United States Environmental Protection 1=1 m m Agency EPA/690/R-09/063F Final 9-03-2009 Provisional Peer-Reviewed Toxicity Values for 2,4,6-Tribromophenol (CASRN 118-79-6) Superfund Health Risk Technical Support Center National Center for Environmental Assessment Office of Research and Development U.S. Environmental Protection Agency Cincinnati, OH 45268 ------- COMMONLY USED ABBREVIATIONS BMD Benchmark Dose IRIS Integrated Risk Information System IUR inhalation unit risk LOAEL lowest-observed-adverse-effect level LOAELadj LOAEL adjusted to continuous exposure duration LOAELhec LOAEL adjusted for dosimetric differences across species to a human NOAEL no-ob served-adverse-effect level NOAELadj NOAEL adjusted to continuous exposure duration NOAELhec NOAEL adjusted for dosimetric differences across species to a human NOEL no-ob served-effect level OSF oral slope factor p-IUR provisional inhalation unit risk p-OSF provisional oral slope factor p-RfC provisional inhalation reference concentration p-RfD provisional oral reference dose RfC inhalation reference concentration RfD oral reference dose UF uncertainty factor UFa animal to human uncertainty factor UFC composite uncertainty factor UFd incomplete to complete database uncertainty factor UFh interhuman uncertainty factor UFl LOAEL to NOAEL uncertainty factor UFS subchronic to chronic uncertainty factor 1 ------- FINAL 9-3-2009 PROVISIONAL PEER-REVIEWED TOXICITY VALUES FOR 2,4,6 TRIBROMOPHENOL (CASRN 118-79-6) Background On December 5, 2003, the U.S. Environmental Protection Agency's (U.S. EPA) Office of Superfund Remediation and Technology Innovation (OSRTI) revised its hierarchy of human health toxicity values for Superfund risk assessments, establishing the following three tiers as the new hierarchy: 1) U.S. EPA's Integrated Risk Information System (IRIS). 2) Provisional Peer-Reviewed Toxicity Values (PPRTVs) used in U.S. EPA's Superfund Program. 3) Other (peer-reviewed) toxicity values, including ~ Minimal Risk Levels produced by the Agency for Toxic Substances and Disease Registry (ATSDR), ~ California Environmental Protection Agency (CalEPA) values, and ~ EPA Health Effects Assessment Summary Table (HEAST) values. A PPRTV is defined as a toxicity value derived for use in the Superfund Program when such a value is not available in U.S. EPA's IRIS. PPRTVs are developed according to a Standard Operating Procedure (SOP) and are derived after a review of the relevant scientific literature using the same methods, sources of data, and Agency guidance for value derivation generally used by the U.S. EPA IRIS Program. All provisional toxicity values receive internal review by two U.S. EPA scientists and external peer review by three independently selected scientific experts. PPRTVs differ from IRIS values in that PPRTVs do not receive the multiprogram consensus review provided for IRIS values. This is because IRIS values are generally intended to be used in all U.S. EPA programs, while PPRTVs are developed specifically for the Superfund Program. Because new information becomes available and scientific methods improve over time, PPRTVs are reviewed on a 5-year basis and updated into the active database. Once an IRIS value for a specific chemical becomes available for Agency review, the analogous PPRTV for that same chemical is retired. It should also be noted that some PPRTV documents conclude that a PPRTV cannot be derived based on inadequate data. Disclaimers Users of this document should first check to see if any IRIS values exist for the chemical of concern before proceeding to use a PPRTV. If no IRIS value is available, staff in the regional Superfund and Resource Conservation and Recovery Act (RCRA) program offices are advised to carefully review the information provided in this document to ensure that the PPRTVs used are appropriate for the types of exposures and circumstances at the Superfund site or RCRA facility in question. PPRTVs are periodically updated; therefore, users should ensure that the values contained in the PPRTV are current at the time of use. 1 ------- FINAL 9-3-2009 It is important to remember that a provisional value alone tells very little about the adverse effects of a chemical or the quality of evidence on which the value is based. Therefore, users are strongly encouraged to read the entire PPRTV document and understand the strengths and limitations of the derived provisional values. PPRTVs are developed by the U.S. EPA Office of Research and Development's National Center for Environmental Assessment, Superfund Health Risk Technical Support Center for OSRTI. Other U.S. EPA programs or external parties who may choose of their own initiative to use these PPRTVs are advised that Superfund resources will not generally be used to respond to challenges of PPRTVs used in a context outside of the Superfund Program. Questions Regarding PPRTVs Questions regarding the contents of the PPRTVs and their appropriate use (e.g., on chemicals not covered, or whether chemicals have pending IRIS toxicity values) 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), or OSRTI. INTRODUCTION No RfD, RfC, or carcinogenicity assessment for 2,4,6-tribromophenol is available on IRIS (U.S. EPA, 2009). There are no entries for 2,4,6-tribromophenol in the Chemical Assessments and Related Activities list (CARA; U.S. EPA, 1991a, 1994), the Health Effects Assessment Summary Tables (HEAST; U.S. EPA, 1997), or the Drinking Water Standards and Health Advisories lists (U.S. EPA, 2006). The Agency for Toxic Substances Disease Registry (ATSDR, 2009) has not published a Toxicological Profile for 2,4,6-tribromophenol. The World Health Organization (WHO, 2005) has published a Concise International Chemical Assessment Document (CICAD) for 2,4,6-tribromophenol and other simple brominated phenols, but found the existing toxicity data inadequate for derivation of either oral or inhalation criteria to protect human health. 2,4,6-Tribromophenol has been reviewed under the HPV Challenge Program, and robust summaries from sponsors (e.g., Great Lakes Chemical Corporation, 2002) are available online (U.S. EPA, 2008). Data summaries for 2,4,6-tribromophenol have been prepared under the Organization for Economic Cooperation and Development-Screening Information Data System (OECD-SIDS, 2006) program and are available online. The International Agency for Research on Cancer (IARC, 2009) has not reviewed 2,4,6-tribromophenol with respect to chronic toxicity or carcinogenicity in humans or animals. The chronic toxicity and carcinogenicity of 2,4,6-tribromophenol have not been assessed by the National Toxicology Program (NTP, 2005, 2009). The Occupational Safety and Health Administration (OSHA, 2009), National Institute for Occupational Safety and Health (NIOSH, 2005), and the American Conference of Governmental Industrial Hygienists (ACGIH, 2001, 2007) have not established occupational exposure limits for 2,4,6-tribromophenol. The California Environmental Protection Agency (CalEPA, 2002, 2005a, 2005b) has not derived a Recommended Exposure Limit (REL) or cancer potency factor for 2,4,6-tribromophenol. 2 ------- FINAL 9-3-2009 Except as noted, literature searches were conducted from 1960s through June 2007 for studies relevant to the derivation of provisional toxicity values for 2,4,6-tribromophenol. Databases searched include MEDLINE, TOXLINE (Special), BIOSIS (August 2000 through June 2007), TSCATS 1/TSCATS 2, CCRIS, DART/ETIC, GENETOX, HSDB, RTECS, and Current Contents (December through June 2007). A final search of the literature was conducted for the period from June, 2007 thru July, 2009. REVIEW OF PERTINENT DATA Human Studies No studies regarding the toxicity of 2,4,6-tribromophenol in humans were identified. Animal Studies Oral Exposure Subchronic Studies—Cij :CD Sprague-Dawley rats (12/sex/dose) were exposed orally to 2,4,6-tribromophenol (in corn oil) by gavage at daily doses of 0 (vehicle), 100, 300, or 1000 mg/kg-day in a combined repeat-dose and developmental/reproductive screening toxicity test (OECD Test Guideline 422) (Tanaka et al., 1999). Males were exposed for 14 days prior to mating (plus an additional 34 days; 48 days total) and females were exposed 14 days prior to mating through Day 3 of lactation (41-45 days total). Females that did not mate successfully were exposed for a total of 48 days. Only one female (from the control group) did not mate successfully. Animals were observed daily, and clinical signs of toxicity, body weight, food consumption, and water consumption were assessed for all animals throughout the study. Comprehensive hematological and serum chemistry variables were assessed at study termination for all males, but not for females. Absolute and relative organ weights were evaluated for brain, thymus, liver, spleen, kidneys, adrenals, and reproductive tissues (testes and epididymis for males; ovaries for females). Gross necropsies were conducted