Document 15DG3yEMjEyX7EVQNgR6QyaEX

1. DISPOSITION AND PHARMACOKINETICS The disposition and pharmacokinetics of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and related compounds have been investigated in several species and under various exposure conditions. Several reviews on this subject focus on TCDD and related halogenated aromatic hydrocarbons (Neal et al., 1982; Gasiewicz et al., 1983a; Olson et al., 1983; Birnbaum, 1985; van den Berg et al., 1994). The relative biological and toxicological potency of TCDD and related compounds depends not only on the affinity of these compounds for the aryl hydrocarbon receptor (AhR), but on the species-, strain-, and congener-specific pharmacokinetics of these compounds (Neal et al., 1982; Gasiewicz et al., 1983a; Olson et al., 1983; Birnbaum, 1985; van den Berg et al., 1994, DeVito and Birnbaum, 1995). 2,3,7,8-TCDD and other similar compounds discussed here are rapidly absorbed into the body and slowly eliminated, making body burden (bioaccumulation) a reliable indicator of timeintegrated exposure and absorbed dose. Because of the slow elimination kinetics, it will be shown in this section that lipid or blood concentrations, which are often measured, are in dynamic equilibrium with other tissue compartments in the body, making the overall body burden and tissue disposition relatively easy to estimate. Finally, it will be shown that body burdens can be correlated with adverse health effects (Hardell et al., 1995; Leonards et al., 1995), further leading to the choice of body burden as the optimal indicator of absorbed dose and potential effects. 1.1. ABSORPTTON/BTOAVATLABTLTTY FOLLOWING EXPOSURE Gastrointestinal, dermal, and transpulmonary absorptions represent potential routes for human exposure to this class of persistent environmental contaminants. Parenteral absorption is a route of exposure that has been used to generate disposition and pharmacokinetic data on these compounds. 1.1.1. Oral 1.1.1.1. Gastrointestinal Absorption in Animals Much of human exposure to TCDD and related compounds is thought to be through the diet. Experimentally, these compounds are commonly administered in the diet or by gavage in an oil vehicle. Gastrointestinal absorption is usually estimated as the difference between the administered dose (100%) and the percent of the dose that was not absorbed. The unabsorbed fraction is estimated as the recovery of parent compound in feces within 24 to 48 hours of a single oral exposure by gavage. Table 1-1 summarizes gastrointestinal absorption data on TCDD and related compounds. December 2003 1-1 DRAFT--DO NOT CITE OR QUOTE In Sprague-Dawley rats given a single oral dose of 1.0 pg [14C]-2,3,7,8-TCDD/kg bw in acetone:corn oil (1:25, v/v), the fraction absorbed ranged from 66% to 93%, with a mean of ~84% (Rose et al., 1976). With repeated oral dosing of rats at 0.1 or 1.0 pg/kg/day (5 days/week for 7 weeks), gastrointestinal absorption of TCDD was observed to be approximately that observed for a single oral exposure (Rose et al., 1976). Oral exposure of Sprague-Dawley rats to a larger dose of TCDD in acetone:corn oil (50 pg/kg) resulted in an average absorption of 70% of the administered dose (Piper et al., 1973). More recently, Diliberto et al. (1996a) reported 88% absorption of TCDD in male Fischer 344 rats following oral exposure in Emulphor/95% ethanol/water (1:1:3). (Emulphor EL-620 is a polyoxyethylated vegetable oil preparation [GAF Corp., New York, NY]). One study in the guinea pig reported that ~50% of a single oral dose of TCDD in acetone:corn oil was absorbed (Nolan et al., 1979). The gastrointestinal absorption of TCDD was also examined in the hamster, the species most resistant to the acute toxicity of this compound (Olson et al., 1980). Hamsters were given a single, sublethal, oral dose of [1,6-3H]2,3,7,8-TCDD in olive oil (650 pg/kg), and an average of 75% of the dose was absorbed. When TCDD was administered to rats in the diet at 7 or 20 ppb (0.5 or 1.4 pg/kg/day) for 42 days, 50% to 60% of the consumed dose was absorbed (Fries and Marrow, 1975). These findings indicate that oral exposure to TCDD in the diet or in an oil vehicle results in the absorption of >50% of the administered dose. The intestinal absorption of [3H]-2,3,7,8-TCDD has also been investigated in thoracic duct-cannulated rats (Lakshmanan et al., 1986). The investigators concluded that TCDD was absorbed into chylomicrons and transported through the lymphatic system before entering the systemic circulation. The absorption of 2,3,7,8-TBDD in male Fischer 344 rats was studied after oral exposure by gavage at 5 pg/kg in Emulphor:ethanol:water (1:1:3) (Diliberto et al., 1990). The percent of the dose absorbed for this study was defined as 100% (% total oral dose in feces on days 1 and 2 minus % total intravenous dose in feces on days 1 and 2) using the intravenous pharmacokinetic data of Kedderis et al. (1991a). The relative absorbed dose or bioavailability of 2,3,7,8-TBDD after oral exposure was estimated at 78%, 82%, 60%, and 47% at dose levels of 0.001, 0.01, 0.1, and 0.5 pmol/kg, respectively. These results suggest nonlinear absorption at the higher doses, with maximal oral absorption at an exposure of #0.01 pmol/kg (5 pg/kg). The absorption of 2,3,7,8-TCDF has been investigated after oral exposure by gavage. Approximately 90% of the administered dose (0.1 and 1.0 pmol/kg) of 2,3,7,8-TCDF in Emulphor:ethanol (1:1) was absorbed in male Fischer 344 rats (Birnbaum et al., 1980). Similarly, >90% of the administered dose (0.2 pmol/kg, 6 pg/kg, and 1-15 pg/kg) of 2,3,7,8- December 2003 1-2 DRAFT--DO NOT CITE OR QUOTE TCDF in Emulphor:ethanol:water (1:1:8) was absorbed in male Hartley guinea pigs (Decad et al., 1981a; Ioannou et al., 1983). Thus, 2,3,7,8-TCDF appears to be almost completely absorbed from the gastrointestinal tract. This absorption may be related to the greater relative solubility of 2,3,7,8-TCDF compared with that of 2,3,7,8-TCDD or 2,3,7,8-TBDD. The oral bioavailability of 2,3,4,7,8-PeCDF and 3,3',4,4'-TCB in corn oil was similar to that of 2,3,7,8-TCDD (Brewster and Birnbaum, 1987; Wehler et al., 1989; Clarke et al., 1984). Furthermore, 2,3,4,7,8-PeCDF absorption was independent of the dose (0.1, 0.5, or 1.0 gmol/kg). Incomplete and variable absorption of 1,2,3,7,8-PeCDD was reported in rats, with 19% to 71% of the dose absorbed within the first 2 days after oral exposure (Wacker et al., 1986). Early studies on the pharmacokinetic behavior of OCDD by Williams et al. (1972) and Norback et al. (1975) demonstrated that OCDD was poorly absorbed after oral exposure. More recently, Birnbaum and Couture (1988) also found that the gastrointestinal absorption of OCDD in rats was very limited, ranging from 2% to 15% of the administered dose. Lower doses (50 :g/kg) in an o-dichlorobenzene:corn oil (1:1) vehicle were found to give the best oral bioavailability for this extremely insoluble compound. Recently Mes et al. (1995) have reported PCBs in nondosed infants of dosed rhesus monkeys. Transfer to the infants was from both intrauterine and lactational exposure. Also, the infants showed a larger percentage of heptachorobiphenyls than did the dosed dams. Busbee and Zipring (1994) reported the direct absorption across the gastric mucosa of Aroclor 1232 (DCB) in an ovine. The absorption was rapid and the circulating DCB was not found to be associated with plasma lipid fractions. The researchers report no occurrence in the thoracic duct lymph prior to appearance in the circulating plasma. Also, the DCB did bind with plasma lipids in vitro. These data suggest that this DCB is transported similar to water soluble compounds. 1.1.1.2. Gastrointestinal Absorption in Humans The above animal data indicate that gastrointestinal absorption of TCDD and related compounds is variable, incomplete, and congener- and vehicle-specific. More soluble congeners, such as 2,3,7,8-TCDF, are almost completely absorbed, whereas the extremely insoluble OCDD is poorly absorbed. In some cases, absorption has been found to be dose dependent, with increased absorption occurring at lower doses (2,3,7,8-TBDD, OCDD). The limited database in experimental animals also suggests that there are no major interspecies differences in the gastrointestinal absorption of these compounds. Poiger and Schlatter (1986) investigated the absorption of TCDD in a 42-year-old man after ingestion of 105 ng [3H]-2,3,7,8-TCDD (1.14 ng/kg bw) in 6 mL corn oil and found that >87% of the oral dose was absorbed from the gastrointestinal tract. Following absorption, the half-life for elimination was estimated to be 2,120 days. December 2003 1-3 DRAFT--DO NOT CITE OR QUOTE Schlummer et al. (1998) used a mass balance approach to assess the gastrointestinal absorption of CDDs, CDFs, PCBs, and hexachlorobenzene (HCB) from food in seven individuals, 24 to 81 years of age, with different contaminant body burdens. The difference between the ingested (food concentration) and excreted (fecal concentration) amounts of each compound was defined as net absorption. Three types of net absorption were observed in this study: (1) nearly complete net absorption (e.g. 2,3,7,8-TCDF and 1,2,3,7,8-PeCDF); (2) incomplete net absorption (e.g., TCDD and 1,2,3,7,8-PeCDD in the younger subjects); and (3) net excretion (excretion to a greater extent than ingestion, e.g., 1,2,3,6,7,8-HxCDD and OCDD). In terms of TEQs, the maximum net absorption of CDDs and CDFs was 63% in one individual, while a net excretion of TEQs was found for the three oldest subjects, which also have the highest serum concentration of these congeners. When PCB 126 (3,3'4,4',5), with a TEF of 0.1, is included in the TEF calculation, the TEQ balance was dominated by this congener, resulting in a maximum net TEQ absorption of 80% and a net TEQ absorption in all but the oldest subject. Table 1-1 illustrates that compounds showing nearly complete net absorption had very low or nondetectable levels in the serum lipids, and for other congeners, there was a trend for decreasing net absorption/increasing net excretion with increasing congener concentration in serum lipids. Together, the data support the passive diffusion model for gastrointestinal absorption, where the concentration of the contaminant in the blood is the major factor determining absorption. However, the relatively high absorption levels of many congeners could not be explained on the basis of diffusive gradients estimated from the difference between the lipid-based food and serum concentrations, as the lipid-based food levels were always lower, favoring net excretion. The authors propose a fat-flush theory, which hypothesizes that the fat compartment of the absorbing tissue (gut wall) expands because of the uptake of dietary fat, resulting in a decrease in the lipidbased concentration of the gut wall below that of the food, thus facilitating absorption. Therefore, as food passes through the duodenum and the jejunum, CDDs, CDFs and PCBs experience a diffusion gradient and net absorption as a result of the fat-flush. As the gut contents reach the colon, dietary fat has been absorbed, causing a reduction in the concentration gradient favoring absorption of CDDs, CDFs, and PCBs. Thus, the fat-flush theory supports the hypothesis that absorption and excretion of CDDs, CDFs, and PCBs are distinct processes occurring at different locations in the digestive tract. Duarte-Davidson and Jones (1994) report an average intake of a sum of several PCB congeners in the contemporary United Kingdom population of 0.53 micrograms/person/day. They estimate 97% of exposure can be accounted for by food consumption. This indicates that PCBs can likewise be absorbed through the gastrointestinal system. Because CDDs, CDFs, and PCBs are present in human milk, McLachlan (1993) investigated the net absorption of these compounds in a nursing infant. The contaminant input, December 2003 1-4 DRAFT--DO NOT CITE OR QUOTE through the ingestion of mother's milk, and the contaminant output in the feces were measured to estimate the digestive tract absorption of these compounds. For almost all congeners, more than 90% of the ingested compound was absorbed, indicating that the common assumption of 100% absorption of CDDs, CFFs, and PCBs in nursing infants is reasonable. Dahl et al. (1995) provide further evidence of this, as they report > 95% absorption in postpartum infants (1, 2, 3 months) in Sweden. Abraham et al. (1996) assessed the oral intake and fecal excretion of CDDs and CDFs in one formula-fed and two breast-fed infants at 1 and 5 months of age. The breast-fed infants had significantly more exposure to CDDs and CDFs, with >90% of the 2,3,7,8-TCDD, 2,3,4,7,8PeCDF, 1,2,3,7,8-PeCDD, and 1,2,3,6,7,8-HxCDD (>93% of TEQs) being absorbed from mother's milk. Less complete bioavailability of higher CDDs was observed, with 62% to 88% of 1,2,3,4,6,7,8-HepCDD and 16% to 75% of OCDD absorbed from mother's milk (Abraham et al., 1996). Furst et al. (1994), Hong et al. (1994), Schecter et al. (1994a,b), Georgii et al. (1994), and others provide further evidence of the presence of dioxins, PCPs, and PCBs in human milk. This important route for excretion and exposure is discussed later in the chapter. 1.1.1.3. Bioavailability Following Oral Exposure Oral exposure of humans to TCDD and related compounds usually occurs as a complex mixture of these contaminants in food, soil, dust, water, or other mixtures that would be expected to alter absorption. The influence of dose and vehicle or adsorbent on gastrointestinal absorption has been investigated in rats by Poiger and Schlatter (1980), using hepatic concentrations 24 hours after dosing as an indicator of the amount absorbed (Table 1-2). Administration of TCDD in an aqueous suspension of soil resulted in a decrease in the hepatic levels of TCDD as compared with hepatic levels resulting from administration of TCDD in 50% ethanol. Likewise, Diliberto et al. (1996a) report 24.4% (+ 1.4) in the liver after dosing male Fischer 344 rats with TCDD in a solution of 1:1:3 ratio of Emulphor/95% ethanol/distilled water. The extent of the decrease was directly proportional to the length of time the TCDD had been in contact with the soil. When TCDD was mixed in an aqueous suspension of activated carbon, absorption was almost totally eliminated (<0.07% of the dose in hepatic tissues). Philippi et al. (1981) and Huetter and Philippi (1982) have shown that radiolabeled TCDD becomes progressively more resistant with time to extraction from soil. Similarly, the feeding of fly ash, which contains CDDs, to rats in the diet for 19 days resulted in considerably lower hepatic levels of CDDs than did the feeding of an extract of the fly ash at comparable dietary concentrations of CDDs (van den Berg et al., 1987a). The CDDs were tentatively identified as 2,3,7,8-TCDD, 1,2,3,7,8-PeCDD, 1,2,3,6,7,8-HxCDD, and 1,2,3,7,8,9-HxCDD, and the difference in hepatic levels noted between fly ash-treated and extract-treated rats was greater December 2003 1-5 DRAFT--DO NOT CITE OR QUOTE for the more highly chlorinated isomers than for 2,3,7,8-TCDD. These results indicate the importance of the formulation or vehicle containing the toxin(s) on the relative bioavailability of 2,3,7,8-TCDD, PeCDD, and HxCDDs after oral exposure. Because TCDD in the environment is likely to be absorbed to soil, McConnell et al. (1984) and Lucier et al. (1986) compared the oral bioavailability of TCDD from environmentally contaminated soil with that from TCDD administered in corn oil in guinea pigs and rats, respectively. As indicated by biological effects and the amount of TCDD in the liver, the intestinal absorption from soil from Times Beach and Minker Stout, Missouri, was ~50% less than that from corn oil. Shu et al. (1988a) reported an oral bioavailability of ~43% in rats dosed with three environmentally contaminated soil samples from Times Beach, Missouri. This figure did not change significantly over a 500-fold dose range of 2-1,450 ng 2,3,7,8-TCDD/kg bw for soil contaminated with ~2, 30, or 600 ppb of TCDD TCDD. In studies of other soil types, Umbreit et al. (1986a,b) estimated an oral bioavailability in the rat of 0.5% for soil at a New Jersey manufacturing site and 21% for a Newark salvage yard. These results indicate that bioavailability of TCDD from soil varies between sites and that TCDD content alone may not be indicative of potential human hazard from contaminated environmental materials. Although these data indicate that substantial absorption may occur from contaminated soil, soil type and duration of contact, as suggested from the data that demonstrated decreased extraction efficiency with increasing contact time between soil and TCDD (Philippi et al., 1981; Huetter and Philippi, 1982), may substantially affect the absorption of TCDD from soils obtained from different contaminated sites. 1.1.2. Dermal Absorption Brewster et al. (1989) examined the dermal absorption of TCDD and three CDFs in male Fischer 344 rats (10 weeks old; 200-250 g). The fur was clipped from the intrascapular region of the back of each animal. A single compound in 60 pL of acetone was then applied over a 1.8 cm2area of skin, which then was covered with a perforated stainless steel cap. Table 1-3 summarizes data on the absorption of each compound at 3 days after a single dermal exposure. At an exposure of 0.1 pmol/kg, the absorption of 2,3,7,8-TCDF (49% of administered dose) was greater than that of 2,3,4,7,8-PeCDF, 1,2,3,7,8-PeCDF, and 2,3,7,8-TCDD. For each compound, the relative absorption (percentage of administered dose) decreased with increasing dose, while the absolute absorption (pg/kg) increased nonlinearly with dose. Results also suggest that the majority of the compound remaining at the skin exposure site was associated with the stratum corneum and did not penetrate through to the dermis. In a subsequent study, Banks and Birnbaum (1991a) examined the rate of absorption of TCDD over 120 hours after the dermal application of 200 pmol (1 nmol/kg) to male Fischer 344 rats. The absorption kinetics appeared December 2003 1-6 DRAFT--DO NOT CITE OR QUOTE to be first order, with an absorption rate constant of 0.005 hour-1. With a similar exposure protocol, the dermal absorption of 2,3,7,8-TCDF was found to follow a first-order process, with a rate constant of 0.009 hour-1(Banks and Birnbaum, 1991b). Together, these results on dermal absorption indicate that at lower doses (#0.1 pmol/kg), a greater percentage of this administered dose of TCDD and three CDFs was absorbed. Nonetheless, the rate of absorption of TCDD is still very slow (rate constant of 0.005 hour-1), even following a low-dose dermal application of 200 pmol (1 nmol/kg). Results from Table 1-3 also suggest that the dermal absorption of 2,3,7,8TCDF, 2,3,4,7,8-PeCDF, and 1,2,3,7,8-PeCDF occurs at a very slow rate. Using a similar exposure protocol, the dermal absorption of 2,3,7,8-TBDD was only 30% to 40% of that observed for TCDD (Diliberto et al., 1993a; 1996a). The dermal absorption of several polyhalogenated aromatic hydrocarbons in male F344 rats was also compared with estimates of their respective octanol-water partition coefficients (Jackson et al., 1993). Inverse correlations were found between octanol-water partition coefficient estimates and the single-dose (-1.0 nmol/kg and -0.1 pmol/kg) dermal absorption for most of the compounds studied. The differential dermal absorption of TCDD and 2,3,7,8-TBDD may result, in part, from the diminished ability of the more lipophilic 2,3,7,8-TBDD to partition out of the stratum corneum and into the underlying epidermal and dermal layers. Rahman et al. (1992) and Gallo et al. (1992) compared the in vitro permeation of TCDD through hairless mouse and human skin. In both species, the amount of TCDD permeated increased with the dose, but the percentage of the dose permeated decreased with increasing dose. The permeability coefficient of TCDD in human skin was about one order of magnitude lower than that in mouse skin. The hairless mouse skin does not appear to be a suitable model for the permeation of TCDD through human skin because the viable tissues were major barriers to TCDD permeation in hairless mouse skin, while the stratum corneum layer provided the greater resistance in human skin. A significant increase in TCDD permeation through human skin was observed when the skin was damaged by tape-stripping. Gallo et al. (1992) suggested that washing and tape-stripping of the exposed area might remove most of the TCDD and reduce the potential for systemic exposure and toxicity because most of the TCDD remained within the horny layer of human skin even at 24 hours following exposure. Weber et al. (1991) also investigated the penetration of TCDD into human cadaver skin at concentrations of 65-6.5 ng/cm2. This study found that the stratum corneum acted as a protective barrier, as its removal increased the amount of TCDD absorbed into layers of the skin. With intact skin and acetone as the vehicle, the rate of penetration of TCDD into the dermis ranged from 6 to 170 pg/hour/cm2, while penetration into the dermis and epidermis ranged from 100 to 800 pg/hour/cm2. With mineral oil as the vehicle, there was about a five-to-tenfold reduction in the rate of penetration of TCDD into the intact skin. December 2003 1-7 DRAFT--DO NOT CITE OR QUOTE Wester et al. (1993a) studied the dermal permeation potential of two PCBs, Aroclor 1242 and Aroclor 1254, from soil. Soil is the most common medium of contact for humans; hence any permeation potential is of great interest for risk assessment purposes. This study consisted of experiments conducted in rhesus monkeys (in vivo), in vitro human skin, and powdered human corneum. The monkeys were exposed topically for a 5-week period. Percutaneous absorption was determined by urinary and fecal [14C]-PCB excretion. The percutaneous absorption in the monkey was 13.8% (2.7%) for Aroclor 1242 and 14.1% (1.0%) for Aroclor 1254. The authors report that these absorption rates for soil are similar to those from vehicles such mineral oil, trichlorobenzene, and acetone. These rates are somewhat higher than those for other compounds such as the pesticide butachlor, which was about 5.0% when applied to human skin (Ademola et al., 1993). On the other hand, Wester et al. (1993b) report a much higher absorption for pentachlorophenol (PCP) (24.4% from soil and 29.2% from acetone) in the rhesus monkey. The authors report that PCP is one of the more extensively absorbed compounds they have studied. Thus, at least for these two PCBs (Aroclor 1242 and Aroclor 1254), the absorption in the rhesus monkey appears to be intermediate between that of other compounds tested. In summary, these studies indicate that these two PCBs show significant partitioning into the stratum corneum from soil, a common environmental matrix. 1.1.2.1. Bioavailability Following Dermal Exposure Dermal exposure of humans to TCDD and related compounds usually occurs as a complex mixture of these contaminants in soil, oils, or other mixtures, which would be expected to alter absorption. Poiger and Schlatter (1980) presented evidence that the presence of soil or lipophilic agents dramatically reduces dermal absorption of TCDD compared with absorption of pure compound dissolved in solvents. In a control experiment, 26 ng of TCDD in 50 pL methanol was administered to the skin of rats; 24 hours later the liver contained 14.82.6% of the dose. By comparing this value to the hepatic levels obtained after oral administration in 50% ethanol (in the same study), the amount absorbed from a dermal application can be estimated at -40% of the amount absorbed from an equivalent oral dose. This comparison assumes that hepatic levels are valid estimates of the amount absorbed from both oral and dermal routes and that absorption from methanol is equivalent to absorption from 50% ethanol. The dosedependent distribution of TCDD in the liver is another factor that may limit quantitative conclusions regarding bioavailability that are based solely on hepatic levels following exposure to 2,3,7,8-TCDD. As compared with dermal application in methanol, dermal application to rats of TCDD in vaseline or polyethylene glycol reduced the percentage of the dose in hepatic tissue to 1.4% and 9.3%, respectively, but had no observable effect on the dose of TCDD required to induce skin lesions (-1 pg/ear) in the rabbit ear assay. Application of TCDD in a soil/water December 2003 1-8 DRAFT--DO NOT CITE OR QUOTE paste decreased hepatic TCDD to -2% of the administered dose and increased the amount required to produce skin lesions to 2-3 :g in rats and rabbits, respectively. Application in an activated carbon/water paste essentially eliminated absorption, as measured by percent of dose in the liver, and increased the amount of TCDD required to produce skin lesions to -160 :g. These results suggest that dermal absorption of TCDD depends on the formulation (vehicle or adsorbent) containing the toxin. Shu et al. (1988b) investigated the dermal absorption of soil-bound TCDD in rats. Relative dermal bioavailability was estimated by comparing the level of TCDD in the liver of rats given soil-bound TCDD dermally to that of rats given oral doses of TCDD dissolved in corn oil. The level of TCDD in livers of rats dosed orally with TCDD in corn oil, following correction for unabsorbed TCDD TCDD, is assumed to represent 100% bioavailability. The dermal penetration of TCDD after 4 hours of contact with skin was -60% of that after 24 hours of contact. After 24 hours of contact with the skin, the degree of dermal uptake from contaminated soil was - 1% of the administered dose. The authors observed that the degree of uptake does not appear to be influenced significantly by the concentration of TCDD in soil, by the presence of crankcase oil as a co-contaminant, or by environmentally versus laboratorycontaminated soil. A major limitation of these studies is the uncertainty regarding the extrapolation of dermal absorption data on these compounds from the rat to the human. The in vitro dermal uptake of TCDD has been investigated in hairless mouse and human skin (Gallo et al., 1992; Rahman et al., 1992). In vitro dermal uptake of TCDD from laboratory-contaminated soil indicated that aging of soils (up to 4 weeks) and the presence of additives (2,4,5-trichlorophenol and motor oil) in the soil did not have any significant effect on dermal uptake (Gallo et al., 1992). Because most of the TCDD remained in the stratum corneum layer of human skin, the permeation of TCDD was significantly lower in human than in hairless mouse skin. There are no published quantitative in vivo data on the dermal absorption of TCDD and related compounds in the human, and data on the rhesus monkey are very limited. Brewster et al. (1988) found that 1,2,3,7,8-PeCDF was poorly absorbed in the monkey after dermal application, with <1% of the administered dose being absorbed in 6 hours. This provides further evidence for the very slow rate of dermal absorption of TCDD and related compounds. 