United States Environmental Protection 1=1 m m Agency EPA/690/R-14/003F Final 7-10-2014 Provisional Peer-Reviewed Toxicity Values for 1,3 -Dibromobenzene (CASRN 108-36-1) Superfund Health Risk Technical Support Center National Center for Environmental Assessment Office of Research and Development U.S. Environmental Protection Agency Cincinnati, OH 45268 ------- AUTHORS, CONTRIBUTORS, AND REVIEWERS CHEMICAL MANAGERS Evisabel A. Craig, PhD National Center for Environmental Assessment, Cincinnati, OH Q. Jay Zhao, PhD, DABT National Center for Environmental Assessment, Cincinnati, OH PRIMARY INTERNAL REVIEWERS Paul G. Reinhart, PhD, DABT National Center for Environmental Assessment, Research Triangle Park, NC Ambuja Bale, PhD, DABT National Center for Environmental Assessment, Washington, DC Questions regarding the contents of this document may be directed to the U.S. EPA Office of Research and Development's National Center for Environmental Assessment, Superfund Health Risk Technical Support Center (513-569-7300). 1 ------- TABLE OF CONTENTS COMMONLY USED ABBREVIATIONS AND ACRONYMS iii BACKGROUND 1 DISCLAIMERS 1 QUESTIONS REGARDING PPRTVs 1 INTRODUCTION 2 REVIEW OF POTENTIALLY RELEVANT DATA (NONCANCER AND CANCER) 4 HUMAN STUDIES 7 ANIMAL STUDIES 7 Oral Exposures 7 Inhalation Exposures 7 Short-Term-Duration Studies 7 OTHER DATA (SHORT-TERM TESTS, OTHER EXAMINATIONS) 8 Other Toxicity Studies (Exposures Other than Oral or Inhalation) 10 Acute Studies 10 Short-Term-Duration Studies 11 Toxicokinetics Studies 12 Genotoxicity Studies 13 DERIVATION 01 PROVISIONAL VALUES 13 DERIVATION OF ORAL REFERENCE DOSES 14 Feasibility of Deriving Subchronic and Chronic p-RfDs 14 DERIVATION OF INHALATION REFERENCE CONCENTRATIONS 14 Feasibility of Deriving Subchronic and Chronic p-RfCs 14 CANCER WEIGHT-OF-EVIDENCE DESCRIPTOR 15 DERIVATION OF PROVISIONAL CANCER RISK VALUES 15 APPENDIX A. SCREENING PROVISIONAL VALUES 16 APPENDIX B. DATA TABLES 28 APPENDIX C. BENCHMARK DOSE MODELING RESULTS 29 APPENDIX D. REFERENCES 30 li ------- COMMONLY USED ABBREVIATIONS AND ACRONYMS a2u-g alpha 2u-globulin MN micronuclei ACGIH American Conference of Governmental MNPCE micronucleated polychromatic Industrial Hygienists erythrocyte AIC Akaike's information criterion MOA mode-of-action ALD approximate lethal dosage MTD maximum tolerated dose ALT alanine aminotransferase NAG N-acetyl-P-D-glucosaminidase AST aspartate aminotransferase NCEA National Center for Environmental atm atmosphere Assessment ATSDR Agency for Toxic Substances and NCI National Cancer Institute Disease Registry NOAEL no-observed-adverse-effect level BMD benchmark dose NTP National Toxicology Program BMDL benchmark dose lower confidence limit NZW New Zealand White (rabbit breed) BMDS Benchmark Dose Software OCT ornithine carbamoyl transferase BMR benchmark response ORD Office of Research and Development BUN blood urea nitrogen PBPK physiologically based pharmacokinetic BW body weight PCNA proliferating cell nuclear antigen CA chromosomal aberration PND postnatal day CAS Chemical Abstracts Service POD point of departure CASRN Chemical Abstracts Service Registry POD[adj] duration-adjusted POD Number QSAR quantitative structure-activity CBI covalent binding index relationship CHO Chinese hamster ovary (cell line cells) RBC red blood cell CL confidence limit RDS replicative DNA synthesis CNS central nervous system RfC inhalation reference concentration CPN chronic progressive nephropathy RfD oral reference dose CYP450 cytochrome P450 RGDR regional gas dose ratio DAF dosimetric adjustment factor RNA ribonucleic acid DEN diethylnitrosamine SAR structure activity relationship DMSO dimethylsulfoxide SCE sister chromatid exchange DNA deoxyribonucleic acid SD standard deviation EPA Environmental Protection Agency SDH sorbitol dehydrogenase FDA Food and Drug Administration SE standard error FEV1 forced expiratory volume of 1 second SGOT glutamic oxaloacetic transaminase, also GD gestation day known as AST GDH glutamate dehydrogenase SGPT glutamic pyruvic transaminase, also GGT y-glutamyl transferase known as ALT GSH glutathione SSD systemic scleroderma GST glutathione -S -transferase TCA trichloroacetic acid Hb/g-A animal blood-gas partition coefficient TCE trichloroethylene Hb/g-H human blood-gas partition coefficient TWA time-weighted average HEC human equivalent concentration UF uncertainty factor HED human equivalent dose UFa interspecies uncertainty factor i.p. intraperitoneal UFh intraspecies uncertainty factor IRIS Integrated Risk Information System UFS subchronic-to-chronic uncertainty factor IVF in vitro fertilization UFd database uncertainty factor LC50 median lethal concentration U.S. United States of America LD50 median lethal dose WBC white blood cell LOAEL lowest-observed-adverse-effect level 111 ------- FINAL 7-10-2014 PROVISIONAL PEER-REVIEWED TOXICITY VALUES FOR 1,3-DIBROMOBENZENE (CASRN 108-36-1) 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 Agency guidance on human health toxicity value derivations. All PPRTV assessments receive internal review by a standing panel of National Center for Environment Assessment (NCEA) scientists and an independent external peer review by three scientific experts. 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. The PPRTV review process provides needed toxicity values in a quick turnaround timeframe while maintaining scientific quality. PPRTV assessments are updated approximately on a 5-year cycle for new data or methodologies that might impact the toxicity values or characterization of potential for adverse human health effects and are revised as appropriate. It is important to utilize the PPRTV database (http://hhpprtv.ornl.gov) to obtain the current information available. When a final Integrated Risk Information System (IRIS) assessment is made publicly available on the Internet (www.epa.gov/iris). the respective PPRTVs are removed from the database. 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. Environmental Protection Agency (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. QUESTIONS REGARDING PPRTVs Questions regarding the contents and appropriate use of this PPRTV assessment should be directed to the EPA Office of Research and Development's National Center for Environmental Assessment, Superfund Health Risk Technical Support Center (513-569-7300). 1 1,3-Dibromobenzene ------- FINAL 7-10-2014 INTRODUCTION 1,3-Dibromobenzene (CASRN 108-36-1), also known as meta-dibromobenzene (w-dibromobenzene), appears as a clear, colorless to light-yellow liquid. It is used in various organic syntheses. 1,3-Dibromobenzene is an irritant that can cause inflammation and burns to the eyes and skin. It is stable but flammable and its combustion can lead to irritating, corrosive, and/or toxic fumes. The molecular formula of 1,3-dibromobenzene is CtsFUBri (see Figure 1). Table 1 provides a list of its physicochemical properties. Br Figure 1. Structure of 1,3-Dibromobenzene Table 1. Physicochemical Properties of 1,3-Dibromobenzene (CASRN 108-36-1) Property (unit) Value Boiling point (°C) 218-2193 Melting point (°C) -T Density at 25°C (g/inL) 1.9523 Log P (unitless) 3.75b Vapor pressure (mm Hg at 25 °C) 0.269b pH (unitless) Not available Solubility in water (mg/L at 35°C) 67.5b Relative vapor density (air = 1) 8.16a Molecular weight (g/mol) 235.9a aChemicalBook (accessed on 7-22-2013). bNLM (accessed on 7-22-2013). A summary of available toxicity values for 1,3-dibromobenzene from U.S. EPA and other agencies/organizations is provided in Table 2. 