on all test animals. Comprehensive histological examination of the digestive system was conducted for all test groups. The thymus and urinary systems were evaluated histologically for all male test groups, but only for control and high-dose females. Histological evaluation of the heart, spleen, testes, ovaries, adrenals, and brain was performed only for control and high-dose animals of both sexes. The reproductive variables assessed in the study, as well as conclusions regarding the reproductive endpoints, are discussed in thq Reproductive/Developmental Studies section of this document. Table 1 shows the endpoints relevant to the assessment of systemic toxicity by Tanaka et al. (1999). No parental mortality was observed. In comparison with controls, body weight was statistically significantly decreased in the high-dose males beginning on Day 8 and at every weekly measurement interval through the last measurement on Day 43 of the study (the deficit from controls was approximately 10% throughout this period). Body weights were also decreased among high-dose females relative to controls starting with Day 7 of gestation and persisting through the last measurement on Day 4 of lactation, but the difference was not as pronounced as in the males (deficit of approximately 6% throughout this period). Necropsy body weights were significantly decreased in high-dose males (-12%; p < 0.0001, two-tailed /-test) and females (- 5%;p < 0 .001, two-tailed Mest) with respect to controls (see Table 1). Food consumption was reduced among high-dose animals of both sexes during the first week of the 3 ------- FINAL 9-3-2009 study, but not in subsequent weeks. A dose-related increase in the occurrence of excessive salivation 5-35 minutes after dosing was observed among males and females exposed to 300 and 1000 mg/kg-day (data not shown by the researchers). This reaction to dosing was more common at 1000 mg/kg-day than at 300 mg/kg-day and in males rather than females. No treatment-related adverse effects on hematological variables were observed in males (females were not assessed) (Tanaka et al., 1999). A statistically significant increase in prothrombin clotting time in high-dose males relative to controls (15.6 ±1.2 seconds versus 14.2 ± 0.7 seconds) was not considered toxicologically relevant by the researchers because of the small magnitude of change and because other indicators of potential adverse effects on clotting (APPT [activated partial thromboplastin time] and fibrinogen) were unaffected by treatment. Serum chemistry examination revealed statistically significant, dose-related increases in creatinine in males dosed at 300 and 1000 mg/kg-day (indicative of renal dysfunction), an increase in alkaline phosphatase (ALP) activity in males at 1000 mg/kg-day (possibly indicative of an effect on the liver), and a number of other changes of uncertain biological significance in high-dose males (small 10-15% increases in protein, albumin, albumin-to-globulin ratio [A/G], and chloride [CI], and decreases in total bilirubin and potassium [K]) potentially related to decreased body weight at this dose (Table 1). Creatinine was the only serum chemistry variable that differed significantly from controls at the 300-mg/kg-day dose group. Blood urea nitrogen (BUN) appeared to be elevated in the high-dose males, providing further support for a renal effect, but it was not statistically increased due to high variability in the treated group. Clinical chemistry was not evaluated in females. There were statistically increased relative and absolute organ weights with respect to controls for a number of organs in males and females only at the high dose (Table 1) (Tanaka et al., 1999). With the possible exception of the changes in liver weight (increased absolute and relative weights in both sexes) and thymus weight (decreased absolute weight in males), the observed changes (increased relative organ weights) were consistent with, and likely secondary to, the decrease in body weight in this dose group. Liver enlargement was visible upon gross necropsy of high-dose males. Treatment-related histopathologic changes were noted in thymus, kidney, and liver tissue. A statistically significant increase in the incidence of hepatocellular hypertrophy (severity graded as slight -1 on a scale of 1 to 3) was noted in high-dose males (12/12), but not females (0/11), with respect to controls (0/11). There was also a significant decrease in the incidence of fatty change in liver sections from high-dose males. There were statistically significant increases in the incidences of hyaline casts, tubular dilatation, papillary necrosis, and lymphocyte infiltration of kidneys in high-dose males (slight-to-moderate severity), but not females. Histological evaluation of thymus sections revealed slight atrophy in 3/12 high-dose males, but not in control, low-, or mid-dose males or in control or high-dose females. This finding was not statistically significant, but it appears to be biologically relevant given the statistically significant decrease in thymus weight observed in high-dose males. 4 ------- FINAL 9-3-2009 Table 1. Summary of Significant Findings Following Subchronic Oral (Gavage) Exposure of Rats to 2,4,6-Tribromophenola Endpointb Dose (mg/kg-day) 0 100 300 1000 Male Female Male Female Male Female Male Female No. examined0 12 11 12 12 12 12 12 11 Body weight (g)d 492 ± 34 332 ± 16 478 ±31 317 ±27 478 ± 36 333 ±22 422 ± 25e 307 ±15e Absolute Organ Weights Thymus (mg) 299 ±81 157 ±46 269 ± 52 134 ±48 269 ± 66 168 ±75 201± 57f 137 ±32 Liver (g) 13.99 ± 1.72 13.70 ±0.80 13.18 ± 1.31 13.48 ±2.07 14.20 ± 1.99 14.39 ± 1.76 16.23 ±2.32g 15.74 ± 1.28f Relative Organ Weights (g%) Brain 0.460 ± 0.042 0.613 ±0.034 0.465 ± 0.033 0.657 ± 0.067 0.473 ±0.041 0.602 ±0.038 0.522 ±0.032f 0.665 ± 0.025s Liver 2.834 ±0.218 4.138 ±0.287 2.751 ±0.152 4.230 ±0.396 2.964 ±0.285 4.312 ±0.393 3.837 ±0.447f 5.117 ±0.265f Kidney 0.678 ±0.054 0.649 ± 0.072 0.661 ±0.053 0.694 ± 0.078 0.679 ±0.083 0.666 ± 0.047 0.824 ±0.101f 0.772 ± 0.094f Adrenals 12.257 ± 1.299 23.171 ± 1.572 11.807 ± 1.277 25.991 ±3.418 13.494 ± 1.966 25.988 ±4.091 15.304 ± 1.697f 27.315 ±3.415f Testes 0.721 ±0.062 NA 0.733 ± 0.067 NA 0.729 ± 0.080 NA 0.794 ± 0.046s NA Histological Findings (observed/examined) Liver Fatty change 6/11 2/11 5/12 1/12 3/12 0/12 0/12f 0/11 Hepatocyte hypertrophy 0/11 0/11 0/12 0/12 0/12 0/12 12/12f 0/11 Kidney Hyaline casts'1 1/11 1/11 1/12 NE 0/12 NE 8/12f 0/11 Dilatation, tubules 0/11 0/11 0/12 NE 0/12 NE 7/12f 0/11 Mineralization 1/11 3/11 0/12 NE 0/12 NE 1/12 1/11 Papillary necrosis 0/11 0/11 0/12 NE 0/12 NE 5/12f 0/11 Lymphocyte infiltration 1/11 0/11 1/12 NE 0 NE 6/128 0/11 5 ------- FINAL 9-3-2009 Table 1. Summary of Significant Findings Following Subchronic Oral (Gavage) Exposure of Rats to 2,4,6-Tribromophenola Endpointb Dose (mg/kg-day) 0 100 300 1000 Male Female Male Female Male Female Male Female Thymus (atrophy,) 0/11 0/11 0/12 NE 0/12 NE 3/12 0/11 Blood Chemistry (12 males/dose examined) Protein (g/dL) 5.87 ±0.22 NE 5.84 ±0.14 NE 5.95 ±0.26 NE 6.45 ± 0.51f NE Albumin (g/dL) 3.36 ±0.13 NE 3.33 ±0.09 NE 3.39 ± 0.19 NE 3.88 ± 0.29f NE A/G 1.34 ± 0.06 NE 1.33 ±0.09 NE 1.33 ±0.10 NE 1.51 ± 0.08f NE Creatinine (mg/dL) 0.27 ±0.03 NE 0.30 ±0.04 NE 0.33 ± 0.07s NE 0.47 ± 0.26f NE BUN (mg/dL) 13.3 ± 1.4 NE 13.6 ±2.1 NE 13.2 ±2.3 NE 20.9 ± 11.4 NE Bilirubin (mg/dL) 0.05 ±0.01 NE 0.05 ±0.01 NE 0.04 ±0.01 NE 0.02 ± 0.01f NE ALP (U/L) 354 ±74 NE 440 ± 162 NE 342 ±102 NE 514 ±155s NE K (mmol/L) 4.46 ±0.29 NE 4.40 ±0.25 NE 4.38 ±0.30 NE 4.06 ± 0.25f NE CI (mmol/L) 106.6 ± 1.2 NE 107.6 ± 1.1 NE 107.8 ± 1.5 NE 119.0 ±3.6f NE 3Tanaka et al., 1999 bValues are mean ± SD; NA = Not Applicable; NE = Not Evaluated °The reason why <12 animals per dose were examined for some groups is not clear from the English translation of the study dValues taken from Table 3 of Tanaka et al., 1999; these values differ from body weights depicted in Figures 1 and 2 of Tanaka et al., 1999 "Significant difference from control group, p < 0.001, two-sided t-test performed for this review Significant difference from control group, p < 0.01 reported by Tanaka et al., 1999 Significant difference from control group, p < 0.05 reported by Tanaka et al., 1999 hComposition and specific location within the kidney were not addressed 6 ------- FINAL 9-3-2009 The increase in liver weight and hepatocellular hypertrophy, and the slightly elevated serum ALP levels observed in the high-dose males, are indicative of an adaptive response of the liver to 2,4,6-tribromophenol exposure, but do not suggest an adverse effect. In the thymus, slight atrophy in some individuals and decreased mean absolute organ weight suggest a potential chemical-related effect in high-dose males. The serum chemistry and microscopic evaluation of urinary tract tissues made in this study (Tanaka et al., 1999) suggest that 2,4,6-tribromophenol adversely affects