1.1.3. Transpulmonary Absorption The use of incineration as a means of solid and hazardous waste management results in the emission of contaminated particles that may contain TCDD and related compounds into the environment. Thus, significant exposure to TCDD and related compounds may result from inhalation of contaminated fly ash, dust, and soil. In an attempt to address the bioavailability and December 2003 1-9 DRAFT--DO NOT CITE OR QUOTE potential health implications of inhaling contaminated particles, Nessel et al. (1990) examined the potential for transpulmonary absorption of TCDD in female Sprague-Dawley rats after intratracheal instillation of the compound in a corn oil vehicle or as a laboratory-prepared contaminant of gallium oxide particles. Several biomarkers of systemic absorption were measured, including the dose-dependent effects of TCDD on hepatic microsomal cytochrome P-450 content, AHH activity, and liver histopathology. Significant dose-related effects were observed at an exposure of >0.55 pg TCDD/kg. The authors found that induction was slightly higher when animals received TCDD in corn oil than when animals received TCDDcontaminated particles, and was comparable to induction after oral exposure. The results from Nessel et al. (1990) indicate that systemic effects occur after pulmonary exposure to TCDD, suggesting that transpulmonary absorption of TCDD does occur. The pulmonary bioavailability of TCDD was also examined in female Sprague-Dawley rats following intratracheal instillation of PCDD-contaminated soil from a former 2,4,5trichlorophenoxy-acetic acid manufacturing site (Nessel et al., 1992). A size-dependent enrichment of PCDDs and PCDFs was observed, with the smaller particles being more highly contaminated. TCDD was enriched up to 33-fold in small respirable particles as compared with unfractionated soil. Pulmonary bioavailability of TCDD was assessed by hepatic enzyme induction (AHH activity) and TCDD concentration. The data indicate that the relative pulmonary bioavailability of TCDD on respirable soil particles is 100% as compared with laboratory-recontaminated gallium oxide. The transpulmonary absorption of TCDD was assessed in male Fischer 344 rats following intratracheal instillation of a 1 nmol/kg dose in Emulphor:ethanol:water (1:1:3) (Diliberto et al., 1996a). Transpulmonary absorption was 95%, suggesting that there was almost complete absorption of TCDD by inhalation under these conditions. Similar results also were observed for the transpulmonary absorption of 2,3,7,8-TBDD under similar exposure conditions (Diliberto et al., 1993a,b). Recent studies (Diliberto et al., 1996a) further show the importance of transpulmonary absorption for 2,3,7,8-TCDD. Tissue distributions were measured 3 days after administration via different routes. Comparisons show that the percentage of dose distributed to the liver after intratracheal (itr) injection is similar to that after intravenous administration (iv) (33% for itr, 37% for iv). Also, both the iv and itr routes show a preference for greater sequestration in the liver over fat when compared to the oral (po) route. These results suggest that the transpulmonary absorption of 2,3,7,8-TCDD and 2,3,7,8-TBDD was similar to that observed following oral exposure. December 2003 1-10 DRAFT--DO NOT CITE OR QUOTE 1.1.4. Parenteral Absorption In an effort to obtain more reproducible and complete absorption of TCDD and related compounds for pharmacokinetic studies, Abraham et al. (1989a, b) used various vehicles to investigate the absorption of TCDD after parenteral application in rats. These investigators observed optimal results with the subcutaneous injection of TCDD with a mixture of toluene:DMSO (1:2) as the vehicle. At 3 and 5 days after treatment, the percentages of administered dose remaining at the injection site under the skin of the back were -10% and 2%, respectively. The vehicle did not cause adverse effects at an applied volume of 0.2 mL/kg bw. The absorption of a defined mixture of CDDs and CDFs in the rat was also examined after subcutaneous injection with toluene:DMSO (1:2) as a vehicle. Of the 97 congeners analyzed, 70 were >95% absorbed 7 days after exposure; 21 were 90%-95% absorbed; and 1,2,3,9-TCDD, 1.2.3.6.7.9- /1,2,3,6,8,9-HxCDD, 1,2,3,4,6,7,9-HpCDD, OCDD, 1,2,4,6,8,9-HxCDF and 1.2.3.7.8.9- HxCDF were 84%-89% absorbed. Greater than 90% absorption of CDDs and CDFs also was observed under these conditions in the marmoset monkey, with the exception of 1.2.3.4.7.8.9- HpCDF, OCDF, and OCDD, which had -50%-80% of the administered dose absorbed (Neubert et al., 1990; Abraham et al., 1989a). Although the absorption of CDDs and CDFs after subcutaneous administration in toluene:DMSO (1:2) is somewhat slow in rats and monkeys, absorption of most congeners was >90% within 7 days. Even for highly chlorinated insoluble congeners, such as OCDD and OCDF, subcutaneous absorption was >84% in the rat and >50% in the monkey. Less complete and slower absorption of CDDs and CDFs was observed after subcutaneous injection of these compounds using an oil-containing vehicle (Brunner et al., 1989; Abraham et al., 1989a). Using a corn oil:acetone vehicle (24:1, v/v), Lakshmanan et al. (1986) observed that only 7% of the administered dose of TCDD was absorbed 24 hours after subcutaneous injection and that only 35% was absorbed after intraperitoneal injection. Brunner et al. (1989) also reported that intraperitoneal administration of CDDs and CDFs revealed a delayed absorption from the abdominal cavity that varied for the different congeners. Therefore, concentrations measured in abdominal adipose tissue after intraperitoneal administration may not represent average values of adipose tissue in the whole body, particularly at early time points following exposure. This may also be true following oral exposure because of different perfusion rates of different fat depots (McKinley et al., 1993). 1.2. DISTRIBUTION 1.2.1. Distribution in Blood and Lymph Once a compound is absorbed, its distribution is regulated initially by its binding to components in blood and its ability to diffuse through blood vessels and tissue membranes. December 2003 1-11 DRAFT--DO NOT CITE OR QUOTE Lakshmanan et al. (1986) investigated the absorption and distribution of TCDD in thoracic ductcannulated rats. The results suggest that after gastrointestinal absorption, TCDD is absorbed primarily by the lymphatic route and is transported predominantly by chylomicrons. Ninety percent of the TCDD in lymph was associated with the chylomicron fraction. The plasma disappearance of TCDD-labeled chylomicrons followed first-order decay kinetics, with 67% of the compound leaving the blood compartment very rapidly (t1/2=0.81 minutes), whereas the remainder of the TCDD had a t/ of 30 minutes. TCDD was then found to distribute primarily to the adipose tissue and the liver. In vitro studies have investigated the distribution of TCDD in human whole blood. Henderson and Patterson (1988) found -80% of the compound associated with the lipoprotein fraction, 15% associated with protein (primarily human serum albumin), and 5% associated with cellular components. A subsequent in vivo investigation reported a similar distribution of TCDD in the various fractions of human whole blood (Patterson et al., 1989). Theoretical and limited experimental data also suggest that TCDD and related compounds may be associated with plasma prealbumin (McKinney et al., 1985; Pedersen et al., 1986). The distribution of [3H]-2,3,7,8-TCDD among lipoprotein fractions from three fasting, normolipemic donors indicated a greater percentage associated with LDL (55.3% 9.03% SD) than with VLDL (17.4% 9.07% SD) or HDL (27.3% 10.08% SD). The distribution of TCDD among the lipoprotein fractions was similar to that reported earlier by Marinovich et al. (1983). When the binding of TCDD was calculated per mole of lipoprotein, it was suggested that the maximal binding capacity was exerted by VLDL, followed by LDL and HDL (Marinovich et al., 1983). The results also suggest that variations in the amounts of each lipoprotein class may alter the distribution of TCDD among lipoproteins in a given subject. Significant species differences also exist; in the case of the rat, which has markedly lower plasma lipids compared to humans, TCDD was distributed almost equally among the lipoprotein fractions (Marinovich et al., 1983). Congener-specific differences have been observed for the in vivo binding of the 2,3,7,8substituted PCDDs and PCDFs to different serum fractions in human blood (Patterson et al., 1989). Binding to the lipoproteins gradually decreased with increasing chlorine content, with about 75% of TCDD bound to lipoproteins while approximately 45% of OCDD was bound to this fraction. In contrast, binding to other serum proteins increased with chlorine content, from approximately 20% for TCDD to 50% for OCDD. The results indicate that the higher chlorinated PCDDs and PCDFs do not partition according to the lipid content of these blood fractions. However, Busbee and Zipring (1994) report that at least one lower chlorinated PCB, dichlorobiphenyl, absorbs and distributes with the nonlipid plasma fractions, rather than the lipid fractions as might be expected. December 2003 1-12 DRAFT--DO NOT CITE OR QUOTE In addition, there is indirect evidence that suggests that the binding of TCDD to lipoproteins may alter the pharmacokinetics and toxic potency of the compound. Marinovich et al. (1983) found that experimentally induced hyperlipidemia in rats delayed the development of overt toxicity (lethality). However, the disposition of TCDD was not investigated under these conditions. These investigators suggest that the release of lipoprotein-bound TCDD is related to the metabolic turnover of lipoproteins. In hyperlipidemic rats, the turnover of VLDL and LDL is delayed significantly compared to that in normolipidemic animals, and this may contribute to the plasma lipoprotein binding modifying the toxicity of TCDD in hyperlipidemic rats. The time- and temperature-dependent cellular uptake of lipoprotein-associated TCDD by cultured human fibroblasts was greatest from LDL, intermediate from HDL, and least from serum (Shireman and Wei, 1986). Decreased cellular uptake of LDL and TCDD was observed in mutant fibroblasts, which lack the normal cell membrane receptor for LDL. This provides some evidence that specific binding of LDL and the LDL receptor pathway may account for some of the rapid early uptake of TCDD with LDL entry. The results suggest that the entry of TCDD into cells may not be solely by simple diffusion. However, nonspecific binding of the LDL and transfer of TCDD from LDL to the cell membranes are probably also important, as significant time- and temperature-dependent uptake of TCDD and LDL occurred in the mutant fibroblasts. Thus, upon absorption, TCDD and probably related compounds are bound to chylomicrons, lipoproteins, and other serum proteins that assist in distributing these uncharged, lipophilic compounds throughout the vascular system. These compounds then partition from blood components into cellular membranes and tissues, probably largely by passive diffusion. In addition, cellular uptake may be facilitated partly through the cell membrane LDL receptor, the hepatic receptor for albumin (Weisiger et al., 1981), and other systems. 1.2.2. Tissue Distribution Once absorbed into blood, TCDD and related compounds readily distribute to all organs. Tissue distribution within the first hour after exposure parallels blood levels and reflects physiological parameters such as blood flow to a given tissue and relative tissue size. For example, high initial concentrations of TCDD, 1,2,3,7,8-PeCDF, and 3,3',4,4'-TCB were observed in highly perfused tissue such as the adrenal glands during the 24-hour period after a single exposure (Birnbaum et al., 1980; Olson et al., 1980; Pohjanvirta et al., 1990; Brewster and Birnbaum, 1988; Durham and Brouwer, 1990). A high percentage of the dose of 2,3,7,8-TCDF and 1,2,3,7,8-PeCDF was also found in muscle within the first hour after intravenous exposure because of the large volume of this tissue (Birnbaum et al., 1980; Birnbaum, 1985; Brewster and Birnbaum, 1988). Nevertheless, within several hours the liver, adipose tissue, and skin become the primary sites of disposition, when expressed as percent of administered dose per gram tissue December 2003 1-13 DRAFT--DO NOT CITE OR QUOTE and as percent of dose per organ. Liver, adipose tissue, skin, and thyroid were the only tissues to show an increase in the concentration of TCDD during the initial 4 days after a single intra peritoneal exposure of rats (Pohjanvirta et al., 1990). In this study, a similar general pattern of disposition was observed in Han/Wistar and Long-Evans rats, which are, respectively, most resistant and susceptible to the acute toxicity of TCDD (Pohjanvirta et al., 1990). Table 1-4 illustrates the tissue distribution of TCDD in female Wistar rats 7 days after a single subcutaneous exposure (Abraham et al., 1988). This general pattern of distribution is similar to that observed in mice, rats, rhesus monkeys, hamsters, and guinea pigs, where liver and adipose tissue consistently have the highest concentrations of TCDD (Piper et al., 1973; Fries and Marrow, 1975; Rose et al., 1976; Allen et al., 1975; van Miller et al., 1976; Kociba et al., 1978a,b; Gasiewicz et al., 1983b; Manara et al., 1982; Olson et al., 1980; Gasiewicz and Neal, 1979; Birnbaum, 1986; Pohjanvirta et al., 1990; Abraham et al., 1988). A similar pattern of disposition also was observed for 2,3,7,8-TCDF in the guinea pig, rat, C57BL/6J and DBA/2J mouse, and rhesus monkey, with 2,3,7,8-TCDF concentrations highest in liver and adipose tissue (Decad et al., 1981b; Birnbaum et al., 1980, 1981). In summary, there do not appear to be major species or strain differences in the tissue distribution of TCDD and 2,3,7,8-TCDF, with the liver and adipose tissue being the primary disposition sites. For the PCBs, Ness et al. (1994) provide evidence of accumulation of PCB congeners in rat brain. Dams were dosed with Aroclor 1242 and pups from litters were euthanized at weaning. No differences in PCB concentrations between sexes or among brain regions were found, but the different congeners differed from each other in degree of bioaccumulation. The tissue distribution of the coplanar PCBs and PBBs also appears to be similar to that of TCDD and 2,3,7,8-TCDF. Limited studies in rats and mice found that 3,3',4,4'-TCB, 3,3',4,4'-TBB, and 3,3',4,4',5,5'-HxBB distributed preferentially to adipose tissue and liver (Clarke et al., 1983, 1984; Millis et al., 1985; Wehler et al., 1989; Clevenger et al., 1989). Lindenau et al. (1994) studied the distribution of PCBs in the reproductive tissues of female rabbits after long-term low-dose exposures. Chlorinated hydrocarbons were found to accumulate especially in oviductal and uterine tissues and in follicular and uterine fluids. Although the liver and adipose tissue contain the highest concentrations of TCDD and 2,3,7,8-TCDF, there are some congener-specific differences in the relative tissue distribution of related compounds. 2,3,7,8-TBDD and 1,2,3,7,8-PeCDD disposition in the rat was very similar to that of TCDD (Kedderis et al., 1991a; Wacker et al., 1986). The hepatic concentration of OCDD and 2,3,4,7,8-PeCDF in the rat, however, was approximately 10- to 20-fold greater than that in adipose tissue, which generally contains the second highest levels of these compounds (Birnbaum and Couture, 1988; Norback et al., 1975; Williams et al., 1972; Brewster and Birnbaum, 1987). The tissue distribution of a defined mixture of CDDs and CDFs (28.8 :g December 2003 1-14 DRAFT--DO NOT CITE OR QUOTE CDDs+CDFs/kg bw containing 120 ng TCDD/kg bw) was measured in marmoset monkeys 7 days after a single subcutaneous exposure (Abraham et al., 1990). For most of the 2,3,7,8substituted congeners, the highest concentrations were detected in hepatic and adipose tissue, with correspondingly lower values detected in kidney, brain, lung, heart, thymus, or testes. The hepatic and adipose tissue concentrations were similar for TCDD, 1,2,3,7,8-PeCDD, 2,3,7,8TCDF, and 1,2,3,7,8-/1,2,3,4,8-PeCDF. Nonetheless, the hepatic concentrations were approximately tenfold or more greater than those of adipose tissue for 1,2,3,4,7,8-HxCDD, 1.2.3.6.7.8- HxCDD, 1,2,3,7,8,9-HxCDD, 1,2,3,4,6,7,8-HpCDD, OCDD, 2,3,4,7,8-PeCDF, 1.2.3.4.7.8- /1,2,3,4,7,9-HxCDF, 1,2,3,6,7,8-HxCDF, 1,2,3,7,8,9-HxCDF, 2,3,4,6,7,8-HxCDF, 1.2.3.4.6.7.8- HpCDF, 1,2,3,4,7,8,9-HpCDF, and OCDF. The lungs and thymus contained higher concentrations of all of these congeners than were detected in kidney, brain, heart, and testes. Unexpectedly, the concentrations of these HxCDDs, HpCDDs, OCDD, and HxCDFs were similar in the adipose tissue, lungs, and thymus. In the case of HpCDFs and OCDF, the concentrations were greater in the lungs than in the adipose tissue. The enhanced disposition of highly chlorinated congeners to the lungs and thymus is of interest and deserves further investigation. For example, it is possible that the high concentration in the lungs could be related to the insolubility of these compounds. Further, Diliberto et al. (1995) report that in mice, the ratio of liver to adipose tissue concentration of TCDD changes with dose. As the dose increases, the adipose (and other nonhepatic tissues) tissue dose/g decreases while it increases in the liver. Section 1.2.5 of this document discusses induction of hepatic cytochrome P-450 1A2 (CYP1A2) as the primary factor responsible for the hepatic sequestration of TCDD and related compounds. It is also to be expected that exposure to a mixture of these compounds can cause effects in the uptake and distribution. Darnerud et al. (1993) report on the effects of pretreatment of various PCBs on the hepatic uptake of 2,3,7,8-TCDF in mice. After a 4-hour pretreatment, several mono- and di-ortho PCB congeners and Aroclor 1254 increased the hepatic uptake of TCDF. Non-ortho congeners decreased the uptake of TCDF. At longer pretreatment times (48 hours), both non-ortho and mono-ortho PCBs increased the hepatic uptake of TCDF. These studies illustrate the complexity of the pharmacokinetics of mixtures. Whole-body autoradiography of mice and rats after intravenous administration of [14C]-2,3,7,8-TCDD showed a selective localization of radioactivity in the liver and nasal olfactory mucosa (Appelgren et al., 1983; Gillner et al., 1987). The selective localization of TCDD in the nasal olfactory mucosa was apparently overlooked by other distribution studies that only examined selected organs. Gillner et al. (1987) found no TCDD-derived radioactivity in the olfactory mucosa after solvent extraction of sections, suggesting that TCDD was not covalently bound in this tissue. In addition, Gillner et al. (1987) reported induction of mRNA coding for P4501A2 (CYP1A2) in the absence of P-4501A1(CYP1A1) induction in olfactory mucosa of rats. December 2003 1-15 DRAFT--DO NOT CITE OR QUOTE The selective distribution of 2,3,7,8-TCDD in the liver and olfactory mucosa correlates with the tissue-specific localization of CYP1A2, which represents a potential sequestration (binding) protein (see Section 1.2.5). Increases in the incidence of squamous cell carcinoma of nasal turbinates and carcinoma of the liver were observed in rats after a 2-year exposure to TCDD in rat chow (Kociba et al., 1978a); however, this effect was not observed in nasal tissues of mice or rats intubated with TCDD. Evidence has also been reported that suggests that TCDD uptake and retention by the liver is dependent on the cell type within the liver. Hakansson et al. (1989) found that at 4 days after exposure of rats to TCDD, 60% of the dose distributed to hepatocytes and 12% was retained by stellate cells. Half-lives for TCDD in hepatocytes and stellate cells were calculated to be 13 and 50 days, respectively, suggesting that TCDD is more persistent in nonparenchymal cells. The induction of CYP1A2 in hepatocytes is the primary factor responsible for the hepatic sequestration of TCDD and related compounds (see Section 1.2.5 of this document). 1.2.2.1. Tissue Distribution in Humans Fachetti et al. (1980) reported tissue concentrations of TCDD at levels of 1-2 ng/g in adipose tissue and pancreas, 0.1-0.2 ng/g in the liver, and #0.1 ng/g in thyroid, brain, lung, kidney, and blood in a woman who died 7 months after potential exposure to TCDD from the Seveso accident. This pattern of TCDD distribution, however, may not be representative for humans because the woman at the time of death had an adenocarcinoma (which was not considered related to the accident) involving the pancreas, liver, and lung. Ryan et al. (1985a) examined the distribution of TCDD in two humans at autopsy. They determined on a weight basis that TCDD distributed in descending order to fat (-6 ppt) and liver (-2 ppt), with levels in muscle and kidney below detection; however, TCDD levels compared on a per lipid basis were similar between tissues. These data should be interpreted with caution because only two subjects were examined and one of the subjects was suffering from fatty liver syndrome; therefore, the data cannot be generalized to the entire population. Poiger and Schlatter (1986) estimated that -90% of the body burden of TCDD was sequestered in the fat after a volunteer ingested [3H]-2,3,7,8-TCDD in corn oil at a dose of 1.14 ng/kg. During this 135-day study, elevated radioactivity was detected in the blood only during the first 2 days after treatment. The data would be consistent with the high lipid bioconcentration potential of TCDD in humans, as calculated by Geyer et al. (1986) from daily intake assumptions, levels in human adipose tissue, and pharmacokinetic models. Geyer et al. (1986) estimated a BCF of between 104 and 206 for TCDD in human adipose tissue. In human adipose tissue, levels of TCDD averaging 5-10 ppt have been reported for background populations in St. Louis, Missouri, by Graham et al. (1986), in Atlanta and Utah by December 2003 1-16 DRAFT--DO NOT CITE OR QUOTE Patterson et al. (1986), and in Canada by Ryan et al. (1985b). Sielken (1987) evaluated these data and concluded that the levels of TCDD in human adipose are log-normally distributed and positively correlated with age. Among the observed U.S. background levels of TCDD in human adipose tissue, more than 10% were >12 ppt. Patterson et al. (1987) developed a high-resolution gas chromatographic/high-resolution mass spectrometric analysis for TCDD in human serum. The arithmetic mean of the individual human serum samples was 47.9 ppt on a whole-weight basis and 7.6 ppt on a lipid-weight basis. Paired human serum and adipose tissue levels of TCDD have been compared by Patterson et al. (1988), Kahn et al. (1988), and Schecter et al. (1990a). All three laboratories reported a high correlation between adipose tissue and serum TCDD levels when the samples were adjusted for total lipid content. This correlation indicates that serum TCDD is a valid estimate of the TCDD concentration in adipose tissue. Congener-specific partitioning of 2,3,7,8-substituted PCDDs and PCDFs between adipose tissue and plasma lipids has also been reported in a study of 20 Massachusetts Vietnam veterans (Schecter et al., 1990b). The distribution ratio between plasma lipid and adipose tissue increased with chlorine substitution on the PCDDs and PCDFs. Whereas 2,3,7,8-substituted TCDD, TCDF, PeCDD, PeCDF, HxCDD, and HxCDF had a plasma lipid-to-adipose tissue ratio of about 1.0, OCDD had a ratio of 2.0. On the other hand, whole blood PCDDs and PCDFs seem to be found at the same concentrations as in adipose tissue on a lipid basis (Schecter, 1991). The disposition of 2,3,7,8-substituted PCDDs and PCDFs in human liver and adipose tissue was assessed in a study of 28 people from the Munich area (Thoma et al., 1989; 1990). Table 1-5 summarizes these results, which are expressed both on a lipid and wet-weight basis. The concentrations of PCDDs and PCDFs in adipose tissue and liver are not the same when calculated on a lipid basis. This is in contrast to the high correlation that was reported between adipose tissue and serum TCDD levels when expressed on a lipid-weight basis (Patterson et al., 1988; Kahn et al., 1988; Schecter et al., 1990a). Furthermore, the liver/adipose tissue ratio increased with the higher chlorinated PCDDs and PCDFs. The congener-specific hepatic disposition is also similar to that observed in rats and marmoset monkeys exposed to a complex mixture of PCDDs and PCDFs (Abraham et al., 1989b; Neighbored et al., 1990). Therefore, it is important to consider congener- and tissue-specific differences in disposition of PCDDs and PCDFs when blood levels are used to estimate tissue levels or body burdens. Schecter et al. (1998a) assessed the disposition of PCDDS, CDFS, and coplanar PBS in the blood, milk, adipose tissue, placenta, and cord blood from five American women. When expressed on a pg/g lipid basis, the mean total TEQs were 11.6, 12.1, 10.5, 5.8, 10.0, and 10.2 in adipose tissue, predelivery blood, placenta, cord blood, postpartum blood, and breast milk, respectively. The results suggest that CDDs, CDFs, and PCBs, when expressed as total TEQs on December 2003 1-17 DRAFT--DO NOT CITE OR QUOTE a lipid basis, partition to a similar extent between these tissues. Even though 2,3,4,7,8-PeCDF and PCB 126 were lower in cord blood than other tissues, the levels of TCDD were similar in these tissues. In a study of potentially heavily exposed Vietnam veterans, MMWR (1988) reviewed an Air Force study of Ranch Hand veterans who were either herbicide loaders or herbicide specialists in Vietnam. The mean serum TCDD levels of 147 Ranch Hand personnel was 49 ppt in 1987, based on total lipid weight, while the mean serum level of the 49 controls was 5 ppt. In addition, 79% of the Ranch Hand personnel and 2% of the controls had TCDD levels >10 ppt. The distribution of TCDD levels in this phase of the Air Force health study indicates that only a small number of Ranch Hand personnel had unusually heavy TCDD exposure. Similar results were obtained by Kahn et al. (1988), who compared TCDD levels in blood and adipose tissue of Agent Orange-exposed Vietnam veterans and matched controls (Kahn et al., 1988). This study also examined moderately exposed Vietnam veterans who handled herbicides regularly while in Vietnam. Although this study can distinguish moderately exposed men from others, the data do not address the question of identifying persons whose exposures were relatively low and who constitute the bulk of the population, both military and civilian, that may have been exposed to greater than background levels of TCDD. 1.2.3. Time-Dependent Tissue Distribution 2,3,7,8-TCDD and related compounds exhibit congener-specific disposition, which depends on tissue, species, and time after a given exposure. In general, these compounds are cleared rapidly from the blood and distributed to liver, muscle, skin, adipose tissue, and other tissues within the first hour(s) after exposure. This is followed by redistribution to the liver and adipose tissue, which exhibit increasing tissue concentrations over several days after exposure. Elimination from tissues then occurs at rates that are congener-, tissue-, and species-specific. Thus, the ratio of the concentration of TCDD and related compounds in different tissues (i.e., liver/adipose) may not remain constant over an extended period after a single exposure. Abraham et al. (1988) examined the concentrations of TCDD in liver and adipose tissue of female Wistar rats over a 91-day period after a single subcutaneous exposure at a dose of 300 ng/kg bw (Figure 1-1). The maximum concentration of TCDD in the liver and adipose tissue was reached at 3 and 7 days after exposure, respectively. The liver/adipose tissue concentration ratio does not remain constant over time because the concentration of TCDD decreases more rapidly in the liver than in the adipose tissue. For example, the liver/adipose tissue concentration ratio (for TCDD) was 10.3 at 1 day after exposure and 0.5 at 91 days after exposure (Figure 1-1). Results from other disposition studies also indicate that the ratio of the concentration of TCDD and related compounds in liver, adipose tissue, and other tissues does not remain constant over December 2003 1-18 DRAFT--DO NOT CITE OR QUOTE an extended period after a single exposure (Pohjanvirta et al., 1990; Birnbaum, 1986; Birnbaum et al., 1980; Decad et al., 1981a; Birnbaum and Couture, 1988; Olson et al., 1980; Kedderis et al., 1993a; Brewster and Birnbaum, 1987, 1988; Neubert et al., 1990). This relationship is important in attempting to correlate dose-response data with tissue concentrations of TCDD and related compounds. In an attempt to maintain constant TCDD levels in tissues to study long-term effects, Krowke et al. (1989) investigated several loading-dose/maintenance-dose exposure regimens. They found that similar liver/adipose tissue concentrations ranging from 5 to 8 could be maintained in rats over a 22-week period with a loading dose of 25 pg/kg followed by weekly maintenance doses of 5 pg/kg. A large body of data on the tissue concentrations of TCDD and related compounds over time after exposure can be evaluated by estimating congener-specific half-life values for a given tissue and species. Table 1-6 summarizes pharmacokinetic elimination parameters for TCDD and related compounds from major tissue depots. Data from Abraham et al. (1988) (see Figure 1-1) were used to estimate the half-life for TCDD in the liver and adipose tissue of rats (Table 1-6). The decrease in the TCDD concentration in adipose tissue is a linear function in the semilogarithmic plot in Figure 1-1 (log concentration versus time), which indicates apparent first-order elimination kinetics with a half-life of 24.5 days (Table 1-6). Liver tissue exhibits a biphasic (two-component) exponential decay pattern, with a half-life of 11.5 days for the first component (days 10-49) and a half-life of 16.9 days for the second component (days 49-91) (see Figure 1-1 and Table 1-6). Santostefano et al. (1997) reported a similar half-life of approximately 10 days in female Sprague-Dawley rats administered a 10 pg/kg oral dose of TCDD/kg. Results of Abraham et al. (1988) and Lakshmanan et al. (1986) indicate that in the rat, TCDD is more persistent in the adipose tissue than in the liver. This is in contrast to the mouse, where liver and adipose tissue have similar half-lives (Birnbaum, 1986). TCDD is exceptionally persistent in the adipose tissue of the rhesus monkey, with a half-life approximately ten- to fortyfold