2 1,3-Dibromobenzene ------- FINAL 7-10-2014 Table 2. Summary of Available Toxicity Values for 1,3-Dibromobenzene (CASRN 108-36-1) Source/Parameter3 Value (Applicability) Notes Reference Date Accessed Noncancer ACGIH NV NA ACGIH (2013) N/A ATSDR NV NA ATSDR (2013) N/A Cal/EPA NV NA Cal/EPA (2014. 2013) 4-17-2014b NIOSH NV NA NIOSH (2010) NA OSHA NV NA OSHA (2011. 2006s) NA IRIS NV NA U.S. EPA 4-17-2014 Drinking water NV NA U.S. EPA (2012a) NA HEAST NV NA U.S. EPA (2011a) NA CARA HEEP NV Noncancer toxicity values were not derived due to inadequate noncancer data and lack of carcinogenicity studies on the chemical. U.S. EPA (1994) NA WHO NV NA WHO 4-14-2014 Cancer IRIS NV NA U.S. EPA 4-14-2014 HEAST NV NA U.S. EPA (2011a) NA IARC NV NA IARC (2013) NA NTP NV NA NTP (2011) NA Cal/EPA NV NA Cal/EPA (2013. 2011) NA aSources: ACGIH = American Conference of Governmental Industrial Hygienists; ATSDR = Agency for Toxic Substances and Disease Registry; Cal/EPA = California Environmental Protection Agency; CARA = Chemical Assessments and Related Activities; HEAST = Health Effects Assessment Summary Tables; HEEP = Health and Environmental Effects Profile; 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. bThe Cal/EPA Office of Environmental Health Hazard Assessment (OEHHA) Toxicity Criteria Database (http ://oehha. ca. gov/tcdb/index. asp) was also reviewed and found to contain no information on 1,3 -Dibromobenzene. NA = not applicable; NV = not available. 3 1,3-Dibromobenzene ------- FINAL 7-10-2014 Literature searches were conducted on sources published from 1900 through April 2014 for studies relevant to the derivation of provisional toxicity values for 1,3-dibromobenzene (CASRN 108-36-1). The following databases were searched by chemical name, synonyms, or CASRN: ACGM, ANEUPL, AT SDR, BIOSIS, Cal/EPA, CCRIS, CDAT, ChemlDplus, CIS, CRISP, DART, EMIC, EPIDEM, ETICBACK, FEDRIP, GENE-TOX, HAPAB, HERO, HMTC, HSDB, I ARC, INCHEM IPCS, IP A, ITER, IUCLID, LactMed, NIOSH, NTIS, NTP, OSHA, OPP/RED, PESTAB, PPBIB, PPRTV, PubMed (toxicology subset), RISKLINE, RTECS, TOXLINE, TRI, U.S. EPA IRIS, U.S. EPA HEAST, U.S. EPA HEEP, U.S. EPA OW, and U.S. EPA TSCATS/TSCATS2. The following databases were searched for toxicity values or exposure limits: ACGM, AT SDR, Cal EPA, U.S. EPA IRIS, U.S. EPA HEAST, U.S. EPA HEEP, U.S. EPA OW, U.S. EPA TSCATS/TSCATS2, NIOSH, NTP, OSHA, and RTECS. REVIEW OF POTENTIALLY RELEVANT DATA (NONCANCER AND CANCER) Table 3 provides an overview of the relevant databases for studies on 1,3-dibromobenzene and includes all potentially relevant, repeated-dose, short-term-duration studies (no sub chronic-duration or longer-term-duration studies have been located). All statistical comparisons were made at the 5% level of statistical significance (p < 0.05), unless noted otherwise. 4 1,3-Dibromobenzene ------- FINAL 7-10-2014 Table 3. Summary of Potentially Relevant Data for 1,3-Dibromobenzene (CASRN 108-36-1) Category Number of Male/Female, Strain, Species, Study Type, Study Duration Dosimetry Critical Effects NOAEL BMDL/ BMCL LOAEL Reference Comments Human 1. Oral No data 2. Inhalation No data Animal 1. Oral Short-term 4-6 female Wistar rats, gavage, 7 days 0, 70, 135, 270, or 400 mg/kg Increased ALA-D (135- and 270-mg/kg dose groups only) and increased ALA-S (all dose groups except for 70 mg/kg) activities ND NC ND Szvmanska (1996) No hepatic lesions were found. Short-term 4-6 female Wistar rats, gavage, 28 days 0, 5, 25, or 125 mg/kg Increased serum GGT activity (25- and 125-mg/kg dose groups only), and porphyrinuria ND NC ND Szvmanska (1996) Increased GSH level as a compensatory effect. No hepatic lesions were observed. Subchronic No data Chronic No data Developmental No data Reproductive No data Carcinogenic No data 5 1,3-Dibromobenzene ------- FINAL 7-10-2014 Table 3. Summary of Potentially Relevant Data for 1,3-Dibromobenzene (CASRN 108-36-1) Category Number of Male/Female, Strain, Species, Study Type, Study Duration Dosimetry Critical Effects NOAEL BMDL/ BMCL LOAEL Reference Comments 2. Inhalation No data ALA-D = delta-aminolevulinate dehydratase; ALA-S = delta-aminolevulinate synthase; GGT = L-y-glutamyltransferase; GSH = glutathione; NC = not calculated; ND = not determined. 6 1,3-Dibromobenzene ------- FINAL 7-10-2014 HUMAN STUDIES No studies have been identified. ANIMAL STUDIES No sub chronic-duration, chronic-duration, developmental toxicity, reproductive toxicity, or carcinogenicity studies on 1,3-dibromobenzene exposure (via the oral or inhalation route) were identified. A few short-term-duration gavage studies were located and are discussed below. Oral Exposures The effects of oral exposure of animals to 1,3-dibromobenzene via gavage have been evaluated in two short-term-duration, repeated-dose toxicity studies (Szymanska. 1996). Inhalation Exposures No studies have been identified. Short-Term-Duration Studies Szymanska (1996) In a 7-day repeated-dose study, Szymanska (1996) administered daily doses of 1,3-dibromobenzene via gavage. Female Wistar rats (4-6 per dose group) were treated with 0, 70, 135, 270, or 400 mg/kg of 1,3-dibromobenzene or l,3-dibromo[3H]-benzene (purity not specified) in sunflower oil. The control group received either sunflower oil or no gavage. Animals were sacrificed 24 hours after the administration of the seventh dose and the liver was excised for histological examination as well as to determine enzyme levels. The activity of alanine aminotransaminase (ALT) and L-y-glutamyltransferase (GGT) were determined in serum. The levels of glutathione (GSH) and malondialdehyde (MDA) as well as the activity of 5-aminolevulinate dehydratase (ALA-D) and synthase (ALA-S) were determined in liver. The study author reported a 50% increase in liver GSH for the 70-mg/kg dose group. In addition, ALT activity did not correlate with the change in GSH levels. There was a statistically significant increase of ALT in the 270-mg/kg dose group only. The activities of ALA-D (135- and 270-mg/kg dose groups only) and ALA-S (all dose groups except for 70 mg/kg) also increased. There were no statistically significant changes in GGT activity or MDA levels. No hepatic lesions were observed. In another short-term-duration (28-day repeated-dose) study, Szymanska (1996) administered daily doses of 1,3-dibromobenzene via gavage. Female Wistar rats (4-6 per dose group) were treated with 0, 5, 25, or 125 mg/kg of 1,3-dibromobenzene (purity not specified) in each dose group. The control group received either sunflower oil alone or no gavage. Control animals were kept in two types of cages: (1) metabolic cages (n = 14; Control Group 1) and (2) normal breeding cages (n = 21; Control Group 2). Treated animals were kept in metabolic cages beginning at 24 hours before the start of the investigation. Animals were sacrificed 24 hours after the administration of 7, 14, 21, or 28 doses. Livers were excised for histological examination and to determine enzyme levels. Increased GSH levels were observed in the 125-mg/kg dose group starting on Day 7. There was also a statistically significant increase in GGT activity for the mid- and high-dose groups. ALT activity did not change significantly in any