the kidney in male rats. Significant incidences of several types of kidney lesions, including hyaline casts, tubular dilatation, lymphocyte infiltration, and papillary necrosis, were observed in the high-dose males only. The kidney observations appear congruent with the chemically induced globulin accumulation (CIGA alpha2u) nephropathy specific to male rats. However, chemicals known to produce alpha2u nephropathy typically produce minimal changes in clinical chemistry and have little-to-no effect on glomerular function (U.S. EPA, 1991b). A dose-related and statistically significant increase in serum creatinine was observed in males exposed to 300 (22%) and 1000 (74%) mg/kg-day, suggesting a treatment-related adverse effect on glomerular function, which, in turn, suggests that alpha2u nephropathy may not be responsible for the observed kidney damage. Unfortunately, no serum chemistries were assessed in females. Based on the dose-related increase in serum creatinine in males and clinical signs (salivation) in both sexes, this study identifies a LOAEL of 300 mg/kg-day for 2,4,6-tribromophenol. The NOAEL is 100 mg/kg-day. Chronic Studies—No chronic studies of 2,4,6-tribromophenol were identified. Reproductive/Developmental Studies—As discussed in the Subchronic Studies section, Tanaka et al. (1999) exposed groups of male and female rats by gavage to 2,4,6-tribromophenol (0, 100, 300, or 1000 mg/kg-day) in a combined repeated-dose/reproductive/developmental toxicity screening study (OECD Test Guideline 422). The reproductive variables assessed in the study included numbers of pairs copulated, numbers of pregnant females, copulation index, fertility index, mean days of estrous cycle, numbers of dams delivering live pups, mean duration of gestation, mean number of total corpora lutea, mean number of total implants, mean number of total pups born, mean number of total live pups born, mean sex ratio, mean stillbirths (cannibalism evaluated separately), gestation index, mean implantation index, mean liver birth index, and mean viability index (male and female) on Day 4. All of the reproductive variables were presented as totals per dose-group as well as means per litter per dose-group where appropriate. There were no treatment-related adverse effects on any endpoint except for neonatal growth (decreased body weight relative to controls; both sexes; Days 0 and 4 of lactation) and viability (Day 4 of lactation) in the 1000 mg/kg-day test group. Table 2 summarizes the results for the viability endpoints. Data for pup body weight were not reported. Based on these observations, the LOAEL for reproductive toxicity was 1000 mg/kg-day for decreased neonatal viability (both sexes) on Day 4 of lactation and decreased neonatal body weight on Days 0 and 4 of lactation. The NOAEL is 300 mg/kg-day. 7 ------- FINAL 9-3-2009 Table 2. Summary of Significant Reproductive/Developmental Endpoints Following Oral (Gavage) Exposure of Rats to 2,4,6-Tribromophenol Prior to Mating and Throughout Gestation and Early Lactationa Dose (mg/kg-day) 0 100 300 1000 No. Live Pups on Day 4 (per litter mean ± SD) Male 83 (7.3 ± 1.2) 87 (7.3 ± 2.8) 86 (7.2 ± 2.3) 42 (3.5 ± 2.4)b'° Female 72 (6.5 ± 1.9) 84 (7.0 ± 2.2) 80 (6.7 ± 1.2) 49(4.1 ±2.9)b'd Mean ± SD Day 4 Viability Index6 Male 92.6 ±8.6 88.6 ±23.7 97.4 ±6.3 53.3 ± 34.2° Female 97.6 ±5.4 92.7 ± 15.5 94.0 ±9.6 50.4 ± 35.1° "Tanaka et al., 1999 bNot clear whether the investigator's assessment of statistical significance applies to the total numbers, the means or both Significant difference from control group, p < 0,01 Significant difference from control group, p < 0.05 ePer litter: (Number of live pups on Day 4 number live pups born) x 100 A pilot teratology study was conducted in which 2,4,6-tribromophenol (purity not reported) in corn oil was administered by gavage to groups of five Charles River CD female rats at doses of 0, 10, 30, 100, 300, 1000, or 3000 mg/kg-day on Gestation Days (GD) 6-15 (International Research and Development Corporation, 1978a). The dams were observed for clinical signs, changes in body weight, and mortality and were sacrificed on GD 20 for uterine observations. Numbers of viable and nonviable fetuses, early and late resorptions, total implantations, and corpora lutea were recorded, but the fetuses were not examined for skeletal or visceral malformations. All rats in the 3000-mg/kg-day group died by GD 7. Significant effects at the 1000-mg/kg-day dose included a 12.7% decrease, relative to controls, in maternal weight gain between GD 6 and 12 and a 16% decrease in the number of live fetuses (13.7, 12.0, 13.6, 13.0, 13.6, and 11.5% at 0, 10, 30, 100, 300, and 1000 mg/kg-day, respectively; standard deviations [SDs] not reported). Postimplantation loss was increased 500% at 1000 mg/kg-day, but this effect was not clearly dose-related (increased 433, 100, 100, and 33% at 10, 30, 100, and 300 mg/kg-day, respectively). The LOAEL for maternal and fetal toxicity in this study is 1000 mg/kg-day for decreased maternal weight gain and decreased fetal viability. The NOAEL is 300 mg/kg-day. Inhalation Exposure Subchronic Studies—Groups of five male and five female Charles River COBS rats were exposed via whole-body inhalation to 2,4,6-tribromophenol (purity 99.5 to 99.1%) dust at nominal concentrations of 0, 0.1, or 1 mg/L (mean analytical concentrations of 0, 0.10, or 0.92 mg/L (0, 100, or 920 mg/m3); mean gravimetric concentrations of 0, 0.15, or 0.98 mg/L) for 2 or 6 hours/day, 5 days/week, for 3 weeks (Industrial Biotest Laboratories, Inc., 1977). Particle sizes ranged from 1-74 microns, with 78% of the particles <10 microns and 65% = 1-5 microns. A mass median aerodynamic diameter (MMAD) was not reported and one cannot be estimated from the data presented. Clinical signs, body weight, mortality, hematology (three/sex/group), clinical chemistry (three/sex/group), urinalysis endpoints (three/sex/group), and gross pathology 8 ------- FINAL 9-3-2009 were evaluated in all groups. Animals were observed daily, body weights were measured weekly, and clinical chemistry, urinalysis, and hematological variables were assessed on Study Day 0 and at study termination. Histopathology was assessed in surviving animals in the control and high-concentration groups. No statistical analyses were reported by the researchers. Deaths occurred in the high-dose group (1/5 males and 1/5 females) after 10-11 exposures (Industrial Biotest Laboratories, Inc., 1977). No control or low-dose animals died. Clinical signs of toxicity (primarily hypoactivity, salivation, lacrimation, and red nasal discharge) were observed at both exposure concentrations. Hypoactivity and salivation were observed in all high- and low-dose animals on every exposure day of the study. Mean terminal body weights were markedly reduced in high-dose males (-30%) and females (-25%), in comparison to controls; the animals in these groups actually lost weight during the course of the study (the loss in weight occurred during the third week of the study). In the low-dose group, terminal body weights did not differ significantly from controls, but body weight gain over the course of the study was marginally reduced in females only (p = 0.045, two-tailed t-test performed for this review). Hematology and urinalysis findings were unremarkable. Although the researchers suggested that serum chemistry findings were likewise normal, there were statistically significant (p < 0.05, two-tailed t-tests performed for this review) 1.5 to 3-fold increases in BUN (males and females) and alanine aminotransferase (ALT) (males) in the high-dose group; small group sizes (n = 2 or 3) limit the reliability of these data, however. At gross necropsy, 4/5 males and 5/5 females in the high-dose group were visibly emaciated. Pathologic changes were observed in the liver and kidneys of high-dose animals. Histologic alterations included dilatation of renal tubules in 3/5 rats of each sex and a solitary area of submassive hepatic necrosis in one female. Proteinaceous casts (unilateral) were observed in the renal tubules of 2/5 high-dose males, but in none of the control males and in none of the females (either control or high-dose). There were no treatment-related effects on the lungs or trachea of high-dose rats in comparison with controls. The LOAEL for this study is 100 mg/m3 (lowest dose tested) based on clinical signs of toxicity (especially hypoactivity and salivation) in males and females and marginally decreased body weight gain in females. A NOAEL was not identified. Chronic Studies—No chronic inhalation studies were identified. Reproductive/Developmental Studies—Pregnant Wistar rats (25/dose) were exposed via whole-body inhalation to research-grade 2,4,6-tribromophenol (no further characterization) at -3 nominal concentrations of 0, 0.03, 0.1, 0.3, or 1.0 mg/m , 24 hr/day, 7 days/wk, on Days 1-21 of gestation (Lyubimov et al., 1998). No details