greater than that observed in the rat and mouse (Bowman et al., 1989). Thus, the relative persistence of TCDD is tissue-specific and exhibits marked interspecies variability. Most of the pharmacokinetic data on the relative persistence of other congeners in Table 1-6 have been reported in rat studies, which limits interspecies comparisons. Results in the rat suggest that the distribution and elimination of 2,3,7,8-TBDD from tissue are similar to those of 2,3,7,8-TCDD. The most persistent congeners are OCDD, 2,3,4,7,8-PeCDF, and 1,2,3,6,7,8HxCDF, which distribute almost entirely to the liver. OCDD and 2,3,4,7,8-PeCDF also exhibit similar elimination kinetics, with a relative half-life in the liver more than twofold greater than that in adipose tissue. The least persistent congeners are 2,3,7,8-TCDF, 1,2,3,7,8-PeCDF, and 3,3',4,4'-TCB. These congeners exhibit similar elimination kinetics in the rat, with half-lives in December 2003 1-19 DRAFT--DO NOT CITE OR QUOTE the adipose tissue greater than those in liver. The relative tissue distribution of these congeners varies, however, with 2,3,7,8-TCDF and 1,2,3,7,8-PeCDF distributing primarily to the liver while 3,3',4,4'-TCB distributes predominantly to the adipose tissue. Viluksela et al. (1997) conducted a subchronic/chronic toxicity study with 1,2,3,4,6,7,8HpCDD (13 weeks of dosing, 13 week off-dosing) in Sprague-Dawley rats. Liver concentrations during the study provided a hepatic half-life estimate for 1,2,3,4,6,7,8-HpCDD of 237 days in male rats and 314 days in female rats. Under similar exposure conditions, TCDD had half-lives of 12.4 and 15.8 days in male and female rats, respectively. Liver concentrations of CDDs were also assessed in a subchronic/chronic toxicity study in rats administered a mixture of 2,3,7,8TCDD, 1,2,3,7,8-PeCDD, 1,2,3,4,7,8-HxCDD, and 1,2,3,4,6,7,8-HpCDD orally in corn oil (13 weeks of dosing, 13 weeks of off-dosing) (Viluksela et al., 1998). Average liver half-lives were 14.5, 29.3, 45.6, and 100 days, respectively, in males and 17.8, 38.2, 63.3, and 195.8 days, respectively, in females. When administered individually, 1,2,3,4,7,8-HxCDD had liver half lives of 36.2 days in males and 55.5 days in females and 1,2,3,7,8-PeCDD had a liver half-life of 36.1 days in male rats. Together, these results indicate that CDDs are more persistent in female rats and that the hepatic half-life of these compounds is similar following exposure to individual CDDs and mixtures of CDDs. The experimental tissue distribution and elimination data in Table 1-6 were obtained after exposure to a single congener, but real-world exposure to TCDD and related compounds occurs as a complex mixture of congeners. Neubert et al. (1990) examined the persistence of various CDDs and CDFs in hepatic and adipose tissue of male and female marmoset monkeys. Animals received a single subcutaneous exposure to a defined CDD/CDF mixture (total dose of 27.8 pg/kg bw), which contained 0.12 pg 2,3,7,8-TCDD/kg bw. Using the international toxicity equivalency (I-TE) factors (NATO, 1988; U.S. EPA, 1989), the total administered dose corresponded to 0.464 pg I-TE/kg bw. The concentrations of specific congeners in liver and adipose tissue were measured at 1, 6, 16, or 28 weeks after exposure, and elimination constants and half-lives were estimated assuming first-order kinetics (Table 1-7). Data in Table 1-7 were determined from pregnant and nonpregnant female and male marmosets (total of 12 animals), because no obvious differences in tissue concentrations were observed among these groups. All 2,3,7,8-substituted CDDs and CDFs were consistently more persistent in the adipose tissue than in the liver of marmoset monkeys. In general, the persistence in adipose tissue was from -1.3- to 2.0-fold greater than that in liver, with the exception of 1,2,3,4,7,8-/1,2,3,4,7,9-HxCDF, HpCDFs, and OCDF, which were even more persistent in adipose tissue. For the latter congeners and OCDD, there was marked variance in half-life values, which may be due to delayed and incomplete absorption of the exceptionally persistent congeners and the relatively short (28 weeks) period of investigation. A significant species difference exists for OCDD and December 2003 1-20 DRAFT--DO NOT CITE OR QUOTE 2,3,4,7,8-PeCDF, which, in contrast to the results in the marmoset monkey, was found to be more persistent in the liver of the rat, with half-lives more than twofold greater than that in adipose tissue (Birnbaum and Couture, 1988; Brewster and Birnbaum, 1987) (see Table 1-6). Further comparison of tissue elimination data in the rat (Table 1-6) and monkey (Table 1-7) indicates that 2,3,7,8-TCDD, OCDD, 2,3,7,8-TCDF, 1,2,3,6,7,8-HxCDF, and 2,3,4,7,8-PeCDF (adipose tissue only) are more persistent in the marmoset monkey than in the rat. The exception to this relationship is 2,3,4,7,8-PeCDF, which is more persistent in rat liver compared to the monkey. The exposure of marmoset monkeys to a complex mixture of CDDs and CDFs included exposure to both 2,3,7,8- and non-2,3,7,8-substituted congeners (Neubert et al., 1990). One week after exposure to this complex mixture, the non-2,3,7,8-substituted CDDs and CDFs were present in liver and adipose tissue in relatively minor quantities when compared with 2,3,7,8substituted congeners; however, non-2,3,7,8-substituted compounds represented a considerable percent of the exposure mixture. In this study, none of the non-2,3,7,8-substituted TCDDs, PeCDDs, TCDFs, or PeCDFs could be detected in the liver. Some of the hexa- and heptacongeners were detected in adipose tissue and liver, but after 1 week, the total amount in the liver was >5% of the administered dose only in the case of 1,2,4,6,8,9-HxCDF. Similar results were obtained in rats after exposure to a defined, complex mixture of CDDs and CDFs (Abraham et al., 1989c). Additional short-term studies in rats provide evidence that the low tissue concentrations of non-2,3,7,8-substituted congeners, measured 1 week after exposure, were the result of rapid elimination, as these congeners were detected at higher levels in the liver 13 to 14 hours after exposure (Abraham et al., 1989d). These results in monkeys and rats are compatible with data from analysis of human tissue samples and milk, in which the non-2,3,7,8-substituted congeners have also not been shown to be present in significant concentrations when compared with the 2,3,7,8-substituted congeners (Schecter et al., 1985, 1986; Ryan, 1986; Rappe et al., 1986; Beck et al., 1987, 1989; Thoma et al., 1989). Several human studies also show the importance of time dependence and age on tissue distribution within the body. Duarte-Davidson and Jones (1994) reported that in a population in the United Kingdom adipose tissue concentrations of PCBs increased at a slower rate as age increased. The researchers attributed this to the dilution effect caused by the increase in body fat with age. Wolfe et al. (1994) reported similar findings with TCDD. They found significant increases in half-life with increases in percent body fat and with age. As will be discussed in a later section, body burden levels could also be decreasing with time because of reduced exposure levels (Furst et al., 1994). Other recent studies also show that age is an important factor affecting distribution of these compounds within the body. Pegram et al. (1995) show that the hepatic concentration of December 2003 1-21 DRAFT--DO NOT CITE OR QUOTE TCDD was about 25% greater in young mice than in old mice. No age-related differences in nuclear TCDD concentration was found in the liver fat tissue concentration ratios, adipose tissue, or blood concentrations. In old mice kidney, skin, and muscle concentrations were about twofold higher than in younger mice at a dose of 15 :g/kg. The authors suggest that these age-related differences represent differences in Ah receptor-mediated enzyme induction. A potential problem of tissue distribution and elimination studies after exposure to a complex mixture of CDDs and CDFs is the possible interaction of the mixture during the uptake and elimination of specific congeners from tissues. A similar hepatic distribution (-25% of dose) and liver/adipose tissue concentrations ratio (-2) for TCDD were observed in rats 7 days after exposure to TCDD (100 ng/kg bw) when the compound was administered alone or in combination with a large amount of other CDDs/CDFs (total 23,222 ng/kg bw) (Abraham et al., 1988, 1989d). This suggests that under these experimental conditions, the tissue distribution of TCDD was not altered when the exposure included a complex mixture of CDDs/CDFs. van den Berg et al. (1989) studied the hepatic disposition and elimination of CDFs administered individually (see Table 1-6) and as mixtures. Co-administration of 1,2,3,7,8- and 2,3,4,7,8PeCDF resulted in 46% of the dose of 1,2,3,7,8-PeCDF distributing to the liver, while 70% was distributed to the liver after administration of the single-compound (see Table 1-6). Nevertheless, this combined exposure did not alter the rate of elimination of 1,2,3,7,8-PeCDF from the liver. Co-administration of 2,3,4,7,8-PeCDF and 1,2,3,6,7,8-HxCDF did not alter the hepatic uptake of either congener or the hepatic elimination of 2,3,4,7,8-PeCDF but increased the hepatic half-life of 1,2,3,6,7,8-HxCDF to 156 days from the single-compound exposure half-life of 73 days (see Table 1-6). However, these values must be considered rough estimates because the experimental period of 42 days was too short to calculate half-lives accurately. Although there are few investigations of potential interactions of mixtures of CDDs and CDFs on the uptake and elimination of individual congeners, the limited available data suggest that exposure to complex mixtures (see Table 1-7) may alter the tissue disposition of individual congeners. Several studies have investigated potential pharmacokinetic interactions following exposure to two or more PCDDs, PCDFs, or PCBs (DeJongh et al., 1992, 1993a, 1993b, van Birgelen et al., 1996a). Interactive effects on the hepatic disposition of these compounds may in some cases explain, in part, the potentiation and antagonistic effects on CYP1A1/1A2 activities observed with combined exposure to some of these compounds. In the case of 1,2,3,7,8-PeCDD and 2,4,5,2',4',5'-HxCB, no pharmacokinetic bases were found to explain the antagonistic effects of the combined exposure on CYP1A1/1A2 activities in mice (DeJongh et al., 1992). In a subsequent study the hepatic disposition of 1,2,3,7,8-PeCDD in mice was increased with combined exposures to 1,2,3,6,7,8-HxCDD, 2,3,4,7,8-PeCDF, and/or 2,4,5,2',4',5' HxCB (De Jongh et al., 1993b). December 2003 1-22 DRAFT--DO NOT CITE OR QUOTE De Jongh et al. (1995) report that TCDD co-administration with 2.2',4,4'5,5'hexachlorobiphenyl (HxCB) in mice results in an increased disposition of TCDD in the liver. At the same time, the results do not show any effect of the TCDD on the HxCB disposition. The distribution of TCDD (0, 0.1, 1.0, or 10 pg/kg) and/or 2.2',4,4',5,5'-hexachlorobiphenyl (PCB 153; 0, 3.58, 35.8, or 358 mg/kg) were investigated alone and in all possible combinations of these doses following oral administration in corn oil to female B6C3F1 mice (van Birgelen et al., 1996b). Coadministration of the low doses of TCDD and PCB 153 had little or no effect on the distribution of either compound. Interactive effects on the pharmacokinetic behavior occurred only at high doses. For example, the hepatic disposition of TCDD increased when it was administered in combination with the highest dose of PCB 153. Although there is evidence that PCDDs, PCDFs, and PCBs may influence each other's pharmacokinetics when administered in mixtures, this area needs investigation. 1.2.4. Dose-Dependent Tissue Distribution Recent evidence suggests that the tissue distribution of TCDD and related compounds is dose-dependent. Abraham et al. (1988) investigated the distribution of TCDD in liver and adipose tissue of rats 7 days after a single subcutaneous exposure to TCDD at doses of 1-3,000 ng/kg bw. More than 97% of the administered TCDD was absorbed at all doses, with the exception of the 3,000 ng/kg group, where 84% of the dose was absorbed. Figure 1-2 illustrates the dose-dependent disposition of TCDD in liver and adipose tissue (% dose/g) 7 days after exposure. A sharp increase in TCDD concentration in liver was observed at exposure levels >10 ng/kg bw. Disposition in the liver increased from -11% of the administered dose at an exposure level of 1-10 ng/kg bw to -37% of the dose at an exposure level of 300 ng/kg bw. The increase in distribution to the liver was accompanied by a dose-related decrease in the concentration of TCDD in the adipose tissue. As a result, the liver/adipose tissue concentration ratio for TCDD at 7 days after exposure increased with increasing doses, starting at an exposure level of 30 ng/kg bw (Table 1-8). Thus, the tissue-specific disposition of TCDD is regulated by a complex relationship, which includes species, time after a given exposure, and dose (see Figures 1-1 and 1-2; Tables 1-6 and 1-7). Other studies on the tissue disposition of TCDD and related compounds report similar dose-dependent behavior with disproportionately greater concentrations in the liver at high doses compared with low doses. Poiger et al. (1989) observed a dose-related increase in distribution to the liver (% of dose/liver) and an increase in the liver/adipose tissue concentration ratio for 2,3,7,8-TCDD, 2,3,4,7,8-PeCDF, 1,2,3,7,8-PeCDF, and 1,2,3,6,7,8-HxCDF in the rat. Kedderis et al. (1991a) also observed a dose-related increase in hepatic disposition (1.27% versus 10.05% of dose/liver) and an increase in the liver/adipose tissue concentration ratio (0.16 versus 2.59) for December 2003 1-23 DRAFT--DO NOT CITE OR QUOTE 2,3,7,8-TBDD at 56 days after exposure at doses of 0.001 and 0.1 pmol/kg bw, respectively. In a related study, pretreatment of mice with TCDD (5 or 15 pg/kg) produced a dose-related, enhanced hepatic accumulation of a subsequent oral dose of TCDD (Curtis et al., 1990). Diliberto et al. (1993b) also observed a dose-dependent tissue distribution of TCDD in female B6C3F1 mice. In all tissues except the liver, the relative percent of the total dose of TCDD decreased while it increased in the liver with higher doses. Similarly, a dose-related increase in hepatic uptake of [125I]-2-iodo-3,7,8-trichlorodibenzo-p-dioxin was observed after pretreatment of mice with TCDD (Poland et al., 1989a; Leung et al., 1990a). Shen and Olson (1987) also observed an increase in the uptake of TCDD by isolated hepatocytes from TCDD-pretreated mice. Chronic studies also support dose-dependent alterations in the tissue distribution of these compounds. Kociba et al. (1978a,b) found that female rats maintained on a daily dietary TCDD intake of 100 ng for 2 years had an average TCDD content of 8,100 ppt in fat and 24,000 ppt in the liver. Rats given 10 ng/kg/day had an average of 1,700 ppt TCDD in the fat and 5,100 ppt in the liver. For both of these exposures the liver/adipose tissue concentration ratio of TCDD was -3. At the lowest dose level of 1 ng/kg/day, both fat and liver contained an average of 540 ppt TCDD. Kociba et al. (1976) presented evidence that steady state had been reached by <13 weeks of feeding of TCDD. Other studies do not support the dose-dependent tissue distribution of TCDD and related compounds described above. Rose et al. (1976) reported a lack of a dose-dependent accumulation of [14C]-TCDD in male and female rat liver and adipose tissue following 7, 21, and 49 days of exposure at 0.01, 0.1, or 1.0 pg/kg/day, Monday through Friday. The rates of accumulation of TCDD-derived radioactivity were similar in fat, liver and whole body; however, the concentration in the liver was about fivefold greater than in fat. Brewster and Birnbaum (1987) also observed similar concentrations (% dose/g) of 2,3,4,7,8-PeCDF in liver, adipose tissue, and other tissues 3 days after oral exposure at doses of 0.1, 0.5, or 1.0 pmol/kg bw. These results conflict with the above studies, which support the dose-dependent tissue distribution of these compounds. An explanation for the lack of a dose-dependent accumulation is that all of the doses administered in the studies of Rose et al. (1976) and Brewster and Birnbaum (1987) were inducing hepatic CYP1A2, which is now known to be primarily responsible for the hepatic sequestration of these compounds (see Section 1.2.5). The dose-dependent tissue distribution of TCDD and related compounds is a critical factor that must be considered in estimating the concentration of these compounds in human tissues after chronic low-level exposure. This is particularly important because the general human population is exposed to much smaller daily doses (possibly 0.3 pg TCDD/kg/day) than those used in experimental disposition studies. Owing at least partly to the long half-life of December 2003 1-24 DRAFT--DO NOT CITE OR QUOTE TCDD in humans, however, this exposure results in concentrations of 3-18 pg/g in human adipose tissue (Leung et al., 1990b). Similar levels of TCDD in adipose tissue (14 pg/g) were observed in rats 7 days after subcutaneous exposure to 3 ng/kg bw (see Table 1-8) (Abraham et al., 1988). Under these experimental conditions, the liver/adipose tissue TCDD concentration was 0.74. Nonetheless, steady state was definitely not reached under these conditions, and with increasing time after exposure, this ratio may decrease, based on the observation that TCDD was more persistent in adipose tissue than in liver in rats exposed to 300 ng/kg bw (see Figure 1-1 and Table 1-6) (Abraham et al., 1988). Human data on the liver/adipose tissue concentration ratio of TCDD and related compounds are limited, but suggest that the ratio may vary by at least an order of magnitude between individuals. Leung et al. (1990b) observed a geometric mean adipose tissue TCDD concentration of 7.78 ppt in 26 individuals and a concentration in liver at about one-tenth of that in adipose tissue on a whole-weight basis. When measured on a total lipid basis, the concentrations of TCDD in both tissues were approximately the same. Thoma et al. (1990) reported a liver/adipose tissue TCDD concentration on a wet weight basis of 0.14, whereas on a lipid basis the ratio was 2.05 (Table 1-5). Considerable variability in CDD and CDF concentrations in liver and adipose tissues was also observed between individual marmoset monkeys (Neubert et al., 1990), suggesting that individual variability may also contribute to the difficulty in assigning a constant liver/adipose tissue ratio for CDDs and CDFs in humans and nonhuman primates. 1.2.5. Potential Mechanisms for Dose-Dependent Tissue Distribution The observation that exposure to higher doses of TCDD and related compounds results in a disproportionately greater hepatic concentration of these compounds may be explained by a hepatic binding species that is induced by TCDD and other agonists for the Ah receptor. The studies of Voorman and Aust (1987, 1989) and Poland et al. (1989a, b) provide evidence that this binding species is CYP1A2. Poland et al. (1989a, b) reported that TCDD and other Ah agonists (2,3,7,8-TCDF, $naphthoflavone, 3,3',4,4',5,5'-hexabromobiphenyl) act through the Ah receptor to increase a liver binding species that increases the hepatic uptake of [125I]-2-iodo-3,7,8-trichlorodibenzo-pdioxin (a radiolabeled isosteric analogue of TCDD) in vivo and binding of this radioligand to liver homogenate in vitro. Twenty-four hours after the administration of a non-AHH-inducing dose (1 x 10-10mol/kg) of [125I]-2-iodo-3,7,8-trichlorodibenzo-p-dioxin to C576BL/6J mice, the hepatic concentration of radioactivity was 1%-2% of the administered dose, whereas in mice pretreated 48 hours earlier with an AHH-inducing dose of TCDD (1 x 10-7mol/kg), the hepatic accumulation of radiolabel was 25%-30% of that administered. A similar, though less dramatic, effect was observed in vitro, with liver homogenate from TCDD-treated mice binding about four December 2003 1-25 DRAFT--DO NOT CITE OR QUOTE times more [125I]-2-iodo-3,7,8-trichlorodibenzo-p-dioxin than homogenate from control mice. The administration of TCDD to C57BL/6J mice produced a dose-related stimulation of in vivo hepatic uptake of [125I]-2-iodo-3,7,8-trichlorodibenzo-p-dioxin, increased binding of radioligand to liver homogenate, and induction of hepatic activity, with an ED50ranging from 1.5 to 4.0 x 10-9mol/kg. In congenic C57BL/6J (Ahd/Ahd) mice, which express the lower affinity Ah receptor, the ED50values for all three responses were shifted to doses that were about tenfold higher. The observed effects on hepatic disposition were tissue-specific, with no remarkable dispositional changes being observed in kidney, lung, spleen, small intestine, or muscle. This is significant in that TCDD and other agonists for the Ah receptor induce CYP1A1 in liver and other tissues, whereas CYP1A2 is apparently inducible only in liver (Tuteja et al., 1985; Gillner et al. 1987). Furthermore, the changes in hepatic disposition were not species-specific; similar responses were observed in guinea pigs, rats, mice, and hamsters (Poland et al., 1989a). The following evidence reported by Poland et al. (1989b) supports the hypothesis that the TCDD-inducible hepatic binding protein is CYP1A2: the TCDD-induced hepatic binding species was found predominantly in the microsomal fraction and was inactivated by heating at 60C, trypsin, and mercurials; the TCDD-induced hepatic binding species was specific for the liver, with a large pool size (Bmaxof 22 5 nmol/g liver); the major microsomal binding species covalently labeled with the photoaffinity ligand [125I]-2-iodo-3-azido-7,8-dibromodibenzo-pdioxin migrates with that immunochemically stained with polyclonal antiserum, which binds to CYP1A2. One observation of Poland et al. (1989a,b) does not support the hypothesis that the TCDD-inducible hepatic protein is CYP1A2. These investigators found that dietary administration of isosafrole did not stimulate hepatic uptake of [125I]-2-iodo-3,7,8trichlorodibenzo-p-dioxin or the in vitro binding of this ligand to liver homogenate. Isosafrole is not an agonist for the Ah receptor, but it selectively induces CYP1A2 (Ryan et al., 1980). Poland et al. (1989a,b) suggest that this may be attributable to the high affinity binding of an isosafrole metabolite to the protein, which might inhibit the binding of [125I]-iodo-3,7,8-trichlorodibenzo-pdioxin to CYP1A2 at or near the active site of the enzyme. This does not explain why TCDD, which also has high affinity for CYP1A2, cannot displace some of the isosafrole metabolite from the protein, which should produce enhanced hepatic disposition of TCDD. A more recent study used pretreatment with isosafrole to investigate the role of CYP1A2 in the hepatic disposition of TCDD (Kedderis et al., 1993a). Although isosafrole is an inducer of CYP1A2, it also has high affinity for the protein and thus is a selective inhibitor of CYP1A2. A greater than threefold decrease in the hepatic disposition of TCDD in isosafrole-pretreated rats supports the conclusion that TCDD is bound to CYP1A2 in the liver. December 2003 1-26 DRAFT--DO NOT CITE OR QUOTE Olson et al. (1994) have reported the results of in-vitro studies with isolated hepatocytes and liver slices of rats pretreated with TCDD. Pretreatment increased the uptake of TCDD but not TCDF. There was an increase in both CYP1A1 and CYP1A2. The authors interpret these results as consistent with CYP1A2 being a hepatic binding protein for TCDD but not TCDF. Voorman and Aust (1987, 1989) support further the hypothesis that CYP1A2 is the TCDD-inducible hepatic binding species. These investigators found that 3,3',4,4',5,5'-HxBB, an agonist for the Ah receptor, was associated only with CYP1A2 through the immunoprecipitation of CYP1A1 and 1A2, which were induced in 3,3',4,4',5,5'-HxBB-treated rats. In addition, they found that 3,3',4,4',5,5'-HxBB inhibited estradiol 2-hydroxylase activity of purified CYP1A2. A similar association of PAHs with immunoprecipitated CYP1A2 was observed for other agonists for the Ah receptor, including TCDD, 3,3',4,4'-TCB, 3,3',4,4',5-PeCB, and 3,3',4,4',5,5'-HxCB. The association of TCDD with CYP1A2 occurred within 2 minutes, with maximum inhibition of estradiol 2-hydroxylase occurring at a concentration comparable to the concentration of the enzyme (50 nm). CYP1A2 was inhibited (complexed) by TCDD with nearly 1:1 stoichiometry, and the K for TCDD was calculated to be 8 nM. Therefore, TCDD can be considered a higher binding inhibitor of CYP1A2. Santostefano et al. (1996) assessed the subcellular and tissue-specific disposition of TCDD in rats and mice. TCDD was equally distributed between the hepatic P9 (mitochondrial, lysosomal, and nuclear) and S9 (cytosol and microsomal) fractions, with the microsomal fraction retaining the TCDD present in the S9 fraction. In contrast, TCDD was retained in the P9 fractions of lung and liver at all doses tested. The lack of pulmonary or renal sequestration coupled with the lack of localization of TCDD to pulmonary and renal microsomes supports the role of CYP1A2 as a hepatic microsomal binding protein involved in the hepatic sequestration of TCDD. Recently, Santostefano et al. (1999) assessed the intralobular hepatic distribution of TCDD in rats and observed that centrilobular hepatocytes had a 2.7- to 4.5-fold higher concentration of TCDD than did periportal hepatocytes. The enhanced centrilobular distribution of TCDD was associated with elevated CYP1A2-mediated MROD activity and CYP1A2 mRNA in centrilobular hepatocytes. The TCDD-induced binding species was found to have an apparent equilibrium dissociation constant, KD, for [125I]-2-iodo-3,7,8-trichlorodibenzo-p-dioxin of 56 16 nM, and a pool size, Bmax, of 225 nmol/g of liver in C57BL/6J mice (Poland et al., 1989b). The induced microsomal binding species has an affinity about 104times less than the Ah receptor but a pool size that is -2 x 103greater. Thus, agonists for the Ah receptor may significantly affect their disposition through a dose-related enhancement of hepatic uptake, which should correlate with induction of CYP1A2. December 2003 1-27 DRAFT--DO NOT CITE OR QUOTE More recently, Diliberto et al. (1997) used transgenic mice lacking the CYP1A2 gene to study the influence of CYP1A2 in the hepatic sequestration and distribution of TCDD, 2,3,4,7,8PeCDF, and PCB 153 (2,2',4,4',5,5'-HexCB), a non-dioxin-like PCB. The liver/fat concentration ratios of these compounds in the parental lineage (C57BL6N and 129/Sv) were approximately 3.6, 18, and 0.07, respectively, indicating a high degree of hepatic sequestration for TCDD and 2.3.4.7.8- PeCDF. Under identical exposure conditions, the 1A2 knockout mice had liver/fat concentration ratios of 0.17, 0.34, and 0.10, respectively. Thus, in the absence of the CYP1A2 gene, mice exhibited no hepatic sequestration of these compounds. This study and a related study in CYP1A2 knockout mice by Diliberto et al. (1999) provide direct confirmation of the hypothesis that CYP1A2 is the dioxin-inducible hepatic binding protein responsible for the hepatic sequestration of TCDD and related compounds, such as 2,3,4,7,8-PeCDF. The disposition and pharmacokinetics of 2,2',4,4',5,5'-HxCB and -HxBB have been investigated in several species (Tuey and Matthews, 1980; Lutz et al., 1984). These lipophilic compounds are similar to TCDD in that they are slowly metabolized and that metabolism is required for urinary and biliary elimination; however, they do not exhibit dioxin-like activity. 2,2',4,4',5,5'-HxCB and -HxBB distribute primarily to adipose tissue, with partition coefficients (tissue/blood ratio) ranging from 300 to 500 in the mouse, rat, monkey, dog, and human. The liver is not a major site for the disposition of 2,2',4,4',5,5'-HxCB and -HxBB, in contrast to TCDD and related compounds. Partition coefficients in the liver range from 10 to 30 in these species. 