of the dose groups. There was no statistically significant change in ALA-D in any of the dose groups on Day 28 (some statistically significant changes on Day 7 only). For ALA-S, a statistically significant decrease was observed in the 5-mg/kg dose group only. For liver MDA 7 1,3-Dibromobenzene ------- FINAL 7-10-2014 concentrations, the results were ambiguous in terms of the types of control groups used. Control Group 1 had much higher MDA levels than Control Group 2. All test groups had lower MDA concentrations than Control Group 1 and higher MDA concentrations than Control Group 2. The study author concluded that the change in MDA concentrations was likely a stress-induced effect due to the changing of cages rather than treatment related. No hepatic lesions were observed. In addition to the liver parameters mentioned above, Szymanska (1996) also examined liver iron levels and concentrations of 5-animolevulinic acid (ALA-U) and porphyrins in urine. There were no significant changes in iron concentrations in the rat liver in any of the dose groups. At the end of Week 4, however, there was a statistically significant decrease of excreted ALA-U in urine for the 25-mg/kg dose group. The study author also observed that several urinary porphyrins (i.e., tetracarboxy-, pentacarboxy-, and heptacarboxyporphyrins) were increased following repeated exposure to 1,3-dibromobenzene (Day 28), mostly at the higher doses. Because increased porphyrins were not accompanied by an increased excretion of ALA-U, the study author concluded that short-term exposure to 1,3-dibromobenzene produced porphyrinuria only and not porphyria in rats. Due to the short duration and differences observed between the two control groups, the 7-day and 28-day studies by Szymanska (1996) are not suitable for the derivation of a provisional reference dose (p-RfD). The study author did not identify any effect levels or median lethal dose (LD50) values, and neither a NOAEL nor a LOAEL are determined for this PPRTV assessment. OTHER DATA (SHORT-TERM TESTS, OTHER EXAMINATIONS) Other studies that examined 1,3-dibromobenzene but are not appropriate for the selection of a point of departure (POD) are described here. These studies are not adequate for the determination of p-RfD, provisional reference concentration (p-RfC), provisional oral slope factor (p-OSF), or provisional inhalation unit risk (p-IUR) values but provide supportive data supplementing a weight-of-evidence (WOE) approach. These may include genotoxicity, metabolism/toxicokinetic, and studies using routes of exposure other than the oral or inhalation route (see Table 4). 8 1,3-Dibromobenzene ------- FINAL 7-10-2014 Table 4. Other Studies Test Materials and Methods Results Conclusions References Other toxicity studies (exposures other than oral or inhalation) Male BALB/c mice (4-12 per dose group); single i.p. injection (0, 150, 300, or 600 mg/kg) of 1,2-, 1,3- ,or 1,4-dibromobenzene. Decreased glutathione (GSH) and increased malondialdehyde (MDA) levels in liver; increased gamma-glutamyltransferase (GGT) activities with all three isomers. Increased serum glutamate pyruvate transaminase (GPT) and hepatic necrosis in the central lobular zone with 1,2- and 1,3-dibromobenzene but not 1,4-dibromobenzene. 1,2- and 1,3-Dibromobenzene appear to have more pronounced effects on the liver than 1,4-dibromobenzene. Szvmanska et al. (1996) Female Wistar rats (4-6 per dose group); single i.p. injection (0, 40, 100, 300, or 600 mg/kg) of 1,3 -dibromobenzene. Reduced GSH levels, increased alanine aminotransferase (ALT) and GGT activities, and increased MDA concentration (two highest dose groups only). This study did not indicate whether the liver was histologically examined. 1,3-Dibromobenzene induces liver enzyme changes in rats. Szvmanska (1996) Male Outbred Imp BALB/c mice (4-12 per dose group); single i.p. injection (0, 150, 300, or 600 mg/kg) of 1,2,4- or 1,3,5-tribromobenzene, 1,2,4,5 -tetrabromobenzene, bromobenzene, or hexabromobenzene. Results from this study were compared to those from a previous studv with dibromobenzenes (Szvmanska et al.. 1996). All brominated compounds decreased liver GSH levels and increased GGT activity as well as MDA levels in the liver, but these effects were more pronounced with dibromobenzenes and bromobenzene. The acute hepatotoxicity of bromobenzenes decreases with the increase in the number of bromine atoms. Szvmanska (1998) Male BALB/c mice (4-5 per dose group); i.p. injection (0, 30, 60, 80, or 140 mg/kg) of 1,3-dibromobenzene or seven other brominated benzenes for 7 days. Increased relative liver weight (liver-to-body weight ratio) and increased MDA concentration in all brominated benzenes. Steatosis was observed for some brominated benzenes but not for 1,3-dibromobenzene. Liver effects were observed following repeated exposure to brominated benzenes. Szvmanska et al. (1998) Metabolism/ Toxicokinetic Female Outbred IMP:Wist rats (4 per dose group);single i.p. injection (100 or 300 mg/kg) of 1,3-dibromobenzene (no controls identified). An average of 79.3% of 1,3-dibromobenzene and its metabolites was excreted in urine. The highest concentration of 1,3-dibromobenzene was found in the liver, kidneys, and fat tissue. Urine is the main route of excretion for 1,3-dibromobenzene. Saoota et al. (1999) 9 1,3-Dibromobenzene ------- FINAL 7-10-2014 Other Toxicity Studies (Exposures Other than Oral or Inhalation) The effects of intraperitoneal (i.p.) exposure of animals to 1,3-dibromobenzene have been evaluated in three acute, single-injection studies (Szymanska. 1998. 1996; Szymanska et al.. 1996) and one short-term-duration, repeated-injection study. The results are reported in Szymanska et al. (1998) and Szymanska (1996). Acute Studies Szymanska et al. (1996) Szymanska et al. (1996) conducted an acute toxicity study of 1,2-, 1,3-, and 1,4-dibromobenzene isomers in male BALB/c mice. Single doses of 0, 150, 300, or 600 mg/kg in sunflower oil (purity not reported) were administered to 4-12 male mice per dose group by i.p. injection. The control group (n = 28-30) received no injections or were injected with sunflower oil only. Livers were removed and blood was collected at different time intervals: 2, 4, 12, 24, 48, 72, and 120 hours after injection. Treatment-related effects included decreased GSH levels in the first 24 hours after administration at the two highest doses (up to 90% decrease), a statistically significant increase in MDA in the liver, and increases in GGT in all three isomers. Increased GPT activities and an increase in the incidence of hepatic necrosis (as determined in histopathology) were observed with 1,2- and 1,3-dibromobenzene but not 1,4-dibromobenzene. The results of this study indicated that all three isomers are acutely hepatotoxic, with 1,3- and 1,2-dibromobenzene being more toxic (based on more pronounced incidences of hepatic necrosis in the central lobular zone) than 1,4-dibromobenzene (no statistically significant change from the control group; caused necrosis only in individual hepatocytes). Neither effect levels nor LDsos were determined by the study authors. Szymanska (1996) In addition to the short-term-duration oral studies in rats, Szymanska (1996) conducted an acute single-dose study. Female Wistar rats (4-6 per dose group) were administered 0, 40, 100, 300, or 600 mg/kg of 1,3-dibromobenzene (purity not specified) in sunflower oil by i.p. injection. The control rats (n = 21-22) received either sunflower oil alone or no injection. Heavy depletion of liver GSH was observed at the high doses. Even at the low doses, there was a statistically significant decrease in GSH levels. However, GSH levels eventually went up in all dose groups, and in some cases, increased above control levels after 24 hours of administration. The study author stated that these increases in GSH may indicate either an adaptation or compensatory effect. Serum ALT increased slightly within a short time after the administration of 1,3-dibromobenzene but then fluctuated above and below control levels between 4 and 72 hours after administration. There was a statistically significant increase in GGT activity within 4 hours. A statistically significant increase in liver MDA concentration was observed in the highest dose group only (up to 12 hours after administration). The study author did not indicate whether morphological examinations were conducted to detect hepatic lesions (necrosis), as previously observed in the mouse study (Szymanska et al.. 1996). In terms of acute hepatotoxicity, a species-specific difference (rats vs. mice) could exist; however, the study author did not speculate on reasons for this possible difference. Szymanska (1998) Szymanska (1998) conducted another acute single-dose hepatotoxicity study for multiple brominated benzenes (bromobenzene, 1,2,4-tribromobenzene, 1,3,5-tribromobenzene, 1,2,4,5-tetrabromobenzene, and hexabromobenzene) in male Outbred Imp BALB/cJ mice in an 10 1,3-Dibromobenzene ------- FINAL 7-10-2014 attempt to find a relationship between chemical structure and hepatotoxic effects. Single doses of 0, 150, 300, or 600 mg/kg in sunflower oil (purity not reported) were administered to 4-12 male mice per dose group by i.p. injection. The control mice received either sunflower oil alone or no injection. Similar to the other acute single-dose i.p. studies (Szymanska. 1996; Szymanska et al.. 1996). Szymanska (1998) found that all examined compounds (i.e., bromobenzene, 1,2,4-tribromobenzene, 1,3,5-tribromobenzene, 1,2,4,5-tetrabromobenzene, and hexabromobenzene) lowered liver GSH levels shortly after the administration. The results were compared with data for dibromobenzenes (1,2-, 1,3-, and 1,4-dibromobenzene) obtained from an earlier study (Szymanska et al.. 1996). Statistically significant changes (30- to 120-fold increases) in ALT activity were observed for bromobenzene, 1,2-dibromobenzene, 1.3-dibromobenzene, and 1,2,4-tribromobenzene. All brominated compounds produced an increase in GGT activity in serum and MDA concentration in the liver. As stated in the Szymanska et al. (1996) study and summarized in Szymanska (1998). increased incidence of hemorrhagic necrosis in the liver central lobular zone was observed for 1,3-dibromobenzene. Finally, Szymanska (1998) concluded that in mice ".. .acute toxicity of bromobenzenes decreases with the increase of the number of bromine atoms in the molecule" (p. 97). Neither effect levels nor LDsos were determined. Short-Term-Duration Studies Szymanska et al. (1998) In a follow-up study, Szymanska et al. (1998) conducted another short-term-duration (7-day repeated dose) study in male BALB/c mice. Szymanska et al. (1998) reported the effects on selected indicators of liver impairment after repeated administration of mono- and polybromobenzenes. Szymanska et al. (1998) administered 1,3-dibromobenzene along with seven other brominated benzenes (i.e., bromobenzene, 1,2-dibromobenzene, 1.4-dibromobenzene, 1,2,4-tribromobenzene, 1,3,5-tribromobenzene, 1,2,4,5-tetrabromobenzene, and hexabromobenzene; purity not specified) daily via i.p. injection in sunflower oil in volumes of 0.2 ml per 20 g body weight of mice. The mice (4-5/group) were exposed to 0, 30, 60, 80, or 140 mg/kg in each dose group. The control mice received either sunflower oil only or no injection. Because the study authors found no difference between the control groups, they chose the pooled controls that received no injection for comparison with other treatment groups. Changes in relative liver weight (liver-to-body weight ratio) were observed for all brominated benzenes. For exposure to 1,3-dibromobenzene, relative liver weight increased in the 60-, 80-, and 140-mg/kg dose groups, but these changes were not dose dependent. For histopathological changes, distinct steatosis in the peripheral lobular zone was observed for 1,4-dibromobenzene, 1,2,4-tribromobenzene, 1,3,5-tribromobenzene, 1,2,4,5-tetrabromobenzene, and hexabromobenzene but not 1,3-dibromobenzene. For exposure to 1,3-dibromobenzene, the GSH level was statistically significantly increased in the 80-mg/kg dose group only; no depletion of the GSH level was observed in any of the dose groups (the study authors stated that this may explain the lack of hepatic necrosis). There was also a statistically significant increase in liver MDA level at the highest dose of 1,3-dibromobenzene (140 mg/kg). ALA-S was significantly decreased after exposure to 1,2-dibromobenzene and 1,3-dibromobenzene. 1,4-Dibromobenzene decreased ALA-S in the lower doses and increased ALA-S in the higher doses. For ALA-D, the study authors claimed there were no statistically significant changes for any of the brominated benzenes. The activity of ALT—another key indicator for necrotic changes of hepatocytes—was not affected by any of the brominated benzenes. 11 1,3-Dibromobenzene ------- FINAL 7-10-2014 Szymanska et al. (1998) stated that there was an apparent "shift" from single exposure to repeated exposure in terms of hepatotoxic effects, with necrosis occurring after single-dose exposure and the less severe steatosis occurring after repeated exposure (observed for other brominated benzenes but not observed for 1,3-dibromobenzene directly). These findings are consistent with the interpretation by Chakrabarti (1991) that, . .repeated doses of bromobenzene may by means of inducing microsomal enzymes and GSH levels accelerate the process of bromobenzene metabolism and/or may intensify repair/regeneration processes in the cell" (as cited in Szymanska et al.. 1998. p. 28). In addition, instead of necrosis in the liver and associated increases in ALT and GGT activities in the serum observed in the acute single-dose studies in mice, the study authors found changes in heme synthesis and subsequent porphyrogenic effects in the repeated dose studies. Neither effect levels nor LDsos have been identified or determined in any of the studies in this section, and none of the studies are suitable for the derivation of a p-RfD due to the route of administration (i.p.), study duration (<90 days), and study design (e.g., metabolic cage vs. normal breeding cage). Toxicokinetics Studies Sapota et al. (1999) investigated the toxicokinetic (distribution and excretion) properties of 1,3-dibromobenzene in rats by radiotracing. Four female Outbred IMP:Wist rats per group were administered 100 or 300 mg/kg of l,3-dibromobenzene-[3H] (purity not specified) dissolved in olive oil via a single i.p. injection. The use of a control group was not reported. Metabolites were identified and quantified by gas chromatography-mass spectrometry (GC-MS) technique. Urine was the main route of excretion, where an average of 79.3% was excreted in urine