regarding the methods for generating the test atmosphere, particle sizes, or measurement of test concentrations were reported. CNS effects were monitored in pregnant dams (Day 21 of gestation) and pups (postnatal days [PND] 30 and 60 by assessing skin pain threshold (SPT) and behavior in an open field (mobility, orientation, horizontal and vertical movement, etc.), but these methods were not described in detail. Maternal body weight, rectal temperature, lipid peroxidation (liver and placenta), total amino nitrogen in the urine and blood, phenol excretion (urine), and serum hormone and enzyme levels were recorded. Nonspecific immunological status of the dams was assessed by evaluating phagocytosis and antimicrobial activity (test not specified) of the blood. On Day 21 of pregnancy, 15 dams per dose group were sacrificed and examined for corpora lutea, numbers of implants, resorptions, and live and dead fetuses. Half of the fetuses were examined for skeletal variations and half were examined for visceral malformations. Groups of 10 dams per dose were 9 ------- FINAL 9-3-2009 allowed to deliver pups, which were then observed for two months and evaluated in the aforementioned SPT and behavioral tests. These pups were sacrificed on PND 60 and relative organ weights were determined for heart, liver, kidneys, spleen, adrenals, and reproductive organs (ovaries and testes). No mortality and no effects on maternal body-weight gain were reported (data not shown by researchers) (Lyubimov et al., 1998). Dams exposed to the highest concentration had significant decreases in mean orientation reactions (i.e., vertical head movements), relative to controls (3.8 ± 0.59 versus 11.11 ± 2.73 in controls), but no other behavioral changes. Significant increases in serum ALP (65.0 versus 29.0 mEQ in controls, no measure of error reported), serum progesterone (93.5 ± 7.3 versus 61.3 ± 7.1 mg/L in controls), urinary total amino nitrogen (-22% higher than controls), and total excretion of phenols (-23% higher than controls) were also observed in dams exposed at the highest concentration. Data were not shown for the aforementioned variables at the other exposure concentrations. There were no treatment-related effects on immune function, serum corticosterone levels, or serum estradiol concentrations (data not shown). Statistically significant dose-related increases in embryolethality (combined pre- and postimplantation loss) were found at test concentrations of "3 0.1 mg/m and above (approximately 7, 8, 18, 22, and 36% embryolethality for the control, 0.03, 0.1, 0.3, and 1.0 mg/m3 groups, respectively; data presented graphically in the original report). "3 Fetal weight was significantly decreased at concentrations of 0.1 and 1 mg/m , but not at 0.3 mg/m . Delayed sternal ossification was reported in exposed groups; this manifest as a significant decrease in the number of centers for sternal ossification (6.5 ± not reported, 3.85 ± 0.42, 4.98 ± 0.27, and 4.95 ± 0.27 in control, 0.1, 0.3, and 1.0 mg/m3 groups; data for "3 0.03 mg/m were not reported). No other meaningful treatment-related findings regarding skeletal or visceral malformations or other variables that were assessed are apparent from the data presented in the report. A somewhat detailed discussion of postnatal development and neurobehavioral effects is presented in the report (Lyubimov et al., 1998), but the meaning of these findings is unclear. The reported behavioral changes that had statistical significance (grooming behavior and" emotionality") were not dose-related, and, in the case of'emotionality," which is defined by the investigators as a measure of the number of defecations made during the observation period, the mean and SDs were extremely small. Changes in relative organ weights are discussed for 2-month-old neonates, but no data are presented. With the possible exception of reduced relative testes weight in high-dose males, the reported changes do not appear to be dose-related. In addition to the absence of data to discern the magnitude of the reported changes, the lack of absolute organ weights and the lack of organ histopathology preclude the assignment of meaning to any of the reported findings with respect to organ weights. Deficiencies in reporting of the study (Lyubimov et al., 1998) limit the utility of these data for toxicity assessment. Using the data reported, the LOAEL for maternal toxicity in this "3 study is 1.0 mg/m on the basis of increases in serum ALP and progesterone and urinary amino nitrogen, and the NOAEL is 0.3 mg/m3. For fetotoxicity, the study appears to identify a LOAEL 3 3 of 0.1 mg/m for embryolethality and delayed sternal ossification, with a NOAEL of 0.03 mg/m . These findings suggest that the developing fetus may be a sensitive target for 2,4,6-tribromophenol. However, the reliability of these data is uncertain due to inadequate reporting of study methods and results. 10 ------- FINAL 9-3-2009 Other Studies Toxicokinetics Absorption, distribution, and elimination were evaluated in 11 rats (strain not reported) that were treated with a single dose of 14C-2,4,6-tribromophenol (purity 99.98%) in aqueous ethanol by gavage (Velsicol Chemical Corporation, 1977). Groups of 2-3 females and 2 males were given 4.0-5.3 mg/kg and sacrificed 8, 17, 48, and 96 hours after treatment, respectively, for measurement of radiocarbon in tissues. Blood radiocarbon was measured in four rats at 1, 2, 4, 8, 17, 24, and 48 hours after treatment. 2,4,6-Tribromophenol appears to have been rapidly absorbed and readily excreted. Radiocarbon in the blood peaked 1 hour after dosing and then decreased log-linearly to negligible levels within 24 hours. Blood level kinetics are apparently first order and the elimination rate constant (ke) and half-life are calculated to be 0.3 and 2.03 hours, respectively. In tissues, the levels of radiocarbon reached maximum values 8 hours following dosing, with the highest levels occurring in the blood, muscle, fat, kidneys, liver, and lungs. The only tissues containing detectable residues after 48 hours were kidneys, liver, and lungs. Tissues retained approximately 4.9% and 0.01% of the administered radiocarbon at 8 and 48 hours following treatment, respectively. Most of the administered radioactivity was eliminated via urine (-70-90%) and feces (-4-6%) within 48 hours. Elimination of radioactivity in the urine was proportional to the decreasing concentrations in the blood. Accumulation of 2,4,6-tribromophenol (purity 99.3%) in adipose tissue was evaluated in groups of 8 male Charles River rats (3 control and 5 test rats/group) by gas chromatography (Industrial Biotest Laboratories, Inc., 1975). Rats were fed a diet containing 1000 ppm of 2,4,6-tribromophenol and then sacrificed after 7 days of exposure followed by 0, 7, or 14 days of recovery or 21 days of exposure followed by 0, 14, or 42 days of recovery. Compared to control values ranging from not detectable (<0.01 ppm) to 0.016 ppm, the fat tissue analysis showed, increased 2,4,6-tribromophenol concentration at the end of the 7- and 14-day exposure periods (0.56 and 0.30 ppm, respectively), as well as an increase (0.06 ppm) in 2/5 animals given 7 days recovery after 7 days of treatment. None of the animals given 14-day or longer recovery periods had detectable residue. No treatment-related changes in food consumption or body weight were observed during the study. Acute/Short-term Toxicity Cij:CD Sprague-Dawley rats (5/sex/dose) were given a single oral dose of 2,4,6-tribromophenol (99.8% purity by weight) in corn oil by gavage at doses of 1000, 1300, 1690, 2197, or 2856 mg/kg and observed for 14 days (Tanaka et al., 1999). Hypoactivity was observed at all doses and excessive salivation was observed in most treated animals. Mortality, convulsions, tremors, and prone or lateral body position were observed in both sexes at doses >1300 mg/kg. All deaths occurred within 1 day of exposure. The combined LD50 for both sexes is 1486 mg/kg. No macroscopic abnormalities were observed at necropsy. Spartan rats (5/sex/dose) were treated by gavage with a single dose of 2,4,6-tribromophenol (suspended in 0.5% Methocel) at 1585, 2512, 3980, 6308, 10,000, or 15,848 mg/kg and were observed for 14 days (International Research and Development Corporation, 1974a). LD50 values of 5012 mg/kg for males and 5012 mg/kg for females were calculated based on zero, one (male), one (female), nine (five males, four females), nine (four males, five females) and nine (four males, five females) deaths in the low- to high-dose groups. Clinical signs of toxicity, including nasal and ocular discharge (clear and porphyrin-containing), lacrimation, decreased motor activity, tachypnea, and/or tachycardia, were observed in all dose 11 ------- FINAL 9-3-2009 groups. Ataxia, tremors, flaccidity, prostration, and/or cyanosis occurred at >6308 mg/kg. Gross necropsy revealed a dose-related increase in congestion