assessment. The 28-day oral exposure study by JECDB (2011) is limited by unpublished status, lack of peer review, and use of Japanese language with only tables and figures in English. There was enough material presented in English, however, to ascertain that the study appeared to be adequately designed and conducted, and to provide dose-response information on a wide range of endpoints suitable for use in quantitative toxicity assessment, including body weight, food consumption, clinical observations, functional observational battery (FOB), hematology, serum chemistry, urinalysis, and selected organ weight and histopathology (see study summary in Section 2.2.1 for more details). Liver effects were a sensitive target for dibenzothiophene in the JECDB (2011) study. Dose-related and biologically significant (>10%) increases in relative liver weight were reported in both male and female rats at >10 mg/kg-day and were a primary basis for the study reported no-observed-adverse-effect level (NOAEL)/lowest-observed-adverse- effect level (LOAEL) values (NOAEL = 3 mg/kg-day and LOAEL =10 mg/kg-day). The relative liver weight changes were accompanied by increased incidence of hepatocyte hypertrophy in both sexes at >10 mg/kg-day (1/6 females at 10 mg/kg-day and 6/6 males and females at 30 mg/kg-day) and possible evidence of structural degeneration (slight necrosis in 1/6 animals) and changes in serum markers of liver damage (decreased albumin protein fraction and albumin/globulin [A/G] ratio]) in males at 30 mg/kg-day. Although increases (>10%) in absolute liver weights were observed in rats at >3 mg/kg-day, the changes at the lowest dose 25 Dib enzothi ophene ------- EPA/690/R-22/002F were not supported by corroborative evidence of liver toxicity and overall pattern of effects lacked a dose-response relationship (see Table B-l). The findings across organ weight, histopathology, and clinical chemistry measures provide coherent evidence of liver toxicity after short-term oral exposure to dibenzothiophene. Other treatment-related effects observed at the JECDB (2011) study LOAEL of 10 mg/kg-day included significant reductions in motor activity, although there was no corroborative evidence from other FOB assays evaluating reactivity (visual, touch, auditory, pain, proprioceptive), righting reflex, or grip strength. Additionally, significant increases in prothrombin time (PT) were reported in males at >10 mg/kg-day, accompanied by significant increases in activated partial thromboplastin time (APTT) at 30 mg/kg-day in these animals. Prolonged clotting times (i.e., increased PT and APTT) are consistent with findings of dibenzothiophene-induced platelet aggregation in vitro (Chaudhurv et al.. 1988). and decreased motor activity is consistent with findings of decreased viability of differentiated SK-N-SH human neuroblastoma cells with in vitro dibenzothiophene exposure (Sarma et al.. 2017). However, there is limited in vivo evidence to determine the biological significance of the changes in motor activity and prolonged clotting times in males. Male rats in the JECDB (2011) study exhibited biologically significant increases (>10%) in relative kidney weights at 30 mg/kg-day. Dose-related increases in the incidence of hyaline droplets and eosinophilic bodies in the proximal tubular epithelium of the kidney (one of six, two of six, and six of six animals at 3, 10, and 30 mg/kg-day, respectively) also occurred in males. Accumulation of hyaline droplets (also described as cytoplasmic eosinophilic bodies containing protein) are commonly associated with alpha 2u-globulin (a2u-g)-mediated nephropathy (Hard et al.. 1999). a male rat-specific nephropathy not considered relevant to humans. According to (U.S. EPA. 1991). three criteria are required for evaluating the relevance of kidney lesions in males based on possible involvement of a2u-g: (1) observation of an increase in number and size of hyaline droplets only in male kidneys; (2) identification of the protein contained in the hyaline droplets as a2u-g; and (3) observation of additional events in the pathological sequence of lesions associated with a2u-g disease (i.e., single cell necrosis, exfoliation of epithelial cells into tubular lumen, and granular casts). The evidence for dibenzothiophene is limited to increases in hyaline droplets occurring only in male rats in the 28-day JECDB (2011) study. Given that the study is in Japanese, it is unclear whether the study authors performed any specialized staining for detection of a2u-g and no additional observations were made regarding other events in the pathological sequence of the development of a2u-g disease. Further, there is no supporting evidence for a2u-g-, including the 165-day study by Thomas et al. (1942). Thomas et al. (1942) reported slight-to-moderate, light brown, granular pigmentation in the epithelial cells of the proximal convoluted tubules of male rats (with no evidence of cell destruction) but no other details were provided; therefore, the toxicological significance of the findings is unknown. The limitations in the database for dibenzothiophene prevent further interpretation of the relevance of the male rat kidney lesions in the JECDB (2011) study. Given the uncertainty and lack of information for further evaluation, these kidney lesions in male rats (hyaline droplets and eosinophilc bodies) were not further considered for dose-response analysis. 26 Dib enzothi ophene ------- EPA/690/R-22/002F The 165-day dietary exposure study in rats by Thomas et al. (1942) had outstanding limitations such as the lack of a concurrent control group, instead making inferences based on historical control groups. Other limitations included the use of males only, lack of reporting on the number of test animals per group, and incomplete data reporting for histopathological outcomes. The major study findings were increases in liver weight that reached 35% at 27 mg/kg-day and 115% at 63 mg/kg-day (over body-weight-matched laboratory historical controls). Histopathological lesions in the liver (i.e., fat accumulation, irregular vacuolation of the parenchymal cells [hepatocytes] throughout the lobules, and indications that adjacent cells had fused) were also observed at all doses; however, incidence of lesions was not provided, and severity was described as much less in the low- and mid-dose groups (13 and 27 mg/kg-day) compared to the high-dose group (63 mg/kg-day). Although the study limitations add considerable uncertainty to the interpretation of the findings or the determination of NOAEL/LOAEL values, these observations are consistent with the liver effects in the JECDB (2011) study, providing supportive evidence of dibenzothiophene-induced liver toxicity. Overall, the increases in relative liver weight and liver lesions (primarily hypertrophy) and decreases in serum markers of liver function (albumin protein fraction and A/G ratio) provide coherent evidence of liver effects in rats at >10 mg/kg-day after 28-day exposure (JECDB. 2011). Although the relevance of male kidney lesions reported in the JECDB (2011) study is unclear, the changes in relative kidney weights in males at 30 mg/kg-day were considered biologically significant (>10%). Therefore, both the liver effects and relative kidney weight changes from this study were considered further for the derivation of screening p-RfDs. Other treatment-related effects (decreased motor activity, increased PT and APTT and increased hyaline droplets in males) in the JECDB (2011) study were not advanced for dose-response analysis due to the limitations in the database for dibenzothiophene, which prevent further determination of the toxicological significance of the findings. Derivation of Screening Subchronic Provisional Reference Dose Data for liver effects in male and female rats and increased relative kidney weights in male rats from the JECDB (2011) study were modeled using the U.S. Environmental Protection Agency (U.S. EPA) Benchmark Dose Software (BMDS, Version 3.2). Despite the non-peer-reviewed status and lack of full English language report, the study used an adequate design (28-day rat study), included multiple doses and a comprehensive array of toxicity endpoints, and identified sensitive health effects that are suitable for the derivation of the screening subchronic p-RfD (JECDB. 