after 72 hours at a dose of 300 mg/kg, with 10% in feces. Similar (slightly lower) absorption and excretion rates were also found at a dose of 100 mg/kg. Tissues examined included the liver, kidneys, lung, adrenals, sciatic nerve, spleen, heart, brain, and fat. The highest concentrations (radioactivity) of l,3-dibromobenzene-[3H] after administration at a dose of 100 mg/kg were found in the liver, kidneys, and fat tissue. A similar pattern was observed for the 300-mg/kg dose group. Several metabolites isolated from urine were identified by GC-MS: unchanged (unconjugated) 1,3-dibromobenzene (18%), dibromophenols (34%), dibromothiophenols (28%), dibromothioanisole (1.8%), bromophenol (5.5%), bromohydroxythiophenols (5%), and bromohydroxythioanisole (7.5%). The study authors concluded that there are three different metabolic pathways of 1,3-dibromobenzene: (1) ring hydroxylation (dibromophenols), (2) glutathione conjugation (dibromothiophenols and dibromothioanisole), and (3) hydrolytic dehalogenation (bromophenol, bromohydroxythiophenol and bromohydroxythioanisole). The study authors observed that approximately half of the metabolism products contain sulfur, and this finding is consistent with earlier observations (Szymanska et al.. 1996) in which the hepatic necrotic action of 1,3-dibromobenzene was accompanied by decreased hepatic GSH levels. The study authors also concluded that 1,3-dibromobenzene has a relatively high turnover rate (e.g., high level of excretion in urine) with minor levels of radiotracer 3H in the tissues for longer time periods. Finally, they stated that 1,3-dibromobenzene is an acute hepatotoxicant in rats and is also a potential nephrotoxicant (Sapota et al.. 1999). 12 1,3-Dibromobenzene ------- FINAL 7-10-2014 In a follow-up toxicokinetic study, Szymanska et al. (2002) compared the metabolism and tissue distribution of 1,2- and 1,4-dibromobenzene isomers in female Outbred IMP:Wist rats. The study used a similar protocol as detailed in Sapota et al. (1999). As with 1.3-dibromobenzene, urine is also the main route of excretion for both the 1,2- and 1.4-dibromobenzene isomers. Several metabolites isolated from urine were identified by GC-MS for the 1,2- and 1,4-dibromobenzene isomers; they included unchanged (unconjugated) parent compound (11 and 5%), dibromophenols (73 and 84%), dibromothiophenols (10 and 5%), and bromophenols (0.7 and 1.9%). The study authors concluded that 1,2-dibromobenzene (82.0%) excreted in urine after 72 hours for the 100-mg/kg dose group) was similar to 1,3-dibromobenzene (66.5%> excreted in urine for the 100-mg/kg dose group) (Sapota et al.. 1999) in having a higher turnover rate than 1,4-dibromobenzene, which had a longer retention time in the body (29.6%> excreted in urine for the 70-mg/kg dose group). Genotoxicity Studies No studies investigating the genotoxic effects of 1,3-dibromobenzene have been identified. DERIVATION OF PROVISIONAL VALUES Table 5 below presents a summary of noncancer screening oral provisional reference values derived using a surrogate approach (see Appendix A for details). Table 6 presents a summary of cancer values. The toxicity values have been converted to human equivalent dose (HED) units where appropriate. 13 1,3-Dibromobenzene ------- FINAL 7-10-2014 Table 5. Summary of Noncancer Reference Values for 1,3-Dibromobenzene (CASRN 108-36-1) Toxicity Type (units) Species/Sex Critical Effect Screening p-Reference Value POD Method PODhed UFc Principal Study Screening subchronic p-RfD (mg/kg-day)a Rat/Male Increased relative liver weight and hepatic microsomal enzyme induction 4 x 1(T3 NOAEL 1.2 300 Carlson and Tardiff (\ 977) Screening chronic p-RfD (mg/kg-day)'' Rat/Male Increased relative liver weight and hepatic microsomal enzyme induction 4 x 1(T4 NOAEL 1.2 3,000 Carlson and Tardiff (1977) Subchronic p-RfC (mg/m3) NDr Chronic p-RfC (mg/m3) NDr aA surrogate approach was applied. See Appendix A. NDr = not determined Table 6. Summary of Cancer Values for 1,3-Dibromobenzene (CASRN 108-36-1) Toxicity Type Species/Sex Tumor Type Cancer Value Principal Study p-OSF None p-IUR None DERIVATION OF ORAL REFERENCE DOSES Feasibility of Deriving Subchronic and Chronic p-RfDs No sub chronic-duration, chronic-duration, developmental toxicity, reproductive toxicity, or carcinogenicity studies on 1,3-dibromobenzene exposure via the oral route were identified. However, Appendix A of this document contains screening values (screening subchronic and chronic p-RfDs) using a surrogate (e.g., structural and metabolic) approach, which may be of use under certain circumstances. Please see Appendix A for details regarding the screening values. DERIVATION OF INHALATION REFERENCE CONCENTRATIONS Feasibility of Deriving Subchronic and Chronic p-RfCs No sub chronic-duration, chronic-duration, developmental toxicity, reproductive toxicity, or carcinogenicity studies on 1,3-dibromobenzene exposure via the inhalation route were identified. No inhalation toxicity data have been identified for the derivation of a p-RfC for 1,3-dibromobenzene. Furthermore, no inhalation toxicity data were identified for any of the 14 1,3-Dibromobenzene ------- FINAL 7-10-2014 potential surrogates for 1,3-dibromobenzene, thus precluding derivation of screening subchronic and chronic p-RfCs. CANCER WEIGHT-OF-EVIDENCE DESCRIPTOR Table 7 identifies the cancer WOE descriptor for 1,3-dibromobenzene (in bold). Table 7. Cancer WOE Descriptor for 1,3-Dibromobenzene (CASRN 108-36-1) Possible WOE Descriptor Designation Route of Entry (Oral, Inhalation, or Both) Comments "Carcinogenic to Humans " NA NA No studies pertaining to the carcinogenicity of 1,3-dibromobenzene in humans are available. "Likely to be Carcinogenic to Humans " NA NA No studies pertaining to the carcinogenicity of 1,3-dibromobenzene in multiple species of animals are available. "Suggestive Evidence of Carcinogenic Potential" NA NA No data are available regarding the carcinogenic potential of 1,3-dibromobenzene even in a single animal species. "Inadequate Information to Assess Carcinogenic Potential" Selected Both There is no pertinent information available to assess the carcinogenic potential of 1,3-dibromobenzene. "Not Likely to be Carcinogenic to Humans " NA NA No data are available to suggest that 1,3-dibromobenzene is not likely to be a carcinogen in humans following oral or inhalation exposure. NA= not applicable. DERIVATION OF PROVISIONAL CANCER RISK VALUES The lack of quantitative data on the carcinogenicity of 1,3-dibromobenzene precludes the derivation of a quantitative estimate of cancer risk for either oral (p-OSF) or inhalation (p-IUR) exposures. 