with some hemorrhage in lung, stomach, and liver. Charles River CD rats (5/sex/dose) were given a single oral dose (by gavage) of 631, 1000, 1585, 2512, 3980, or 6308 mg/kg and were observed for 14 days (International Research and Development Corporation, 1978b). One female at 1585 mg/kg and all animals in the higher-dose groups died. LD50 values were estimated to be 1995 mg/kg for males and 1819 mg/kg for females. No information on clinical signs of toxicity or pathology was reported. Mortality was observed in guinea pigs given a single oral dose of 2,4,6-tribromophenol at 3000 mg/kg, but no mortality was observed following similar exposure to 1000 mg/kg (Dow Chemical Company, 1946). No additional information (e.g., numbers tested and deceased, observation period, animal sex, and strain) was reported. "3 Spartan rats (5/sex) that were exposed by inhalation to 50 mg/L (50,000 mg/m ) of 2,4,6-tribromophenol for 4 hours and observed for 14 days showed clinical signs that included decreased motor activity, slight dyspnea, erythema, ocular porphyrin discharge, and clear nasal discharge (International Research and Development Corporation, 1974b). Clinical signs of toxicity observed in Charles River rats (5/sex) during inhalation exposure to 1.63 mg/L "3 (1630 mg/m ) for 4 hours included ptosis (drooping eyelids) and red nasal discharge; the nasal discharge continued for 8-18 hours following exposure (Industrial Biotest Laboratories, Inc., 1977). Genotoxicity 2,4,6-Tribromophenol was not mutagenic in assays conducted by three different laboratories with Salmonella typhimurium, Escherichia coli, or Saccharomyces cerevisiae. 2,4,6-Tribromophenol dissolved in dimethyl sulfoxide (DMSO) is not mutagenic in preincubation assays with Salmonella typhimurium strains TA98, TA100, TA1535, and TA1537 and Escherichia coli strain WP2uvrA (Tanaka et al., 1999). Assays were conducted both with and without metabolic activation (rat liver S9) at six different concentrations up to 500 [j,g/plate in Salmonella strains TA98, 100, and 1535, and up to 1000 [j,g/plate in TA1537. Toxicity was observed at higher test concentrations, and the results at these concentrations are not reported. In E. coli, assays were conducted with six different concentrations up to 5000 [j,g/plate, and toxicity was observed only at 5000 [j,g/plate. All test results were negative. Positive and negative controls responded appropriately. 2,4,6-Tribromophenol is not mutagenic in preincubation assays with S. typhimurium strains TA98, TA100, TA1535, and TA1537 (Zeigler et al., 1987). These assays were conducted with and without Aroclor 1254-induced rat and hamster liver homogenate. 2,4,6-Tribromophenol was also not mutagenic in plate incorporation assays with S. typhimurium strains TA98, TA100, TA1535, TA1537, TA1538, and Saccharomyces cerevisiae strain D4 (Litton Bionetics, Inc., 1978). These assays were conducted with, and without, Aroclor 1254-induced rat liver homogenate. 2,4,6-Tribromophenol dissolved in DMSO induced chromosomal aberrations in Chinese hamster lung (CHL/IU) cells following short-term treatment (Tanaka et al., 1999). The lowest effective concentrations are 0.050 mg/L in the absence of metabolic activation and 0.10 mg/L in the presence of metabolic activation (phenobarbital and 5,6-benzoflavone-induced rat liver S9). 12 ------- FINAL 9-3-2009 These were the highest concentrations tested. No polyploidy was observed. 2,4,6-Tribromophenol did not induce chromosome fragmentation in Allium cepa root cells (Levan and Tjio, 1948). DERIVATION OF PROVISIONAL SUBCHRONIC AND CHRONIC ORAL RfD VALUES FOR 2,4,6-TRIBROMOPHENOL Subchronic p-RfD Derivation The toxicity database for 2,4,6-tribromophenol is limited to a combination repeated-dose reproductive/developmental screening toxicity study (Tanaka et al., 1999) and a pilot teratology study (International Research and Development Corporation, 1978a), both of which were conducted with rats. The repeated-dose study was conducted for 41-48 days. None of the available studies examined fetuses for malformations. There are no human studies. Table 3 shows the dose-response information from the available animal studies. 13 ------- FINAL 9-3-2009 Table 3. Summary of Oral Noncancer Dose-Response Information Species Sex Dose (mg/kg-day) Exposure Regimen NOAEL (mg/kg-day) LOAEL (mg/kg-day) Responses at the LOAEL Comments Reference Subchronic Exposure Rat M,F 0, 100, 300, or 1000 mg/kg-day Gavage, 14 days prior to mating through Day 3 of lactation 100 300 Increased serum creatinine (M), clinical signs (salivation) OECD Guideline Study for combined repeated-dose, reproductive, and developmental toxicity screening. Tanaka et al., 1999 Reproductive/Developmental Toxicity Rat M,F 0, 100, 300, or 1000 mg/kg-day Gavage, 14 days prior to mating through Day 3 of lactation 300 1000 Decreased neonatal viability (both sexes) on Day 4; decreased neonatal body weight (Days 0 and 4) OECD Guideline Study for combined repeated-dose, reproductive, and developmental toxicity screening; no exams for fetal malformations. Tanaka et al., 1999 Rat F 0, 10, 30, 100, 300, 1000, or 3000 mg/kg-day Gavage, Days 6-15 gestation 300 1000 Decrease in maternal weight gain between Gestation Days 6 and 12 and a 16% decrease in the mean number of live fetuses per litter Pilot study; no exams for fetal malformations; 100% mortality by GD 7 at 3000 mg/kg-day. International Research and Development Corporation, 1978a 14 ------- FINAL 9-3-2009 The observed treatment-related effects following the lowest repeated exposure in the animal database were increased serum creatinine and clinical signs of toxicity (excessive salivation) (Tanaka et al., 1999). These effects were observed at a concentration of 300 mg/kg-day. The next highest dose tested (1000 mg/kg-day) was associated with a larger increase in serum creatinine, greater occurrence of salivation, histopathologic evidence of kidney damage (males), increased absolute and relative liver weight, hepatocellular hypertrophy, increased serum ALP, other serum chemistry changes in males (increased protein, albumin, chloride, and decreased potassium), possible thymic atrophy in males (decreased thymus weight and histological evidence of atrophy), and decreased body weight in both sexes. The observed liver changes (increased liver weight, hepatocellular hypertrophy, and increased serum ALP levels) appear to represent an adaptive response to exposure, rather than toxicity. Despite the similarity in lesions and pattern of occurrence, the observed effects on the kidney in males may not be alpha2U-related nephropathy; increased serum creatinine is indicative of an effect on glomerular function that is not typical of alpha2U nephropathy. Unfortunately, no serum chemistries were assessed in females, making further characterization of potential effects on the kidney in females impossible. Data from the reproductive phase of the combined study and from the pilot teratology study are supportive of adverse effects at the 1000 mg/kg-day level of exposure. Decreases in maternal weight gain during exposure and in neonatal growth and viability were observed in both the screening reproductive and pilot teratology studies at 1000 mg/kg-day (Tanaka et al., 1999; International Research and Development Corporation, 1978a;). No treatment-related effects were observed at 300 mg/kg-day in either the reproductive phase of the combined study or in the pilot teratology study. These observations suggest that 300 mg/kg-day is a LOAEL for short-term oral exposure on the basis of increased serum creatinine in male rats. The dose-related statistically significant (p < 0.05) increase in creatinine in male rats (Tanaka et al., 1999) is the most sensitive treatment-related endpoint in the database. Although clinical signs (salivation) were observed at 300 mg/kg-day, the data are not presented. The serum creatinine data are used as the basis for benchmark dose (BMD) modeling. Table 4 summarizes the data set used in the BMD modeling. Appendix B presents the results of the BMD model. A linear model with modeled variance provided adequate fit to the data set and yields a BMDLisd of 92 mg/kg-day. Table 4. Data Set for Increased Serum Creatinine in Male Ratsa Dose (mg/kg-day) 0 100 300 1000 Mean 0.27 0.30 0.33 0.47 Standard Deviation 0.08 0.04 0.07 0.26 Number Animals 12 12 12 12 3Tanaka et al., 1999 15 ------- FINAL 9-3-2009 Applying a composite uncertainty factor (UF) of 1000 to the BMDLisd of 92 mg/kg-day yields a subchronic p-RfD for 2,4,6-tribromophenol as follows: Subchronic p-RfD = BMDLisd ^ Composite UF = 92 mg/kg-day 1000 = 0.092, rounded to 0.09 mg/kg-day or 9 x 10"2 mg/kg-day The Composite UF of 1000 is composed of the following: • A full UF of 10 is applied for interspecies extrapolation to account for potential pharmacokinetic and pharmacodynamic differences between rats and humans. • A full UF of 10 is applied for intraspecies differences