2,2',4,4',5,5'-HxCB and -HxBB do not induce hepatic CYP1A1 or 1A2 and do not exhibit dioxin-like activity. The lack of induction of hepatic CYP1A2 may explain the lack of a dose-dependent hepatic disposition of these compounds. Kedderis et al. (1991b) assessed the dose-response relationship for the induction of hepatic CYP1A1 and 1A2 in male Fischer 344 rats exposed to 2,3,7,8-TBDD at doses as low as 0.1 nmol/kg. They reported that induction of P-4501A2 by 2,3,7,8-TBDD appeared to be a more sensitive response than P-4501A1 induction over the dose range studied. In addition, comparison of hepatic P-4501A2 levels and liver:adipose tissue concentration ratios suggested that induction of P-4501A2 alone would not directly account for the preferential hepatic accumulation of 2,3,7,8-TBDD and that additional factors must be involved. One explanation may be that at low 2,3,7,8-TBDD concentrations, endogenous substrates bind to CYP1A2, not allowing 2,3,7,8-TBDD to be sequestered by the protein (Kedderis et al., 1993b). At higher 2.3.7.8- TBDD concentrations, new protein is formed and 2,3,7,8-TBDD can compete for binding to CYP1A2, resulting in the increased hepatic disposition observed at higher exposures of 2.3.7.8- TBDD (Kedderis et al., 1991b). The structure-activity relationship for the disposition and hepatic sequestration of CDDs, CDFs, and PCBs was investigated by DeVito et al. (1998). Female B6C3F1 mice were treated December 2003 1-28 DRAFT--DO NOT CITE OR QUOTE for 13 weeks with different oral doses of 2,3,7,8-TCDD, 1,2,3,7,8-PeCDD, 2,3,7,8-TBDD, 2.3.7.8- TCDF, 1,2,3,7,8-PeCDF, 2,3,4,7,8-PeCDF, OCDF, PCB126 (3,3',4,4',5-PeCB), PCB169 (3,3'4,4',5,5'HxCB), PCB105 (2,3,3',4,4' PeCB), PCB118 (2,3',4,4',5-PeCB), and PCB156 (2,3,3',4,4',5-HxCB). All of these compounds exhibited dose-dependent increases in the liver/fat concentration except PCBs 105, 118, and 156. 4-PeCDF, PeCDD, OCDF, TCDF, and PCB126 were sequestered in hepatic tissue to a greater extent than was TCDD. Together, the results support the presence of an inducible protein (CYP1A2) and the congener-specific binding of some dioxin-like compounds to this hepatic sequestration protein. Other factors may also regulate the intracellular distribution of TCDD and related compounds. The possible role of hepatic lipoproteins as intracellular carriers in the transport of TCDD has been assessed by in vitro and in vivo studies (Soues et al., 1989a,b). TCDD and 2.3.7.8- TCDF were bound to lipoproteins in mouse and rat liver, which subsequently underwent rapid and pronounced degradative processing, possibly catalyzed by lipoprotein lipase, to heavier entities. The in vitro incubation of TCDD-lipoprotein complex with separated Ah receptor demonstrated that a passive transfer occurred. The authors suggest a carrier role for lipoproteins in the intracellular transport of TCDD and related compounds. 1.3. METABOLISM AND EXCRETION Although early in vivo and in vitro investigations were unable to detect the metabolism of TCDD (Vinopal and Casida, 1973; van Miller et al., 1976), there is evidence that a wide range of mammalian and aquatic species are capable of biotransforming TCDD to polar metabolites (Ramsey et al., 1979, 1982; Poiger and Schlatter, 1979; Olson et al., 1980; Olson, 1986; Gasiewicz et al., 1983a; Poiger et al., 1982; Sijm et al., 1990; Kleeman et al., 1986a,b, 1988). Although metabolites of TCDD have not been directly identified in humans, recent data regarding feces samples from humans in a self-dosing experiment suggest that humans can metabolize TCDD (Wendling et al., 1990). Table 1-9 summarizes data on the metabolism and excretion of TCDD and related compounds after exposure to a single radiolabeled congener. Investigations of TCDD in rats, mice, guinea pigs, and hamsters found that >90% of the radiolabeled material excreted in urine and bile represented polar metabolites. Similar results were also observed for other congeners (see Table 1-9), with the exception of OCDD; however, studies were often limited to the rat. OCDD is apparently not metabolized by the rat, or metabolized to a very minimal extent (Birnbaum and Couture, 1988). For all of the congeners in Table 1-9, essentially all of the CDD, BDD, CDF, or PCB-derived radioactivity in liver, adipose tissue, and other tissues represented parent compound, suggesting that metabolites of these compounds were readily excreted. Thus, with the exception of OCDD, the metabolism of TCDD and related compounds is required for December 2003 1-29 DRAFT--DO NOT CITE OR QUOTE urinary and biliary elimination and therefore plays a major role in regulating the rate of excretion of these compounds. In addition, direct intestinal excretion of parent compound is another route for excretion of TCDD and related compounds that is not regulated by metabolism. 1.3.1. Structure of Metabolites Several metabolites of TCDD have recently been identified. Sawahata et al. (1982) investigated the in vitro metabolism of TCDD in isolated rat hepatocytes. The major product was deconjugated with $-glucuronidase, derivatized with diazomethane, and separated into two compounds by high-performance liquid chromatography. These metabolites were subsequently identified as 1-hydroxy-2,3,7,8-TCDD and 8-hydroxy-2,3,7-trichlorodibenzo--p-dioxin. Poiger et al. (1982) identified six metabolites in the bile of dogs that were given a lethal dose of [3H]-2,3,7,8-TCDD. The major metabolite was 1,3,7,8-tetrachloro-2-hydroxydibenzo-p-dioxin; however, 3,7,8-trichloro-3-hydroxydibenzo-p-dioxin and 1,2-dichloro-4,5-hydroxybenzene were identified as minor metabolites. The structures of the three remaining metabolites were not determined; however, two appeared to be trichlorohydroxydibenzo-p-dioxins and the third was apparently a chlorinated 2-hydroxydiphenyl ether. Poiger and Buser (1984) reported differences in the relative amounts of various TCDD metabolites in dog and rat bile. Trichlorodihydroxydibenzo-p-dioxin and tetrachlorodihydroxydiphenyl ether appear to be major metabolites in rat bile. Furthermore, conjugates, presumably glucuronides, were formed in the rat but not in the dog. The investigators also observed a generally higher metabolism rate of TCDD in the dog. This finding is in good agreement with the unique ability of the dog to readily metabolize persistent PCBs such as 2,4,5,2',4',5'-HxCB (Sipes et al., 1982). Biliary metabolites of 2,3,7,8-TCDF have been investigated by Poiger et al. (1984); however, unequivocal structure assignment of the metabolites could not be made using gas chromatography/mass spectroscopy. With the use of synthetic standards and GC/MS, Burka et al. (1990) identified 4-hydroxy-2,3,7,8-TCDF and 3-hydroxy-2,7,8-TCDF as major biliary metabolites of 2,3,7,8-TCDF in rats. Small amounts of 3-hydroxy-2,4,7,8-TCDF and 2,2'-dihydroxy-4,4',5,5'-TCB were also detected. 4-Hydroxy-2,3,7,8-TCDF was also the major TCDF metabolite formed by hepatic microsomes from TCDD-pretreated rats (Tai et al., 1993). This suggests that the preferred site of metabolism of 2,3,7,8-TCDF is near the furan oxygen, with oxygenation at C4 predominating over C3. The authors speculate that epoxidation of the C4-C4a bond or the C3-C4 bond could lead to formation of the above metabolites. The results of Burka et al. (1990) and Sawahata et al. (1982) suggest that oxygenation of the unsubstituted carbon nearest the bridging oxygen in both 2,3,7,8-TCDF and TCDD is the major route of metabolism of these compounds in the rat. Furthermore, data on the rate of elimination of these compounds summarized in Tables 1-6 and 1-8 indicate that this reaction occurs at a faster rate for December 2003 1-30 DRAFT--DO NOT CITE OR QUOTE the furan, since the rate of urinary and biliary elimination and resulting persistence of these compounds depend on metabolism. Data summarized in Tables 1-6 and 1-9 indicate that 1,2,3,7,8-PeCDF is metabolized and eliminated at a greater rate than 2,3,4,7,8-PeCDF. The preference for oxygenation at C4 in 2,3,7,8-TCDF offers an explanation for the observation that 2,3,4,7,8-PeCDF is metabolized at a much slower rate than 1,2,3,7,8-PeCDF because one of the preferred sites for metabolism is blocked in the 2,3,4,7,8-substituted compound. The rate of metabolism of these compounds and their resulting relative persistence in rodents correlate with analysis of human tissues from the Yusho cohort, where the relative concentrations were 2,3,4,7,8-PeCDF > 1,2,3,7,8PeCDF > 2,3,7,8-TCDF (Masuda et al., 1985). Pluess et al. (1987) investigated the structure of 1,2,3,7,8-PeCDF metabolites in rat bile. A dihydroxy-tetra-CDF was identified as the major metabolite. The authors propose that this compound could be formed either via further oxidation of the hydroxy-tetra-CDF or possibly via hydrolytic dechlorination of a hydroxy-penta-CDF. Minor metabolites include dihydroxy-triCDF, hydroxy-tetra-CDF, and hydroxy-penta-CPF. Pluess et al. (1987) also investigated the metabolites of 2,3,4,7,8-PeCDF in rat bile. A total of 10 metabolites were detected, with dihydroxy-penta-CB and hydroxy-penta-CDF representing the major metabolites. The biphenyl metabolite indicates that cleavage of the ether bridge of the furan is an important pathway for metabolism of this congener. Other less abundant metabolites of 2,3,4,7,8-PeCDF include hydroxy-tetra-CDF, dihydroxy-tri-CDF, dihydroxy-tetraCDF, and thio-tetra-CDF. Sulfur-containing metabolites were also identified as minor metabolites of 2,3,7,8-TCDF and 1,2,3,7,8-PeCDF in rats (Kuroki et al., 1990). These sulfurcontaining metabolites probably arise from CDF-glutathione conjugates. In another study, a dihydroxy-PeCDF was identified as the only detectable biliary metabolite of 1,2,3,6,7,8-HxCDF, while no metabolites of 1,2,3,4,6,7,8-HpCDF were detected in the bile of rats treated with this congener (Poiger et al., 1989). Several in vivo and in vitro studies have investigated the metabolism of 3,3',4,4'-TCB. Rat feces were found to contain 5-hydroxy-3,3',4,4'-TCB and 4-hydroxy-3,3',4',5-TCB as major metabolites (Yoshimura et al., 1987) and 2,5-dihydroxy-3,3',4,4'-TCB, 4,4'-dihydroxy-3,3',5,5'TCB, 5,6-dihydroxy-3,3',4,4'-TCB, 4-hydroxy-3,3',4-TCB, and 4-hydroxy-4,,5'-epoxy-3,3,,4',5TCB as minor metabolites (Koga et al., 1989). Mouse feces were found to contain 5-hydroxy- and 6-hydroxy-3,3',4,4'-TCB and 4-hydroxy-3,3',4',5-TCB, whereas urine contained 2-hydroxy-3,3',4,4'-TCB in addition to these metabolites (Wehler et al., 1989). 3,3',4,4'-TCB was the major compound present in mouse liver and a minor portion was due to 4-hydroxy-3,3',4,4'-TCB (Wehler et al., 1989). Darnerud et al. (1986) found 2-hydroxy-3,3',4,4'-TCB and methylsulphonyl-TCB as major metabolites in the December 2003 1-31 DRAFT--DO NOT CITE OR QUOTE mouse fetus. Sulfur-containing metabolites of non-co-planar, nondioxin-like PCBs have also been reported to accumulate in the bronchial mucosa and uterine luminal fluid of mice (Bergman et al., 1979; Brandt et al., 1982) and in human lung, liver, and adipose tissue (Haraguchi et al., 1986, 1989). PCB methyl sulfones are stable lipophilic metabolites formed by the mercapturic acid pathway. The toxicological significance of these metabolites of nondioxin-like PCBs remains generally unknown. 1.3.2. Toxicity of Metabolites The discussion above indicates that metabolism of TCDD and related compounds is required for urinary and biliary elimination and thus plays a major role in regulating the rate of excretion of these compounds. At present, metabolism is also generally considered a detoxification process. Data on the metabolism of TCDD suggest that reactive epoxide intermediates may be formed. Poland and Glover (1970) investigated the in vivo binding of [1,6-3H]-2,3,7,8-TCDDderived radioactivity to rat hepatic macromolecules and found maximum levels equivalent to 60 pmol of TCDD/mol of nucleotide in RNA and 6 pmol of TCDD/mol of nucleotide in DNA. This corresponds to one TCDD-DNA adduct per 35 cells. These investigators suggest that it is unlikely that TCDD-induced oncogenesis is through a mechanism of covalent binding to DNA and somatic mutation. Further studies of TCDD and related compounds are needed to confirm these results and assess the relationship between covalent binding and the short- and long-term toxicity of these compounds. Weber et al. (1982a) investigated the toxicity of TCDD metabolites by administering extracts of bile from TCDD-treated dogs to male guinea pigs in single oral doses equivalent to 0.6, 6.0, and 60 :g/kg of parent compound. Other groups of guinea pigs were given bile extract from untreated dogs or TCDD itself. A comparison of the mortality data at 5 weeks after dosing indicated that the acute toxicity of TCDD to guinea pigs was at least 100 times higher than the acute toxicity of its metabolites. Mason and Safe (1986) synthesized 2-hydroxy-3,7,8-TCDD and 2-hydroxy-1,3,7,8TCDD, which are metabolites of TCDD, and assessed the toxicity of these compounds in male Wistar rats. The compounds produced no significant effect on body weight gain and thymus, liver, or spleen weights after exposure to a dose of #5,000 :g/kg bw. 2-Hydroxy-3,7,8-TCDD induced hepatic microsomal AHH, EROD, and 4-chlorobiphenylhydroxylase activity at exposures of 1,000 and 5,000 :g/kg bw, whereas 2-hydroxy-1,3,7,8-TCDD was inactive as an inducer. Thus, while 2-hydroxy-3,7,8-TCDD has dioxin-like activity as an inducer of the hepatic monooxygenase system, the potency of the metabolite is more than three orders of magnitude December 2003 1-32 DRAFT--DO NOT CITE OR QUOTE less than that of TCDD. Furthermore, results are consistent with the expected rapid conjugation and excretion of these TCDD metabolites (Weber et al., 1982b). Metabolism of co-planar PCBs and PBBs also appears to be a detoxification process. 5-Hydroxy-3,3',4,4'-TCB and 4-hydroxy-3,3',4',5-TCB did not produce liver hypertrophy, induction of hepatic AHH or DT-diaphorase activities, or thymus atrophy (Yoshimura et al., 1987). Thus, monohydroxy metabolites of 3,3',4,4'-TCB are much less toxic than the parent compound. Further evidence for metabolism as a detoxification process comes from comparison of the metabolism and toxicity of two co-planar PBBs. Millis et al. (1985) found that 3,3',4,4',5,5'-HxBB exhibited greater toxic potency in rats than 3,3',4,4'-TBB, even though 3,3',4,4'-TBB had about a 10-fold greater affinity for the Ah receptor. Although receptor binding affinities imply that 3,3'4,4'-TBB should be more toxic than 3,3',4,4'5,5'-HxBB, it was less toxic than the HxBB because 3,3',4,4'-TBB was metabolized at a much greater rate than 3,3',4,4',5,5'-HxBB. In addition to supporting metabolism as a detoxification process, the results of Millis et al. (1985) also suggest that receptor binding and in vitro AHH induction do not accurately reflect toxicity for PAHs, which are more readily metabolized, presumably because continued occupation of the receptor is required for toxicity. Structure-activity studies of TCDD and related compounds support the widely accepted principle that this parent compound is the active species. The relative lack of activity of readily excreted monohydroxylated metabolites of TCDD and 3,3'4,4'-TCB suggests that metabolism is a detoxification process necessary for the biliary and urinary excretion of these compounds. This concept has also been generally applied to TCDD-related compounds, although data are lacking on the structure and toxicity of metabolites of other CDDs, BDDs, CDFs, BDFs, PCBs, and PBBs. It is possible that low levels of unextractable and unidentified metabolites may contribute to one or more of the toxic responses of TCDD and related compounds. Further studies on the nature of the biotransformation products of these compounds will help address this uncertainty. 1.3.3. Autoinduction of Metabolism Accurate rate constants for metabolism are important in developing pharmacokinetic models that describe the disposition of TCDD and related compounds. Metabolism plays a major role in regulating the excretion and relative persistence of these compounds because metabolism is required for urinary and biliary excretion. Although the relative rate of metabolism of TCDD and related compounds can be estimated from tissue and excretion half-life data (see Tables 1-6 and 1-9), other factors such as relative body composition, hepatic and extrahepatic binding proteins, and direct intestinal elimination of the parent compound can also regulate the excretion of TCDD and related compounds. Therefore, in vivo disposition data (see December 2003 1-33 DRAFT--DO NOT CITE OR QUOTE Tables 1-6 and 1-9) provide only a limited approximation of the relative rate of metabolism of a specific congener in a given species. In vivo disposition data were also obtained at exposures that were associated with induction of CYP1A1 and 1A2 and other potentially adverse responses that could alter metabolism and disposition. Therefore, it may not be appropriate to directly extrapolate these data to predict the pharmacokinetics at low levels of exposure. Low-dose extrapolations can be assisted by assessments of the potential for autoinduction of metabolism that may occur at exposures associated with enzyme induction. Characterization of the dosedependent disposition of TCDD and related compounds is particularly important in the extrapolation of high-exposure animal data to low-exposure human data. The excretion of metabolites of TCDD and related compounds into bile represents a direct means for estimating the rate of metabolism because biliary elimination depends on metabolism and is the major route for excretion of these compounds. The rate of metabolism of CDFs was estimated from the relative abundance of metabolites in rat bile (Poiger et al., 1989). The rates of biotransformation of 2,3,7,8-TCDF, 1,2,3,7,8-PeCDF, 2,3,4,7,8-PeCDF, and 1,2,3,6,7,8-HxCDF were characterized as fairly high, moderate, low, and very low, respectively. Kedderis et al. (1991b, 1993a) observed 10% of the dose of [3H]-2,3,7,8-TBDD excreted in bile 5 hours after intravenous administration of 1 nmol/kg to male Fischer 344 rats. All biliary radioactivity was attributable to metabolites. This rate of elimination is similar to the fecal excretion (-8% of the dose) 24 hours after intravenous administration of 1 nmol/kg [3H]-2,3,7,8TBDD (Kedderis et al., 1991a) and reflects the effect of intravenous bolus versus oral administration on distribution and elimination. The large percent dose excreted within the first few days may also be due to a rapidly excreted impurity in the radiolabeled 2,3,7,8-TBDD (Kedderis et al., 1993a). To assess the ability of TCDD and 2,3,7,8-TBDD to induce their own metabolism (biliary elimination), rats were pretreated with 100 nmol/kg, per of, of each compound 3 days prior to intravenous injection of 1 nmol/kg of the respective [3H]-congeners. Biliary excretion of the radiolabeled dose was quantitatively and qualitatively unaffected by pretreatment, despite a twofold increase in hepatic levels of [3H] in the pretreated animals and significant induction of CYP1A1 and 1A2 (Kedderis et al., 1991b). Therefore, under these conditions, autoinduction of TCDD and 2,3,7,8-TBDD metabolism did not occur in the rat in vivo at doses that elicited enhanced hepatic uptake. Similarly, Curtis et al. (1990) observed no change, or even an apparent decrease, in gastrointestinal contents and fecal elimination of TCDD equivalents in pretreated versus naive mice 24 hours after oral administration of [14C]-2,3,7,8TCDD, despite significantly enhanced levels of TCDD in the livers of pretreated mice. Although the above studies suggest that autoinduction of TCDD metabolism does not occur, other results indicate that metabolism may be induced under certain conditions. Poiger and Buser (1984) observed a small yet significant increase in biliary excretion over a 72-hour December 2003 1-34 DRAFT--DO NOT CITE OR QUOTE period, with pretreated rats (10 pg/kg, intraperitoneal) excreting 9.71.9% of the radiolabeled dose of TCDD (200-300 pg/kg, per of) compared with 7.00.9% excreted by naive animals. In addition to being small changes, these results were obtained using a dose of TCDD in excess of the LD50in the rat. Poiger and Schlatter (1985) examined the influence of pretreatment with phenobarbital and TCDD on the biliary excretion of [3H]-2,3,7,8-TCDD metabolites in a dog given a single oral dose of the [3H]-congener (31 or 33.8 ng/kg). Without pretreatment, 24.5% of the absorbed dose was excreted in the bile within 110 hours. Phenobarbital did not alter this rate, whereas pretreatment with TCDD (10 pg/kg) 9 days earlier resulted in a doubling of the amount of metabolites excreted in bile (47.4%). Although this observation is limited to one dog and requires further investigation, the results suggest that significant autoinduction of TCDD metabolism and biliary excretion may occur in the dog. Nonetheless, the small increase in metabolism and biliary excretion of TCDD in the rat observed by Poiger and Buser (1984) and the negative results of Kedderis et al. (1991b; 1993a) and Curtis et al. (1990) suggest that autoinduction of TCDD metabolism and biliary excretion in the rat may not occur or occurs to an extent that is not biologically relevant. More recently, Jackson et al. (1998) investigated the effects of age, sex, and pretreatments with phenobarbital, dexamethasone, and 1-aminobezotriazole (a CYP450 inhibitor) to modulate the rate of metabolism-dependent biliary elimination of TCDD in F344 rats. Biliary excretion of TCDD-derived radioactivity was measured over a 6-hour period following iv administration. Male adult and juvenile rats and female juvenile rats excreted from 0.63% to 0.68% of the administered dose, while adult females, male senescent, and male adults pretreated with the above drugs excreted from 0.28% to 0.45% of the dose of TCDD in bile. The results suggest some variability in the metabolism-dependent biliary excretion of TCDD-derived radioactivity; however, the differences between groups were not great. Limited data suggest that autoinduction of metabolism and biliary excretion does occur for CDFs. Pretreatment of rats with 2,3,7,8-TCDF (1.0 pmol/kg, 3 days earlier) significantly increased the biliary excretion of a subsequent dose of [14C]-2,3,7,8-TCDF (McKinley et al., 1993). The naive rats excreted 5.692.35% of the dose over the initial 8 hours, while the pretreated rats excreted 13.183.15% of the [14C]-2,3,7,8-TCDF. Similarly, pretreatment of rats with 2,3,4,7,8-PeCDF (500 pg/kg, per of, 3 days earlier) resulted in a twofold increase in the biliary elimination of a subsequent dose of [14C]-2,3,4,7,8-PeCDF (Brewster and Birnbaum, 1987). These results suggest that pretreatment with 2,3,7,8-TCDF and 2,3,4,7,8-PeCDF induces the metabolism of these congeners. 3,3',4,4'-TCB and 3,3',4,4'-TBB appear to be metabolized by a 3-methylcholanthreneinducible form of hepatic CYP(1A1 or 1A2), which is also induced by 3,3',4,4'-TCB (Shimada December 2003 1-35 DRAFT--DO NOT CITE OR QUOTE and Sawabe, 1983; Mills et al., 1985; McKinley et al., 1993). This suggests that these compounds can induce their own rate of metabolism and subsequent excretion. Isolated hepatocytes in suspension culture have been demonstrated to provide a useful in vitro system for studying the hepatic metabolism of TCDD under the same conditions in species that have a wide range of sensitivity to the compound (Olson et al., 1981). The in vitro rate of metabolism of TCDD in guinea pig, rat, C57BL/6J mouse, DBA/2J mouse, and hamster hepatocytes was estimated to be 0.2, 1.2, 1.1, 0.9, and 1.2 pmol/mg protein/hour, respectively (Wroblewski and Olson, 1985, 1988; Shen and Olson, 1987). These results indicate that TCDD is metabolized by the guinea pig liver at a rate about fivefold less than that observed for the rat, mouse, and hamster. The limited ability of the guinea pig to metabolize TCDD can explain the limited excretion of TCDD metabolites in feces, which represents the major route for TCDD excretion (Olson, 1986). In addition, the limited metabolism in the guinea pig may partly explain the relatively long excretion half-life for TCDD in the guinea pig and may contribute to the remarkable sensitivity of the guinea pig to the acute toxicity of this agent (Olson, 1986). Isolated hepatocytes in suspension culture have been used as an in vitro system for studying the autoinduction of metabolism of TCDD and related compounds. Wroblewski and Olson (1988) investigated the metabolism of [14C]-2,3,7,8-TCDD (2.2 :M ) in hepatocytes isolated from untreated TCDD-, 3-MC-, isosafrole-, and phenobarbital-pretreated rats and hamsters. In both species, TCDD and 3-MC pretreatments elevated the rate of TCDD metabolism by five- to sixfold, while phenobarbital pretreatment had no effect. Isosafrole produced a 1.8- to 2.5-fold increase in metabolism. These in vitro results at a high substrate concentration (2.2 :M ) indicate that TCDD can induce its own rate of metabolism in the rat and hamster. In contrast, TCDD was not able to induce its own rate of metabolism in guinea pig and mouse hepatocytes (Wroblewski and Olson, 1985; Shen and Olson, 1987). Together, these results indicate that TCDD is metabolized in the liver by a TCDD-inducible enzyme, which is expressed in the rat and hamster but not in the guinea pig and mouse. More recently, the kinetics of TCDD metabolism were investigated in isolated rat hepatocytes incubated with [3H]-2,3,7,8TCDD at concentrations of 0.01, 0.1, and 1.0 :M (Olson et al., 1994). Lower TCDD concentrations in the media result in concentrations in hepatocytes that are more similar to the levels in the liver after in vivo exposure. For example, the concentration of TCDD in hepatocytes incubated at 0.01 :M is similar to hepatic levels after in vivo exposure of rats at a dose of -10 :g/kg. At 0.01 and 0.1 :M , the rate of metabolism of [3H]-2,3,7,8-TCDD was December 2003 1-36 DRAFT--DO NOT CITE OR QUOTE similar in hepatocytes isolated from control and TCDD-pretreated rats, whereas at 1.0 :M , [3H]2,3,7,8-TCDD metabolism was greater in hepatocytes isolated from TCDD-pretreated rats. The results indicate that TCDD can induce its own rate of metabolism in the rat, but only at high hepatic concentrations, which are generally not attained after in vivo exposure. Therefore, in vitro studies of the hepatic metabolism of TCDD (at 0.01 and 0.1 :M ) are consistent with the lack of autoinduction of TCDD metabolism and biliary excretion observed in vivo in the rat (Kedderis et al., 1991b; Curtis et al., 1990). The metabolism of [3H]-2,3,7,8-TCDF was also investigated in isolated rat hepatocytes incubated at concentrations of 0.01, 0.1, and 1.0 :M (Olson et al., 1994). At all concentrations, hepatocytes from TCDD-pretreated rats metabolized 2,3,7,8-TCDF at a rate from 4- to 25-fold greater than that observed in hepatocytes from control rats. The results indicate that 2,3,7,8TCDF is metabolized in rat liver by a TCDD-inducible enzyme, possibly CYP1A1 or 1A2. These in vitro results support the in vivo autoinduction of 2,3,7,8-TCDF metabolism and biliary elimination observed in the rat (McKinley et al., 1993). 