2011). For liver effects, dose-related increases in relative liver weight in males and females at >10 mg/kg-day were modeled as continuous data using a benchmark response (BMR) of 10% relative deviation (RD) because a 10% change in liver weight is considered a minimally biologically significant response in adult animals. Hepatocyte hypertrophy was modeled in females as dichotomous data, applying a standard BMR of 10% extra risk (ER). Hepatocyte hypertrophy in males was not modeled given that the effects were only observed in the high-dose group. Although the decreases in some serum markers of liver function (albumin protein fraction and A/G ratio) in male rats provide supporting evidence for dibenzothiophene-induced liver effects, these endpoints were not considered for dose-response assessment since more sensitive and relevant markers of liver toxicity were available (i.e., relative liver weight and hepatocyte hypertrophy). Increased relative kidney weight in males were modeled as continuous data using a BMR of 10%, which is considered biologically significant. Human equivalent doses (HEDs) in mg/kg-day were used as the dose metric for BMD analysis. 27 Dib enzothi ophene ------- EPA/690/R-22/002F Table A-l shows the data for liver and kidney endpoints that were considered for dose- response assessment and Table A-2 summarizes the BMD modeling results and provides candidate points of departure (PODs) for the derivation of the screening subchronic p-RfD. Details of model fit for each data set are presented in Appendix C. Candidate PODs that could not be evaluated via BMD analysis (i.e., hepatocyte hypertrophy in males) are presented as NOAEL/LOAEL values. Table A-l. Data for Sensitive Endpoints in Male and Female Sprague Dawley Rats After Oral Treatment with Dibenzothiophene for 28 Days" Endpoint ADD [HED] in mg/kg-db 0 3 [0.68 female, 0.75 male] 10 [2.2 female, 2.5 male] 30 [6.7 female, 7.4 male] Increased relative liver weight in males0 3.233 ±0.247 3.512 ±0.271 (+9%) 3.578 ±0.153* (+11%) 4.465 ± 0.208** (+38%) Increased relative liver weight in females0 3.123 ±0.170 3.355 ±0.145 (+7%) 3.450 ±0.299* (+10%) 3.970 ±0.187** (+27%) Increased hepatocyte hypertrophy in males'1 0/6 (0%) 0/6 (0%) 0/6 (0%) 6/6 (100%) Increased hepatocyte hypertrophy in females'1 0/6 (0%) 0/6 (0%) 1/6 (17%) 6/6 (100%) Increased relative kidney weight in males0 0.732 ±0.040 0.735 ±0.023 (+0%) 0.798 ±0.052* (+9%) 0.823 ±0.031** (+12%) aJECD6 (2011). bADDs were converted into HEDs (HED = ADD x DAF) using DAFs of 0.250, 0.248, and 0.247 for low-, mid-, and high-dose males and 0.226, 0.223, and 0.222 for low-, mid-, and high-dose females calculated as follows: DAF = (BWa1/4 + BWh1'4), where BWa = animal body weight, and BWh = human body weight. Study-specific TWA animal body weights of 0.272, 0.264, and 0.259 kg for low-, mid-, and high-dose males, and 0.182, 0.174, and 0.171 kg for low-, mid-, and high-dose females were used. For humans, the reference value of 70 kg was used for body weight, as recommended by U.S. EPA (1988). Data are means ± SD; n = 6 for all data points; value in parentheses is % change relative to control = ([treatment mean - control mean] + control mean) x 100. dData are number of animals showing changes/ total number of animals examined (% incidence). * Significantly different from control (p < 0.05) by Dunnett's test as reported by the study authors. **Significantly different from control (p < 0.01) by Dunnett's test as reported by the study authors. ADD = adjusted daily dose; DAF = dosimetric adjustment factor; HED = human equivalent dose; SD = standard deviation; TWA = time-weighted average. 28 Dib enzothi ophene ------- EPA/690/R-22/002F Table A-2. Comparison of Candidate POD Values in Male and Female Sprague Dawley Rats After Oral Treatment with Dibenzothiophene for 28 Days" Endpoint Best-Fitting Model BMR BMDL (HED) (mg/kg-d) POD type POD (HED) (mg/kg-d) Increased relative liver weight in males Exponential 3 (constant variance) 10% RD from control (0.1 RD) 2.01 BMDL 2.01 Increased relative liver weight in females Linear (constant variance) 10% RD from control (0.1 RD) 2.19 BMDL 2.19 Increased hepatocyte hypertrophy in males Data not amenable for BMD modeling13 NOAEL 2.5 Increased hepatocyte hypertrophy in females Probit (constant variance) 10% ER from control (0.1 ER) 1.04 BMDL 1.04 Increased relative kidney weight in males Exponential 4 (constant variance) 10% RD from control (0.1 RD) 1.36 BMDL 1.36 aJECD6 (2011). bData were not considered amenable for BMD modeling given that incidence was 100% at the highest dose and 0% at lower doses. BMD = benchmark dose; BMDL = benchmark dose lower confidence limit; BMR = benchmark response; ER = Extra Risk; HED = human equivalent dose; NOAEL = no-observed-adverse-effect level; POD = point of departure; RD = relative deviation. The 10% benchmark dose lower confidence limit (BMDLio) (HED) of 1.04 mg/kg-day for increased hepatocyte hypertrophy in female rats in the JECDB (2011) study is the lowest POD in the available database and is expected to be protective of other health effects associated with dibenzothiophene oral exposure. The significance of dibenzothiophene-induced liver effects is based on coherent evidence across organ weights (increased relative liver weight), histopathology (primarily hypertrophy with some evidence of necrosis), and serum markers of liver function (decreased albumin protein fraction and A/G ratio) in rats at >10 mg/kg-day after 28-day oral exposure (JECDB. 2011). Supportive evidence of potential liver toxicity was also found after dietary exposure for 165 days in males rats (increased liver weight and fatty accumulation in the liver at >27 mg/kg-day) (Thomas et al.. 1942) and acute gavage range- finding and median lethal dose (LD50) experiments in mice (centrilobular degeneration and necrosis across 260-1,609 mg/kg) (Leighton. 1989). Altogether, the weight of evidence suggests that the liver is a primary target for dibenzothiophene via oral exposure and the BMDLio [HED] of 1.04 mg/kg-day for increased hepatocyte hypertrophy in female rats exposed for 28 days (JECDB. 