in order to account for potentially susceptible individuals in the absence of information on the variability of response in humans. • A full UF of 10 is applied to account for database deficiencies. The toxicological database for oral exposure to 2,4,6-tribromophenol is composed solely of two studies conducted on only one species and lacks true subchronic, reproductive, and developmental toxicity studies. Neither of the existing studies examined fetuses for malformations. A UF for LOAEL-to-NOAEL extrapolation is not applied because BMD modeling is used to identify the POD; a UF for subchronic-to-chronic extrapolation is not applied because a subchronic study was available. Confidence in the principal study (Tanaka et al., 1999) is medium because the rats were exposed for only 41-48 days (approximately half of the typical 90-day duration of a subchronic toxicity study in rats), and females were not evaluated for clinical chemistry and hematology. Confidence in the database is low. Toxicity is investigated in only one species—rats—and the existing studies were designed as screening-level and, as such, employed small numbers of animals and less-than-complete analyses. Also, as discussed above, neither of the existing studies examined fetuses for malformations. Consequently, confidence in the subchronic p-RfD is low. Chronic p-RfD Derivation There are no chronic toxicity studies for 2,4,6-tribromophenol, and, as discussed in the previous section, there are only screening-level, repeated-dose, reproductive and developmental toxicity studies. It is a commonly accepted practice to use a subchronic p-RfD as the basis for a chronic RfD by applying an additional UF of 10 to account for the use of a subchronic study to approximate a chronic duration of exposure. However, given that the subchronic p-RfD for 2,4,6-tribromophenol derived in the previous section already incorporates an UF of 1000 for intraspecies variability, interspecies variability, and database uncertainties, employing an additional UF of 10 to account for less-than-chronic duration would yield a total UF of 10,000. Provisional reference values are usually not developed for studies that exceed a composite UF of 3000 because of the high level of uncertainty. Therefore, no chronic p-RfD is derived for 2,4,6-tribromophenol. However, Appendix A of this document contains a screening value that may be useful in certain instances. Please see the Appendix A for details. 16 ------- FINAL 9-3-2009 FEASIBILITY OF DERIVING PROVISIONAL SUBCHRONIC AND CHRONIC INHALATION p-RfC VALUES FOR 2,4,6-TRIBROMOPHENOL The relevant inhalation studies for 2,4,6-tribromophenol are restricted to two 21-day studies (one with gestational exposure); neither of which is suitable for quantitative risk assessment. Table 5 summarizes the data from these studies. The study by Industrial Biotest Laboratories, Inc. (1977) used only two test concentrations and does not establish a NOAEL. Clinical signs of toxicity, most notably hypoactivity and excessive salivation, were observed at both test concentrations in males and females. Body weight gain was marginally decreased in females at the low concentration. At the high concentration, body weights were markedly reduced in both sexes and most of the animals were visibly emaciated. Histopathologic examination and serum chemistry analyses suggested effects on the liver and kidneys in rats at the high concentration. Histological examination was not performed for the low-exposure animals. There are two further deficiencies in this study that preclude its use in quantitative human risk assessment. Only the lungs and trachea are examined microscopically, limiting the usefulness of the study in determining critical respiratory endpoints. In addition, no information on aerodynamic particle sizes is reported, making it impossible to determine the mass aerodynamic diameter variables necessary to extrapolate from a particulate animal exposure concentration to a human equivalent concentration (HEC). The developmental toxicity study by Lyubimov et al. (1998) has many deficiencies in reporting. The most important deficiency with respect to the quantification of dose is a complete lack of discussion about how the test atmosphere was generated and whether the nominally reported exposure concentrations were validated. If the reported nominal concentrations are correct, then the LOAELs for maternal toxicity (1 mg/m3) and embryolethality (0.1 mg/m3) observed in this study occur at concentrations 18 times and 180 times lower, respectively, than the duration-adjusted LOAEL for adult toxicity (18 mg/m3) reported for rats in the 21-day Industrial Biotest Laboratories, Inc. (1977) study. As in the previous study, no information on particle sizes is reported. Therefore, it is not possible to know—with confidence—the conditions and concentrations for animal exposure, and it is, therefore, not possible to extrapolate a HEC from the available information. Based on these deficiencies, the study by Lyubimov et al. (1998) is not useful for quantitative human health risk assessment. In conclusion, there are no suitable inhalation data from which to derive a subchronic or a chronic p-RfC for 2,4,6-tribromophenol. 17 ------- FINAL 9-3-2009 Table 5. Summary of Inhalation Noncancer Dose-Response Information Species Sex Exposure Concentration3 (mg/m3) Exposure NOAEL (mg/m3) LOAEL (mg/m3) Responses Comments Reference Short-term Exposure Rat M, F Mean analytical concentrations of dust at 0, 100, or 920 mg/m3 adjusted to 0, 18 or 164 mg/m3 (mg/m3 x 6/24 x 5/7) Whole- body, 6 hr/day, 5 days/wk, for 3 wk Not established 18 Clinical signs of toxicity (hypoactivity and excessive salivation) in males and females and marginally decreased body-weight gain in females No information on MMAD or data from which to generate MMAD were provided. Histopathologic examination of the respiratory tract included only lungs and trachea. Industrial Biotest Laboratories, Inc., 1977 Reproductive/Developmental Toxicity Rat F Nominal: 0, 0.03, 0.1, 0.3, and 1.0 mg/m3 Whole body continuous, Days 1-21 of gestation Maternal 0.3 Fetal 0.03 Maternal 1.0 Fetal 0.1 Maternal: Increased serum ALP. serum progesterone, urinary total amino nitrogen, and urinary excretion of total phenols Fetal: Embrvolethalitv (combined pre- and postimplantation loss), delayed sternal ossification No information on generation of the test atmosphere, measurement of test concentrations or particle size distribution was reported; many deficiencies in methodology and data reporting. Lyubimov et al., 1998 Concentrations cannot be adjusted to human equivalent concentrations (HEC) due to the lack of information on mean aerodynamic particle diameters for these studies 18 ------- FINAL 9-3-2009 PROVISIONAL CARCINOGENICITY ASSESSMENT FOR 2,4,6-TRIBROMOPHENOL Weight-of-Evidence Descriptor No data have been located on the carcinogenicity of 2,4,6-tribromophenol in humans or animals. The available toxicity studies were conducted for very short durations and, as such, are not useful for assessing potential carcinogenicity. Mutagenicity data in bacteria and yeast are negative—although positive results have been obtained for chromosomal aberrations in Chinese hamster lung cells. In accordance with Guidelines for Carcinogen Risk Assessment and Supplemental Guidance for Assessing Susceptibility from Early-Life Exposure to Carcinogens (U.S. EPA, 2005), there is"Inadequate Information to Assess [the] Carcinogenic Potential" of 2,4,6-tribromophenol in humans. REFERENCES ACGIH (American Conference of Governmental Industrial Hygienists). 2001. Documentation of the threshold limit values for chemical substances. 7th Edition. Cincinnati, OH. ACGIH (American Conference of Governmental Industrial Hygienists). 2007. Threshold limit values for chemical substances and physical agents and biological exposure indices. Cincinnati, OH. ATSDR (Agency for Toxic Substances and Disease Registry). 2009. Toxicological Profile Information Sheet. U.S. Department of Health and Human Services, Public Health Service. Online, http://www.atsdr.cdc.gov/toxprofiles/index.asp. CalEPA (California Environmental Protection Agency). 2002. Hot Spots Unit Risk and Cancer Potency Values. Online, http://www.oehha.ca.gov/air/hot spots/pdf/TSDlookup2002.pdf. CalEPA (California Environmental Protection Agency). 2005a. OEHHA/ARB Approved Chronic Reference Exposure Levels and Target Organs. Online. http ://www. arb. ca. gov/toxics/healthval/chronic.pdf. CalEPA (California Environmental Protection Agency). 2005b. Air Chronic Reference Exposure Levels Adopted by OEHHA as of February 2005. Dow Chemical Company. 1946. Preliminary tests on the toxicity of 2,4,6-tribromophenol. TSCA 8D Submission OTS0522208. Great Lakes Chemical Corporation. 2002. IUCLID Data Set for 2,4,6-Tribromophenol. Submitted to U.S. EPA under the HPV Challenge Program. Online. http://www.epa.gov/chemrtk/pubs/summaries/tribomop/cl4177rs.pdf. IARC (International Agency for Research on Cancer). 