2,3,7,8-TCDF metabolism was also investigated in rat liver, kidney, and lung microsomes in the presence and absence of selective chemical inhibitors and antibodies to CYP1A1 and CYP1A2 (Tai et al., 1993). Together, the results of this investigation indicate that CYP1A1 is the primary enzyme responsible for the metabolism of 2,3,7,8-TCDF. 2,3,7,8-TCDF was also metabolized by recombinant yeast microsomes expressing human CYP1A1 and reductase. However, based on EROD activity, a marker of CYP1A1, the relative rate of 2,3,7,8-TCDF metabolism was about 100-fold greater in TCDD-induced rat liver microsomes than in yeast microsomes expressing human CYP1A1 and reductase (Tai et al., 1993). Although 2,3,7,8TCDF was metabolized by rat and human CYP1A1, the results indicated that there are marked quantitative differences in metabolism that suggest that 2,3,7,8-TCDF will be more persistent in humans. In summary, there are in vivo and in vitro data suggesting that autoinduction of TCDD and 2,3,7,8-TBDD metabolism does not occur in the rat after exposure to sublethal doses of these agents. This is in contrast to 2,3,7,8-TCDF and 2,3,4,7,8-PeCDF, where in vivo and in vitro results support the autoinduction of metabolism and biliary elimination of these compounds in the rat. 1.3.4. Excretion in Animals Data regarding the excretion of 2,3,4,7-TCDD and related compounds after exposure to a single radiolabeled congener (see Table 1-9) support the assumption of a first-order elimination process consisting of one or more components. These studies show that TCDD was excreted slowly from all species tested, with half-lives ranging from 11 days in the hamster to 2,120 days December 2003 1-37 DRAFT--DO NOT CITE OR QUOTE in humans. TCDD is exceptionally persistent in humans relative to other animal models. Elimination data in tissues (see Tables 1-6 and 1-7) also indicate that TCDD and related compounds are exceptionally persistent in nonhuman primates (Bowman et al., 1989; Neubert et al., 1990). These differences may also be in part related to the dose dependency of the excretion of these compounds. In general, the congener- and species-specific rates of elimination of TCDD and related compounds from major tissue depots (see Table 1-6) are similar to the excretion data summarized in Table 1-9. In the Syrian Golden hamster, the mammalian species least sensitive to the acute toxicity of TCDD, excretion occurred readily through both the urine (35% of administered dose, 41% of total excreted radioactivity) and feces (50% of the administered dose, 59% of total excreted radioactivity) (Olson et al., 1980). A similar excretion pattern, with significant urinary elimination, was observed in mice, although there was significant strain variability (Gasiewicz et al., 1983b; Birnbaum, 1986). In all the other species, excretion occurred mainly through the feces, with relatively minor amounts of TCDD metabolites found in the urine (Piper et al., 1973; Allen et al., 1975; Olson, 1986; Rose et al., 1976; Gasiewicz and Neal, 1979; Pohjanvirta et al., 1990). Results in Table 1-9 also indicate that fecal elimination was the primary route for the excretion of 1,2,3,7,8-PeCDD, OCDD, 2,3,7,8-TBDD, 2,3,7,8-TCDF, 1,2,3,7,8-PeCDF, 2.3.4.7.8- PeCDF, and 3,3',4,4'-TCB. Only Piper et al. (1973) reported the excretion of metabolites in the expired air. During 21 days following administration of a single oral dose of [14C]-2,3,7,8-TCDD to rats, 3.2% of the administered radioactivity (4.6% of the excreted radioactivity) was recovered in the expired air. Studies in the rat, guinea pig, hamster, and mouse have found that essentially all of the TCDD-derived radioactivity excreted in the urine and bile corresponds to metabolites of TCDD (see Table 1-9). The apparent absence of TCDD metabolites in liver and fat suggests that, once formed, the metabolites of TCDD are excreted readily. Thus, urinary and biliary elimination of TCDD depends on metabolism of the toxin. The more limited data for other compounds also suggest that this relationship may be true for 1,2,3,7,8-PeCDD, 2,3,7,8-TBDD, 2,3,7,8-TCDF, 1.2.3.7.8- PeCDF, 2,3,4,7,8-PeCDF, and 3,3',4,4'-TCB (see Table 1-9). Although urine and bile appear to be free of unmetabolized TCDD, data indicate that TCDD and its metabolites are excreted in the feces of guinea pigs, rats, mice, and hamsters treated with [3H]- or [14C]-2,3,7,8-TCDD (see Table 1-9). Whereas 15% to 35% of the TCDDderived radioactivity in rat, mouse, and hamster feces represents unchanged TCDD, 81% of the radioactivity in guinea pig feces represents unmetabolized TCDD (Olson, 1986; Neal et al., 1982; Gasiewicz et al., 1983b; Olson et al., 1980). The daily presence of unchanged TCDD in feces and its absence in bile suggest that direct intestinal elimination may be the source for the fecal excretion of TCDD. Data also suggest that direct intestinal elimination of parent compound December 2003 1-38 DRAFT--DO NOT CITE OR QUOTE contributes to the fecal excretion for 2,3,7,8-TBDD (Kedderis et al., 1991a). Direct intestinal elimination of the parent compound may occur for other congeners (see Table 1-9), but this conclusion cannot be made at this time because of the lack of experimental data. Nonetheless, the species-specific fecal excretion of 2,3,7,8-TCDF is very similar to that observed for TCDD, with >90% of the 2,3,7,8-TCDF-derived radioactivity excreted in guinea pig feces representing parent compound (Decad et al., 1981a). In addition, the excretion of unchanged CDDs and CDFs was detected in rat feces after subcutaneous exposure to a defined mixture of congeners (Abraham et al., 1989d). Studies in lactating rats have also found that unchanged TCDD may be excreted in the milk of lactating animals (Moore et al., 1976; Lucier et al., 1975; Nau et al., 1986). Lactation, direct intestinal elimination, and perhaps sebum may serve as routes for excretion of TCDD that do not depend on metabolism of the toxin. These data suggest that the in vivo half-life for elimination of TCDD and related compounds provides only an approximation of the rate of metabolism of these compounds in a given animal. The results in Table 1-9 suggest that 2,3,7,8-TCDF, 1,2,3,7,8-PeCDF, and 3,3',4,4'-TCB are metabolized and excreted more rapidly than 2,3,7,8-TCDD, 2,3,7,8-TBDD, 1,2,3,7,8-PeCDD, 2,3,4,7,8-PeCDF, and OCDD. The rate of excretion of TCDD and related compounds is species- and congener- specific (see Table 1-9). TCDD is most persistent in human and nonhuman primates. In the hamster, the least sensitive species to the acute toxicity of TCDD, the mean t1/2was 10.8 days (Olson et al., 1980), and in the guinea pig, the most sensitive species to the acute toxicity of TCDD, the mean t/ was 94 days (Olson, 1986). 2,3,7,8-TCDF was also most persistent in the guinea pig, with a t/ of 20 to 40 days (Decad et al., 1981a; Ioannou et al., 1983). Furthermore, results indicate that the relatively limited ability of the guinea pig to metabolize TCDD and -TCDF may contribute to the greater persistence and greater acute toxicity of these congeners in the guinea pig. The tissue distribution, metabolism, and excretion of TCDD were also investigated in Han/Wistar and Long-Evans rats, which were, respectively, more resistant (LD50>3,000 pg/kg) versus more susceptible (LD50 -10 pg/kg) to the acute toxicity of TCDD (Pohjanvirta et al., 1990). The results suggest that the metabolism and disposition of TCDD do not have a major role in explaining the strain differences in toxicity. The intraspecies differences in the t/2of TCDD in three mouse strains may be due to the finding that the DBA/2J strain possesses about twofold greater adipose tissue stores than the C57BL/6J and B6D2FX/J strains (Gasiewicz et al., 1983b). The sequestering of the lipophilic toxin in adipose tissue stores of the DBA/2J mouse may contribute to the greater persistence of TCDD in this strain. Birnbaum (1986) examined the effect of genetic background on the distribution and excretion of TCDD in two sets of congenic mouse strains in which the congenic pairs differed only at the Ah locus. The Ah locus had no effect on the tissue distribution or excretion of TCDD. Thus, the distribution and excretion of TCDD were primarily governed by December 2003 1-39 DRAFT--DO NOT CITE OR QUOTE the total genetic background rather than the allele present at the Ah locus. These findings are consistent with the in vitro results of Shen and Olson (1987), who found that the hepatic uptake and metabolism of TCDD did not correlate with genetic differences at the murine Ah locus. However, it is important to note that all of these are relatively high-dose studies, which may not allow for detection of Ah receptor-mediated effects on disposition. Although the dose-related tissue distribution of TCDD and related compounds has been described recently, very limited data are available on the dose-related excretion of these compounds. Rose et al. (1976) investigated the elimination of [14C]-2,3,7,8-TCDD in rats given repeated oral doses of 0.01, 0.1, or 1.0 :g/kg/day Monday through Friday for 7 weeks or a single dose of 1.0 :g/kg. In the single-dose study, no 14C was excreted in the urine or expired air; in the repeated-dose study, however, 3% to 18% of the cumulative dose was excreted in the urine by 7 weeks. This study indicated that steady-state concentrations will be reached in the bodies of rats in -13 weeks. The rate constant defining the approach to steady-state concentrations was independent of the dose of TCDD over the range studied. Relatively small changes in the excretion of 2,3,7,8-TBDD were also observed after exposures at 1 and 100 nmol/kg (Kedderis et al., 1991a). These results are consistent with the in vivo and in vitro evidence suggesting that autoinduction of TCDD and 2,3,7,8-TBDD metabolism does not occur in the rat after exposure to sublethal doses of these compounds (Kedderis et al., 1991b; Curtis et al., 1990; Olson et al., 1994). In contrast to these compounds, 2,3,7,8-TCDF and 2,3,4,7,8-PeCDF can induce their own rate of metabolism and biliary excretion (Brewster and Birnbaum, 1987; McKinley et al., 1993; Olson et al., 1994). Autoinduction of metabolism suggests that these compounds may exhibit dose-related excretion, with longer half-lives for elimination at lower doses, which are not associated with enzyme induction. Further data are needed to test this hypothesis. 1.3.5. Excretion in Humans Poiger and Schlatter (1986) investigated the excretion of TCDD in a 42-year-old man (92 kg) after ingestion of 105 ng (1.14 ng/kg) [3H]-2,3,7,8-TCDD in 6 mL corn oil (see Tables 1-9 and 1-10). The half-life for elimination was estimated to be 2,120 days based on fecal excretion over a 125-day period following the single exposure. The concentration of 3H-TCDD-derived radioactivity was also measured in adipose tissue in the same individual over a 6-year period following exposure. A more accurate estimate of TCDD half-life of 9.7 years was calculated based on adipose tissue concentrations over a 6-year period (Schlatter, 1991). Table 1-10 summarizes additional half-life estimates for TCDD and related compounds in humans, on the basis of serum and adipose tissue concentrations at two or more time points. The Air Force is conducting a 20-year prospective study of veterans of Operation Ranch Hand, the unit responsible for the aerial spraying of herbicides, contaminated with TCDD, in December 2003 1-40 DRAFT--DO NOT CITE OR QUOTE Vietnam from 1962 to 1971. A subset of the Ranch Hand cohort has had a series of up to four serum TCDD analyses conducted to investigate the elimination of TCDD in humans. Initially, the half-life of TCDD in humans was estimated to be -7 years on the basis of TCDD levels in serum samples taken in 1982 and 1987 from 36 of the Ranch Hand personnel who had TCDD levels >10 ppt in 1987 (Pirkle et al., 1989). Subsequently, Wolfe et al. (1994) investigated the half-life of TCDD in an expanded cohort of 337 Air Force veterans of Operation Ranch Hand that also included the 36 subjects of the earlier half-life study by Pirkle et al. (1989). Based on paired TCDD measurements from serum collected in 1982 and in 1987, the authors reported a mean predicted half-life of 11.6 years and a median observed half-life of 11.3 years with a nonparametric 95% confidence interval of 10.0 to 14.1 years. The authors also investigated how the TCDD half-life varied with percent body fat (PBF), relative changes in PBF from 1982 to 1987, and age. They found that the TCDD half-life increased significantly with a high PBF, suggesting that persons with more body fat tend to eliminate TCDD more slowly. In contrast, increasing age was associated with a shorter half-life. The redistribution of fat stores from subcutaneous to abdominal areas with aging, resulting in greater mobilization of TCDD, could in part explain the shorter half-life observed in older veterans. An increase in PBF from 1982 to 1987 was also associated with a decrease in half-life, which can be explained by a dilution of the existing body burden of TCDD into the increasing adipose tissue mass. More recently, Michalek et al. (1996) estimated the half-life of TCDD in 213 veterans of Operation Ranch Hand on the basis of TCDD serum analyses conducted in 1982, 1987, and 1992. Of the 278 subjects with complete data in all 3 years, 213 were included for analysis of half-life 3 on the basis of TCDD levels greater than 22.3 ppt in 1982, >14.9 ppt in 1987, and >10 ppt in 1992. All TCDD levels were background-corrected by subtracting 4 ppt, and the logarithm of the background-corrected levels were modeled as a linear function of time to estimate decay rates using first-order kinetics. Using the Toeplitz assumption, the unadjusted estimated decay rate is 0.0797 per year (95% CI of 0.0727 to 0.0868), giving an unadjusted half life estimate of 8.7 years (95% CI of 8.0 to 9.5 years). The adjusted half-life was found to increase significantly with an increase in PBF in 1982, but the half-life did not vary with age or relative changes in PBF. Most recently, Michalek and Tripathi (1999) estimated the half-life of TCDD in 97 veterans of Operation Ranch Hand on the basis of TCDD serum analyses conducted in 1982, 1987, 1992, and 1997. Of the 244 subjects with complete data at all four time points, only 97 were included for analysis of half-life based on the criteria of TCDD levels greater than 39.5 ppt in 1982, >25.0 ppt in 1987, >15.8 ppt in 1992, and >10 ppt in 1997. With increasing time since the initial exposure to TCDD, a greater proportion of the population was excluded from the analysis, as more subjects approached background body burdens of TCDD. Using the methods of the previous report (Michalek et al., 1996), the unadjusted estimated elimination rate December 2003 1-41 DRAFT--DO NOT CITE OR QUOTE was 0.0915 per year (95% CI of 0.0844 to 0.0986), giving an unadjusted half-life estimate of 7.6 years (95% CI of 7.0 to 8.2 years). Because of the smaller sample size, the current elimination rate estimate, based on four measurements per subject, has less precision than the earlier estimate of Michalek et al. (1996), which was based on three measurements per subject. Once again, the elimination rate decreased slightly but significantly as PBF increased, supporting the hypothesis that individuals with more body fat tend to eliminate TCDD more slowly than those with less body fat. Michalek and Tripathi (1999) also reported no significant change in the elimination rate with age or with relative changes in PBF. The half-life of TCDD has also been investigated in two additional populations. FleschJanys et al. (1996) studied a group of 43 German herbicide plant workers that had initial TCDD serum levels from 15.6 to 300 ppt. A median half-life estimate of 7.2 years was reported for this occupational cohort, which received an initial exposure to TCDD similar to that of the Ranch Hand veterans. A similar half-life estimate of 8.2 years was reported in 27 victims of the accident in Seveso, Italy (Needham et al., 1994). This cohort had a greater initial exposure, resulting in serum levels of 130 to 3,830 ppt TCDD. This study also included the early and later portions of the TCDD decay curve, as the initial blood sampling began immediately following exposure and continued for 15.9 years. Thus, based on results from the Ranch Hand, the German, and the Seveso studies, the estimated half-life of TCDD in humans is from 7.2 to 8.7 years (Table 1-10). Half-life estimates for other CDDs and CDFs have been estimated to range from 0.8 to 19.6 years (Table 1-10). Some of the half-life values in Table 1-10 are rough estimates based on a small number of individuals and analysis at as few as two time points. Phillips (1989) discusses this issue. Estimates also assume a simple, single-compartment, first-order elimination process. In the largest and most comprehensive study, Flesch-Janys et al. (1996) investigated the elimination of 2,3,7,8-chlorine substituted CDDs and CDFs in a cohort of workers from a herbicide-producing plant in Germany (summarized in Table 1-10). The study group consisted of 45 males and 3 females with a mean duration of employment of 13.1 years. Mean time between end of employment and first blood sample was 5.4 years (median 2 years) and mean time between first and last blood sample was 5.6 yr (median 6.3 years). A total of 43 subjects with two serum samples and 5 subjects with three serum samples were included in the study. For each congener, only those subjects whose congener serum levels exceeded 95% of German background concentration were included in the analysis. The mean background concentration was also subtracted from every original measurement before analysis. For 2,3,7,8-TCDF, 1,2,3,7,8-PeCDF and OCDF, no half-life was estimated because no person in the study passed the above inclusion criteria. 2,3,7,8-TCDF and 1,2,3,7,8-PeCDF are excreted in animals much December 2003 1-42 DRAFT--DO NOT CITE OR QUOTE more rapidly than other congeners (van den Berg et al., 1994), suggesting that these congeners may also be excreted more rapidly in humans. Conversely, 2,3,4,7,8-PeCDF is far more persistent in animal models than is TCDD, which supports the estimated 19.6-year half-life of 2.3.4.7.8- PeCDF and 7.2-year half-life of TCDD in humans. However, this estimate was based on only five subjects who met the criteria for inclusion in the study. With the exception of 1.2.3.7.8- PeCDD and 1,2,3,6,7,8-HxCDD, the median half-lives of the CDDs are generally similar. The estimates for these two congeners may be somewhat unstable because of variable individual rate constants for elimination and the fact that about 25% of the population showed no decrease in serum levels over the sampling period. Furthermore, the investigation found that increasing age and PBF were associated with increasing half-life for most congeners. Finally, it is important to note that the half-life data reflect only the elimination of CDDs and CDFs from blood lipid and may not reflect elimination from different storage sites for all congeners. In the case of TCDD, it can be assumed that the half-life estimate reflects elimination from the main storage site, because about 90% of the body burden is sequestered in fat and the blood fat/adipose tissue concentration is about 1 (Patterson et al., 1987; Kahn et al., 1988; Schecter et al., 1990a; van den Berg et al., 1994). Data are more limited on the relative amount of other congeners stored in adipose tissue in humans, and limited and somewhat conflicting data suggest that the blood fat/adipose tissue concentration ratio may increase up to a factor of 2 for OCDD (Schecter et al., 1990a; Gum et al., 1995). Thus, some uncertainties remain regarding the extent that the observed decrease in serum levels of higher CDDs and CDFs reflects the elimination of these compounds from the body. Ryan and Masuda (1991) reported on their continuing investigation into the elimination of CDFs in humans from the Yusho and Yu-Cheng rice oil poisonings. Yu-Cheng individuals had CDF blood levels on a lipid basis of 1-50 :g/kg, whereas Yusho patients had levels of 0.1-5 :g/kg. In the Yu-Cheng individuals, half-lives for three CDFs were 2 to 3 years, whereas elimination from Yusho individuals was more variable and slower, with half-lives >5 years (see Table 1-10) and, in several cases, no measurable elimination during the 7 years in which samples were available. The limited results suggest that clearance of these CDFs in the human is biphasic, with faster elimination at higher exposure. Schecter et al. (1990b) and Ryan and Masuda (1989) also reported longer half-life values for CDFs in humans at later time points after exposure, when concentrations are closer to the background levels of individuals with no unusual exposure. While results from animal studies (summarized in Table 1-9) suggest that direct fecal excretion of unmetabolized CDDs and CDFs represents a significant mechanism for the elimination of these lipophilic compounds, human data have been limited until recently. The mass balance study of Schlummer et al. (1998) provided the experimental human data in support December 2003 1-43 DRAFT--DO NOT CITE OR QUOTE of the two-step model of CDD and CDF transfer in the gastrointestinal tract, where absorption and excretion are distinct processes occurring at the small and large intestine, respectively (see Section 1.1.1.2). Rohde et al. (1999) conducted a digestive tract mass balance study of six German men (age 41 to 73 years) with occupational exposure to CDDs and CDFs. Blood lipid levels of the subjects in 1996 ranged from 84 to 505 pg/g lipid for TCDD and 270 to 640 pg/g lipid for TEQs, compared with background levels in unexposed individuals of 5.2 and 32 pg/g lipid, respectively. The daily quantity of nonmetabolized 2,3,7,8-chlorine-substituted CDDs and CDFs excreted in the feces exceeded the daily uptake from food, indicating significant clearance across the gastrointestinal tract. The concentration of these compounds in feces was also found to be highly correlated with that in blood, demonstrating that the fecal CDD and CDF content was directly related to the body burden of these compounds. No significant clearance (excretion via feces at least fourfold greater than uptake by food) was observed for congeners, including 2,3,7,8-TCDF, 1,2,3,7,8-PeCDF, 1,2,3,4,7,8,9-HepCDF, or OCDF, which were not markedly elevated in the serum lipids. Together, these results support the relationship that fecal excretion is regulated by the lipid-based blood concentration of these compounds. Because of fecal clearance of nonmetabolized congeners, the half-lives in these subjects were estimated from the excretion rate and current body burden and ranged from 10 years for OCDD to 22 years for TCDD to 33 years for 2,3,4,7,8-PeCDF. Congener-specific half-lives, similar to those reported by Flesch-Janys et al. (1996), were also calculated based on the decrease in serum lipid level of congeners between 1990/92 and 1996. The fecal clearance of nonmetabolized CDDs and CDFs contributed on average between 37% (2,3,7,8-TCDD) and 90% (OCDD) to the total elimination. Thus, fecal clearance plays an important role in the overall elimination of most congeners, with the daily fecal excretion estimated to be equivalent to the amount of TEQ present in about 1.7 g of blood lipids (Rohde et al., 1999). Because direct fecal excretion is a significant route for the excretion of nonmetabolized CDDs and CDFs (Schlummer et al., 1998; Rohde et al., 1999), two recent studies investigated whether Olestra, a nonabsorbable sucrose-polyester synthetic fat substitute, may enhance the elimination of these compounds in humans. Moser and McLachlan (1999) compared the fecal excretion of CDDs, CDFs, PCBs, and HCB with background exposures in three subjects who ate an Olestra-free diet and who ate a diet supplemented with 25 g/day of Olestra. The fecal excretion while on the Olestra diet was 1.5 to 11-fold higher, depending on the congener. If fecal excretion is estimated to contribute 40% of the overall elimination of TCDD, and the Olestra diet enhanced fecal excretion 5.7-fold, the overall rate of elimination of TCDD would be more than doubled on the Olestra diet. Geusau et al. (1999) investigated the effect of an Olestrasupplemented diet on the excretion of TCDD in two patients with chloracne and very high serum TCDD levels of 144,000 and 26,000 pg/g lipid. A diet supplemented with fat-free potato chips December 2003 1-44 DRAFT--DO NOT CITE OR QUOTE (33 to 66 g Olestra/day) enhanced the fecal excretion of TCDD by up to eight- to ten-fold. Results suggest that the increase in fecal excretion of TCDD was due mainly to an increase in the amount of fat (dietary fat plus Olestra) excreted via the feces. The resulting elimination half lives of TCDD due to fecal excretion were estimated to be 1.4 years in the more highly exposed patient and 1.9 years in the other individual. However, half-lives of 200 days and 230 days, respectively, were determined through analysis of serum and adipose tissue TCDD levels over the 8-month observation period. The observed half-lives were far shorter than could be explained by an enhanced fecal or other elimination mechanism. In addition to the enhanced fecal excretion with Olestra, the authors speculate that the high levels of TCDD may have also induced the metabolism of TCDD in these subjects, resulting in the shorter observed half-lives. In related studies, Morita et al. (1993, 1995, 1997, 1999) investigated the role of dietary fiber or Chlorella in the fecal excretion of CDDs and CDFs in rats. Chlorella is a unicellular green algae, sold as a health food or health supplement. Chlorella in the diet of rats also enhanced the fecal excretion of CDDs from 0.8- to 5.6-fold and CDFs from 0.9- to 11.1-fold above that of rats on a control diet (Morita et al., 1999). Rice bran fibers enhanced the fecal excretion of CDDs from 0.6- to 2.3-fold and CDFs from 0.5- to 10.4-fold above that of rats on a control diet (Morita et al., 1997). Dietary fiber, chlorophyll, and/or lipid in the Chlorella may be factors responsible for the enhanced fecal excretion of CDDs and CDFs observed in this study. Thus, fiber and/or Chlorella may be other dietary factors capable of increasing the fecal excretion of CDDs and CDFs. Because of the lipophilic nature of milk, lactation can provide a relatively efficient mechanism for decreasing the body burden of TCDD and related compounds in females. As discussed by Graham et al. (1986), this elimination of TCDD through mother's milk can result in high exposure levels in the infant. The relatively high bioavailability of CDDs and CDFs from mother's milk in nursing infants was discussed earlier in Section 1.1.1.2. Further discussion of lactation as a route for excretion of CDDs and CDFs in women and exposure in infants is given in Section 1.4.5. 1.4. PHARMACOKINETICS AND EXPOSURE 1.4.1. Introduction Pharmacokinetic models, often simple models that represent the human body or a specific tissue in the human body as a storage compartment for a chemical, can be used to relate doses to internal tissue concentrations or to some other metric of toxic action. These models are represented by a differential equation that accounts for the mass balance about the compartment. In the simplest case, the change in mass of a chemical within the storage compartment is equal to December 2003 1-45 DRAFT--DO NOT CITE OR QUOTE the difference between the mass entering the compartment and the mass leaving the compartment over a specified time period. Physiologically based pharmacokinetic (PBPK) models are special extensions of the simple pharmacokinetic model. These models utilize multiple compartments to represent the different tissues or physiological regions of the body. They incorporate known or estimated anatomical, physiological, and physicochemical parameters to describe quantitatively the disposition of a chemical between different tissues. PBPK models are useful to extrapolate between high- to low-dose kinetics within a species, to estimate equivalent doses by different routes of administration, and to extrapolate doses across species (Scheuplein et al., 1990). Given some knowledge of possible exposure patterns and scenarios, PBPK models can be used in an inverse fashion to estimate exposures on the basis of internal body doses. Some examples of how to use simple PK and PBPK models to estimate exposure to TCDD and related compounds will be given here. In addition, this section will utilize model estimations to examine some special cases of exposure, in particular, exposure to lactating infants and the elderly. 1.4.2. Estimating Daily Intake of TCDD Because TCDD is highly lipophilic, it has been shown that a majority of the TCDD in any body tissue is stored in the fat (van der Molen et al., 1996). As a first approximation, a onecompartment pharmacokinetic model with first-order elimination may be used to compute the daily intake of TCDD based on steady-state concentrations in the fat. The mass balance model is: dCF V F -- dt = D ~ k eA F where: Vf CF D Af ke volume of fat concentration of TCDD in fat daily dose (mass/time) mass of TCDD in fat first order elimination constant (time-1) Concentration, CF, is given by: (1-1) December 2003 1-46 DRAFT--DO NOT CITE OR QUOTE (1-2) Then, at steady state (dCF/dt = 0), daily dose exactly balances elimination. D = k eA,,F = k e VFj : ,,F Note that kecan be expressed in terms of half-life: k = ln2 e t 1/2 (1-3) (1-4) Substituting Equation 1-4 into Equation 1-3, one obtains the following expression for daily dose in terms of fat concentration: ln2 D= ' 1/2 (1-5) It is important to recall the two assumptions implicit in the derivation of the above formula for daily uptake. First, steady-state conditions are assumed. Given that the half-life of some of these compounds is long (e.g., for TCDD the half-life is 7 years), steady-state levels would only be approached if the level of exposure were constant for 15-30 years. Pinsky and Lorber (1998) compiled data from several studies that indicate that environmental concentrations of TCDD and related compounds have been decreasing over the past 20-30 years. They use a single-compartment model similar to the one presented above, but with a time-varying exposure profile rather than the constant input. The profile was determined statistically on the basis of previously recorded environmental trends. Using the prior exposure knowledge, Pinsky and Lorber found that the pharmacokinetic model was better able to predict body burdens that have been recorded over time than was the steady-state model. By manipulating the non-steady-state model and comparing results to the steady-state approximation, it can be shown for a given body burden measurement, the steady-state approximation would result in an overestimate of daily intake. Using some estimates of the decreasing exposure function presented by Pinsky and Lorber (1998), it appears that the overestimate of daily intake could be 20% or more with the steady-state model. December 2003 1-47 DRAFT--DO NOT CITE OR QUOTE Another assumption of the simple model presented in this section is that the elimination kinetics are assumed to be constant over the entire life of the individual. Because TCDD and related compounds are stored primarily in fat, sudden weight loss and lactation would result in alterations of the TCDD elimination rate. Again, it would be assumed that for calculation of daily intake due to background exposure, the body burden data from such individuals would be identified and calculations handled accordingly. Figure 1-3 shows a sample calculation for TCDD using Equation 1-5. A fat volume of 14 L was chosen, representing 20% of the body weight. Also, for the purposes of this example, 1 mL of tissue was assumed to be equivalent to 1 g. Table 1-11 shows the estimated daily intake of TCDD at several conditions. The range of daily intakes calculated is in agreement with those reported elsewhere (Frst et al., 1991). Thomaseth and Salvan (1998) developed a minimal PBPK model for TCDD in humans and utilized this model to estimate occupational exposures to TCDD. The model was reduced to one compartment for ease of solution and was based on the following assumptions: (1) dynamic equilibrium of TCDD concentration between different body lipid distribution volumes, (2) first order elimination proportional to TCDD liver content, and (3) daily intake proportional to body weight. The best parameter estimates based on Ranch Hand data were obtained with logtransformed data under a mixed-effects model, with liver elimination k = 0.022 days-1(95% CI of 0.02-0.024), and background input = 0.125 pg/kg/day (95% CI of 0.071-0.179). The model accounts for changes in body mass index (BMI) over time, with higher BMI being related to a longer half-life for TCDD. The model was then used to estimate occupational exposure of 253 U.S. chemical plant workers for whom one measure of serum TCDD was available. The estimated exposure of the NIOSH cohort was 233 pg/kg/day (95% CI of 192-273). This model is much more rigorous than the simple steady-state approximation, and if a PK model is to be used to attempt to estimate intake, this model would be preferable. However, solving this model for daily intake is more complex and requires some assumption of the pattern of exposure over time. Alternatively, one could estimate daily intake by direct exposure calculations, that is, by examining the interaction of humans with environmental media containing the highest concentrations of dioxins. More information on this is provided in the chapter on exposure (Part II, Chapter 4). 