2011) is selected as the most sensitive POD for the derivation of the subchronic p-RfD. 29 Dib enzothi ophene ------- EPA/690/R-22/002F The screening subchronic p-RfD of 3 x 10 3 mg/kg-day for dibenzothiophene is derived by applying a composite uncertainty factor (UFc) of 300 (reflecting an interspecies uncertainty factor [UFa] of 3, an interindividual variability uncertainty factor [UFh] of 10, and a database uncertainty factor [UFd] of 10) to the selected POD of 1.04 mg/kg-day, as follows: Screening Subchronic p-RfD = POD (HED) UFc = 1.04 mg/kg-day -^300 = 3 x 10"3 mg/kg-day Table A-3 summarizes the uncertainty factors for the screening subchronic p-RfD for dib enzothi ophene. Table A-3. Uncertainty Factors for the Screening Subchronic p-RfD for Dibenzothiophene (CASRN 132-65-0) UF Value Justification UFa 3 A UFa of 3 (100 5) is applied to account for uncertainty in characterizing the toxicodynamic differences between rats and humans following oral dibenzothiophene exposure. The toxicokinetic uncertainty has been accounted for by calculation of an HED through application of a DAF as outlined in the U.S. EPA's Recommended Use of Body Weight4 as the Default Method in Derivation of the Oral Reference Dose (U.S. EPA, 2011c). UFd 10 A UFd of 10 is applied to account for deficiencies and uncertainties in the database. The repeat-dose oral database for dibenzothiophene includes a non-peer-reviewed, 28-day rat study in Japanese and a chronic rat study with significant limitations (primarily lack of a concurrent control and reporting on the number of test animals). No reproductive or developmental toxicity studies are available by any route of exposure. UFh 10 A UFh of 10 is applied for interindividual variability to account for human-to-human variability in susceptibility in the absence of quantitative information to assess the toxicokinetics and toxicodynamics of dibenzothiophene in humans. UFl 1 A UFl of 1 is applied because the POD is a BMDL. UFS 1 A UF s of 1 is applied because the POD was derived from a study of suitable duration (28 days) for a subchronic value. UFC 300 Composite UF = UFa x UFd x UFh x UFl x UFs. DAF = dosimetric adjustment factor; HED = human equivalent dose; POD = point of departure; BMDL = benchmark dose lower confidence limit; p-RfD = provisional reference dose; UF = uncertainty factor; UFa = interspecies uncertainty factor; UFC = composite uncertainty factor; UFD = database uncertainty factor; UFh = intraspecies uncertainty factor; UFL = LOAEL-to-NOAEL uncertainty factor; UFS = subchronic-to-chronic uncertainty factor. Derivation of Screening Chronic Provisional Reference Dose The POD used for derivation of the screening subchronic p-RfD based on increased hepatocyte hypertrophy in female rats (BMDLio [HED] of 1.04 mg/kg-day) from the 28-day study by JECDB (2011), cannot be used directly for derivation of the screening chronic p-RfD, due to the short duration of the critical study. The available 165-day chronic study by Thomas et al. (1942) reported increases in liver weight and liver histopathology at >27 mg/kg-day, which are similar to the doses associated with liver effects in the 28-day JECDB (2011) study (>10 mg/kg-day). However, the limitations in the study design and data reporting in Thomas et 30 Dib enzothi ophene ------- EPA/690/R-22/002F al. (1942) raise significant concerns regarding the interpretation of the study findings. Overall, the lack of adequate data to inform whether the liver or other health effects associated with dibenzothiophene worsen with chronic exposure prevent the derivation of a screening chronic p-RfD. APPLICATION OF AN ALTERNATIVE ANALOGUE APPROACH (METHODS) The analogue approach allows for the use of data from related compounds to calculate screening values when data for the compound of interest are limited or unavailable. Details regarding searches and methods for analogue analysis are presented in Wang et al. (2012). Three types of potential analogues (structural, metabolic, and toxicity-like) are identified to facilitate the final analogue chemical selection. The analogue approach may or may not be route-specific or applicable to multiple routes of exposure. All information was considered together as part of the final weight-of-evidence (WOE) approach to select the most suitable analogue both toxicologically and chemically. An expanded analogue identification approach was developed to collect a more comprehensive set of candidate analogues for the compounds undergoing a U.S. EPA PPRTV screening-level assessment. As described below, this method includes application of a variety of tools and methods for identifying candidate analogues that are similar to the target chemical based on chemical structure and key features; metabolic relationships; or related toxic effects and mechanisms of action. To identify structurally-related compounds, an initial pool of analogues is identified using automated tools, including ChemlDplus (ChcmlDplus. 2021), CompTox Chemicals Dashboard (U.S. HP A. 202 lb), and Organisation for Economic Co-operation and Development (OECD) Quantitative Structure-Activity Relationship (QSAR) Toolbox (OECD, 2021). to conduct structural similarity searches. Additional analogues identified as ChemlDplus-related substances, parent, salts, and mixtures, and CompTox-related substances are considered. CompTox GenRA analogues are collected using the methods available on the publicly available GenRA Beta version, which may include Morgan fingerprints, Torsion fingerprints, ToxPrints and ToxCast, Tox21, and ToxRef data. For compounds that have very few analogues identified by structure similarity using a similarity threshold of 0.8 or 80%, substructure searches in the QSAR Toolbox may be performed, or similarity searches may be rerun using a reduced similarity threshold (e.g., 70 or 60%). The compiled list of candidate analogues is batch run through the CompTox Chemicals Dashboard where QSAR-ready simplified molecular-input line-entry system (SMILES) are collected and toxicity data availability is determined (e.g., from the Agency for Toxic Substances and Disease Registry [ATSDR], Office of Environmental Health Hazard Assessment [OEHHA), California Environmental Protection Agency [CalEPA], U.S. EPA Integrated Risk Information System [IRIS], PPRTVs). The batch output information is then uploaded into the Chemical Assessment Clustering Engine (ChemACE) (U.S. HP A. 201 la), which clusters the chemicals based on chemical fragments and displays the toxicity data availability for each candidate. The ChemACE output is reviewed by an experienced chemist, who narrows the list of structural analogues based on known or expected structure-toxicity relationships, reactivity, and known or expected metabolic pathways. 31 Dib enzothi ophene ------- EPA/690/R-22/002F Toxicokinetic studies identified from the literature searches performed for this PPRTV assessment were used to identify metabolic analogues (metabolites and metabolic precursors). Metabolites were also identified from the two OECD QSAR Toolbox metabolism simulators (in vivo rat metabolism simulator and rat liver S9 metabolism simulator). Targeted PubMed searches were conducted to identify metabolic precursors and other compounds that share any of the observed or predicted metabolites identified for the target chemical. Metabolic analogues are then added to the pool of candidate analogues and toxicity data availability is determined (e.g., from AT SDR, OEHHA, CalEPA, U.S. EPA IRIS, PPRTVs). In vivo toxicity data for the target chemical (if available from the literature searches) are evaluated to determine whether specific or characteristic toxicity was observed (e.g., cholinesterase inhibition, inhibition of oxidative phosphorylation). In addition, in vitro mechanistic data identified from the literature searches or obtained from tools including GenRA, ToxCast/Tox21, and Comparative Toxicogenomics Database (CTD) (Davis et al.. 2021) were evaluated for this purpose. Data from CompTox Chemicals Dashboard ToxCast/Tox21 are collected to determine bioactivity of the target chemical in in vitro assays that may indicate potential mechanism(s) of action. The GenRA option within the Dashboard also offers an option to search for analogues based on similarities in activity in ToxCast/Tox21 in vitro assays. Using the ToxCast/Tox21 bioactivity data, nearest neighbors identified with similarity indices of >0.5 may be considered potential candidate analogues. The CTD (Davis et al.. 2021) is searched to identify compounds with gene interactions similar to interactions induced by the target chemical; compounds with gene interactions similar to the target chemical (with a similarity index >0.5) may be considered potential candidate analogues. These compounds are then added to the pool of candidate analogues, and toxicity data availability is determined (e.g., from AT SDR, OEHHA, CalEPA, U.S. EPA IRIS, PPRTVs). The