2009. Search IARC Monographs. Online. http://monographs.iarc.fr/ENG/Monographs/PDFs/index.php. 19 ------- FINAL 9-3-2009 Industrial Biotest Laboratories, Inc. 1975. Report to Michigan Chemical Corporation. Bioaccumulation study in fat tissue with 2,4,6-tribromophenol in albino rats. TSCA 8D Submission 0TS0523301. Industrial Biotest Laboratories, Inc. 1977. Report to Michigan Chemical Corporation. 21-Day subacute dust inhalation toxicity study with 2,4,6-tribromophenol in albino rats. TSCA 8D Submission OTS0523305; TSCA 8ECP Submission OTS0536719; TSCA 8E Submission OTS0200291. International Research and Development Corporation. 1974a. Acute oral toxicity (LD50) study in albino rats. TSCA 8D Submission OTS0523299. International Research and Development Corporation. 1974b. Acute inhalation toxicity in the albino rat. TSCA 8D Submission OTS0523297. International Research and Development Corporation. 1978a. Pilot teratogenicity study in rats. TSCA 8D Submission OTS0523308. International Research and Development Corporation. 1978b. Acute oral toxicity (LD50) study in rats. TSCA 8E Submission OTS0200382; TSCA 8D Submission OTS0523310. Levan, A. and J.H. Tjio. 1948. Induction of chromosome fragmentation by phenols. Hereditas. 34:453-484. Litton Bionetics, Inc. 1978. Mutagenicity evaluation of 2,4,6-tribromophenol Lot #3287 in the Ames Salmonella!microsome plate test. TSCA 8D Submission OTS0523309. Lyubinov, A.V., V.V. Babin and A.I. Kartashov. 1998. Developmental neurotoxicity and immunotoxicity of 2,4,6-tribromophenol in Wistar rats. Neurotoxicity. 19(2):303-312. NIOSH (National Institute for Occupational Safety and Health). 2005. NIOSH Pocket Guide to Chemical Hazards. Online, http://www.cdc.gov/niosh/npg/. NTP (National Toxicology Program). 2005. 11th Report on Carcinogens. Online. http://ntp.niehs.nih.gov/index.cfm?objectid=32BA9724-FlF6-975E-7FCE50709CB4C932. NTP (National Toxicology Program). 2009. Management Status Report. Online. http://ntp.niehs.nih.gov/?obi ectid=96A77AlC-123F-7908-7BA79AB04E206892. OECD-SIDS. 2006. IUCLID Data Set. 2,4,6-Tribromophenol. Revised May 2003, Updated January 2006, Printed February 2006. OSHA (Occupational Safety and Health Administration). 2009. OSHA Standard 1910.1000 Table Z-l. Part Z, Toxic and Hazardous Substances. Online. https://www.osha.gov/pls/oshaweb/owadisp.show docuinent?p table=STANDARDS&p id=999 2. Tanaka, R., R. Yamada, K. Oba, et al. 1999. Combined repeat dose and reproductive/developmental toxicity screening test of 2,4,6-tribromophenol by oral administration in rats (Japanese) Toxicity testing reports of environmental chemicals no. 7. Shizuoka, Japan: Ministry of Health & Welfare, Biosafety Research Centre. 20 ------- FINAL 9-3-2009 U.S. EPA. 1991a. Chemical Assessments and Related Activities (CARA). Office of Health and Environmental Assessment, Washington, DC. April. U.S. EPA. 1991b. Alpha2u-Globulin: Association with chemically-induced renal toxicity and neoplasia in the male rat. Risk Assessment Forum. Washington, D.C. EPA/625/3-91.019F. U.S. EPA. 1994. Chemical Assessments and Related Activities (CARA). Office of Health and Environmental Assessment, Washington, DC. December. U.S. EPA. 1997. Health Effects Assessment Summary Tables. FY-1997 Update. Prepared by the Office of Research and Development, National Center for Environmental Assessment, Cincinnati OH for the Office of Emergency and Remedial Response, Washington, DC. July. EPA/540/R-97/036. NTIS PB97-921199. U.S. EPA. 2000. Benchmark Dose Technical Guidance Document [external review draft], EPA/630/R-00/001. Available at http://www.epa.gov/raf/publications/benchmark-dose-doc- d raft, htm. U.S. EPA. 2005. Guidelines for Carcinogen Risk Assessment and Supplemental Guidance for Assessing Susceptibility from Early-Life Exposure to Carcinogens. Risk Assessment Forum, Washington, DC. EPA/630/P-03/001F. Online. http://www.epa.gov/raf/publications/pdfs/childrens supplement final.pdf. U.S. EPA. 2006. 2006 Edition of the Drinking Water Standards and Health Advisories. Office of Water, Washington, DC. EPA/822/R-06/013. Washington, DC. Online. http://water.epa.gov/drink/standards/hascience.cfm. U.S. EPA. 2009. Integrated Risk Information System (IRIS). Online. Office of Research and Development, National Center for Environmental Assessment, Washington, DC. Online. http ://www. epa. gov/iris/. U.S. EPA. 2008. High Production Volume (HPV) Challenge Robust Summaries for 2,4,6-Tribromophenol. Online. http ://www. epa. gov/chemrtk/pub s/summaries/tribomop/c 14177tc.htm. Velsicol Chemical Corporation. 1977. Pharmacokinetic study of 2,4,6-tribromophenol in rats. OTS0523314, 8D. WHO (World Health Organization). 2005. Concise International Chemical Assessment Document (CICAD) 66: 2,4,6-Tribromophenol and other simple brominated phenols. Published under the joint sponsorship of the United Nations Environment Programme, the International Labour Organization, and the World Health Organization, and produced within the framework of the Inter-Organization Programme for the Sound Management of Chemicals. Online. http://www.who.int/ipcs/publications/cicad/cicad 66 web version.pdf. Zeigler, E., B. Anderson, S. Haworth et al. 1987. Salmonella mutagenicity tests: III. Results from the testing of 255 chemicals. Environ. Mutagen. 9(Suppl 9): 1-110. 21 ------- FINAL 9-3-2009 APPENDIX A. DERIVATION OF A CHRONIC SCREENING RfD FOR 2,4,6-TRIBROMOPHEN OL For reasons noted in the main PPRTV document, it is inappropriate to derive a chronic p-RfD for 2,4,6-tribromophenol. However, 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 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. Tanaka et al. (1999) observed that 300 mg/kg-day is a LOAEL for short-term oral exposure on the basis of increased serum creatinine in male rats. The dose-related statistically significant (p < 0.05) increase in creatinine in male rats (Tanaka et al., 1999) is the most sensitive treatment-related endpoint in the database. The serum creatinine data are used as the basis for benchmark dose (BMD) modeling. Table 4 of the main text summarizes the data set used in the BMD modeling. Appendix B presents the results of the BMD model. A linear model with modeled variance provided adequate fit to the data set and yields a BMDLisd of 92 mg/kg-day. Applying a composite uncertainty factor (UF) of 10,000 to the BMDLisd of 92 mg/kg-day yields a chronic screening p-RfD for 2,4,6-tribromophenol as follows: Chronic Screening p-RfD = BMDLisd Composite UF = 92 mg/kg-day ^ 10,000 = 0.0092 mg/kg-day or 9 x 10 3 mg/kg-day The Composite UF of 10,000 is composed of the following: • A full UF of 10 is applied for interspecies extrapolation to account for potential pharmacokinetic and pharmacodynamic differences between rats and humans. • A UF of 10 is applied for intraspecies differences in order to account for potentially susceptible individuals in the absence of information on the variability of response in humans. • A UF of 10 is applied for extrapolation from sub chronic-to-chronic exposures in order to account for additional effects that may be observed with longer exposure periods. • A full UF of 10 is applied to account for database deficiencies. The toxicological database for oral exposure to 2,4,6-tribromophenol is composed solely of two studies conducted on only one species and lacks true subchronic, reproductive, and developmental toxicity studies. Neither of the existing studies examined fetuses for malformations. 22 ------- FINAL 9-3-2009 A UF for LOAEL-to-NOAEL extrapolation is not applied because BMD modeling was used to identify the POD. 23 ------- FINAL 9-3-2009 APPENDIX B. DETAILS OF BENCHMARK DOSE MODELING FOR CHRONIC SCREENING p-RfD The data have been analyzed using all available models for continuous data in the BMD dose software (BMDS) program (version 2.1) developed by the U.S. EPA (2000). Risk was calculated as extra risk. For the continuous data, the original data were modeled with all the continuous models available within the software with a default BMR of 1 SD. An adequate fit was judged based on the goodness of fit p-walue (p > 0.1), scaled residual at the range of BMR, and visual inspection of the model fit. In addition to the three criteria forjudging the adequate model fit, whether the variance needed to be modeled, and if so, how it was modeled, also determined final use of the model results. If a homogenous variance model was recommended based on statistics (Test 2) provided from the BMD model runs, the final BMD results would be estimated from a homogenous variance model. If the test for homogenous variance (Test 2) was negative (i.e. ,p< 0. 