1.4.3. Daily Intake of Congeners Steady-state average dose calculations may be performed for other congeners in a manner similar to that presented for TCDD. Three pieces of information are necessary. First, concentrations in the adipose tissues must be known. Second, the half-lives of the compounds within the body must be known. Third, some understanding of the kinetics and exposure December 2003 1-48 DRAFT--DO NOT CITE OR QUOTE conditions is required to ensure that steady-state conditions were achieved at the time of monitoring. Concentrations of various congeners in adipose tissues can be found in several sources (Stanley et al., 1986; Schecter, 1991). Values range from around 2 ppt for 2,3,7,8-TCDF to several hundred ppt for 1,2,3,4,6,7,8,9-OCDD. Half-lives could be determined from elimination data, if available. Methods have been suggested to determine the half-life of such compounds from uptake data relative to TCDD. Schlatter (1991) has proposed one such method. The following has been adapted from that proposed method. Manipulation of Equation 1-5 results in: n TCDD ^ T C D D ^1/2, TCDD fin 2 For some other congener x: D x t.1/2,x V ln2 ( 1 6) (-) (1-7) Thus, the ratio of concentrations of TCDD to x can be described by: c ( D tTCDD _ TCDD 1/2, TCDD ' c ( V ln2 ) X ( V ln2 ' ^ D x t l/2,xy Which, with algebraic manipulation and simplification, becomes: D t, 1 TCDD 1/2, TCDD V (7TCDD,, ^ CX \ Dx) (1-8) (1-9) Assuming intake D to be mostly from food, especially animal-fat products, it can be related to absorption from these foods according to: ^ T C D D ~ a, TCDD) TCDD) (1-10) where: ka,TCDD = ATCDD = absorption rate constant for TCDD concentration of TCDD in animal fat (diet) December 2003 1-49 DRAFT--DO NOT CITE OR QUOTE and D = ,k a, x) A (1-11) where: ka,x = Ax = absorption rate constant for x concentration of x in animal fat (diet) As a result, the half-life for compound x can be described by: t 1/2,x ( t 1 / 2 , TCDD \ A) TCDD CX 1 A \ x)T C D D ^ \ k a , TCDD k a ,x (1-12) When the absorption rate constants for each are equal or when the difference between them is small compared to differences in other parameters (concentration, half lives), Equation 1-12 can be further simplified to: 1 / 2 , TCDD ^ TCDD 1 CX ) c,, TCDD ^ A \ x) (1-13) It should be noted that for some of these substances exposure is expected from other than food sources. For such cases Equation 1-12 would be modified to include these other sources as follows. ^1/2,x ( lr A a, i, TCDD i, TCDD ^ 1 / 2 , TCDD ----------z - ------- A. k. a, i,x x'x/ (1-14) where: k^a,i,TCDD absorption rate constants for TCDD from each of the i media AjCDD ka,i,x = concentration of TCDD in each of the i media = absorption rate constants for x from each of the i media Ax = concentration of x in each of the i media Other symbols: as previously defined December 2003 1-50 DRAFT--DO NOT CITE OR QUOTE Again, if the differences between the absorption rate constants for TCDD and x are judged to be small, then the following variation of Equation 1-14 can be used: \ C X )) A. 1,X (1-15) Table 1-12 shows the results of some half lives calculated in this manner. The half lives calculated using Equation 1-15 for the first three compounds in Table 1-12 agree with those calculated by Schlatter (1991). The large difference in the two calculations for OCDD is due to significant differences in absorption rates between TCDD and OCDD. Schlatter notes in his paper that for some compounds, including OCDD, corrections were made of differences in absorption. No explanation was offered on how this was done. However, FleschJanys et al. (1996) report a half-life of 6.7 years for OCDD. In addition, Flesch-Janys et al. (1996) report longer half-lives for 1,2,3,7,8-PeCDD (15.7 years) and 2,3,4,7,8-PeCDF (19.6 years). These differences could be due to a variety of factors including the dietary concentrations, absorption rates, other background sources, etc. Table 1-13 summarizes the results reported by Flesch-Janys. When using this method to estimate daily intake of TCDD and congeners, it should be noted that studies (Kim and Dubin, 1996; Shirai and Kissel, 1996) have indicated that there is considerable uncertainty associated with half-life measurements published in the literature. 1.4.4. Induction of Liver Binding Proteins and Resultant Distribution Andersen et al. (1997a) developed a multicompartment geometric model of the liver in relation to regional induction of cytochrome P-450s. The model was based on five morphological and functional zones of the liver acinus: a concentric periportal zone, a fenestrated periportal region that interconnects among multiple functional units, and three concentric centrilobular areas. At low doses, TCDD exhibits centrilobular expression of CYP1A1, and with increasing dose, adjacent areas radiating out from the centrilobular region also begin to express CYP1A1. To create realistic total induction curves that are relatively smooth, the differences in Kd(dissociation constant) values between adjacent subcompartments must be less than fivefold. Because of the high n (Hill constant) values, the low-dose induction characteristics predicted with the multicompartment liver model differ significantly from those predicted with a model that considers the liver as a single homogeneous compartment. A physiologically based pharmacokinetic (PBPK) model for TCDD was then combined with a five-compartment geometric model of hepatic zonation to predict both total and regional induction of CYPs within the liver (Andersen et al., 1997b). The model predicts an 81-fold difference in the affinity of the December 2003 1-51 DRAFT--DO NOT CITE OR QUOTE AhR-TCDD complex binding to DNA response elements for CYP1A1 between the centrilobular and the periportal regions. The PBPK analysis based on the multicompartment liver model suggests that the low-dose behavior for hepatic CYP1A1/1A2 induction by TCDD is highly nonlinear. 1.4.5. Pregnancy and Lactation (Exposure of Offspring) The distribution and excretion of [14C]-2,3,7,8-TCDD (30 :g/kg) and [14C]-2,3,7,8-TCDF (800 :g/kg) were studied in pregnant C57BL/6N mice after oral exposure on gestation day 11 (Weber and Birnbaum, 1985). The distribution and excretion of TCDD and 2,3,7,8-TCDF in pregnant mice were similar to those of males of the same strain (Gasiewicz et al., 1983b; Decad et al., 1981b) (see Tables 1-6 and 1-9), although elimination rates were higher in the pregnant mice for both congeners. For TCDD, liver, urinary, and fecal elimination was 3.0, 3.4, and 14.4 times faster than that reported for males. For 2,3,7,8-TCDF, liver, urinary, and fecal elimination was 1.3, 1.8, and 1.8 times faster than that observed for males. Elimination data from pregnant mice were based on only three time points (gestation days 12, 13, and 14) and thus represent only rough estimates. In addition, the greater fecal excretion could have been due to incomplete absorption of TCDD after oral exposure. Although these results need further substantiation, it is conceivable that the sex of the animal, pregnancy, and the route of exposure could have a significant impact on the pharmacokinetics of these compounds. In a related study, Krowke (1986) compared the TCDD concentrations in the livers of pregnant and nonpregnant NMRI mice exposed subcutaneously to 12.5 or 25 nmol/kg/day on gestation days 9-11. At 7 days after exposure to the lower dose, the hepatic TCDD concentrations were 7 and 32 ng/g in pregnant and nonpregnant mice, respectively. At the higher exposure, 5.5 times lower concentrations of TCDD were found in the livers of pregnant animals on gestation day 18. A similar effect on hepatic TCDD levels was observed in combined exposure, which contained 1,2,3,7,8-PeCDD, 1,2,3,4,7,8-HxCDD, or 2,3,7,8-TCDF. The decreased hepatic levels of TCDD in pregnant mice are consistent with the Weber and Birnbaum (1985) observation of more rapid elimination of TCDD in pregnant mice. Further investigations are necessary to better characterize the apparently significant effects of pregnancy on the disposition of TCDD and related compounds. Weber and Birnbaum (1985) also investigated the distribution of [14C]-2,3,7,8-TCDD (30 :g/kg) and [14C]-2,3,7,8-TCDF (800 :g/kg) to the embryos of pregnant C57BL/6N mice after oral exposure on gestation day 11. On gestation days 12, 13, and 14, the percent of the maternal dose in the embryo remained constant at 0.032%-0.037%/embryo, while the concentrations in the embryo were 0.34%, 0.17%, and 0.15% of the dose/g embryo, respectively. Embryos had approximately 11-fold higher concentrations of TCDD than 2,3,7,8-TCDF when exposed on a December 2003 1-52 DRAFT--DO NOT CITE OR QUOTE percent of total dose/g tissue basis. This may be due to the more rapid metabolism and excretion of 2,3,7,8-TCDF compared with TCDD. Assuming that all radioactive material found in embryos was parent compound, at most 2.6 ng (8 pmol) of TCDD and 6.4 ng (21 pmol) of 2,3,7,8-TCDF/g tissue were detected under these conditions. The transfer of [14C]-2,3,7,8-TCDD to the embryo during early gestation was assessed in NMRI mice given a dose of 25 pg/kg by intraperitoneal injection on days 7, 8, 9, 10, 11, or 13 of gestation (Nau and Bass, 1981). The mice were sacrificed after 48 hours, and TCDD concentrations were determined by liquid scintillation counting of solubilized tissue and by GCECD and GC/MS. Similar results were given by these methods, suggesting that TCDD-derived [14C] in maternal and embryonic tissue was the parent compound. The maternal liver contained from 4% to 8% of the dose/g, or 40-80 ng/g. TCDD in embryonic tissue from gestation days 11-15 ranged from 0.04% to 0.1% of the dose/g, or 0.4-1.0 ng/g. In contrast, higher levels were found earlier in gestation, with 10 ng/g embryo on gestation day 9 and 2 ng/g on day 10. The higher levels may be related to placentation, which occurs at approximately gestation days 10-11 in this mouse strain. The affinity of fetal liver for TCDD was relatively low, as compared to maternal liver; however, TCDD levels in fetal livers were two to four times higher than levels in other fetal organs. Nau and Bass (1981) also attempted to correlate TCDD levels in the fetuses with the observed incidence of cleft palate. Three groups of mice were given either a single intraperitoneal exposure to 25 pg/kg TCDD on gestation day 7 or 10 or 5 pg/kg/day, intraperitoneally, on gestation days 7-11. On gestation day 13, TCDD concentrations in maternal tissues were very similar in the three exposure groups. At day 13, however, the embryo contained 0.0380.011% (0.36 ng/g), 0.0960.027% (0.92 ng/g), and 0.120.05% (1.1 ng/g) of the dose (meanSD) in the 7-, 10-, and 7- to 11-day exposure groups, respectively. Cleft palate incidence on gestation day 18 was 16%, 84%, and 65% for the 7-, 10-, and 7- to 11-day exposure groups, respectively. Although further studies are needed, these results suggest that cleft palate incidence is generally related to the TCDD concentration in the embryo. In a related study, Couture et al. (1990) found that gestation day 12 was the peak period of sensitivity for TCDDinduced cleft palate in C57BL/6N mice; however, tissue levels were not investigated. In the same laboratory, Abbott et al. (1989) investigated the distribution of TCDD in the C57BL/6N mouse fetus following maternal exposure on gestation day 11 to 30 pg/kg. 2,3,7,7TCDD was detected in the gestation day-11 embryo at 3 hours postexposure and was equally distributed between the embryonic head and body. At 72 hours postexposure, 0.035% of the total dose was in fetal tissues and 1% of the TCDD in the fetus (1.4-3.5 pg) was found in the palatal shelf. More recently, Abbott et al. (1996) found that TCDD was detected in maternal blood, liver, and fat and in the placenta, embryonic liver, and palate within 30 min after oral exposure of mice on gestation day 12. The levels peaked in blood and placenta at 3 hours and in December 2003 1-53 DRAFT--DO NOT CITE OR QUOTE the other tissues at 8 hours. At 24 hours following a single oral dose of 24 :g TCDD /kg, the above respective tissues contained 0.25, 98.8, 72.9, 1.22, 1.03, and 0.44 ng TCDD/g. Krowke (1986) also measured the concentration of TCDD in the placenta, amniotic fluid, and fetus of NMRI mice exposed to 2.5 nmol/kg by subcutaneous injection on days 9-11 of gestation. Similar concentrations of TCDD were observed in the placenta, amniotic fluid, and fetus (-0.5 ng/g) on day 16 of gestation. Fetal liver TCDD concentrations were at least five times greater than in other fetal tissue. Krowke (1986) reported slightly lower TCDD levels in the fetal head relative to other extrahepatic fetal tissue, while Weber and Birnbaum (1985) found a slightly higher TCDD concentration in the head relative to other extrahepatic fetal tissue. Nau et al. (1986) investigated the transfer of TCDD via the placenta and milk in NMRI mice exposed to 25 pg/kg on day 16 of gestation. The authors confirmed the relatively low fetal tissue levels with prenatal exposure to TCDD (Nau and Bass, 1981) and found that postnatally TCDD was transferred efficiently to mouse neonates and offspring by lactating mothers. During the first 2 postnatal weeks, the pups were given doses of TCDD via the milk that were, on a body-weight basis, similar to those that had been administered prenatally to their mothers. TCDD levels in the tissue of lactating mothers decreased within the first 3 postnatal weeks by two to three orders of magnitude to reach levels that were only -2% of the corresponding levels in the pups that these mothers had nursed. Thus in mice, excretion into milk represents a major pathway for maternal elimination of TCDD and for the subsequent exposure of pups. The disposition of TCDD in rat pups was assessed after the prenatal (via placental transfer) and postnatal (via milk) exposure from pregnant Wistar rats given a single dose of 3, 30, or 300 ng/kg, subcutaneously, on day 19 of gestation (Korte et al., 1990). Lactation resulted in the rapid elimination of TCDD from maternal tissues, with the half-life of TCDD in the liver of lactating rats estimated to be -7 days. This compares to a half-life of 13.6 days in the liver of nonlactating rats (Abraham et al., 1988). At postnatal day 7, exposure via the milk resulted in pup liver TCDD concentrations that were greater than the corresponding levels in maternal liver. In cross-fostering experiments, the concentrations of TCDD in the liver of offspring at postnatal day 7 were 0.47, 2.59, and 4.16 ng/g in the 300 ng/kg groups exposed through the placenta only, via the milk only, and through the placenta and via the milk, respectively. These results support the earlier observations that the placental transfer of TCDD in rats and mice is relatively limited compared with the efficient transfer via maternal milk. Van den Berg et al. (1987b) investigated the transfer of CDDs and CDFs to fetal and neonatal rats. Prenatal exposure of the fetus was assessed in pregnant Wistar rats fed a diet containing a fly ash extract from a municipal incinerator on days 10-17 of gestation. Postnatal exposure of 10-day-old pups was assessed through feeding lactating mothers the same contaminated diet for the first 10 days after delivery. Although the fly ash extract contained December 2003 1-54 DRAFT--DO NOT CITE OR QUOTE almost all of the 136 tetra- to octa-CDDs and -CDFs, only 17 CDD and CDF congeners were detected as major compounds in the tissue of fetuses, pups, and dams. All of the congeners were 2.3.7.8- substituted with the exception of 2,3,4,6,7-PeCDF. TCDD had the highest retention (0.0092% of the dose/g) in the fetus, while 2,3,7,8-TCDF, 1,2,3,7,8-PeCDF, and hepta- and octa- CDDs and -CDFs were not detected in the fetus. In the liver of offspring, the highest retention was found for TCDD, 1,2,3,7,8-PeCDD, and the three 2,3,7,8-substituted HxCDDs (0.74%-1.13% dose/g). The 2,3,7,8-substituted penta- and hexachlorinated congeners showed the highest retention in the livers of dams (2.05%-5.17% of dose/g liver), while 2,3,7,8-TCDF, 1.2.3.7.8- PeCDF, and 2,3,4,6,7-PeCDF had the lowest retention. A linear relationship was found between the retention of CDDs and CDFs in the livers of pregnant and lactating rats. Furthermore, a linear relationship was found between the retention of CDDs and CDFs in the livers of the lactating rats and livers of the offspring. In a related study, Hagenmaier et al. (1990) investigated the transfer of CDDs and CDFs through the placenta and via milk in a marmoset monkey. A defined mixture of CDDs and CDFs was given as a single subcutaneous injection to a pregnant marmoset monkey at the end of the organogenesis period (week 10 of gestation, 11 weeks prior to delivery). Transfer of CDDs and CDFs through the placenta was investigated in a newborn 1 day after birth, and transfer through the placenta and via milk was assessed in an infant of the same litter after a lactation period of 33 days. Tissue concentrations of the offspring were compared with those of the mother at the end of the lactation period and with data from other adult marmosets obtained at this time of maximum absorption (1 week after injection) and 6 weeks after injection. Deposition of CDDs and CDFs into the newborn liver was very low, suggesting very little transplacental transport and hepatic accumulation of these compounds. TCDD and 1,2,3,7,8-PeCDD were found at the highest concentration in the liver of the newborn (-0.15% of dose/g). For all other congeners, the concentrations in the liver of the newborn were <10% of the corresponding concentrations in adults. In contrast to liver, concentrations of 2,3,7,8-substituted congeners in the adipose tissue of the newborn were at least 33% of the levels in adults, and in the case of OCDD and OCDF, levels were threefold higher in the newborn than in the adult. The adipose tissue/liver concentration ratios for 2,3,7,8-substituted congeners in the newborn ranged from 2.2 for 1.2.3.4.6.7.8- HpCDF to 10.9 for 2,3,7,8-TCDF. Furthermore, the concentration of these congeners in the newborn was highest in the adipose tissue, followed by the skin and liver. This is in contrast to the relative distribution in the adult, where the liver generally contains the highest levels of these congeners. The results indicate that hepatic concentrations in the fetus may not be representative of the rate of placental transfer of CDDs and CDFs. In the marmoset monkey, substantial placental transfer into fetal adipose tissue can be observed for most of the 2.3.7.8- substituted congeners during the fetal period. As expected from rodent studies, the December 2003 1-55 DRAFT--DO NOT CITE OR QUOTE transfer of CDDs and CDFs via mothers' milk was considerable, resulting in hepatic concentrations of 2,3,7,8-TCDD, 1,2,3,7,8-PeCDD, and 1,2,3,6,7,8-HxCDD in the suckled infant (postnatal day 33) higher than those in the dam. The hepatic concentration of TCDD in the 33day-old infant was -0.9% of the dose/g tissue. Transfer of hepta- and octa-CDDs and CDFs to the suckled infant was rather low, only -10% of the levels in the dam. When total exposure of the mother and offspring at the end of the 33-day nursing period was assessed in terms of I-TE factors (U.S. EPA, 1989), the liver of the mother contained 2,494 pg I-TE/g, whereas the offspring liver contained 2,022 pg I-TE/g. This approach is necessary to assess total exposure due to the congener-specific transfer via lactation. The pre- and postnatal transfer of TCDD to the offspring of rhesus monkeys was investigated by Bowman et al. (1989). Animals were fed a diet containing TCDD at concentrations of 5 or 25 ppt for -4 years and were on a TCDD-free diet for -18 months prior to parturition. Maternal TCDD levels (meanSE) in adipose tissue were 4911 (n=7) and 17381 (n=3) ppt in the 5 and 25 ppt groups, respectively. Corresponding levels in the adipose tissue of offspring at weaning (4 months) were 18758 and 847298 ppt in the 5 and 25 ppt groups, respectively. From these data, a TCDD BCF of 4.29 was estimated from mother to nursing infant. This value is similar to that observed for TCDD in the marmoset monkey (Hagenmaier et al., 1990). The milk of the rhesus monkeys in the 25 ppt group contained from 4 to 14 ppt of TCDD, which corresponds to 150-500 ppt on a lipid basis. The authors calculated that the three mothers in the 25 ppt group excreted from 17% to 44% of their TCDD body burden by lactation. They also concluded that the results are generally consistent with overall triglyceride movement as mediating the excretion of TCDD in milk. In a subsequent study, Bowman et al. (1990) reported the relative persistence of TCDD in the offspring of rhesus monkeys that were exposed earlier to 5 or 25 ppt of TCDD in the diet. The concentration of TCDD in adipose tissue was measured in offspring at -4-5, 12, and 24 months of age. The decrease of TCDD levels in adipose tissue of seven young monkeys departed somewhat from first-order, single-compartment kinetics, but with the limited data and an assumption of first-order kinetics, a half-life of 121 days was estimated. When the data were adjusted within each animal for body weight gain and for average fat content at each age, the adjusted data apparently followed first-order, single-compartment kinetics, with a half-life of -181 days. Thus, young monkeys apparently eliminate TCDD from adipose tissue at a faster rate than adult rhesus monkeys, which had individual half-lives ranging from 180 to 550 days (Bowman et al., 1989). Several studies provide data in support of the transplacental transport of CDDs and CDFs to the human fetus. Kreuzer et al. (1997) measured the concentration of CDDs and CDFs in the lipids of adipose tissue and livers of 3 stillborn infants and detected 16 of a possible 17 December 2003 1-56 DRAFT--DO NOT CITE OR QUOTE congeners, with the exception of 1,2,3,7,8,9-HxCDF. TEQ levels ranged from 6.2 to 10.8 pg/g lipid, and TCDD levels ranged from 0.8 to 2.1 pg/g lipid for human infants that died at birth. These levels are similar to those reported in the lipid fractions of maternal tissues, suggesting that prenatal exposure to CDDs and CDFs reflects the levels present in maternal tissues. Similar findings were reported earlier by Schecter et al. (1990c), who detected TCDD (1.3 to 4.3 pg/g lipid) in the livers of three stillborn infants. Thus, significant prenatal exposure to CDDs and CDFs occurs, with the concentration of these compounds in the lipids of the newborn infants generally reflecting that in maternal lipids. A significant source of postnatal exposure of human infants to CDDs and CDFs is through the ingestion of human milk. Several investigators have quantified the levels of TCDD in human milk samples. Many of the milk samples were pooled (Jensen, 1987). Rappe (1984) reported levels of 1-3 ppt TCDD in milk fat (lipid adjusted) from five volunteers in West Germany, and in a later report, Rappe et al. (1985) reported an average level of 0.6 ppt TCDD in milk fat from four volunteers in northern Sweden. Furst et al. (1986) reported an average level of 9.7 ppt TCDD in milk fat from three individuals in the Netherlands and <1.0 ppt TCDD in milk fat from two individuals in Yugoslavia. Nygren et al. (1986) reported average levels of TCDD in human milk samples from four subjects in Sweden to be 0.6 ppt in milk fat, in five subjects from West Germany to be 1.9 ppt in milk fat, and in four subjects from Vietnam to be <0.5 ppt in milk fat. High levels of TCDD have been detected in the milk of mothers exposed to high levels of TCDD in the environment. Reggiani (1980) reported levels between 2.3 and 28.0 ppt TCDD in whole milk from mothers in Seveso. Baughman (1975) reported levels between 40.0 and 50.0 ppt TCDD in whole milk from mothers in South Vietnam. Schecter et al. (1987) also found high ppt levels of TCDD in human milk samples from South Vietnam. These authors found that levels from samples taken in 1985 from South Vietnamese mothers were comparable to the level of TCDD currently found in North American human milk samples (5 ppt). Furst et al. (1989) examined the levels of CDDs and CDFs in human milk and the dependence of these levels on the period of lactation. The mean concentrations of CDDs in human milk (on a fat basis) ranged from 195 ppt for OCDD to 2.9 ppt for TCDD, with the levels of the other congeners decreasing with decreasing chlorination. This is in contrast to the generally lower levels of CDFs in human milk, which range from 25.1 ppt for 2,3,4,7,8-PeCDF to 0.7 ppt for 1,2,3,7,8-PeCDF. An evaluation of the CDD and CDF levels in relation to the number of breast-fed children found that the concentrations in milk generally decreased with the greater number of children. The CDD and CDF levels in milk from mothers nursing their second child are on average 20%-30% lower than those for mothers breast-feeding their first child. CDD and CDF levels were also analyzed in one mother over a period of 1 year after delivery of her December 2003 1-57 DRAFT--DO NOT CITE OR QUOTE second baby to assess the effect of duration of lactation. After breast-feeding for 1 year, the mother had CDD and CDF levels that were 30%-50% of the starting concentration. Levels in milkfat (ppt) at 1, 5, and 52 weeks after delivery were 251, 132, and 119 for OCDD; 7.9, 5.9, and 1.4 for TCDD; and 33.1, 24.5, and 10 for 2,3,4,7,8-PeCDF, respectively. The results suggest a more rapid mobilization of CDDs and CDFs and excretion into human milk during the first few weeks postpartum. Although further studies are necessary, the limited data suggest that there are time-dependent, isomer-specific differences in the excretion of CDDs and CDFs in human milk. Schecter et al. (1998a) assess the decrease in the levels of CDDs, CDFs, PCBs, DDE, and HCB in the blood and milk lipid in a mother that nursed twins over a 38-month period. During the first 23 months of nursing, the CDD and CDF TEQ decreased 68% (15.7 to 5.0 ppt, lipid) for blood and decreased 77% (13.6 to 3.1 ppt, lipid) for breast milk. Thus, lactation results in a similar reduction in CDD and CDF concentrations in the lipid fractions of blood and milk. During the first 23 months of nursing, the PCB 126 (3,3',4,4',5-PeCB) milk concentration also decreased 71% (21.0 to 6.1 ppt, lipid). The authors estimate that approximately 115 ng TEQ (CDDs, CDFs, coplanar PCBs) was ingested by each infant from breast feeding for this extended period of time. Abraham et al. (1996) investigated the intake, fecal excretion, and blood levels of CDDs, CDFs, and PCB 126 in two breast-fed and two formula-fed infants. At 1 month, the concentrations of CDDs and CDFs in breast milk were 19.7 and 22.2 TEQ (pg/g lipid), while the formula diet contained only 0.38 TEQ (pg/g