tools used for the expanded analogue searches were selected because they are publicly available, which allows for transparency and reproducibility of the results, and because they are supported by U.S. and OECD agencies, updated regularly, and widely used. The application of a variety of different tools and methods to identify candidate analogues serves to minimize the limitations of any individual tool with respect to the pool of chemicals included, chemical fragments considered, and methods for assessing similarity. Further, the inclusion of techniques to identify analogues based on metabolism and toxicity or bioactivity expands the pool of candidates beyond those based exclusively on structural similarity. Analogue Search Results for Dibenzothiophene Candidate analogues for dibenzothiophene were identified based on structural relationships, metabolic relationships, and toxicity/mechanisms/mode-of-action (MOA) relationships. For candidates identified through these approaches, U.S. EPA (IRIS and PPRTV), ATSDR, and CalEPA sources were searched for subchronic, intermediate, and chronic inhalation toxicity values. No candidate analogues with inhalation toxicity values were identified. Details are provided below. 32 Dib enzothi ophene ------- EPA/690/R-22/002F Identification of Structural Analogues with Established Toxicity Values Dibenzothiophene is not a member of an existing OECD or New Chemical category. Candidate structural analogues for dibenzothiophene were identified using similarity searches in the OECD Toolbox, U.S. EPA CompTox Chemicals Dashboard, and ChemlDplus tools. A total of 24 unique structural analogues were identified for dibenzothiophene in the Dashboard, OECD QSAR Toolbox, and ChemlDplus (>80% similarity threshold) (NLM, 2021a; OECD, 2020). The list of potential analogues was manually reviewed and the following criteria were applied to select candidate analogues for further evaluation based on the structural features expected to influence toxicokinetics and/or toxicity,: • Contains one thiophene ring fused with 1-3 benzene rings, and • Only methyl, ethyl, or propyl alkyl substituents are present. Using these criteria, all 24 structural analogues initially identified were considered candidate analogues for dibenzothiophene (see Table A-4). No inhalation toxicity values were identified for any of the candidate structural analogues. Table A-4. Candidate Structural Analogues Identified for Dibenzothiophene c£o Tool (Method)3 Analogue (CASRNs) Selected for Toxicity Value Searches Structure Dashboard (Tanimoto), OECD Toolbox, and ChemlDplus (method not described) Benzo|/> |naphtho[2,1 -c/|thiophcne (239-35-0) Benzo|/) |naphtho|2.3-t/|thiophene (243-46-9) Dashboard (Tanimoto) ChemlDplus (method not described) 2,8-Dimethyldibenzo[b,c/|thiophene (1207-15-4) 2-Methyldibenzothiophene (30995-64-3) OECD Toolbox, and ChemlDplus (method not described) 3-Methyldibenzothiophene (16587-52-3) Dashboard (Tanimoto) Naphtho(2,l-6)thiophene (233-02-3) 33 Dib enzothi ophene ------- EPA/690/R-22/002F Table A-4. Candidate Structural Analogues Identified for Dibenzothiophene Tool (Method)3 Analogue (CASRNs) Selected for Toxicity Value Searches Structure Naphtho(1,2-6)thiophene (234-41-3) NaplUho(2.3-/;)thiophcnc (268-77-9) % Anthra(2.3-/))thiophcnc (22108-55-0) CCXI> Anthra(2.1 -/>)thiophcnc (227-56-5) Anthra(l,2-/>)thiophene (227-86-1) Bcnzo(/))naphtho(2.3-t/)thiophcnc. 8-methyl- (24964-07-6) Bcnzo(/>)naplUho(2.3-t/)thiophcnc. 9-methyl- (41895-72-1) Bcnzo(/))naphtho(2.1 -c/)thiophcnc. 2-methyl- (4567-43-5) Bcnzo(/))naphtho(2.1 -c/)thiophcnc. 3-methyl- (4567-45-7) Bcnzo(/))naphtho(2.3-t/)thiophcnc. 2-methyl- (83656-84-2) C—^ Bcnzo(/>)naphtho(2.1 -c/)thiophcnc. 8-methyl- (83821-53-8) / \ /~ 34 Dib enzothi ophene ------- EPA/690/R-22/002F Table A-4. Candidate Structural Analogues Identified for Dibenzothiophene Tool (Method)3 Analogue (CASRNs) Selected for Toxicity Value Searches Structure ChemlDplus (method not described) Dibenzothiophene, 2-methyl- (20928-02-3) CHj Dibenzothiophene, 1-methyl- (31317-07-4) CH3 cn6 Dimethyldibenzothiophene (70021-47-5) H;C Dibenzothiophene, 4-methyl- (7372-88-5) ch3 1 -Ethyldibcnzo|/),t/|thiophene (79313-22-7) h3c 4-Ethyldibenzothiophene (89816-99-9) HjC— 1 -Propyldibenzo |/>,c/|thiophcnc (79313 -23 -8) Cp© CH3 a80% similarity threshold was applied. OECD = Organisation for Economic Co-operation and Development. Identification of Toxicokinetic Precursors or Metabolites with Established Toxicity Values The main metabolite in urine from a rabbit exposed orally to dibenzothiophene was mono-hydroxy-diphenylene sulfone (Thomas et al.. 1942). In rat liver microsomes incubated with dibenzothiophene, the identified metabolites were dibenzothiophene-5-oxide and dibenzothiophene-5-dioxide (dibenzothiophene sulfone) (Jacob et al.. 1991; Vignier et al.. 1985). Predicted metabolites were collected from the OECD QSAR Toolbox. PubMed searches (searching "dibenzothiophene" or "132-65-0" and "metabolite") were conducted to identify metabolic precursors to dibenzothiophene. No metabolic precursors were identified. PubMed was also searched to identify other compounds that are metabolized to any of the observed or predicted metabolites of dibenzothiophene (searching the metabolite name or [CASRN if 35 Dib enzothi ophene ------- EPA/690/R-22/002F available] and "metabolite"). No compounds that share at least one metabolite with dibenzothiophene were identified in these searches. Table A-5 summarizes the 18 candidate metabolic analogues for dibenzothiophene (3 observed metabolites and an additional 15 unique predicted metabolites). Searches for relevant toxicity values for the candidate metabolic analogues of dibenzothiophene did not identify inhalation toxicity values for any of the observed/predicted metabolites. Table A-5. Candidate Metabolic Analogues of Dibenzothiophene Relationship to Dibenzothiophene Compound Metabolic precursor None identified Metabolite Mono-hydroxy-dibenzothiophene sulfone (location of hydroxy group not specified) Dibenzothiophene 5-oxide (CASRN 1013-23-6) Dibenzothiophene sulfone (CASRN 1016-05-3) 2-Hydroxydibenzothiophene (CASRN 22439-65-2) 3-Hydroxydibenzothiophene (CASRN 69747-77-9) 4-Hydroxydibenzothiophene (CASRN 24444-75-5) 2,3 -Dihydroxy dibenzothiophene3 3,4 -Dihyroxy dibenzothiophene3 3,7 -Dihydroxy dibenzothiophene3 3,4,7-Trihydroxy dibenzothiophene3 2,3,7-Trihydroxy dibenzothiophene3 2-Hydroxydibenzothiophene 5-oxide3 3-Hydroxydibenzothiophene 5-oxide3 4-Hydroxydibenzothiophene 5-oxide3 2,3-Hydroxydibenzothiophene 5-oxide3 3,4-Hydroxydibenzothiophene 5-oxide3 3,7-Hydroxydibenzothiophene 5-oxide3 1-Hydroxydibenzothiophene (CASRN 69747-83-7) Shares common metabolite(s) None identified aCASRN not available for this metabolite. Identification of Analogues on the Basis of Toxicity/Mechanistic/Mode-of-Action Information and Established Toxicity Values Available toxicity and mechanistic data for dibenzothiophene were evaluated to determine whether these data would suggest candidate analogues. The data were reviewed to determine whether there were in vivo toxicity data suggesting specific, characteristic toxicity (e.g., cholinesterase inhibition, inhibition of oxidative phosphorylation) that could be used to identify candidate analogues. The limited available in vivo animal data on dibenzothiophene administered orally indicate that the liver is the primary target organ and increased hepatocyte 36 Dib enzothi ophene ------- EPA/690/R-22/002F hypertrophy in female rats exposed for 28 days. JECDB (2011) was used as a critical effect for the derivation of the screening subchronic p-RfD value (see "DERIVATION OF SCREENING PROVISIONAL REFERENCE DOSES" section for more details). However, the available information was not sufficient to suggest