15 1,3-Dibromobenzene ------- FINAL 7-10-2014 APPENDIX A. SCREENING PROVISIONAL VALUES For reasons noted in the main provisional peer-reviewed toxicity value (PPRTV) document, it is inappropriate to derive provisional toxicity values for 1,3-dibromobenzene. However, information is available for a related 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 Superfund Health Risk Technical Support Center 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 PPRTV documents 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 is considerably more uncertainty associated with the derivation of 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 Superfund Health Risk Technical Support Center. APPLICATION OF AN ALTERNATIVE SURROGATE APPROACH The surrogate 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 surrogate analysis are presented in Wang et al. (2012). Three types of potential surrogates (structural, metabolic, and toxicity-like) are identified to facilitate the final surrogate chemical selection. The surrogate approach may or may not be route-specific or applicable to multiple routes of exposure. In this document, it is limited to the oral noncancer effects only, based on the available toxicity data. All information was considered together as part of the final weight-of-evidence (WOE) approach to select the most suitable surrogate both toxicologically and chemically. Structural Surrogates (Structural Analogs) 1,3-Dibromobenzene is a halogenated compound belonging to the brominated benzene class. Thus, an initial surrogate search focused on the identification of brominated benzenes with toxicity values from the Integrated Risk Information System (IRIS), PPRTV, and Health Effects Assessment Summary Tables (HEAST) databases to take advantage of the well-characterized chemical-class information. Four brominated benzenes were found to have oral toxicity values listed on IRIS: bromobenzene (U.S. EPA. 2009); 1,4-dibromobenzene (U.S. EPA. 1988a); 1,2,4-tribromobenzene (U.S. EPA. 1993); and hexabromobenzene (U.S. EPA. 1988b) (see Tables A-l and A-3). Similarity scores for these chemicals were identified by searching for structural analogs at least 50% similar to 1,3-dibromobenzene using the National Library of Medicine's ChemlDplus database (NLM ). Out of the four brominated benzenes initially obtained from IRIS, only 1,2,4-tribromobenzene had a similarity match of >50% to 1,3-dibromobenzene (55.92%>, see Table A-l). The remaining three potential surrogates had a similarity score less than 50%. However, all four brominated benzenes were retained because of the chemical-class specific information (e.g., common target organ and effect[s]). Table A-l summarizes their physicochemical properties and structural similarity. Although 1,2,4-tribromobenzene was found to be the most structurally similar to 1.3-dibromobenzene based on the ChemlDplus similarity score, 1,2,4-tribromobenzene was not as similar to 1,3-dibromobenzene with regard to physicochemical properties. Instead, 1.4-dibromobenzene displayed the most similarity to 1,3-dibromobenzene with regard to 16 1,3-Dibromobenzene ------- FINAL 7-10-2014 physicochemical properties, followed by bromobenzene and 1,2,4-dibromobenzene (see Table A-l). Hexabromobenzene displayed physicochemical properties that were the least similar to 1,3-dibromobenzene (see Table A-l). Based on this information, the top three chemicals considered as structural surrogates are bromobenzene, 1,4-dibromobenzene, and 1,2,4-tribromobenzene (see details below). 17 1,3-Dibromobenzene ------- FINAL 7-10-2014 Table A-l. Structural Similarity and Physicochemical Properties of 1,3-Dibromobenzene and Potential Surrogates3 Characteristic 1,3-Dibromobenzene Bromobenzene 1,4-Dibromobenzene 1,2,4-Tribromobenzene Hexabromobenzene Structure Br ^^^Br Br Br^^j^^Br Br CASRN 108-36-1 108-86-1 106-37-6 615-54-3 87-82-1 Similarity score (%) 100 <50 <50 55.92 <50 Molecular formula C6H4Br2 C6H5Br C6H4Br2 C6H3Br3 CeBr6 Molecular weight 235.9 157 235.9 314.8 551.5 Melting point (°C) -7.00 -30.6 87.3 44.5 326 Boiling point (°C) 218 156 219 275 - Vapor pressure (mm Hg at 25°C) 0.269 4.18 0.058 5.48 x 10-3 1.63 x 10-8 Henry's law constant (atm-m3/mole at 25 °C) 1.24 x 10-3 2.47 x 10-3 8.93 x 10-4 3.41 x 10-4 2.81 x 10"5 Water solubility (mg/L) 67.5 446 20 4.9 1.60 x 10-4 Log Kowb 3.78 2.99 3.89 4.54 6.07 aNLM (accessed 7-22-2013). bLu et al. (2000). 18 1,3-Dibromobenzene ------- FINAL 7-10-2014 Metabolic Surrogates Toxicokinetic Data The specific metabolism information for 1,3-dibromobenzene and the four potential metabolic surrogates was based on the available metabolic information in the form of metabolites detected in urine; Table A-2 displays a summary of these excretion data. Similar to the analysis of physicochemical properties, hexabromobenzene was the least similar to 1,3-dibromobenzene in terms of metabolite profile; hence, it was concluded that hexabromobenzene is not a suitable candidate to serve as a metabolic surrogate for 1,3-dibromobenzene. Bromobenzene, 1,4-dibromobenzene, and 1,2,4-tribromobenzene had the same common metabolites as 1,3-dibromobenzene (i.e., bromophenols), and they were considered as metabolic surrogates. In a related but indirect metabolism study, Lupton et al. (2009) analyzed brominated diphenyl ethers (BDEs) 47, 99, and 153 and identified their metabolites using human liver microsomes. The study authors found that BDE 99 was metabolized primarily to dihydroxylated BDE 99, but it was also metabolized to 2,4,5-tribromophenol, and 1,3-dibromobenzene to a lesser extent (<2-8%). Because 1,3-dibromobenzene only accounts for a small amount of the metabolites of BDE 99, BDE 99 was not considered as a potential metabolic surrogate for 1,3-dibromobenzene (BDE 99 also has a different toxic endpoint: neurotoxicity). 19 1,3-Dibromobenzene ------- FINAL 7-10-2014 Table A-2. Summary of Brominated Benzene Metabolites Detected in Urine Chemical Route Species Metabolites Reference Bromobenzene i.p. Rabbit Bromophenols Ruzo et al. (1916) i.p. Rat Bromophenols and dihydrodiols Miller et al. CI990s) and Lertratananekoon and Hornine (1987) 1,4 -Dibromobenzene i.p. Rabbit Bromophenols Ruzo et al. (1976) i.p. Rat Bromophenols Szvmariska et al. (2002) 1,2,4-Tribromobenzene i.p. Rabbit Bromophenols Ruzo et al. (1976) Hexabromobenzene oral Rat Bromobenzenes Yamaeuchi et al. (1988) 1,3 -Dibromobenzene i.p. Rabbit Three phenolic products—Due to small amounts of metabolites and unavailability of authentic standards, these metabolites could not be identified. Ruzo et al. (1976) i.p. Rat Bromophenols Saoota et al. (1999) i.p. = intraperitoneal. 20 1,3-Dibromobenzene ------- FINAL 7-10-2014 Toxicity-Like Surrogates Table A-3 summarizes available toxicity data for 1,3-dibromobenzene and the four brominated benzenes identified as potential surrogates. All of the four brominated benzenes induced liver effects (e.g., hepatocellular cytomegaly, increased liver-to-body-weight ratio, hepatic microsomal enzyme induction, etc.). Furthermore, three of the brominated benzenes (bromobenzene, 1,4-dibromobenzene, and 1,2,4-tribromobenzene) had critical effects involving the liver. For hexabromobenzene, liver changes were observed (i.e., increased liver-to-body- weight ratio and increased liver porphyrins at higher doses) but these were not the most sensitive endpoints. From these long-term toxicity data, it is clear that the liver is a common target organ, and liver effects are often times the most sensitive endpoint for the brominated benzene chemical class. Also, the available acute and short-term toxicity studies similarly point to the liver as a target organ for 1,3-dibromobenzene (Szymanska et al.. 1998; Szymanska. 1998. 