lipid). At the age of 11 months, the breast-fed infants' blood CDD and CDF concentrations were 29.2 and 37.5 TEQ (pg/g lipid), whereas the formula-fed infants' blood CDD and CDF concentrations were 2.4 and 2.6 TEQ (pg/g lipid). At this time, the mothers that breast-fed had blood CDD and CDF concentrations of 12.3 and 10.5 TEQ, while the mothers that formula-fed had blood levels of 16.9 and 13.8 TEQ (pg/g lipid). Because PCB 126 has a TEF of 0.1, it is important to note that at 11 months, breast-fed infants also have much higher levels of PCB 126 (287 and 374 pg/g lipid) relative to formula-fed infants (24 and 18 pg/g lipid). PCB 126 levels in mothers that breast-fed were 105 and 86 relative to levels of 193 and 52 (pg/g lipid) in the mothers that formula-fed. Thus, when PCB 126 is included in the TEQ calculation, the breast-fed infants' total TEQ blood concentration (body burden) is more than doubled from that estimated on the basis of CDDs and CDFs alone. The results of this study provide direct, quantitative data showing that the body burden (blood level) of CDDs, CDFs, and PCB 126 is more than 10 times higher in 11-month-old breast-fed infants than in 11-month-old formula-fed infants. Although data are more limited for the co-planar PCBs, 3,3',4,4'-TCB, 3,3',4,4',5-PeCB, and 3,3',4,4',5,5'-HxCB have been detected in human milk from Swedish mothers, at concentrations of 16-32, 72-184, and 46-129 ppt on a fat basis, respectively (Noren et al., 1990). December 2003 1-58 DRAFT--DO NOT CITE OR QUOTE Therefore, lactation appears to be an effective means for the excretion of co-planar PCBs from mothers and a major source of postnatal exposure of nursing infants. Because 3,3',4,4',5-PeCB and other co-planar PCBs are present in human milk at concentrations up to 60-fold higher than TCDD, it is important to consider the relative toxic potency of these dioxin-like compounds and their potential health impact on nursing infants. Kreuzer et al. (1997) measured the levels of CDDs and CDFs in the lipids of adipose tissue and liver of 17 infants (0.43 to 44 weeks of age) who died of sudden infant death syndrome. As expected, the concentrations of these compounds in breast-fed infants were higher than those in non-breast-fed infants; however, the magnitude of this difference varied because of differences in the age of the subjects and the duration of breast-feeding. The TEQ concentrations in the livers of these subjects were slightly, but not significantly, higher than the respective levels measured in the adipose tissue lipids. The results also suggest that the higher chlorinated congeners preferentially accumulate in liver lipids, an observation made earlier for adults in a study by Thoma et al. (1990) (see Table 1-5). Kreuzer et al. (1997) used data from the above study and other published results to validate a physiological toxicokinetic model they developed to describe the body burden of TCDD for the entire human lifetime and the influence of breast-feeding on the body burden. The model includes gender- and age-dependent changes in the following parameters: body weight; volumes of liver, adipose, and muscle tissue; food consumption; and excretion of feces. The model also assumes that TCDD exposure occurs primarily from the ingestion of contaminated food, that TCDD is distributed freely in lipids, and that TCDD is excreted unchanged in the lipids of the feces as well as following hepatic metabolism. More complex biochemical processes such as protein binding, saturation of metabolism at high TCDD concentrations, or induction of metabolism are not part of this model, which considers factors more relevant for low-level or background human exposures. With the basis assumption of this single-compartment model and the free distribution of TCDD in all body lipids, including the gastrointestinal tract, the half-life of the nonmetabolic elimination (tf1/2) is proportional to the ratio of volume of body lipids (V) to the mass of lipids in stool excreted per unit time (dFa/dt). During aging, V increases at least 40 times but dFa/dt only 1.7 times (from 3 g/day in infants to 5 g/day in adults). Consequently, the half-life of the nonmetabolic elimination (tf1/2) is calculated to be only 0.42 years in newborns and 9.5 years in 40-year-old adults. According to this model, most TCDD is eliminated as unchanged compound in children, with the role of metabolism-dependent elimination becoming more important with age. Thus, the half-life increases almost linearly from its starting value of about 4 months in newborns and reaches a value of 5 years at the age of 40. An age dependent elimination of TCDD has also been reported experimentally in the rhesus monkey (Bowman et al., 1989, 1990). Furthermore, the model of Kreuzer et al. (1997) predicts December 2003 1-59 DRAFT--DO NOT CITE OR QUOTE that the relatively high TCDD concentrations that might be reached after 6 months of nursing do not lead to an elevated lifetime body burden of TCDD. In a related study, Patandin et al. (1999) investigated dietary, including lactational, exposure to CDDs, CDFs, and PCBs from early childhood until the early reproductive age of 25 years in order to assess exposure risk to the next generation. Based on the analysis of 83 milk samples, previously reported analysis of food products, and food questionnaire data, the daily TEQ intake per kg body weight is 50 times higher in breast-fed than bottle-fed infants and 3 times higher in toddlers than in adults. Although exposures are relatively high in breast-fed infants, breast-feeding for 6 months contributes only 12% and 14% to the respective body burdens of men and women at the age of 25 years. After babies are weaned, dairy products, processed foods, and meat are major sources of exposure to these compounds. 1.4.6. Pharmacokinetics and Aging The influence of aging on the intestinal absorption of TCDD was studied in 13-week-, 13-month-, and 26-month-old (senescent) male Fischer 344 rats (Hebert and Birnbaum, 1987). Absorption was measured by an in situ intestinal recirculation perfusion procedure. When absorption was calculated in terms of ng TCDD absorbed/g mucosal dry weight/hour, the decrease between the senescent rats and the two younger age groups, from 544 ng/g/hour (young) to 351 ng/g/hour (senescent), was not statistically significant (p<0.05). The results indicate that, as with other molecules that depend on diffusion for their absorption, aging does not affect the intestinal absorption of TCDD. Banks et al. (1990) studied the effect of age on the dermal absorption and disposition of TCDD and 2,3,4,7,8-PeCDF in male Fischer 344 rats. When rats were administered the same dose per body weight, dermal absorption of TCDD at 3 days after exposure decreased from 17.72.7% (meanSD) to 5.62.5% of the administered dose in 10- and 36-week-old rats, respectively. Dermal absorption in the 96-week-old rats was similar to that of the 36-week-old rats. Dermal absorption of 2,3,4,7,8-PeCDF also decreased from 22.20.2% to 14.73.8% of the administered dose in 10- and 36-week-old rats, respectively. Dermal absorption of both compounds was also decreased in older rats given the same total dose per surface area. Older animals may have decreased blood flow in the upper dermis, which will decrease the clearance of these compounds from the application site. Potential age-related changes in the intercellular stratum corneum lipids may also play a role in the decreased dermal absorption observed in older animals. Changes in the percentage of the administered dose detected in various depots reflected age-related changes in dermal absorption, whereas age-related changes in the tissue distribution of the absorbed dose reflected changes in the total mass of these tissues at various ages. Overall elimination of the absorbed dose was not affected by age. Although this investigation was December 2003 1-60 DRAFT--DO NOT CITE OR QUOTE conducted with a lipophilic solvent system and an animal model with skin that is more permeable than human skin, the results suggest that systemic bioavailability after dermal exposure to TCDD or 2,3,4,7,8-PeCDF may be reduced in older age groups. In a similar study, absorption, tissue distribution, and elimination were examined 72 hours after dermal application of a lower dose of 200 pmol (111 pmol/cm2) TCDD to weanling (3-week-old), juvenile (5-week-old), pubescent (8-week-old), young adult (10-week-old), and middle-aged (36-week-old) rats (Anderson et al., 1993). Dermal absorption using acetone as the vehicle was greatest in 3-week-old rats (129 pmol; 64% of the administered dose), decreasing to -80 pmol (40%) in 5-, 8-, and 10-week-old rats and to 45 pmol (22%) in 36-week-old rats. The results indicate that TCDD is absorbed to a greater degree through skin of very young animals and that a significant decrease in potential for systemic exposure may occur during maturation and again during aging. 1.5. SELECTION OF DOSE METRIC Risk assessment requires the scaling of exposure/dose across endpoints and across species. Given the many responses to TCDD and its congeners, the selection of dose metrics for use in quantitative risk assessments is a complex problem. The biochemical and toxicological responses of TCDD and related chemicals are initiated by their interaction with the Ah receptor. Some responses, such as enzyme induction, require short periods (minutes to hours) of Ah receptor activation. Other responses, such as cancer, require prolonged (months to years) activation of this pathway. Still other responses, such as the developmental toxicities, require receptor activation during specific windows of sensitivity. Because of the different mechanisms involved in these diverse responses, it is unlikely that a single dose metric will be adequate for all of these endpoints. A number of studies have proposed a variety of dose metrics for a number of different responses. These studies have taken different approaches ranging from simple curve fitting exercises (Hurst et al., 2000; van Birgelen et al., 1996) to more complex PBPK modeling approaches (Jusko et al., 1995; Andersen et al., 1997; Kohn et al., 1993; Portier and Kohn, 1996). Area under the curve (AUC), a dose metric traditionally used in the pharmaceutical literature, has also been proposed as a dose metric for dioxin cancer risk estimates (Becher et al., 1998). The choice of dose metric not only considers mechanistic data but must also consider pragmatic approaches as well. The use of the dose metric plays a role in its choice. Because of differences in life-span and uncertainties in the window of sensitivities for various endpoints, AUC may not be a useful dose metric for cross species extrapolation. However, AUC has been used in the analysis of the human cancer data (Becher et al., 1998) and may be a useful dose metric when applied to accidental or occupational exposures since cross species scaling is not December 2003 1-61 DRAFT--DO NOT CITE OR QUOTE required. The choice of dose metric is also dependent upon the data available. A number of dose metrics, such as Ah receptor occupancy, induction of CYP1A2, and decreases in EGF receptor have been proposed based on PBPK models (Jusko et al. 1995; Andersen et al.1997; Kohn et al 1993; Portier and Kohn, 1996). While these dose metrics have been useful in hypothesis testing in experimental systems, they are not useful in animal to human extrapolations due to the difficulty in measuring these parameters in humans. In the following section, the strengths and weaknesses of a variety of proposed dose metrics will be presented. 1.5.1 Administered Dose In experimental studies, animals are administered a defined dose through a variety of routes. A default method used by USEPA (USEPA, 1992; 1996) to estimate the human equivalent dose when scaling across species is to use allometric scaling based on the following equation: Dosehuman= Doserat (W ra /^ h u m a i/25 Where BW is the body weight in kg and Dose is the daily administered dose in rats or the scaled human daily dose expressed as ng/kg/d. This method is thought to scale administered dose in such a way as to result in equivalent effective doses in humans and experimental animals (USEPA, 1992). Using this equation a dose of 1 ng TCDD/kg/d in a 0.35 kg rat would result in a scaled human dose of 0.27 ng TCDD/kg/d for a 7 kg human. If this scaling method applies to TCDD and related chemicals, then 1 ng TCDD/kg/d in the rat should produce similar effective dose in a human exposed to 0.27 ng TCDD/kg/d, some 3.8 times lower. Assuming similar sensitivity between rats and humans at the tissue level, effective doses should be a function of tissue concentration. Tissue concentrations of TCDD and related chemicals are directly related to the concentration of TCDD in the body. The steady-state concentration of TCDD in the body, or steady-state body burden, can be estimated in rats and humans using the following equation. Steady-state body burden (ng/kg) = (Dose*F) * (t,/2/Ln(2)) where Dose is the daily administered dose, F is the fraction absorbed, and t/ is the speciesspecific half-life of TCDD. In the present example, we will assume F is 50% and the species specific half-life of TCDD is 25 days for rats and 2593 days for humans. Starting with an administered dose of 1 ng/kg/d in rats and the scaled human dose of 0.27 ng/kg/d, the steady- December 2003 1-62 DRAFT--DO NOT CITE OR QUOTE state body burdens are presented in Table 1-14. The steady-state body burden of TCDD using the scaled human dose is approximately 28 times that of the steady-state body burden in the rat (Table 1-14). Using the equation above, the estimated steady-state body burden in rats exposed to 1 ng TCDD/kg/day is 18 ng/kg. An equivalent administered dose in humans that results in a body burden of 18 ng/kg is estimated at 0.0096 ng/kg/day. It should be noted that when body burden is used as the dose metric, an equivalent human dose is over 100 times less than the human equivalent dose estimated using the EPA default scaling method. Using administered dose as a dose metric has additional uncertainties when route and duration of exposures are varied. Tissue concentrations of a chemical are influenced by the route and duration of exposure. For example, the time course and peak tissue concentrations for the distribution of a dose of TCDD is different when the dose is administered orally, dermally or intratracheally (Diliberto et al., 1994). The dosing vehicle can also influence the absorption and distribution of a chemical (Dix et al., 1997). The amount and rate of absorption of a chemical in an oil vehicle can be very different when the chemical is dissolved or suspended in an aqueous vehicle or bound to soil (Olson et al., 1994). Using administered dose as the metric for exposures occurring through the same route, rate and dosing vehicle within the same species clearly is appropriate. However, in cases where these parameters vary across dosing scenarios, there is increasing uncertainty in the extrapolation. Clearly, the default scaling method results in an estimated human equivalent dose that produces a much greater estimated steady-state body burden (505 ng/kg) than the rat's. One reason for the discrepancy of the scaling method is that the half-life of TCDD in rodents and humans is much larger than is typically observed for other xenobiotics (Bachmann et al., 1996; Sarver et al., 1997). The half-life of TCDD in humans is approximately 100 fold greater than in rats. The EPA default scaling methodology would estimate human half-lives at approximately 3.8 times greater than in rats. This exercise demonstrates that administered dose does not provide a useful dose metric for cross species extrapolation even if the dose is scaled using the EPA default methodology. However, administered dose can be used to compare exposures between human populations in order to describe potential human health risks, because the species differences in half-life would not exist in this case. 1.5.2 Area Under the Curve Area under the curve or AUC is frequently used as a dose metric for reversible responses of pharmaceutical agents. Typically, these agents have half-lives on the order of minutes to hours. In the pharmaceutical literature, AUC is most frequently used for drugs with a rapid onset of action and that are rapidly reversed following cessation of exposure (Goodman and Gilman). In addition, the pharmacological actions of the drugs and the length of time of the response is December 2003 1-63 DRAFT--DO NOT CITE OR QUOTE clearly defined in both animals and humans. In essence, plasma concentrations are readily determined and the time span is easily defined. In cases where repeated doses of a drug is used, the steady-state plasma concentration is the preferred measure of dose. Also, when using drugs with a slow onset (i.e., a week or more), such as the tricyclic antidepressants, dose is typically expressed as steady-state blood concentrations. Mechanistic considerations also suggest that AUC can be a useful dose metric for chemical carcinogenesis. AUC has been applied as a dose metric in chemical carcinogenesis for DNA reactive chemicals. In these cases, the DNA reactive chemicals, increases in the mutational rates are expected to be related to the integrated tissue exposure to the critical DNA reactive species (Krewski et al., 1994). What is less certain about AUC as a dose metric is whether the risk across species for DNA reactive chemicals should be normalized based on integrated daily tissue concentration or an integrated lifetime tissue concentration. In any event, the dose metric would be the integrated tissue concentration of DNA reactive chemical over the appropriate period of the animal or human life span (Krewski et al., 1994). There are some concerns over applying AUC as a cancer dose metric for TCDD. TCDD and related chemicals are thought to induce tumors non-genotoxic mechanisms. The evidence supporting the use of lifetime AUC calculations for cancer risk involves genotoxic chemicals and radiation. TCDD lacks direct acting genotoxicity and is considered a tumor promoter. This promotional process requires sustained tissue concentrations of TCDD sufficient to maintain increased gene expression. It is likely that AUC would be an appropriate dose metric for cancer and may also involve the incorporation of a threshold concentration (Hays et al., 1997). Chemicals for which AUC has been applied as a dose metric are DNA reactive chemicals. DNA reactive chemicals produce DNA adducts and the probability of a mutational event occurring from adduct formations increases with the number of adduct formed. Adduct formation is associated with an integrated measure of dose. Because of the difference in mechanism of action, there no evidence supporting or refuting the use of AUC as a cancer dose metric for TCDD. The need for species extrapolation in toxicology further complicates the use of AUC as a dose metric. While blood or plasma concentrations of TCDD can be determined in both human and animals, the determination of the time span for which the AUC is to be calculated is much less certain. For some of the toxic responses of TCDD, the window of sensitivity is clearly defined in rodents and humans, such as induction of cleft palate. For other responses, such as the developmental reproductive alterations observed in male and female rats, the window of sensitivity has been narrowed to exposures between gestational day 15 and 20 in the rats, but the human window of sensitivity is uncertain. For carcinogenesis, the length of time required to induce the response remains uncertain in both experimental animals and humans. In order to December 2003 1-64 DRAFT--DO NOT CITE OR QUOTE apply AUC for species comparisons of the sensitivity to TCDD, one must have a better understanding of the species differences in the windows of sensitivity to the various biological effects of TCDD. Another of the uncertainties in risk assessment is time scaling across species. The difficulty is that there does not appear to be a consistent method of time scaling between species for different biological processes. If we were to compare the length of time of several common biological phenomena in rats and humans, we would observe different ratios of these time frames. For example, rats live approximately 2-2.5 years, while the average human life span is estimated at 70 years. The gestation period is approximately 21 days in a rat and 270 days in humans. The age at the time of puberty in rats is between postnatal days 29-45 and in humans occurs between 8 and 14 years old. In contrast, some processes, such as diurnal cycles or cell replication, have equivalent time spans between species. Due to uncertainties in how to properly account for the differences in time span for different biological responses, lifetime average daily dose is recommended as the default dose metric (Rhomberg, 1990). In addition, differences in life-span also must be considered. Brody and Reid (1967) proposed that the biological activity of a drug is related to its plasma concentrations. If animals and humans had the same plasma concentrations for their entire lives, the human AUC would be greater because humans have a longer half-life. However, because the plasma concentrations were the same, according to Brody and Reid (1967) the responses should be similar. Hence, in order to use AUC for chronic toxicities, such as cancer, a correction for the difference in life span must be applied. Typically, this involves the derivation of an average daily AUC (Alyward et al., 1996). An estimation of the average daily AUC is directly related to steady-state body burdens. Hence, once the AUC is corrected for life-span differences, these values are equivalent to steady-state body burdens. While AUC may not be an appropriate dose metric for animal to human extrapolations, it is a use tool for comparing populations exposed to high concentrations of dioxins over a short period of time to the background population. Becher et al (1998) successfully used this approach to examine dose response relationships for cancer in an occupationally exposed cohort. One difficulty in determining AUC is the accuracy of the intake measurements. Past exposures through the diet are uncertain, although they have been estimated (Pinsky and Lorber, 1998). Future exposures are thought to be decreasing, although the exact magnitude of this decrease are uncertain. Hence, determination of AUC carries a number of uncertainties that must be considered. 1.5.3 Plasma or Tissue Concentrations December 2003 1-65 DRAFT--DO NOT CITE OR QUOTE Brodie and Reid (1967) have argued that the response to a drug is determined by the amount bound to its biological receptor and since the drug receptor complex is in dynamic equilibrium with the free drug in the plasma, the biological response of a drug will be related to its plasma concentrations. There is no reason to believe that this relationship will not be true for TCDD and related chemicals. However, there are several data gaps that may prohibit the use of plasma or blood concentrations for species extrapolation. First, few animal studies determined blood or plasma concentrations of TCDD, particularly in the subchronic, chronic and lifetime exposures. PBPK models can be used to estimate blood concentrations and should provide reasonable estimates of these values. In contrast, the human exposure data is based predominately on blood, serum or plasma dioxin concentrations. One limitation of the human data is that it is mostly presented on a lipid adjusted basis. Hence in order to compare the human and animal plasma or blood concentrations, one would have to first estimate the blood concentrations in the animals using a PBPK model. Then either the animal data would have to be expressed as a lipid basis or the human data would have to be expressed as a wet weight basis. In either case, assumptions of the percent lipid in the blood would have to be applied as well as a number of assumptions used in the PBPK models. The use of tissue concentrations as a dose metric has been examined by Van Birgelen et al., (1996) and Hurst et al., (1999). Van Birgelen and coworkers (1996) presented data demonstrating tissue that concentrations provided an accurate prediction of enzyme induction regardless of the exposure scenario (i.e. acute vs subchronic). The use of alternative dose metrics, such as AUC and total administered dose, failed to demonstrate predictive relationships for enzyme induction and immunotoxicity (van Birgelen, 1996). Similarly, Hurst et al. (1999) presented data demonstrating that fetal tissue concentrations of TCDD on gestation day 16 predicted decreases in sperm counts, delays in puberty in males, urethra-phallus distance and the incidence of vaginal threads in rats prenatally exposed to TCDD on either gestational day 9 or 15. These data suggest that target tissue concentrations may be a reasonable dose metric for these responses. While tissue concentrations may aid in estimating risks, these data are unlikely to be collected in humans in sufficient numbers to be useful, particularly for fetal concentrations. Plasma concentrations are also a useful tool to compare exposures in different human populations. Application of plasma concentrations as a dose metric for species extrapolation require some level of assumptions as described above, but reasonable comparisons could be made, particularly for comparing steady-state exposures in humans and animals. Comparing plasma or blood concentrations following acute exposures in experimental animals to steadystate human blood or plasma concentrations would not be appropriate. One limitation of the use December 2003 1-66 DRAFT--DO NOT CITE OR QUOTE of either plasma, blood, or target tissue concentrations as dose metrics is the lack of human PBPK models to predict these values based on changes in intake patterns. 1.5.4 Steady-State Body Burdens Body burden is defined as the concentration of TCDD and related chemicals in the body and is typically expressed as ng/kg. Steady-state body burden is defined as the concentration of TCDD and related chemicals in the body under steady-state exposure conditions and is also expressed as ng/kg. Humans generally reach exposures approximating steady-state between the ages of 20 and 30, with body burdens increasing slightly with age over the course of the next several decades. Humans steady-state body burdens and are estimated based on an intake rate and the half-life of TCDD in humans. Alternatively, body burdens in humans are estimated based on lipid adjusted serum or adipose tissue TCDD or TEQ concentrations. In animals, these values are calculated from studies at or approaching steady-state and are associate with either biochemical or toxicological responses. In addition, these values are calculated based on either knowledge of the species-specific half-life and the exposure regimen or they are estimated based on the TCDD tissue concentration, the size of the tissues and the weight of the animal. Steady-state body burdens provide a useful dose metric for several reasons. First, tissue and blood concentrations are directly related to body burdens. Thus, body burdens are surrogates for tissue concentrations. Second, the differences in the half-life of TCDD between species is accounted for because these body burdens are estimated at steady-state conditions. Third, DeVito et al. (1995) have demonstrated that for some biochemical responses, chloracne and cancer, species have similar rates of responses when dose is expressed on a body burden basis. Finally, body burdens provide flexibility because they can be estimated based on either intake rates or on measured tissue concentrations. Body burdens also have some limitations. In order to estimate body burdens from lipid adjusted tissue concentrations, an assumption of the percent body fat must be used. In the reassessment, a value of 25% has been used. It should be noted that there are human populations with body fat compositions less than 10% and greater than 35%. Also when estimating the body burden based on intake rates and half-lives, the uncertainty of these parameters should be considered. In the reassessment, the estimated steady-state body burden of approximately 5 ng TEQ/kg is based on measured serum concentrations from several populations in the mid 1990's. While measured concentrations should eliminate some of the uncertainties around estimates using intake rates and half-life assumptions, it is likely that these measured values represent a past history of higher exposure and we must anticipate a continued downward trend to present a "true" lifetime average concentration. Caution must be used when using body burden in conditions other than steady-state. Under these short-term exposures conditions in experimental December 2003 1-67 DRAFT--DO NOT CITE OR QUOTE animals, the relationship between tissue concentrations and body burden may not be the same as under steady-state conditions. 