specific, characteristic toxicity that could be used to identify candidate analogues. Dibenzothiophene was active in 25 ToxCast/Tox21, 6 EDSP21, and 83 PubChem bioactivity assays reported in the U.S. EPA CompTox Chemicals Dashboard. The GenRA option within the Dashboard offers an option to search for analogues based on similarities in activity in ToxCast in vitro assays. Using the ToxCast bioactivity data, none of the nearest neighbors identified by GenRA had similarity indices >0.5 (the highest index was 0.28 for pentachl oroani sol e). The CTD identified several compounds with gene interactions similar to interactions induced by dibenzothiophene (Davis et at., 2021). In the CTD, similarity is measured by the Jaccard index, calculated as the size of the intersection of interacting genes for chemical A and chemical B divided by the size of the union of those genes (range 0 [no similarity] to 1 [complete similarity]). Among the compounds with gene interactions similar to dibenzothiophene, the numbers of common gene interactions ranged from 23 to 145, and similarity indices ranged from 0.03 to 0.16; the compound with the highest similarity index (0.16) was pyrene. There were no compounds with a similarity index over 0.5. Summary Searches for structural, metabolic, and toxicity/mechanistic analogues for dibenzothiophene yielded a total of 42 unique candidate analogues: 24 structural analogues and 18 metabolites. None of the identified candidate analogues have inhalation toxicity values from authoritative sources such as U.S. EPA, ATSDR, or CalEPA. Because no candidate analogues with inhalation toxicity values were identified for dibenzothiophene, the alternative analogue approach was unable to derive screening reference inhalation concentrations for dibenzothiophene. 37 Dib enzothi ophene ------- EPA/690/R-22/002F APPENDIX B. DATA TABLES Table B-l. Selected Endpoints in Male and Female Sprague Dawley Rats After Oral Treatment with Dibenzothiophene for 28 Days" Males: ADD [HED] in mg/kg-db Endpointc'd 0 3 [0.75] 10 [2.5] 30 [7.4] Motor activity (total count) 1,399.3 ±407.4 1388.3 ±480.2 (-0.8%) 516.7 ±238.8** (-63%) 661.8 ± 322.1** (-53%) PT (sec) 16.62 ±0.87 17.35 ± 1.23 (+4%) 20.48 ±2.37** (+23%) 22.62 ±4.19** (+36%) APTT (sec) 28.23 ±2.79 28.23 ±3.37 (+0%) 33.23 ±2.16 (+18%) 39.92 ±6.67** (+41%) Calcium (mg/dL) 9.55 ±0.1 9.57 ±0.28 (+0.2%) 9.82 ±0.38 (+3%) 10.07 ±0.34 (+5%)* A/G ratio 1.208 ±0.123 1.185 ±0.065 (-2%) 1.175 ±0.049 (-3%) 1.055 ±0.07 (-13%)** Albumin protein fraction (%) 54.65 ± 1.66 54.22 ± 1.35 (-0.8%) 53.97 ± 1.07 (-1%) 51.30 ± 1.67 (-6%)** a2u-g protein fraction (%) 7.93 ±0.35 7.52 ±0.69 (-5%) 8.27 ±0.2 (+4%) 9.03 ±0.41 (+14%)** Beta globulin protein fraction (%) 14.85 ±0.48 15.02 ± 0.44 (+1%) 15.42 ±0.47 (+4%) 16.95 ±0.6 (+14%)** Terminal body weight (g) 341.8 ± 13.7 345.8 ± 19.3 (+1%) 327.3 ±28.7 (-4%) 323.3 ±26.1 (-5%) Absolute liver weight (g) 11.082 ± 1.213 12.170 ± 1.438 (+10%) 11.708 ± 1.117 (+6%) 14.468 ± 1.736** (+31%) Relative liver weight (%) 3.233 ±0.247 3.512 ±0.271 (+9%) 3.578 ±0.153* (+11%) 4.465 ± 0.208** (+38%) Absolute kidney weight (g) 2.498 ± 0.204 2.535 ±0.088 (+1%) 2.615 ±0.355 (+5%) 2.653 ±0.172 (+6%) Relative kidney weight (%) 0.732 ± 0.040 0.735 ± 0.023 (+0%) 0.798 ±0.052* (+9%) 0.823 ±0.031** (+12%) 38 Dib enzothi ophene ------- EPA/690/R-22/002F Table B-l. Selected Endpoints in Male and Female Sprague Dawley Rats After Oral Treatment with Dibenzothiophene for 28 Days" Females: ADD [HED] in mg/kg-d Endpoint 0 3 [0.68] 10 [2.2] 30 [6.7] Terminal body weight (g) 192.7 ±21.4 211.7 ± 7.9 (+10%) 195.7 ± 12.8 (+2%) 195.7 ± 18.3 (+2%) Total cholesterol (mg/dL) 51.8 ± 14.5 58.3 ± 13.8 (+13%) 51.5 ± 11.6 (+0.6%) 79.7 ±23.4* (+54%) Absolute liver weight (g) 6.008 ± 0.644 7.100 ±0.347* (+18%) 6.733 ±0.555 (+12%) 7.767 ±0.795** (+29%) Relative liver weight (%) 3.123 ±0.170 3.355 ±0.145 (+7%) 3.450 ±0.299* (+10%) 3.970 ±0.187** (+27%) Absolute kidney weight (g) 1.548 ±0.141 1.765 ±0.154* (+14%) 1.613 ±0.124 (+4%) 1.593 ±0.154 (+3%) Relative kidney weight (%) 0.808 ±0.059 0.837 ±0.074 (+4%) 0.827 ±0.058 (+2%) 0.818 ±0.058 (+1%) aJECD6 (2011). bADDs were converted into HEDs (HED = ADD x DAF) using DAFs of 0.250, 0.248, and 0.247 for low-, mid-, and high-dose males and 0.226, 0.223, and 0.222 for low-, mid-, and high-dose females calculated as follows: DAF = (BWa1/4 + BWh1'4), where BWa = animal body weight, and BWh = human body weight. Study-specific TWA animal body weights of 0.272, 0.264, and 0.259 kg for low-, mid-, and high-dose males, and 0.182, 0.174, and 0.171 kg for low-, mid-, and high-dose females were used. For humans, the reference value of 70 kg was used for body weight, as recommended by U.S. EPA (1988). Data are means ± SD; n = 6 for all data points, except n = 12 for motor activity in control and high-dose groups. dValue in parentheses is % change relative to control = ([treatment mean - control mean] + control mean) x 100. * Significantly different from control (p < 0.05) by Dunnett's test (motor activity, hematology, serum chemistry and organ weights) or Mann-Whitney U-test (PT time), as reported by the study authors. **Significantly different from control (p < 0.01) by Dunnett's test (motor activity, hematology, serum chemistry and organ weights) or Mann-Whitney U-test (PT time), as reported by the study authors. a2u-g = alpha 2u-globulin; ADD = adjusted daily dose; A/G = albumin/globulin; APTT = activated partial thromboplastin time; DAF = dosimetric adjustment factor; HED = human equivalent dose; PT = prothrombin time; SD = standard deviation; TWA = time-weighted average. 39 Dib enzothi ophene ------- EPA/690/R-22/002F Table B-2. Selected Histopathological Endpoints in Male and Female Sprague Dawley Rats After Oral Treatment with Dibenzothiophene for 28 Days" Males: ADD [HED] in mg/kg-db Endpoint' 0 3 [0.75] 10 [2.5] 30 [7.4] Liver (all lesions graded as slight): Hepatocyte hypertrophy, centrilobular Fatty change, periportal Microgranuloma Necrosis, focal 0/6 (0%) 0/6 (0%) 4/6 (67%) 0/6 (0%) 0/6 (0%) 2/6 (33%) 2/6 (33%) 0/6 (0%) 0/6 (0%) 0/6 (0%) 2/6 (33%) 0/6 (0%) 6/6 (100%) 0/6 (0%) 1/6 (17%) 1/6 (17%) Kidney (proximal tubular epithelium; all lesions graded as slight): Hyaline droplet Eosinophilic body Regeneration 0/6 (0%) 0/6 (0%) 1/6 (17%) 1/6 (17%) 1/6 (17%) 2/6 (33%) 2/6 (33%) 2/6 (33%) 0/6 (0%) 6/6 (100%) 6/6 (100%) 0/6 (0%) Females: ADD [HED] in mg/kg-d Endpoint 0 3 [0.68] 10 [2.2] 30 [6.7] Liver (all lesions graded as slight): Hepatocyte hypertrophy, centrilobular Fatty change, periportal Microgranuloma 0/6 (0%) 3/6 (50%) 4/6 (67%) 0/6 (0%) 2/6 (33%) 3/6 (50%) 1/6 (17%) 0/6 (0%) 3/6 (50%) 6/6 (100%) 0/6 (0%) 2/6 (33%) aJECD6 (20ID. bADDs were converted into HEDs (HED = ADD x DAF) using DAFs of 0.250, 0.248, and 0.247 for low-, mid-, and high-dose males and 0.226, 0.223, and 0.222 for low-, mid-, and high-dose females calculated as follows: DAF = (BWa1/4 ^ BWh1'4), where BWa = animal body weight, and BWh = human body weight. Study-specific TWA animal body weights of 0.272, 0.264, and 0.259 kg for low-, mid-, and high-dose males, and 0.182, 0.174, and 0.171 kg for low-, mid-, and high-dose females were used. For humans, the reference value of 70 kg was used for body weight, as recommended by U.S. EPA (1988). Data are number of animals showing changes/ total number of animals examined (% incidence). ADD = adjusted daily dose; DAF = dosimetric adjustment factor; HED = human equivalent dose; SD = standard deviation; TWA = time-weighted average. 