1996; Szymanska et al.. 1996). Hence, with respect to 1,3-dibromobenzene, the liver is likely to be the target organ and liver effects the most sensitive endpoint for long-term toxicity. Although Szymanska (1998) demonstrated that the acute toxicity of bromobenzenes in mice is reduced when the number of bromine atoms is increased, comparison of median lethal dose (LD50) values among bromobenzenes was inconclusive. The mouse LD50 values for bromobenzene and 1,4-dibromobenzene were slightly higher than the mouse LD50 for 1.3-dibromobenzene, but there were no mouse LD50 data for 1,2,4-tribromobenzene or hexabromobenzene to make a direct comparison (see Table A-3). Furthermore, an opposite trend in the long-term toxicity of brominated benzenes in terms of their effect levels was observed (see Table A-3) (i.e., the higher the number of bromine atoms, the more toxic the chemical is chronically). This trend is consistent with later findings by Szymanska et al. (1998) that there is an apparent "shift" from single exposure to repeated exposure in terms of hepatotoxic effects from necrosis to less severe steatosis and porphyrogenic effects (Szymanska et al.. 1998). This shift may occur because repeated doses of brominated benzenes could accelerate the process of metabolism towards the formation of less toxic metabolites and/or may intensify the repair/regeneration processes in cells (Szymanska et al.. 1998). In vitro Hep ototoxicity Data In addition to the in vivo data on the potential surrogates, in vitro measurements of toxicity such as median lethal concentration (LC50) were also available. The human hepatocyte LC50 of 1,3-dibromobenzene was most similar to the human hepatocyte LC50 of 1,2,4-tribromobenzene (488 vs. 475 [xM; see Table A-3). The rat hepatocyte toxicity data LC50 of 1,3-dibromobenzene was most similar to the rat hepatocyte LC50 of 1,4-dibromobenzene (355 vs. 371 [xM; see Table A-3). The human and rat LC50S of bromobenzene were 2-3 times higher than the LC50S of 1,3-dibromobenzene; these data suggest that 1,3-dibromobenzene could be 2 to 3 times more toxic than bromobenzene in terms of in vitro toxicity. No comparison was possible between hexabromobenzene and 1,3-dibromobenzene because there were no in vitro human or rat hepatocyte toxicity data. Therefore, based on the similarities in in vitro toxicity levels as well as critical effects involving the liver, 1,4-dibromobenzene and 1,2,4-tribromobenzene were considered toxicity-like surrogates. In conclusion, an attempt was made to identify a suitable surrogate to derive toxicity values for 1,3-dibromobenzene. Comparison of the potential surrogates (bromobenzene, 1.4-dibromobenzene, 1,2,4-tribromobenzene, and hexabromobenzene) was made based on their 21 1,3-Dibromobenzene ------- FINAL 7-10-2014 profiles of structural similarity, toxicokinetics, and tissue-specific toxicity. The chronic reference doses (RfDs) for the four potential surrogates range from 2 x io~3 to 1 x 10"2 mg/kg-day, and therefore, use of any of these candidates would have resulted in a comparable screening chronic provisional reference dose (p-RfD) for 1,3-dibromobenzene (see Table A-3). The common target organ among the potential surrogates appears to be the liver, with the kidneys as a likely secondary target organ for some brominated benzenes (e.g., bromobenzene). 22 1,3-Dibromobenzene ------- FINAL 7-10-2014 Table A-3. Comparison of Available Toxicity Data for 1,3-Dibromobenzene and Potential Surrogates Characteristic 1,3-Dibromobenzene Bromobenzene 1,4-Dibromobenzene 1,2,4-Tribromobenzene Hexabromobenzene Structure Br ^^^Br Br^^j^^Br Br^^j^^Br Br Human hepatocyte toxicity LC50 (|iM)a 488 ±48.8 1.150 ±115 560 ± 56 475 ±37.5 NA Rat hepatocyte toxicity LC50 (|iM)a 355 ±35.5 750 ± 75 371 ±37.1 214 ±21.4 NA Mouse LD50 (mg/kg)b 2,250 2,700 3,120 NA NA Chronic RfD (mg/kg-day) NA 8 x 10-3 1 x 10-2 5 x 10-3 2 x 10-3 Critical effect NA Hepatocellular cytomegaly Increased liver-to-body- weight ratio and hepatic microsomal enzyme induction Increased liver-to-body- weight ratio and hepatic microsomal enzyme induction Induced serum carboxylesterase activity POD (mg/kg-day) NA BMDL10: 24.1 NOAEL: 10 NOAEL: 5 NOAEL: 2 UFC NA 3,000 1,000 1,000 1,000 Source NA U.S. EPA (2009) U.S. EPA (1988a) U.S. EPA (1993) U.S. EPA (1988b) aChan et al. (2007). bNLM (accessed on 7-22-2013). BMDL = benclimark dose lower confidence limit; LCso = median lethal concentration; LD50 = median lethal dose; NA = not available; NOAEL = no-observed-adverse- effect level; POD = point of departure; UFC = composite uncertainty factor. 23 1,3-Dibromobenzene ------- FINAL 7-10-2014 Weight-of-Evidence (WOE) Approach To select the best surrogate chemical based on all of the information from the three surrogate types, the following considerations were used in a WOE approach: (1) biological and toxicokinetic data are preferred over the structural data, (2) lines of evidence that indicate pertinence to humans are preferred, (3) chemicals with more conservative/health protective toxicity values may be favored, and (4) if there are no clear indications as to the best surrogate chemical based on the first three considerations, then the candidate surrogate with the highest structural similarity may be preferred. In summary, bromobenzene, 1,4-dibromobenzene, and 1,2,4-tribromobenzene were identified as structural surrogates; bromobenzene, 1,4-dibromobenzene, and 1,2,4-tribromobenzene were identified as metabolic surrogates; and 1,4-dibromobenzene and 1,2,4-tribromobenzene were identified as toxicity-like surrogates. Overall, based on the WOE of all the information presented above, 1,2,4-tribromobenzene appears to be the most appropriate surrogate for 1,3-dibromobenzene because of the following factors: • More similar structurally o Structural similarity of 55.92% (highest similarity score) using the National Library of Medicine's ChemlDplus database (NLM) • Similar toxicokinetic profile and target organ (see Table A-2 and A-3) • Similar in vitro hepatotoxicity data (see Table A-3) (Chan et al.. 2007) • More sensitive effect level (see Table A-3) The 1,2,4-Tribromobenzene IRIS Summary (U.S. EPA. 1993) cited Carlson and Tardiff (1977) as the principal study for the RfD. Six male rats/group were dosed daily with 0, 2.5, 5 or 10 mg 1,2,4-tribromobenzene (TBB)/kg bw for 45 or 90 days. TBB was administered in corn oilp.o. as 0.1% of body weight. Controls received corn oil only. Animals were sacrificed at 45 or 90 days or after an additional 30-day recovery period after 90 days of treatment. Body weight, liver weight, and hepatic microsomal enzyme activity were measured. Liver-to-body weight ratios were increased 12-16% over controls for the rats treated at 10 mg/kg/day. Liver enzyme activities were 1.4- to 3-fold that of controls for the same group. Full recovery to baseline enzyme activity was observed after the 30-day recovery period; liver-to- body weight ratios were only 7% greater than the control values. Similar results were reported by Carlson (1979) in a follow-up study. Although no overt liver toxicity was demonstratedfor TBB, bromobenzene mixtures at higher doses cause acute hepatic necrosis. The mechanism of bromobenzene toxicity has been studied in detail and involves conversion of the parent compound to toxic intermediates by hepatic microsomal enzymes. Induction of these enzymes can potentiate the toxicity of bromobenzenes and other similarly-activated compounds. The uncertainty factor includes factors for interspecies variability, subchronic-to- chronic exposure duration extrapolation, and intrahuman variability. 