1.5.5. Mechanistic Dose Metrics Several groups have proposed a variety of dose metrics based on mechanistic considerations, such as concentration of occupied AhR (Jusko, 1995), induced CYP1A2 (Andersen et al., 1997; Kohn et al., 1993) and reduced epidermal growth factor receptor (EGFR) (Portier and Kohn, 1996). While these dose metrics are intellectually appealing, it must be kept in mind that they are still hypothesized dose metrics and require further research to demonstrate their utility for cross-species extrapolations. In addition, these dose metrics are unlikely to be measured in sufficient human samples to be useful. 1.5.6. Summary A variety of dose metrics have been proposed for estimating potential human health effects following exposure to dioxins. Many of these dose metrics have limitations that prohibit their use, such as tissue concentrations and the mechanistic dose metrics. Other dose metrics, such as AUC have limited utility for species extrapolations because of our limited understanding of the concept of physiological time. Some dose metrics can be used to compare different human exposures, such as AUC and administered dose, but are not suitable for species extrapolations. Other dose metrics, such as steady-state body burdens or blood concentrations, are useful dose metric because they are easily estimated, and are directly related to tissue concentrations and can be estimated in both animals and humans. The use of any of these dose metrics requires a number of assumptions discussed above. The choice of dose metric requires an understanding of the data available and their application of the dose metric. Future research efforts on the issue of dose metrics could provide better guidance in choosing the dose metrics for dioxins and related chemicals. However, in the mean time, the use of steady-state body burdens can provide a basis for species extrapolations. Table 1-1. Gastrointestinal absorption of TCDD and related compounds following a single oral exposure by gavage December 2003 Chemical Species (Sex) Dose (:mol/kg) (:g/kg) Vehicle % Administered Dose Absorbed" ____ fMean (Range)l____ Reference CDDs 2,3,7,8-TCDD Sprague-Dawley rat (M) 0.16 50 acetone:corn oil (1:7) 70 Piper et al., 1973 2,3,7,8-TCDD Sprague-Dawley rat (M/F) 0.003 1.0 acetone:corn oil (1:25) 84 (66-93) Rose et al., 1976 2,3,7,8-TCDD Hartley guinea pig (F) 0.005 1.45 acetone:corn oil (1:45) 50 Nolan et al., 1979 2,3,7,8-TCDD Golden Syrian hamster (M) 2.0 650 olive oil 74 Olson et al., 1980 2,3,7,8-TCDD Human (M) 0.000003 0.001 corn oil 87 Poiger and Schlatter, 1986 1,2,3,7,8-PeCDD Sprague-Dawley rat (M/F) 0.03 9.2 corn oil NR (19-71) Wacker et al., 1986 1-69 DRAFT--DO NOT CITE OR QUOTE OCDD Fischer 344 rat (M) 0.11 1.1 1.1 11 50 500 500 5000 o-dichlorobenzene:Emulphor (1:1) o-dichlorobenzene:corn oil (1:1) corn oil suspension corn oil suspension 12 Birnbaum and 15 Couture, 1988 2 5 December 2003 Table 1-1. Gastrointestinal absorption of TCDD and related compounds following a single oral exposure by gavage (continued) Chemical BDDs Species (Sex) Dose (pmol/kg) (:g/kg) Vehicle % Administered Dose Absorbed" ____IMean (Range)l____ Reference 2,3,7,8-TBDD Fischer 344 rat (M) 0.001 0.01 0.1 0.5 0.5 5 50 500 Emulphor:ethanol:water (1:1:3) 78 Diliberto et al., 82 1990 60 47 CDFs 2,3,7,8-TCDF 2,3,7,8-TCDF 2,3,4,7,8-PeCDF Fischer 344 rat (M) Hartley guinea pig (M) Fischer 344 rat (M) 0.1 30.6 Emulphor:ethanol (1:1) 1.0 306 0.02 6 Emulphor:ethanol:water (1:1:8) 0.1 34 corn oil 0.5 170 1.0 340 90 Birnbaum et al., 90 1980 90 Decad et al., 1981a ~70 Brewster and ~70 Birnbaum, 1987 ~70 1-70 DRAFT--DO NOT CITE OR QUOTE PCBs 3,3'4,4'-T4CB C57BL mouse (F) 34.5 10,000 corn oil 77 Wehler et al., 1989 a"Absorption is generally estimated as the difference between the administered dose (100%) and the percent of the dose that was not absorbed. The unabsorbed fraction is estimated as the recovery of parent compound in feces within 48 hours of exposure. NR = Not reported. December 2003 Table 1-2. Percentage of TCDD in the liver of rats 24 hours after oral administration of 0.5 mL of various formulations containing TCDD_______________________________________________________________ Formulation TCDD Dose (ng) No. of Animals Percentage of Dose in the Liver 50% ethanol 14.7 7 36.7 + 1.2 Aqueous suspension of soil (37%, w/w) that had been in contact with TCDD for: 10-15 hours 8 days 12.7, 22.9 21.2, 22.7 17 10 24.1+4.8 16.0+2.2 Aqueous suspension of activated carbon (25%, w/w) 14.7 6 #0.07 1-71 DRAFT--DO NOT CITE OR QUOTE w/w = Weight by weight. Source: Poiger and Schlatter, 1980. Table 1-3. Dermal absorption of 2,3,7,8-TCDD and related compounds in the rat Chemical Dose (:mol/kg) (:g/kg) % Administered Dose Skin Sitea Absorbed 2,3,7,8-TCDD 0.00015 0.001 0.01 0.1 0.5 1.0 0.05 0.32 3.2 32 160 321 61.73+4.37 59.71 + 1.90 72.60+0.41 82.21+2.85 80.92+2.74 82.68 + 3.69 38.27+4.37 40.29+1.89 27.40+0.41 17.78+2.85 19.08+2.74 17.30 + 3.67 2,3,7,8-TCDF 0.1 31 51.18 + 11.95 48.84 + 11.95 0.5 153 82.14 + 11.22 17.86+11.22 1.0 306 88.70 + 5.17 11.32 + 5.17 1,2,3,7,8-PeCDF 0.1 0.5 1.0 34 74.72 + 3.58 25.27 + 3.58 170 91.67+2.46 8.33+2.46 340 84.23 + 5.44 15.76 + 5.44 2,3,4,7,8-PeCDF 0.1 0.5 1.0 34 65.77+4.80 34.19+4.78 170 75.50+1.81 24.50+1.80 340 81.84 + 1.67 18.16+1.67 "Values are the meanSD of three to four animals and represent the amount of administered dose of radiolabeled congener remaining at the application site 3 days after dermal exposure. Source: Brewster et al., 1989. December 2003 1-72 DRAFT--DO NOT CITE OR QUOTE Table 1-4. Tissue distribution of [14C]-2-3,7,8-TCDD in female Wistar ratsa Tissue Range of 2,3,7,8-TCDD Concentrations (ng/g) Liver 29.23-30.99 Adipose tissue 3.72-4.14 Adrenal glands 0.89-1.08 Ovaries 0.76-0.96 Thymus 0.60-1.05 Skin 0.64-0.68 Lung 0.32-0.33 Kidney 0.27-0.29 Pancreas 0.21-0.31 Spleen 0.18-0.23 Serum 0.16-0.18 Bone (with marrow) 0.16-0.16 Muscle 0.08-0.12 Brain 0.07-0.09 "Distribution was assessed 7 days after a single subcutaneous exposure (3 :g/kg bw). Source: Abraham et al., 1988. December 2003 1-73 DRAFT--DO NOT CITE OR QUOTE Table 1-5. 2,3,7,8-Substituted PCDDs and PCDFs in human liver and adipose tissue Tissue Concentrations on a Lipid Basis (ppt) Fat Liver Liver/Fat Tissue Concentrations on a Wet Weight Basis (ppt) Livera Liver/Fat TCDD 8.0 16.4 2.05 1.1 0.14 PeCDD 16.4 20.1 1.22 1.4 0.09 HxCDD 94.7 166.8 1.76 11.7 0.12 HpCDD 106.7 1,002.4 9.39 70.2 0.66 OCDD 373.2 4,416.2 11.83 309.1 0.83 TCDF 2.5 5.5 2.20 0.4 0.15 PeCDF 35.2 173.7 4.93 12.2 0.35 HxCDF 41.5 389.5 9.38 27.3 0.66 HpCDF 14.2 218.9 15.42 15.3 1.08 OCDF 4.0 29.7 7.43 2.1 0.52 Values are the mean of 28 people from the Munich area. "Estimated from the % fat in the liver (7.02+5.33%, meanSD) Source: Thoma et al., 1990. December 2003 1-74 DRAFT--DO NOT CITE OR QUOTE December 2003 Table 1-6. Elimination of 2,3,7,8-TCDD and related compounds from major tissue depots Chemical CDDs 2,3,7,8-TCDD 2,3,7,8-TCDD 2,3,7,8-TCDD 2,3,7,8-TCDD 2,3,7,8-TCDD 2,3,7,8-TCDD 2,3,7,8-TCDD Species (Sex) Dose Wistar rat (F) 0.3 pg/kg, s.c. Tissue liver liver liver adipose Wistar rat (M) Sprague-Dawley rat (M) Sprague-Dawley rat (F) C57BL/6J mice (M) Ahb/Ahd C57BL/6J mice (M) Ahd/Ahd DBA/2J mice (F) Ahb/Ahd 1.0 pg//kg, i.p. 7 or 20 ppb in diet for 42 days 7 or 20 ppb in diet for 42 days 0.5 pg/kg, i.p. 0.5 pg/kg, i.p. 0.5 pg/kg, i.p. liver adipose liver liver liver adipose skin liver adipose skin liver adipose skin Half-Life (days) Remarks Reference 95% Confidence interval (time period Abraham et investigated): al., 1988 11.5 10.7-12.3 (10-49 16.9 days) 13.6 14.0-21.4 24.5 (49-91 days) 12.8-14.4 (10-91 days) 22.4-26.8 (14-91 days) 37.1 Tissue levels were measured for 20 53.2 weeks following exposure Lakshmanan et al., 1986 11 85% total dose Fries and Marrow, 1975 13 70% o f total dose Fries and Marrow, 1975 Pool size (% o f total dose): 8.5 36.8 10.3 23.6 16.0 7.6 Birnbaum, 1986 Pool size (% o f total dose): 7.1 20.6 7.6 31.3 14.9 10.2 Birnbaum, 1986 Pool size (% o f total dose): 12.4 29.2 13.3 30.9 13.2 21.4 Birnbaum, 1986 1-75 DRAFT--DO NOT CITE OR QUOTE December 2003 Table 1-6. Elimination of 2,3,7,8-TCDD and related compounds from major tissue depots (continued) Chemical 2,3,7,8-TCDD Species (Sex) DBA/2J mice (F) Ahd/Ahd Dose 0.5 :g /k g i.p. 2,3,7,8-TCDD OCDD rhesus monkey (F) Fischer 344 rat (M) 25 ppt in diet 50 :g/kg, i.v. BDDs 2,3,7,8-TBDD Fischer 344 rat (M) 0.5 :g/kg, i.v. (0.001 :mol/kg) CDFs Tissue liver adipose skin adipose liver adipose skin Half-Life (days) 11.9 11.8 12.8 39188 Remarks Pool size (% of total dose): 20.2 42.3 26.6 MeanSE (n=7) Pool size (% of total dose): 84 72.7 38 7.1 3 9.0, 1st 69 component 0.3, 2nd component Reference Birnbaum, 1986 Bowman et al., 1989 Birnbaum and Couture, 1988 liver adipose skin muscle blood 4.5 1st component 16.5 2nd component 57.8 2.5 1st component 57.8 2nd component 1.6 1st component 26.7 2nd component 18.2 Kedderis et al., 1991a 1-76 DRAFT--DO NOT CITE OR QUOTE Table 1-6. Elimination of 2,3,7,8-TCDD and related compounds from major tissue depots (continued) December 2003 Chemical 2,3,7,8-TCDF Species (Sex) Fischer 344 rat (M) Dose 30.6 :g/kg, i.v. (0.1 :mol/kg) 2,3,7,8-TCDF C57BL/6J mice (M) 30.6 :g/kg, i.v. (0.1 :mol/kg) 2,3,7,8-TCDF DBA/2J mice (M) 30.6 :g/kg, i.v. (0.1 :mol/kg) Tissue liver adipose skin muscle blood liver adipose skin muscle liver adipose muscle Half-Life (days) Remarks 0.10 1.25 3.75 0.45 11.09 0.02 0.72 0.02 1.14 1.9 1.6 0.15 4.0 0.015 1.1 1.8 7.0 0.02 4.0 Pool size (% of total dose) 29.09 1st component 31.39 2nd component 17.85 6.84 1st component 1.22 2nd component 24.85 1st component 6.73 2nd component 1.31 1st component 0.89 2nd component 1st component 2nd component 1st component 2nd component 1st component 2nd component Reference Birnbaum et al., 1980 Decad et al., 1981b Brewster and Birnbaum, 1988 1-77 DRAFT--DO NOT CITE OR QUOTE Table 1-6. Elimination of 2,3,7,8-TCDD and related compounds from major tissue depots (continued) December 2003 1-78 DRAFT--DO NOT CITE OR QUOTE Chemical Species (Sex) 1,2,3,7,8-PeCDF Fischer 344 rat (M) Dose 34 pg/kg, i.v. (0.1 pmol/kg) 1,2,3,7,8-PeCDF Sprague-Dawley rat (F) 4.0 pg/kg, p.o. 2,3,4,7,8-PeCDF Fischer 344 rat (M) 34 pg/kg, i.v. (0.1 pmol/kg) Tissue liver adipose skin muscle adrenal blood liver liver adipose skin muscle blood Half-Life (days) 1.36 25.72 12.91 1.32 14.53 0.03 6.96 0.14 2.36 0.07 12.42 3.3 Remarks Pool size (% of total dose): 42.59 1st component 1.27 2nd component 10.19 7.14 1st component 1.49 2nd component 34.81 1st component 7.42 2nd component 0.26 1st component 0.02 2nd component 5.33 1st component 1.29 2nd component 69.8% o f total dose 193 69 0.62 1.23 0.04 0.51 9.84 0.04 1.32 55 Pool size (% of total dose): 67.71 10.53 3.54 1st component 1.37 2nd component 29.40 1st component 2.01 2nd component 0.78 3rd component 3.18 1st component 0.37 2nd component 0.008 3rd component Reference Brewster and Birnbaum, 1988 Van den Berg et al., 1989a,b Brewster and Birnbaum, 1987 Table 1-6. Elimination of 2,3,7,8-TCDD and related compounds from major tissue depots (continued) Chemical Species (Sex) Dose 2,3,4,7,8-PeCDF Sprague-Dawley rat (F) 5.6 pg/kg, p.o. 1,2,3,6,7,8HxCDF PCBs 3,3',4,4'-TCB Sprague-Dawley rat (F) 6.0 pg/kg, p.o. Sprague-Dawley rat (F) 5 mg/kg/day, p.o., for 21 days 3,3',4,4'-TCB ICR mice (M) 8 mg/kg, p.o., every other day for 10 doses i.v. = intravenous; s.c. = subcutaneous; i.p. = intraperitoneal; p.o. = per os. Tissue liver liver liver adipose liver adipose serum Half-Life (days) 108 Remarks 78.3% of total dose 73 63.4% o f total dose Reference Van den Berg et al., 1989b Van den Berg et al., 1989b 0.8 21-day exposure produced steady state Clarke et al., with 300 ng/g in liver and 8 pg/g in 1984 adipose tissue. 2.5 Elimination was assessed over a 22-day postexposure period. Steady-state tissue concentrations: 2.15 1.5 :g /g 2.60 19.2 pg/g 1.07 0.04 pg/mL Clevenger et al., 1989 December 2003 Table 1-7. Elimination constants and half-lives of various 2,3,7,8-Substituted CDDs and CDFs in hepatic and adipose tissue of marmoset monkeysab___________________________________________________ Congener Hepatic Tissue (weKeeks--11) Half-Life (weeks) 95% Conf. Interval (weeks) (weKeeks--11) Adipose Tissue Half-Life (weeks) 95% Conf. Interval (weeks) 1-80 DRAFT--DO NOT CITE OR QUOTE 2,3,7,8-TCDDc 0.08410.0109 8.3 6.6-11.1 0.06580.0072 10.5 8.7-13.4 1,2,3,7,8-PeCDDc 0.06490.0101 10.7 8.2-15.4 0.04900.0057 14.2 11.5-18.3 1,2,3,4,7,8-HxCDD 0.07020.0059 9.9 8.4-11.8 0.04110.0083 16.9 12.1-27.9 1,2,3,6,7,8-HxCDD 0.05580.0046 12.4 10.7-14.9 0.03730.0073 18.6 13.4-30.2 1,2,3,7,8,9-HxCDD 0.07670.0078 9.0 7.5-11.3 0.05250.0089 13.2 9.9-19.7 1,2,3,4,6,7,8-HpCDD 0.05180.0081 13.4 10.2-19.3 0.03720.0060 18.6 14.2-27.2 OCDD 0.00890.0084 78 27--d 0.01220.0093 101 20-d 2,3,7,8-TCDF 0.80120.0549 <0.87e -<1.00 0.49860.0829 1.39 1.05-2.06 1,2,3,7,8-/1,2,3,4,8-PeCDF 0.74760.0294 0.93 0.86-1.00 0.47350.0408 1.46 1.25-1.76 2,3,4,7,8-PeCDF 0.07860.0048 8.8 7.9-10.0 0.05630.0059 12.3 10.2-15.5 1,2,3,4,7,8-/1,2,3,4,7,9-HxCDF 0.03070.0039 23 18-30 0.01030.0074 68 28-d 1,2,3,6,7,8-HxCDF 0.04860.0037 14.3 12.4-16.7 0.02900.0091 24 15-62 1,2,3,7,8,9-HxCDF 0.08480.0057 8.2 7.2-9.4 not analyzedf NA NA 2,3,4,6,7,8-HxCDF 0.03730.0057 18.6 14.3-26.5 0.01820.0082 38 20-327 December 2003 Table 1-7. Elimination constants and half-lives of various 2,3,7,8-Substituted CDDs and CDFs in hepatic and adipose tissue of marmoset monkeysab(continued)_________________________________________ Hepatic Tissue Adipose Tissue Congener (weKeeks--11) Half-Life (weeks) 95% Conf. Interval (weeks) (weKeeks--11) Half-Life (weeks) 95% Conf. Interval (weeks) 1,2,3,4,6,7,8-HpCDF 1,2,3,4,7,8,9-HpCDF OCDF 0.01860.0072 0.00880.0127 0.00400.0096 37 79 174 21-152 20-4d 30-4d -0.01400.0137 0.00110.0112 -0.00420.0148 4d 660 4d 54-4d 30-4d 28-4d 1-81 DRAFT--DO NOT CITE OR QUOTE "Source: Neubert et al., 1990. bAnimals were treated subcutaneously with a single dose of a defined CDD/CDF mixture, and the tissues were analyzed at different times following treatment. Half-lives were calculated from tissue concentrations of the 2,3,7,8-substituted congeners in hepatic and adipose tissue. Values are given as elimination rate constant Keincluding estimated SD and half-life including 95% confidence intervals. "Calculated from the time period: >6 weeks after injection. dCalculated half-life is apparently infinite. Data for OCDD and OCDF are unreliable due to delayed absorption. eNot detected in hepatic tissue 6 weeks after treatment; limits of detection used for calculation. fDue to interference. NA = Not applicable. T ab le 1-8. T C D D co n c en tratio n s in liv er a n d ad ip o se tissu e follow ing d iffe ren t doses an d calculated co ncentration ratios (liver/adipose tissue)a Dose (ng/kg) Number TCDD Concentration Liver (ng/g) TCDD Concentration Adipose Tissue (ng/g) Concentration Ratio: Liver/Adipose Tissue 16 0.00310.0009 ND NA 3 10 30 100 300 1000 3000 6 12 6 6 6 6 5 0.01020.0020 0.04060.0121 0.1620.032 0.6990.130 3.380.22 10.72.2 27.92.4 0.01390.0015 0.04940.0084 0.1390.021 0.3350.065 0.8190.075 2.020.17 3.660.31 0.740.15 0.820.20 1.160.07 2.100.27 4.140.31 5.270.96 7.650.64 aConcentrations were measured 7 days after injection. ND = Not detectable; NA = not applicable. Source: Abraham et al., 1988. December 2003 1-82 DRAFT--DO NOT CITE OR QUOTE December 2003 Table 1-9. Metabolism and excretion of 2,3,7,8-TCDD and related compounds8 Chemical Species Dose Chemical Nature of Excretion Products (% Metabolites) Urine Bile Feces Ratio of % of Dose Excreted (Feces/Urine) Half-Lifeb (days) CDDs 2,3,7,8-TCDD 2,3,7,8-TCDD 2,3,7,8-TCDD 2,3,7,8,-TCDD 2,3,7,8-TCDD 2,3,7,8-TCDD 2,3,7,8-TCDD 2,3,7,8-TCDD 2,3,7,8-TCDD Sprague-Dawley rat (M) Sprague-Dawley rat (M) Sprague-Dawley rat (F) Sprague-Dawley rat (M, F) Sprague-Dawley rat (M, F) Han/Wistar rat (M) Long-Evans rat (M) Sprague-Dawley (M) C57BL/6J mice (M) 50 :g/kg, p.o. 7 or 72 ppb in diet for 42 days 7 or 72 ppb in diet for 42 days 1 . 0 :g/kg, p.o 0.1 and 1.0 :g/kg/day, 5 days/week for 7 weeks 5 :g/kg, i.p. 5 :g/kg, i.p. 500 :g/kg, i.p. 10 :g/kg, i.p. NA NA NA NA NA >90 >90 100 100 NA NA NA NA NA NA NA NA NA NA NA ~ 70-90 NA ~ 20-90 100 NA 100 85 4.0 NA NA 9.9 8.5 14.1 12.0 NA 2.7 17.45.6c 12 15 316d 23.7 21.9 20.8 NA 11.01.2d Comment NC NC NC NC NC NC NC NC NC Reference Piper et al., 1973 Fries and Marrow, 1975 Fries and Marrow, 1975 Rose et al., 1976 Rose et al., 1976 Pohjanvirta et al., 1990 Pohjanvirta et al., 1990 Neal et al., 1982 Gasiewicz et al., 1983a 1-83 DRAFT--DO NOT CITE OR QUOTE 1-83 December 2003 Table 1-9. Metabolism and excretion of 2,3,7,8-TCDD and related compounds" (continued) Chemical Species Dose Chemical Nature of Excretion Products (% Metabolites) Urine Bile Feces Ratio of % of Dose Excreted (Feces/Urine) Half-Lifeb (days) Comment Reference 2,3,7,8-TCDD 2,3,7,8-TCDD 2,3,7,8-TCDD 2,3,7,8-TCDD 2,3,7,8-TCDD 2,3,7,8-TCDD 2-Iodo-3,7,8-TCDD DBA/2J mice (M) io :g/kg, i.p. B6D2F1J mice (M) io :g/kg, i.p. C57BL/6J mice Ahb/Ahd (M) 500 ng/kg, i.p. C57BL/6J mice Ahd/Ahd (M) 500 ng/kg, i.p. DBA/2J Ahb/Ahd 500 ng/kg, i.p. (F) DBA/2J Ahd/Ahd 500 ng/kg, i.p. (F) C57BL/6J mice (F) [125I] 0.1 nmol/kg, i .p. 100 100 NA NA NA NA NA 2-Iodo-3,7,8-TCDD 2,3,7,8-TCDD 2,3,7,8-TCDD 2,3,7,8-TCDD C57BL/6J mice (F) Hartley guinea pig (M) Hartley guinea pig (M) Golden Syrian hamster (M) [125I] 0.1 nmol/kg, i.p., 3 days following pretreatment with 2,3,7,8-TCDD (0.1 pmol/kg, i.p.) 0.5 :g/kg, i.p. NA NA 0.56 :g/kg, i.p. 100 [3H] 650 :g/kg, i .p. NA 100 100 NA NA NA NA NA NA NA 100 NA 82 1.2 86 2.5 NA 3.1 NA 2.1 NA 5.3 NA 6.8 NA NA NA NA NA 15.7 19 11.2 NA 1.4 24.41.0d 12.60.8d 9.42 NC Gasiewicz et al., 1983a NC Gasiewicz et al., 1983a NC Birnbaum, 1986 9.74 NC Birnbaum, 1986 10.40 NC Birnbaum, 1986 11.11 NC Birnbaum, 1986 14.2 whole body counting was Leung et al., used to estimate body 1990b burden over 30-day period 8.0 whole body counting was Leung et al., used to estimate body 1990b burden over 30-day period 30.25.8d 93.715.5d NC Gasiewicz and Neal, 1979 NC Olson, 1986 11.951.95d NC Olson et al., 1980; Neal et al., 1982 1-84 DRAFT--DO NOT CITE OR QUOTE 1-84 December 2003 Table 1-9. Metabolism and excretion of 2,3,7,8-TCDD and related compounds" (continued) Chemical Species Dose Chemical Nature of Excretion Products (% Metabolites) Urine Bile Feces Ratio of % of Dose Excreted (Feces/Urine) Half-Lifeb (days) Comment Reference 2,3,7,8-TCDD 2,3,7,8-TCDD 2,3,7,8-TCDD Golden Syrian [14C] 650 :g/kg, 100 100 55-75 hamster (M) Lp. Golden Syrian hamster (M) [3H] 650 :g/kg, p.o. NA NA NA human (M) 1.14 ng/kg, p.o. NA NA ~50 NA NA >3.1 2,3,7,8-TCDD 2,3,7,8-TCDD 1,2,3,7,8-PeCDD OCDD rainbow trout 494 ppt in diet for 13 weeks NA ~75 yellow perch 494 ppt in diet for 13 weeks NA ~90 Sprague-Dawley rat (M, F) Fischer 344 rat (M) 8.42-10.06 :g/kg, p.o. 50 :g/kg, iv NA <33 100 0 NA NA NA NA NA 12 0 >65 OCDD Fischer 344 rat (M) 50 :g/kg/day, p.o., for 10 days NA NA NA NA BDDs 10.82+2.35 NC Olson et al., 1980; Neal et al., 1982 14.96+2.53 NC Olson et al., 1980; Neal et al., 1982 2120e NC Poiger and Schlatter, 1986; Wendling et al., 1990 105 elimination followed for Kleeman et al., 13 weeks following 1986b exposure 126 elimination followed for Kleeman et al., 13 weeks following 1986a exposure 29.5+2.7 NC Wacker et al., 1986 ~ 7 0 whole body t1/2 estimated Birnbaum and from body burden in liver, Couture, 1988 skin, and adipose tissue over 56-day period ~ 173 whole body t1/2 estimated Birnbaum and from body burden in liver, Couture, 1988 skin, and adipose tissue over 112-day period 1-85 DRAFT--DO NOT CITE OR QUOTE 1-85 December 2003 Table 1-9. Metabolism and excretion of 2,3,7,8-TCDD and related compounds" (continued) Chemical Species Dose Chemical Nature of Excretion Products (% Metabolites) Urine Bile Feces Ratio of % of Dose Excreted (Feces/Urine) Half-Lifeb (days) Comment 2,3,7,8-TBDD 2,3,7,8-TBDD Fischer 344 rat (M) 0.001 pmol/kg, iv NA 100 80-90 Fischer 344 rat 0.1 pmol/kg, iv NA 100 80-90 (M) 11.1 9.2 Pool size (% of dose): 0.7 11.63 1st component 2.9 2.78 2nd component 17.8 1.45 3rd component Pool size (% of dose): 0.6 22.47 1st component 17.8 2.35 2nd component Reference Kedderis et al., 1991a Kedderis et al., 1991a 1-86 DRAFT--DO NOT CITE OR QUOTE 1-86 December 2003 Table 1-9. Metabolism and excretion of 2,3,7,8-TCDD and related compounds" (continued) Chemical Species Dose Chemical Nature of Excretion Products (% Metabolites) Urine Bile Feces Ratio of % of Dose Excreted (Feces/Urine) Half-Lifeb (days) Comment CDFs 2,3,7,8-TCDF 2,3,7,8-TCDF 2,3,7,8-TCDF Fischer 344 rat (M) C57BL/6J mice (M) 0.1 pmol/kg, iv 0.1 pmol/kg, iv DBA/2J mice (M) 0.1 pmol/kg, iv 100 > 9 6 100 NA 100 NA 99 31.4 80 6.5 80 2.8 2,3,7,8-TCDF 2,3,7,8-TCDF 2,3,7,8-TCDF Hartley guinea pig 0.02 pmol/kg, iv (M) >90 NA <10 1.0 Hartley guinea pig 4 :g/kg, p.o. (M) NA NA NA NA rhesus monkey (M) 0.1 pmol/kg, iv 100 > 9 2 >92 5.4 1,2,3,7,8-PeCDF Fischer 344 rat (M) 0.1 pmol/kg, iv ~90 100 NA 12.8 1.8 0.3 2.8 1.8 2.0 4.9 5.4 4.0 20 40 6.24 10.30 ~8 0.92 3.32 1.26 17.32 1.12 6.30 fecal excretion urinary excretion urine feces urine and feces urine feces urine and feces animal exhibited body weight loss no observable toxicity urine feces urine and feces Pool size (% of dose): feces: 57.79 1st component 6.92 2nd component urine: 2.68 1st component 0.16 2nd component feces and urine: 59.97 1st component 2.51 2nd component Reference Birnbaum et al., 1980 Decad et al., 1981b Decad et al., 1981b Decad et al., 1981a Ioannou et al., 1983 Birnbaum et al., 1981 Brewster and Birnbaum, 1988 1-87 DRAFT--DO NOT CITE OR QUOTE 1-87 December 2003 Table 1-9. Metabolism and excretion of 2,3,7,8-TCDD and related compounds" (continued) Chemical Species Dose Chemical Nature of Excretion Products (% Metabolites) Urine Bile Feces Ratio of % of Dose Excreted (Feces/Urine) Half-Lifeb (days) Comment Reference 2,3,4,7,8-PeCDF Fischer 344 rat (M) 0.1 pmol/kg, iv NA > 9 0 >90 >100 2,3,4,7,8-PeCDF rhesus monkey (M) 0.1 pmol/kg, iv NA NA 63-70 --34 1.27 63.82 38-49 Pool size (% of dose): feces 1.22 1st component 0.57 2nd component Brewster and Birnbaum, 1987 t1/2 represents minimum value; all animals lost body weight and exhibited other signs of toxicity Brewster et al., 1988 CBs 3,3'4,4'-TCB 3,3'4,4'-TCB CD rat (M, F) 0.6 mg/kg, iv > 9 0 NA >90 42 --1.3-1.5 rhesus monkey (F) 0.6 mg/kg, iv 97 NA 97 7.2 ~8-10 NC Abdel-Hamid et al., 1981 NC Abdel-Hamid et al., 1981 aAll studies measure the excretion of radiolabeled parent compound and metabolites following exposure to a single congener labeled with 3H, 14C, or 125I. bHalf-life for excretion estimates assume first-order elimination kinetics. c(meanSE). d(m e a n S D ). en = 1. i.p. = intraperitoneal; i.v. = intravenous; NA = not available; NC = no comment; p.o. = per os. 1-88 DRAFT--DO NOT CITE OR QUOTE 1-88 December 2003 Table 1-10. Half-life estimates for 2,3,7,8-TCDD and related compound in humans Chemical Exposure Incident Number of Individuals Sample Time Period Between First and Last Analysis Number of Time Points CDDs 2,3,7 ,8 -T C D D Male volunteer 1 fecal excretion 125 days 28 2,3,7,8-T C D D 2,3,7 ,8 -T C D D 2,3,7 ,8 -T C D D 1 ,2,3,6,7,8-H xC D D 1 ,2,3,4,6,7,8-H p C D D OCDD CDFs 2,3 ,4 ,7 ,8 -P eC D F Male volunteer Ranch Hand Vietnam veterans Ranch Hand Vietnam veterans technical pentachlorophenol in wood of home technical pentachlorophenol in wood of home technical pentachlorophenol in wood of home Binghamton, New York, state office building 1,2,3,4,7,8-H xC D F Binghamton, New York, state office building 1,2,3,6,7,8-H xC D F Binghamton, New York, state office building 1 adipose tissue 6 years 36 serum 5 years 337 serum 5 years 1 adipose tissue 28 months 1 adipose tissue 28 months 1 adipose tissue 28 months 1 adipose tissue initial 43 months blood final 29 months combined total 6 years 1 adipose tissue initial 43 months blood final 29 months combined total 6 years 1 adipose tissue initial 43 months blood final 29 months combined total 6 years 5 2 2 2 2 2 4 4 7 4 4 7 4 4 7 Half-Life (years) 5.8 9.7 7.1a 11.3b 3.5 3.2 5.7 4.7 7.2 4.5 2.9 4.4 4.0 3.5 4.3 4.9 Reference Poiger and Schlatter, 1986 Schlatter, 1991 Pirkle et al., 1989 Wolfe et al., 1994 Gorski et a l., 1984 Gorski et a l., 1984 Gorski et a l., 1984 Schecter et al., 1990b Schecter et al., 1990b Schecter et al., 1990b 1-89 DRAFT--DO NOT CITE OR QUOTE 1-89 December 2003 1-90 DRAFT--DO NOT CITE OR QUOTE Table 1-10. Half-life estimates for 2,3,7,8-TCDD and related compound in humans (continued) Chemical Exposure Incident Number of Individuals Sample Time Period Between First and Last Analysis Number of Time Points Half-Life (years) 1,2,3,4,6,7,8-HpCDF 2,3,4,7,8-PeCDF Binghamton, N ew York, state office building Yu-Cheng 1 adipose tissue initial 43 months blood final 29 months combined total 6 years 4 blood 3 2 initial 2.9 years final 2.7 years total 5.6 years 4 4 7 2 2 3 6.5 4.1 6.8 1.3 2.9 1.7 1,2,3,4,7,8-HxCDF Yu-Cheng 1,2,3,4,6,7,8-HpCDF Yu-Cheng 2,3,4,7,8-PeCDF 1.2.3.4.7.81.2.3.4.6.7.8- Yu-Cheng HxCDF HpCDF 2,3,4,7,8-PeCDF 1,2,3,4,7,8-HxCDF Yusho 1,2,3,4,6,7,8-HpCDF technical pentachlorophenol in wood o f home OCDF technical pentachlorophenol in wood o f home 4 blood 3 2 4 blood 3 2 3 blood initial 2.9 years final 2.7 years total 5.6 years initial 2.9 years final 2.7 years total 5.6 years 9 years 9 blood 7 years 1 adipose tissue 28 months 1 adipose tissue 28 months 2 2.1 2 5.1 3 2.4 2 1.6 2 6.1 3 2.4 5-6 2-3 3-5 >5 2 <1.7 2 1.8 PCBs 3,3',4,4',5-PeCB Yu-Cheng NA blood NA NA <1 3,3',4,4',5,5'-HxCB Yu-Cheng NA blood NA NA 10 Reference Schecter et al., 1990a Ryan and Masuda, 1989 Ryan and Masuda, 1989 Ryan and Masuda, 1989 Ryan and Masuda, 1991 Ryan and Masuda, 1991 Gorski et al., 1984 Gorski et al., 1984 Ryan and Masuda, 1991 Ryan and Masuda, 1991 a95% confidence interval about the median o f 5.8-9.6 years. b95% confidence interval about the median o f 10.0-14.1 years. N A = N ot applicable. 1-90 Table 1-11. Calculated daily intakes for TCDD Half-life (yrs) Fat vol. (L) Fat conc. (ppt) 5.8 14.0 6.72 7.0 14.0 6.72 5.8 14.0 5.00 7.0 14.0 5.00 5.8 7.0 6.72 7.0 7.0 6.72 5.8 7.0 5.00 7.0 7.0 5.00 Calculated daily intake (PG/KG/Day) 0.44 0.37 0.33 0.27 0.22 0.18 0.16 0.14 December 2003 1-91 DRAFT--DO NOT CITE OR QUOTE Table 1-12. Half-life calculations Chem ical 2378-TCDD 2378-TCDF 12378-PeCDD 23478-PeCDF OCDD Food-ppta 0.23 0.84 0.7 1.4 19.2 B od y-p p tb 6.72 3.9 21.5 36.8 653.0 W years 7.0 1.11 7.35 7.94 8.14 t1/2d y ea r s 6.0 1.3 5.0 6.3 50.0 Concentrations in food for TCDD, TCDF, and OCDD were obtained as discussed in Part I, Volume 3; those for PCDD and PCDF were taken from Schlatter (1991). bConcentrations in body (adipose tissue) were all taken from Schecter (1991). Calculated using Equation 1-19, except for TCDD. Calculated by Schlatter (1991), except for TCDD. December 2003 1-92 DRAFT--DO NOT CITE OR QUOTE Table 1-13. Half-life estimates taken from Flesch-Janys et al. (1996) Compound N individuals Median half-life estimate (years) 2,3,7,8-TCDD 48 7.2 1,2,3,7,8-PeCDD 40 15.7 1,2,3,4,7,8-HCDD 41 8.4 1,2,3,6,7,8-HCDD 40 13.1 1,2,3,7,8,9-HCDD 39 4.9 1,2,3,4,6,7,8-HpCDD 26 3.7 OCDD 32 6.7 2,3,4,7,8-PeCDF 5 19.6 1,2,3,4,7,8-HCDF 42 6.2 1,2,3,6,7,8-HCDF 31 6 2,3,4,6,7,8-HCDF 6 5.8 1,2,3,4,6,7,8-HpCDF 22 3 1,2,3,4,7,8,9-HpCDF 6 3.2 December 2003 1-93 DRAFT--DO NOT CITE OR QUOTE Table 1-14. Comparison of administered dose and body burden in rats and humans. (A) Rat Daily Administered Dose/Body Burden (B) Human Scaled Administered Dose/Body Burden1 (C) Human Equivalent Administered Dose/Body Burden2 (A/B) Ratio of Rat to Human Scaled Dose (A/C) Ratio of Rat to Human Equivalent Dose Dose 1 0.27 0.0096 3.7 104 (ng/kg/d) Body Burden 18 505 18 0.036 (ng/kg) 1. Assumes administered dose scales across species as a function of BW 3/4 2. Assumes administered dose scales across species as a function of equivalent body burdens 1 December 2003 1-94 DRAFT--DO NOT CITE OR QUOTE Figure 1-1. Time course of the connection of 14C-TCDD in rat liver and adipose tissue after a single subcutaneous injection of 300 ng TCDD/kg bw to female rats (MSD). Source: Abraham et al., 1988. December 2003 1-95 DRAFT--DO NOT CITE OR QUOTE administered dose of 14C-TCDD/g of tissue recovered in liver and adipose tissue after single subcutaneous doses (values from animals treated with 3000 ng TCDD/kg bw were corrected for 84% adsorption). Concentrations were measured 7 days after the injection. Source: Abraham et al., 1988. December 2003 1-96 DRAFT--DO NOT CITE OR QUOTE D = f l n 2 ' VF CF V t l/2J 10KG) ) \ { ml) { 1 (\D = ln2 5.8y e a r s f 14 L 1000 -- 6.72^ 1 l) V ^ iokg) 1y e a r ' 3 6 5 days) D = 0.44 p g / k g / d a y F ig u re 1-3. S am p le c alc u latio n o f daily in ta k e fo r T C D D . 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