40 Dib enzothi ophene ------- EPA/690/R-22/002F Table B-3. Body, Liver, and Spleen Weights of Male Albino Rats After Dietary Exposure to Dibenzothiophene for 165 Daysa'b Endpoint' Percent in diet ADD [HED] in mg/kg-dd e f 0§ 0.025% 0h 0.050% 0' 0.100% 0 13 [2.9] 0 27 [5.9] 0 63 [13] Terminal body weight (g) 310 310 273 273 212 212 Absolute liver weight (g) 10.00 ±0.11 10.70 ±0.29 (+7%) 9.50 ±0.27 12.80 ±0.48 (+35%) 8.40 ± 0.22 18.10 ±0.74 (+115%) Absolute spleen weight (g) 0.97 ± 0.062 0.69 ±0.015 (-29%) 0.92 ±0.072 0.64 ± 0.067 (-30%) 0.83 ±0.041 0.36 ±0.010 (-57%) "Thomas et al. (1942). Statistical analysis was not reported and is not conducted because number of animals per group was not reported. °Organ weights are expressed as mean ± probable error; value in parentheses is % change relative to matched laboratory historical control = ([treatment mean - control mean] + control mean) x 100. dAnimals were provided dibenzothiophene in the food at 0.25, 0.50, or 1.00% for the first 4 days. Because of low food intakes and decreases in body weight, doses were then decreased to 0.025, 0.050, or 0.100% dibenzothiophene for the remainder of the 165-day study period. The study authors provided the amount of dibenzothiophene consumed. The following equation was used to convert that information to mg/kg-day: ADD = total dibenzothiophene consumption per animal over study duration x (1 + body weight) x (1 -f- days dosed) eADDs were converted to HEDs by multiplying by DAFs of 0.225, 0.219, and 0.208 for low-, mid-, and high-dose rats calculated as follows: DAF = (BWa1/4 + BWh1'4), where BWa = animal body weight, and BWh = human body weight. Study-specific estimated average animal body weights of 0.179, 0.161, and 0.130 kg for low-, mid-, and high-dose rats were used. For humans, the reference value of 70 kg was used for body weight, as recommended by U.S. EPA (1988). fData for each exposure group were compared with data for laboratory historical controls. For the evaluation of organ weights, historical controls were matched according to body weight. gMatched laboratory historical controls for 13-mg/kg-day dose group. hMatched laboratory historical controls for 27-mg/kg-day dose group. 'Matched laboratory historical controls for 63-mg/kg-day dose group. ADD = adjusted daily dose; DAF = dosimetric adjustment factor; HED = human equivalent dose; TWA = time-weighted average. 41 Dib enzothi ophene ------- EPA/690/R-22/002F APPENDIX C. BENCHMARK DOSE MODELING RESULTS MODELING PROCEDURE FOR DICHOTOMOUS DATA The benchmark dose (BMD) modeling of dichotomous data was conducted with the U.S. Environmental Protection Agency (U.S. EPA) Benchmark Dose Software (BMDS; version 3.2). For these data, the Gamma, Logistic, Log-Logistic, Probit, Log-Probit, Hill, Multistage, and Weibull dichotomous models available within the software were fit using a benchmark response (BMR) of 10% extra risk. In general, the BMR should be near the low end of the observable range of increased risk in the study. BMRs that are too low can result in widely disparate benchmark dose lower confidence limit (BMDL) estimates from different models (high model dependence). Adequacy of model fit is judged based on the %2 goodness-of-fitp-value (p > 0.1), magnitude of scaled residuals (absolute value <2.0), and visual inspection of the model fit. Among all models providing adequate fit, the BMDL from the model with the lowest Akaike's information criterion (AIC) is selected as a potential point of departure (POD), if the BMDLs are sufficiently close (less than approximately threefold); if the BMDLs are not sufficiently close (greater than approximately threefold), model dependence is indicated, and the model with the lowest reliable BMDL is selected. MODELING PROCEDURE CONTINUOUS DATA MODELING The BMD modeling of continuous data was conducted with the U.S. EPA BMDS (version 3.2). For these data, the Exponential, Linear, Polynomial, and Power continuous models available within the software were used. The continuous Hill model was not considered for the derivation of a POD because it has five parameters and requires a data set with a minimum of six data points (including control). The continuous models available within the software were fit using a BMR of 1 standard deviation (SD) or alternative BMRs where appropriate as outlined in the Benchmark Dose Technical Guidance (U.S. EPA, 2012). A BMR 10% relative deviation (RD) for liver and kidney weights is considered a minimally biologically significant response in adult animals and was applied in this assessment for benchmark dose (BMD) modeling purposes. An adequate fit was judged based on the %2 goodness-of-fit p-v alue (p> 0.1), magnitude of the scaled residuals in the vicinity of the BMR, and visual inspection of the model fit. In addition to these three criteria forjudging adequacy of model fit, a determination was made as to whether the variance across dose groups was constant. If a constant variance model was deemed appropriate based on the statistical test provided in BMDS (i.e., Test 2; p-v alue > 0.1), the final BMD results were estimated from a constant variance model. If the test for homogeneity of variance was rejected (p-value < 0.1), the model was run again while modeling the variance as a power function of the mean to account for this nonconstant variance. If this nonconstant variance model did not adequately fit the data (i.e., Test 3; p-v alue < 0.1), the data set was considered unsuitable for BMD modeling. Among all models providing adequate fit, the lowest BMDL has been selected if the BMDLs estimated from different models varied more than threefold; otherwise, the BMDL from the model with the lowest AIC has been selected as a potential POD from which to derive the proposed reference value. 42 Dib enzothi ophene ------- EPA/690/R-22/002F BMD MODELING TO IDENTIFY POTENTIAL PODS FOR DERIVATION OF A SCREENING SUBCHRONIC PROVISIONAL REFERENCE DOSE Increased Relative Liver Weight in Male Sprague Dawley Rats After Oral Treatment with Dibenzothiophene for 28 Days (JECDB, 2011) The procedure outlined above for continuous data was applied to the data for increased relative liver weight in male Sprague Dawley rats orally exposed to dibenzothiophene for 28 days (JECDB. 2011). The constant variance model provided an adequate fit to the variance data, and the Exponential models 2 and 3, and the Linear model provided adequate fit to the means. Visual inspection of the dose-response curves suggested adequate fit, BMDLs were not 10 times lower than the lowest nonzero dose, and scaled residuals did not exceed ±2 units at the data point closest to the predefined BMR. BMDLs for models providing adequate fit were sufficiently close (differed by less than threefold), so the model with the lowest AIC was selected (Exponential model 3). The estimated human equivalent benchmark dose associated with 10% relative deviation from the control (BMDo.ird) and benchmark dose lower confidence limit associated with 10% relative deviation from the control (BMDLo.ird) values of 2.33 and 2.01 mg/kg-day, respectively, were selected from this model. The results of the BMD modeling are summarized in Table C-l. Figure C-l shows the fit of the Exponential model 3 model to the data. Table C-l. BMD Modeling Results (Constant Variance) for Relative Liver Weight in Male Sprague Dawley Rats Orally Exposed to Dibenzothiophene for 28 Days" Model Variance />-Valucb Means />-Valucb Scaled Residual at Dose Nearest BMD AIC BMDo.ird (HED, (mg/kg-d) BMDLo.ird (HED, (mg/kg-d) Exponential (model 2)° 0.56101604 0.2269728 -0.767558354 0.935991293 2.330715 2.011481 Exponential (model 3)c d 0.56101604 0.226973 -0.767697226 0.935989576 2.330708 2.011754 Exponential (model 4)° 0.56101604 0.0691553 -1.045102633 3.273097499 2.071878 1.36167 Exponential (model 5)° 0.56101604 NA -1.042495246 5.26907005 2.07426 1.362246 Polynomial (3-degree)6 0.56101604 0.0985958 -0.429504946 2.698252338 1.771026 4.6872224 Polynomial (2-degree)6 0.56101604 0.0890464 -0.53754105 2.861669325 1.761492 4.2908893 Power0 0.56101604 0.0737363 -0.763984146 3.167966089 1.743172 4.5119764 Linear6 0.56101604 0.1921587 -1.042667628 1.269007639 1.737413 2.5442898 aJECDB (2011). bValues <0.10 fail to meet conventional goodness-of-fit criteria. Tower restricted to be >1. dSelected model. "Coefficients restricted to be positive. AIC = Akaike's information criterion; BMD = benchmark dose; BMDL = lower confidence limit on the BMD (subscripts denote BMR: i.e., 0.1RD = dose associated with 10% relative deviation from the control); BMR = benchmark response; NA = test for fit is not valid; HED = human equivalent dose. 43 Dib enzothi ophene ------- EPA/690/R-22/002F 5 4.5 4 3.5 «, 3 u"i c 0 ? 