1), the model was run again while applying the power model integrated into the BMDS to account for nonhomogenous variance (known as nonhomogenous model). If the nonhomogenous variance model did not provide an adequate fit to the variance data (Test 3: p value <0.1), the data set would be considered unsuitable for BMD modeling. Among all the models providing adequate data fit (goodness of fit p-w alue > 0.1), the lowest BMDL will be selected if the BMDLs estimated from different models varies over a wide range (not quantified); otherwise, the BMDL from the model with the lowest AIC would be considered appropriate for the data set. Confidence bounds were automatically calculated by the BMDS using a maximum likelihood profile method. Results of Model Fitting for 2,4,6-Tribromophenol BMD modeling was conducted for the increased incidence of serum creatinine in male rats observed in the Tanaka et al. (1999) study. Table B-l shows the BMD modeling results for the data set. As shown in Table B-l, the linear model with constant variance fit the means but not the variance. Running the linear model with modeled variance provides adequate fit to both the means and the variance and, therefore, is chosen as the basis for BMD derivation. Figure B-l illustrates the best-fitting model. Complete model runs are appended. Table B-l. Model Predictions for Increased Serum Creatinine in Male Rats Model Variance /j-Value" Means /j-Value" AIC bmd1sd (mg/kg-day) BMDL1sd (mg/kg-day) All dose groups Linear (constant variance)13 <0.0001 0.977 -141.006 NA NA Linear (modeled variance)0 0.8892 0.8339 -205.141 143.115 92.1898 aValues <0.10 fail to meet conventional goodness-of-fit criteria bCoefficients restricted to be positive Coefficients restricted to be positive NA = not applicable; model does not fit the data adequately 24 ------- FINAL 9-3-2009 Linear Model with 0.95 Confidence Level Linear 0.65 0.6 0.55 0.5 0.45 0.4 0.35 0.3 0.25 BMDL BMP 200 0 400 600 800 1000 Dose 10:31 02/052008 Figure B-l. Fit of Linear Model with Nonhomogeneous (Modeled) Variance to Data on Increased Serum Creatinine in Male Rats from Tanaka et al., 1999 BMDs and BMDLs indicated are associated with a change of 1 SD from the control and are in units of mg/kg-day. BMDL computation failed for one or more points on the curve, therefore, the BMDL curve is not plotted. 25 ------- FINAL 9-3-2009 BMD Model Runs for Tanaka et al. 1999 Serum Creatinine in Male SD Rats Polynomial Model. (Version: 2.12; Date: 02/20/2007) Input Data File: C:\BMDS\TANAKA99.(d) Gnuplot Plotting File: C:\BMDS\TANAKA99.plt Tue Feb 05 10:28:53 2008 BMDS MODEL RUN The form of the response function is: Y[dose] = beta_0 + beta_l-dose + beta_2-doseA2 + Dependent variable = MEAN Independent variable = dose rho is set to 0 The polynomial coefficients are restricted to be positive A constant variance model is fit Total number of dose groups = 4 Total number of records with missing values = 0 Maximum number of iterations =250 Relative Function Convergence has been set to: le-008 Parameter Convergence has been set to: le-008 Default Initial Parameter Values alpha = 0.01875 rho = 0 Specified beta_0 = 0.273934 beta_l = 0.000195902 Asymptotic Correlation Matrix of Parameter Estimates ( """ The model parameter(s) -rho have been estimated at a boundary point, or have been specified by the user, and do not appear in the correlation matrix ) alpha beta_0 beta_l alpha 1 -2.7e-011 3.8e-011 beta_0 -2.7e-011 1 -0.67 beta_l 3.8e-011 -0.67 1 Parameter Estimates Confidence Interval Vari able Upper Conf. Limit al pha 0.0240865 beta_0 0.323762 Estimate 0.0172037 0.273934 Std. Err. 0.00351169 0.0254227 95.0% Wald Lower Conf. Limit 0.0103209 0.224107 26 ------- FINAL 9-3-2009 beta_l 0.000290919 0.000195902 4.84792e-005 0.000100884 Table of Data and Estimated Values of Interest Dose Res. Obs Mean Est Mean Obs Std Dev Est Std Dev Scaled 0 100 300 1000 12 12 12 12 0.27 0.3 0.33 0.47 0.274 0.294 0.333 0.47 0.03 0.04 0.07 0.26 0.131 0.131 0.131 0.131 -0.104 0.171 -0.0714 0.00433 Model Descriptions for likelihoods calculated Model Al: Model A2: Yij = Mu(i) + e(ij) Var{e(ij)} = SigmaA2 Yij = Var{e(ij) } Mu(i) + e(ij) = Sigma(i)A2 Model A3: Yij = Mu(i) + e(ij) Var{e(ij)} = SigmaA2 Model A3 uses any fixed variance parameters that were specified by the user Model R: Yi = Mu + e(i) Var{e(i)} = SigmaA2 Likelihoods of Interest Model Al A2 A3 fi tted R Log(li keli hood) 73.525750 106.869482 73.525750 73.503155 66.475642 # Param' 5 8 5 3 2 AIC -137.051499 -197.738964 -137.051499 -141.006311 -128.951283 Explanation of Tests Test 1: Do responses and/or variances differ among Dose levels? (A2 vs . R) Test 2 Test 3 Test 4 Are Variances Homogeneous? (Al vs A2) Are variances adequately modeled? (A2 vs. A3) Does the Model for the Mean Fit? (A3 vs. fitted) (Note: When rho=0 the results of Test 3 and Test 2 will be the same.) Tests of Interest Test -2-log(Likelihood Ratio) Test df p-value Test 1 Test 2 Test 3 Test 4 80.7877 66.6875 66.6875 0.0451889 6 3 3 2 <.0001 <.0001 <.0001 0.9777 The p-value for Test 1 is less than .05. There appears to be a 27 ------- FINAL 9-3-2009 difference between response and/or variances among the dose levels It seems appropriate to model the data The p-value for Test 2 is less than .1. Consider running a non-homogeneous variance model The p-value for Test 3 is less than .1. You may want to consider a different variance model The p-value for Test 4 is greater than .1. The model chosen seems to adequately describe the data Benchmark Dose Computation Specified effect = 1 Risk Type = Estimated standard deviations from the control mean Confidence level = 0.95 BMD = 669.534 BMDL = 464.636 Linear Model with 0.95 Confidence Level 0.65 0.6 0.55 CD £ 0.5 o §" 0.45 cd | 0.4 0.35 0.3 0.25 0 200 400 600 800 1000 Dose 10:28 02/05 2008 l Linear BIVD Lower Bound BIVDL BIVD 28 ------- FINAL 9-3-2009 Polynomial Model. (Version: 2.12; Date: 02/20/2007) Input Data File: C:\BMDS\TANAKA99.(d) Gnuplot Plotting File: C:\BMDS\TANAKA99.plt Tue Feb 05 10:31:17 2008 BMDS MODEL RUN The form of the response function is: Y[dose] = beta_0 + beta_l-dose + beta_2-doseA2 + ... Dependent variable = MEAN Independent variable = dose The polynomial coefficients are restricted to be positive The variance is to be modeled as Var(i) = exp(lalpha + log(mean(i)) Total number of dose groups = 4 Total number of records with missing values = 0 Maximum number of iterations = 250 Relative Function Convergence has been set to: le-008 Parameter Convergence has been set to: le-008 - rho) Default Initial Parameter Values lalpha = -3.97656 rho = 0 beta_0 = 0.273934 beta_l = 0.000195902 Asymptotic Correlation Matrix of Parameter Estimates 1alpha rho beta_0 beta_l 1alpha 1 0.98 -0.0046 0.016 rho 0.98 1 -0.0034 0.014 beta_0 -0.0046 -0.0034 1 -0.49 beta_l 0.016 0.014 -0.49 1 Confidence Interval Vari able Upper Conf. Limit 1alpha 4.93273 rho 9.48954 beta_0 0.287028 beta_l 0.000303115 Estimate 2.85154 7.61417 0.272686 0.000206555 Parameter Estimates Std. Err. 1.06185 0.95684 0.00731748 4.92665e-005 95.0% Wald Lower Conf. Limit 0.770343 5.7388 0.258344 0.000109994 29 ------- FINAL 9-3-2009 Table of Data and Estimated Values of Interest Dose Res. Obs Mean Est Mean Obs Std Dev Est Std Dev Scaled 0 100 300 1000 12 12 12 12 0.27 0.3 0.33 0.47 0.273 0.293 0.335 0.479 0.03 0.04 0.07 0.26 0.0296 0.039 0.0645 0.253 -0.315 0.591 -0.25 -0.127 Model Descriptions for likelihoods calculated Model Al: Model A2: Model A3: Yij = Var{e(ij) } Mu(i) + e(ij) = SigmaA2 Yij = Mu(i) + e(ij) Var{e(ij)} = Sigma(i)A2 Yij, Mu(i) + e(ij) Var{e(ij)} = exp(lalpha + rho-1n(Mu(i))) Model A3 uses any fixed variance parameters that were specified by the user Model R: Yi = Mu + e(i) Var{e(i)} = SigmaA2 Likelihoods of Interest Model Al A2 A3 fi tted R Log(li keli hood) 73.525750 106.869482 106.752006 106.570308 66.475642 # Param 5 8 6 4 2 AIC -137.051499 -197.738964 -201.504012 -205.140616 -128.951283 Explanation of Tests Test 1: Do responses and/or variances differ among Dose levels? (A2 vs . R) Test 2 Test 3 Test 4 Are Variances Homogeneous? (Al vs A2) Are variances adequately modeled? (A2 vs. A3) Does the Model for the Mean Fit? (A3 vs. fitted) (Note: When rho=0 the results of Test 3 and Test 2 will be the same.) Tests of Interest Test -2-log(Likelihood Ratio) Test df Test Test Test Test 1 2 3 4 80.7877 66.6875 0.234952 0.363396 6 3 2 2 p-value <.0001 <.0001 0.8892 0.8339 The p-value for Test 1 is less than .05. There appears to be a difference between response and/or variances among the dose levels It seems appropriate to model the data The p-value for Test 2 is less than .1. A non-homogeneous variance 30 ------- FINAL 9-3-2009 model appears to be appropriate The p-value for Test 3 is greater than .1. The modeled variance appears to be appropriate here The p-value for Test 4 is greater than .1. The model chosen seems to adequately describe the data Benchmark Dose Computation Specified effect = Risk Type Confidence level = Estimated standard deviations from the control mean 0.95 1 BMD 143.115 BMDL 92.1898 BMDL computation failed for one or more point on the BMDL curve. The BMDL curve will not be plotted 31 ------- |