24 1,3-Dibromobenzene ------- FINAL 7-10-2014 Low confidence levels were assigned to both the study and the database because of the lack of adequate toxicity parameters in the critical study, the lack of chronic toxicity data in general, and a degree of uncertainty about the significance of the effects. Low confidence in the RfD follows. An updated literature search from 2004 to 2013 was performed for 1,2,4-tribromobenzene and one additional subchronic study was identified. Dodd et al. (2012) treated 10 male Sprague-Dawley rats/dose with 1,2,4-tribromobenzene via gavage for either 5 days, 2, 4, or 13 weeks at 0, 2.5, 5, 10, 25, or 75 mg/kg-day. This study only focused on liver effects and did not evaluate any other possible endpoints of toxicity. The study authors identified a no-observed-adverse-effect level (NOAEL) of 5 mg/kg-day based on increased liver weight and increased incidence of hepatocyte hypertrophy. This coincides with the NOAEL of 5 mg/kg-day identified from Carlson and Tardiff (1977) which is used as the point of departure (POD) in the IRIS assessment of 1,2,4-tribromobenzene. ORAL TOXICITY VALUES Derivation of Screening Subchronic Provisional Reference Dose (Screening Subchronic p-RfD) Based on the overall surrogate approach presented in this PPRTV assessment, the Integrated Risk Information System (IRIS) POD for 1,2,4-tribromobenzene (a NOAEL of 5 mg/kg-day) established in 1993 and based on increased relative liver weight (liver-to-body- weight ratio) and hepatic microsomal enzyme induction in male Sprague-Dawley rats from a 90-day study (Carlson and Tardiff. 1977) is recommended as the surrogate POD for 1,3-dibromobenzene. No duration adjustment was performed for the doses reported in the principal study because Carlson and Tardiff (1977) did not report the treatment schedule used. The data are not amenable to BMD modeling; thus, calculation of a BMDL is precluded. As described in the EPA's Recommended Use of Body Weight314 as the Default Method in Derivation of the Oral Reference Dose (U.S. EPA. 201 lb), the POD from the 90-day 1,2,4-tribromobenzene study by Carlson and Tardiff (1977) in rats is converted to a human equivalent dose (HED) through an application of a dosimetric adjustment factor (DAF) derived as follows: DAF = (BWa1/4 - BWh1/4) where DAF = dosimetric adjustment factor BWa = animal body weight BWh = human body weight Using a BWa of 0.25 kg for rats and a default BWh of 70 kg for humans (U.S. EPA. 1988c). the resulting DAF is 0.24. Applying this DAF to the NOAEL identified in the rat study yields a surrogate PODhed as follows: 25 1,3-Dibromobenzene ------- FINAL 7-10-2014 Surrogate PODhed = NOAEL (mg/kg-day) x DAF = NOAEL (mg/kg-day) x 0.24 = 5 mg/kg-day x 0.24 = 1.2 mg/kg-day As described by Wang et al. (2012). the uncertainty factors typically applied to the chemical of concern are the same as those applied to the surrogate unless additional information is available. The IRIS assessment for the 1,2,4-tribromobenzene surrogate was performed prior to the recommended use of BW3/4 scaling for noncancer effects (U.S. EPA. 2011b) and prior to the application of a database uncertainty factor (UFd). Thus, the composite UF (UFc) for 1,3-dibromobenzene has been adjusted and differs from that of the surrogate. To derive a screening subchronic p-RfD for 1,3-dibromobenzene, a UFc of 300 has been applied to the surrogate PODhed. The screening subchronic p-RfD for 1,3-dibromobenzene is derived as follows: Screening Subchronic p-RfD = Surrogate PODhed ^ UFc = 1.2 mg/kg-day -^300 = 4 x 10"3 mg/kg-day Table A.4 summarizes the uncertainty factors for the screening subchronic p-RfD for 1,3 -dibromobenzene. Table A-4. Uncertainty Factors for the Screening Subchronic p-RfD for 1,3-Dibromobenzene UF Value Justification UFa 3 A UFa of 3 (10°5) has been applied to account for uncertainty in characterizing the toxicodynamic differences between rats and humans following oral 1,3-dibromobenzene exposure. The toxicokinetic uncertainty has been accounted for by calculation of a HED through application of a DAF as outlined in the EPA's Recommended Use of Body Weishf/4 as the Default Method in Derivation of the Oral Reference Dose ('U.S. EPA. 2011b). UFd 10 A UFd of 10 has been applied because there are no acceptable two-generation reproductive toxicity or developmental toxicity studies via the oral route. UFh 10 A UFh of 10 has been applied for inter-individual variability to account for human-to- human variability in susceptibility in the absence of quantitative information to assess the toxicokinetics and toxicodynamics of 1,3-dibromobenzene in humans. UFl 1 A UFl of 1 has been applied for LOAEL-to-NOAEL extrapolation because the POD is a NOAEL UFS 1 A UFS of 1 has been applied because a subchronic-duration study was selected as the principal study. UFC 300 Composite UF. Derivation of Screening Chronic Provisional Reference Dose (Screening Chronic p-RfD) The surrogate POD used to derive a screening chronic p-RfD is the same as the surrogate POD (PODhed =1.2 mg/kg-day) used to derive the screening subchronic p-RfD above. To 26 1,3-Dibromobenzene ------- FINAL 7-10-2014 derive a screening chronic p-RfD, a UFc of 3,000 has been applied to the surrogate PODhed. The screening chronic p-RfD for 1,3-dibromobenzene is derived as follows: Screening Chronic p-RfD = Surrogate PODhed ^ UFc = 1.2 mg/kg-day ^ 3,000 = 4 x 10"4 mg/kg-day Table A. 5 summarizes the uncertainty factors for the screening chronic p-RfD for 1,3 -dibromobenzene. Table A.5. Uncertainty Factors for the Screening Chronic p-RfD for 1,3-Dibromobenzene UF Value Justification UFa 3 A UFa of 3 (10°5) has been applied to account for uncertainty in characterizing the toxicodynamic differences between rats and humans following oral 1,3-dibromobenzene exposure. The toxicokinetic uncertainty has been accounted for by calculation of a HED through application of a DAF as outlined in the EPA's Recommended Use of Body Weight3/4 as the Default Method in Derivation of the Oral Reference Dose (U.S. EPA. 201 lb). UFd 10 A UFd of 10 has been applied because there are no acceptable two-generation reproductive toxicity or developmental toxicity studies via the oral route. UFh 10 A UFh of 10 has been applied for inter-individual variability to account for human-to- human variability in susceptibility in the absence of quantitative information to assess the toxicokinetics and toxicodynamics of 1,3-dibromobenzene in humans. UFl 1 A UFl of 1 has been applied for LOAEL-to-NOAEL extrapolation because the POD is a NOAEL. UFS 10 A UFS of 10 has been applied to account for the extrapolation from less than chronic exposure because no chronic-duration toxicity studies are available to evaluate chronic systemic toxicity. UFC 3,000 Composite UF. 27 1,3-Dibromobenzene ------- FINAL 7-10-2014 APPENDIX B. DATA TABLES No data tables are presented. 28 1,3-Dibromobenzene ------- FINAL 7-10-2014 APPENDIX C. BENCHMARK DOSE MODELING RESULTS There are no BMD modeling outputs. 29 1,3-Dibromobenzene ------- FINAL 7-10-2014 APPENDIX D. REFERENCES ACGIH (American Conference of Governmental Industrial Hygienists). (2013). 2013 TLVs and BEIs. Based on documentation of the threshold limit values for chemical substances and physical agents and biological exposure indices. Cincinnati, OH. AT SDR (Agency for Toxic Substances and Disease Registry). (2013). Minimal risk levels (MRLs) for hazardous substances. 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