5 a. in 0J 11 2 1.5 1 0.5 0 01234567 Dose Figure C-l. Fit of Exponential Model 3 to Data for Relative Liver Weight in Male Sprague Dawley Rats Exposed to Dibenzothiophene for 28 Days (JECDB, 2011) BMD Model Output for Figure C-l Data Relative liver weight in males (JECDB 2011) [Add user notes here] Dose N Mean Std. Dev. HED (mg/kg-d) [Custom] [Custom] [Custom] 0 6 3.233 0.247 0.75 6 3.512 0.271 2.5 6 3.578 0.153 7.4 6 4.465 0.208 Model Results Benchmark Dose BMD 2.33070755 BMDL 2.011754168 BMDU 2.774379473 AIC 0.935989576 Test 4 P-value 0.226972953 D.O.F. 2 Frequentist Exponential Degree 3 Model with BMR of 0.1 Rel. Dev. for the BMD and 0.95 Lower Confidence Limit for the BMDL Estimated Probability Response at BMD O Data BMD BMDL 44 Dib enzothi ophene ------- EPA 690 R-22 002F Model Parameters # of Parameters 4 Variable Estimate a 3.291898107 b 0.040893213 d Bounded log-alpha -3.04887724 Goodness of Fit Dose Size Estimated Median Calc'd Median Observed Mean Estimated SD Calc'd SD Observed SD Scaled Residual 0 6 3.291898107 3.233 3.233 0.21774326 0.247 0.247 -0.662570721 0.75 6 3.394424526 3.512 3.512 0.21774326 0.271 0.271 1.322658242 2.5 6 3.64624315 3.578 3.578 0.21774326 0.153 0.153 -0.767697226 7.4 6 4.455209601 4.465 4.465 0.21774326 0.208 0.208 0.110136507 Likelihoods of Interest Model Log Likelihood* # of Parameters AIC A1 4.014929631 5 1.97014074 A2 5.042561625 8 5.91487675 A3 4.014929631 5 1.97014074 fitted 2.532005212 3 0.93598958 R -17.66965809 2 39.3393162 * Includes additive constant of -22.05452. This constant was not included in the LL derivation prior to BMDS 3.0. Tests of Interest Test -2*Log (Likelihood Ratio) Test df p-value 1 45.42443942 6 <0.0001 2 2.055263988 3 0.56101604 3 2.055263988 3 0.56101604 4 2.965848839 2 0.22697295 45 Dib enzothi ophene ------- EPA/690/R-22/002F Increased Relative Liver Weight in Female Sprague Dawley Rats After Oral Treatment with Dibenzothiophene for 28 Days (JECDB, 2011) The procedure outlined above for continuous data was applied to the data for increased relative liver weight in female Sprague Dawley rats orally exposed to dibenzothiophene for 28 days (JECDB. 2011). The constant variance model provided an adequate fit to the variance data and all models provided adequate fit to the means. Visual inspection of the dose-response curves suggested adequate fit, BMDLs were not 10 times lower than the lowest nonzero dose, and scaled residuals did not exceed ±2 units at the data point closest to the predefined BMR. BMDLs for models providing adequate fit were sufficiently close (differed by less than threefold), so the model with the lowest AIC was selected (Linear). The Polynomial and Power models converged to the Linear model. The Linear model estimated human equivalent BMDo.ird and BMDLo.ird values of 2.73 and 2.19 mg/kg-day, respectively. The results of the BMD modeling are summarized in Table C-2. Figure C-2 shows the fit of the Linear model to the data. Table C-2. BMD Modeling Results (Constant Variance) for Relative Liver Weight in Female Sprague Dawley Rats Orally Exposed to Dibenzothiophene for 28 Days" Model Variance />-Valucb Means />-Valucb Scaled Residual at Dose Nearest BMD AIC BMDo.ird (HED, (mg/kg-d) BMDLo.ird (HED, (mg/kg-d) Exponential (model 2)° 0.27880585 0.354743 0.121794767 -3.39315347 2.94462 2.417078 Exponential (model 3)° 0.27880585 0.3547431 0.121823756 -3.393153703 2.944643 2.419279 Exponential (model 4)° 0.27880585 0.2060485 -0.451460518 -1.866901424 2.210648 1.105419 Exponential (model 5)° 0.27880585 0.2060467 -0.45428488 -1.866889072 2.20753 1.105422 Polynomial (3-degree)d 0.27880585 0.3935635 -0.021131946 -3.600850872 2.733293 2.185768 Polynomial (2-degree)d 0.27880585 0.3935635 -0.021131946 -3.600850872 2.733293 2.185768 Power0 0.27880585 0.3935635 -0.02113139 -3.600850872 2.733292 2.185999 Linearde 0.27880585 0.3935635 -0.021131734 -3.600850872 2.733293 2.185768 aJECDB (2011). bValues <0.10 fail to meet conventional goodness-of-fit criteria. Tower restricted to be >1. Coefficients restricted to be positive. "Selected model. AIC = Akaike's information criterion; BMD = benchmark dose; BMDL = lower confidence limit on the BMD (subscripts denote BMR: i.e., 0.1RD = dose associated with 10% relative deviation from the control); BMR = benchmark response; HED = human equivalent dose. 46 Dib enzothi ophene ------- EPA/690/R-22/002F 4.5 4 3.5 3 g 2.5 o Q. w 2 cc 1.5 1 0.5 0 0 1 2 3 4 5 6 Dose Figure C-2. Fit of Linear Model to Data for Relative Liver Weight in Female Sprague Dawley Rats Exposed to Dibenzothiophene for 28 Days (JECDB, 2011) BMD Model Output for Figure C-2 Data Relative liver weight in females (JECDB 2011) [Add user notes here Dose N Mean Std. Dev. HED (mg/kg-day) [Custom] [Custom] [Custom] 0 6 3.123 0.17 0.68 6 3.355 0.145 2.2 6 3.45 0.299 6.7 6 3.97 0.187 Model Results Benchmark Dose BMD 2.733293247 BMDL 2.185768319 BMDU 3.595625864 AIC -3.600850872 Test 4 P-value 0.393563466 D.O.F. 2 Frequentist Linear Model with BMR of 0.1 Rel. Dev. for the BMD and 0.95 Lower Confidence Limit for the BMDL -^Estimated Probability ^—Response at BMD o Data BMD BMDL 47 Dib enzothi ophene ------- EPA 690 R-22 002F Model Parameters # of Parameters 3 Variable Estimate g 3.194580413 betal 0.116876655 alpha 0.039245734 Goodness of Fit Dose Size Estimated Median Calc'd Median Observed Mean Estimated SD Calc'd SD Observed SD Scaled Residual 0 6 3.194580413 3.123 3.123 0.19810536 0.17 0.17 -0.885061798 0.68 6 3.274056538 3.355 3.355 0.19810536 0.145 0.145 1.000831977 2.2 6 3.451709054 3.45 3.45 0.19810536 0.299 0.299 -0.021131734 6.7 6 3.977654002 3.97 3.97 0.19810536 0.187 0.187 -0.094638521 Likelihoods of Interest Model Log Likelihood* # of Parameters AIC A1 5.732938375 5 -1.46587675 A2 7.654954357 8 0.69009129 A3 5.732938375 5 -1.46587675 fitted 4.800425436 3 -3.60085087 R -9.782299848 2 23.5645997 * Includes additive constant of -22.05452. This constant was not included in the LL derivation prior to BMDS 3.0. Tests of Interest Test -2*Log(Likelihood Ratio) Test df p-value 1 34.87450841 6 <0.0001 2 3.844031964 3 0.27880585 3 3.844031964 3 0.27880585 4 1.865025877 2 0.39356347 48 Dib enzothi ophene ------- EPA/690/R-22/002F Increased Hepatocyte Hypertrophy in Female Sprague Dawley Rats After Oral Treatment with Dibenzothiophene for 28 Days (JECDB, 2011) The procedure outlined above for dichotomous data was applied to the data for increased hepatocyte hypertrophy in female Sprague Dawley rats orally exposed to dibenzothiophene for 28 days (JECDB. 2011). All models provided adequate fit (/rvalue > 0.10). However, based on visual inspection, the Multistage degree 1 model was not found to have an adequate fit (estimated probabilities consistently misrepresented the observed responses by -20%). All other models provided adequate fit upon visual inspection and scaled residuals did not exceed ±2 units at the data point closest to the predefined BMR. BMDLs for models providing adequate fit were sufficiently close (differed by less than threefold), so the model with the lowest AIC was selected (Probit). The Probit model estimated a human equivalent BMDo.ier and BMDLio of 2.08 and 1.04 mg/kg-day, respectively. The results of the BMD modeling are summarized in Table C-3. Figure C-3 shows the fit of the Probit model to the data. Table C-3. BMD Modeling Results for Hepatocyte Hypertrophy in Female Sprague Dawley Rats Orally Exposed to Dibenzothiophene for 28 Days" Model />-Valucb Scaled Residual at Dose Nearest BMD AIC BMDo.ier (HED, (mg/kg-d) BMDL io (HED, (mg/kg-d) Dichotomous Hill 0.9994799 -6.91397E-08 11.40673536 2.125982 1.13544 Gamma0 0.9999729 -0.000199249 9.406843019 2.027344 0.974324 Log-Logisticd 0.9971634 -4.82841E-06 11.40675978 2.104989 1.135448 Multistage Degree 3e 0.9957091 -0.085479764 7.528643645 1.781135 0.624666 Multistage Degree 2e 0.8345974 -0.433247713 8.79031287 1.257594 0.564518 Multistage Degree le f 0.2663758 -0.991744547 13.67071077 0.471974 0.252068 Weibull0 0.9999858 0.002899612 7.409558632 1.99808 0.881564 Logistic 1 4.90622E-06 7.406763669 2.121106 1.125562 Log-Probitd 0.9999999 6.34854E-10 9.406734872 2.132271 1.119394 Probit8 1 4.20484E-06 7.406737143 2.083075 1.035344 aJECDB (20ID. bValues <0.10 fail to meet conventional goodness-of-fit criteria. Tower restricted to be >1. dSlope restricted to be >1. "Betas restricted to be >0. fModel did not pass visual fit inspection. gSelected model. AIC = Akaike's information criterion; BMD = benchmark dose; BMDL = 95% benchmark dose lower confidence limit on the BMD (subscripts denote BMR: i.e., 10 = dose associated with 10% extra risk); BMR = benchmark response; NA = test for fit is not valid; HED = human equivalent dose. 49 Dib enzothi ophene ------- EPA 690 R-22 002F 0.9 0.8 0.7 g °"6 I0"5 Of * 0.4 0.3 0.2 0.1 - 00- 0 Figure C-3. Fit of Probit Model to Data for Increased Hepatocyte Hypertrophy in Female Sprague Dawley Rats Exposed to Dibenzothiophene for 28 Days (JECDB, 2011) HMD Model Output for Figure C-3 Data Increased hepatocyte hypertrophy in females [Add user notes here] Dose N Incidence HED (mg/kg-day) [Custom] [Custom] 0 6 0 0.68 6 0 2.2 6 1 6.7 6 6 Model Results Benchmark Dose BMD 2.083075 BMDL 1.035344 BMDU 3.0208465 AIC 7.406737143 P-value 1 D.O.F. 3 Chi2 1.3185E-06 Frequentist Probit Model with BMR of 10% Extra Risk for the BMD and 0.95 Lower Confidence Limit for the BMDL Estimated Probability Response at BMD o Data IBMD —=BMDL 50 Dib enzothi ophene ------- EPA 690 R-22 002F Model Parameters # of Parameters 2 Variable Estimate a -6.877871888 b Bounded Goodness of Fit Dose Estimated Probability Expected Observed Size Scaled Residual 0 3.03766E-12 1.8226E-11 0 6 -4.269E-06 0.68 2.19744E-07 1.31847E-06 0 6 -0.0011482 2.2 0.166666027 0.999996162 1 6 4.205E-06 6.7 1 6 6 6 0 Analysis of Deviance Model Log Likelihood # of Parameters Deviance Test d.f. P Value Full Model -2.703367253 4 - - NA Fitted Model -2.703368572 1 2.637E-06 3 1 Reduced Model -14.48729404 1 23.5678536 3 <0.0001 51 Dib enzothi ophene ------- EPA/690/R-22/002F Increased Relative Kidney Weight in Male Sprague Dawley Rats After Oral Treatment with Dibenzothiophene for 28 Days (JEC I) II. 2011) The procedure outlined above for continuous data was applied to the data for increased relative kidney weight in male Sprague Dawley rats orally exposed to dibenzothiophene for 28 days (JHCDB. 2011). The constant variance model provided an adequate fit to the variance data and only the Exponential degree 4 model provided adequate fit to the means. Visual inspection of the dose-response curve suggested adequate fit and scaled residuals did not exceed ±2 units at the data point closest to the predefined BMR. Therefore, the human equivalent BMDo iRD and BMDLo.ird values of 3.08 and 1.36 mg/kg-day, respectively, for this model were selected. The results of the BMD modeling are summarized in Table C-4. Figure C-4 shows the fit of the Exponential 4 model to the data. Table C-4. BMD Modeling Results (Constant Variance) for Relative Kidney Weight in Male Sprague Dawley Rats Orally Exposed to Dibenzothiophene for 28 Days" Model Variance />-Valucb Means />-Valucb Scaled Residual at Dose Nearest BMD AIC BMDo. ird (HED, (mg/kg-d) BMDLo.ird (HED, (mg/kg-d) Exponential (model 2)° 0.24433599 0.0810132 -0.493677602 -82.13382255 6.069332 4.448717 Exponential (model 3)° 0.24433599 0.0810136 -0.493536392 -82.13383205 6.069183 4.452747 Exponential (model 4)c d 0.24433599 0.1801994 0.64553924 -83.36413069 3.077499 1.364732 Exponential (model 5)° 0.24433599 NA 0.002299382 -83.16003746 2.701189 0.816626 Polynomial (3-degree)6 0.24433599 0.0905573 -0.519162188 -82.35656545 5.918103 4.248669 Polynomial (2-degree)6 0.24433599 0.0905573 -0.519162176 -82.35656545 5.918103 4.248669 Power0 0.24433599 0.0905573 -0.519162289 -82.35656545 5.918105 4.249527 Linear6 0.24433599 0.0905573 -0.519162316 -82.35656545 5.918103 4.248669 •'JHCDB (2011). bValues <0.10 fail to meet conventional goodness-of-fit criteria. Tower restricted to be >1. dSelected model. "Coefficients restricted to be positive. AIC = Akaike's information criterion; BMD = benchmark dose; BMDL = lower confidence limit on the BMD (subscripts denote BMR: i.e., 0. IRD = dose associated with 10% relative deviation from the control); BMR = benchmark response; HED = human equivalent dose. 52 Dib enzothi ophene ------- EPA 690 R-22 002F 0.9 0.8 0.7 0.6 01 £ 0.5 o ZL <2 0.4 0£ 0.3 0.2 0.1 0 Frequentist Exponential Degree 4 Model with BMR of 0.1 Rel. Dev. for the BMD and 0.95 Lower Confidence Limit for the BMDL "4 -Estimated Probability -Response at BMD Data •BMD BMDL Figure C-4. Fit of Exponential Degree 4 Model to Data for Increased Relative Kidney Weight in Male Sprague Dawley Rats Exposed to Dibenzothiophene for 28 Days (JECDB, 2011) BMD Model Output for Figure C-4 Data Increased relative kidney weight in males [Add user notes here Dose N Mean Std. Dev. HED (mg/kg-day) [Custom] [Custom] [Custom] 0 6 0.732 0.04 0.75 6 0.735 0.023 2.5 6 0.798 0.052 7.4 6 0.823 0.031 Model Results Benchmark Dose BMD 3.07749939 BMDL 1.364731935 BMDU 19.93656715 AIC -83.36413069 Test 4 P-value 0.180199393 D.O.F. 1 53 Dib enzothi ophene ------- EPA 690 R-22 002F Model Parameters # of Parameters 4 Variable Estimate a 0.724353256 b 0.356825599 c 1.150036123 log-alpha -6.644715675 Goodness of Fit Dose Size Estimated Median Calc'd Median Observed Mean Estimated SD Calc'd SD Observed SD Scaled Residual 0 6 0.724353256 0.732 0.732 0.03606769 0.04 0.04 0.519318585 0.75 6 0.749871217 0.735 0.735 0.03606769 0.023 0.023 -1.009959186 2.5 6 0.788494711 0.798 0.798 0.03606769 0.052 0.052 0.64553924 7.4 6 0.825280877 0.823 0.823 0.03606769 0.031 0.031 -0.154902775 Likelihoods of Interest Model Log Likelihood* # of Parameters AIC A1 46.58005477 5 -83.1601095 A2 48.66183302 8 -81.323666 A3 46.58005477 5 -83.1601095 fitted 45.68206535 4 -83.3641307 R 36.61850946 2 -69.2370189 * Includes additive constant of -22.05452. This constant was not included in the LL derivation prior to BMDS 3.0. Tests of Interest Test -2*Log(Likelihood Ratio) Test df p-value 1 24.08664712 6 0.00050343 2 4.163556496 3 0.24433599 3 4.163556496 3 0.24433599 4 1.795978855 1 0.18019939 54 Dib enzothi ophene ------- EPA/690/R-22/002F APPENDIX D. REFERENCES ACGIH (American Conference of Governmental Industrial Hygienists). (2020). 2020 TLVs and BEIs: Based on the documentation of the threshold limit values for chemical substances and physical agents & biological exposure indices. Cincinnati, OH. Ant at. A; Pfohl-l.cszkowicz. A; Castegnaro. M. (2004). Genotoxic activity of thiophenes on liver human cell line (HepG2). Polycycl Aromat Compd 24: 733-742. http://dx.doi.org/10.1080/1040663049Q472473 ATSDR (Agency for Toxic Substances and Disease Registry). (2021). Toxic substances portal: Toxicological profiles [Database], Atlanta, GA. Retrieved from https://www.atsdr.cdc.gov/toxprofiledocs/index.html Blunter. GP; Collin. G; Hoke. H. 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