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TABLE 1-7
2,3,7,8-TCDD Concentrations in Liver and Adipose Tissue Following Different Doses and Calculated
Concentration Ratios (Liver/Adipose Tissue) '
Dose
(ng/kg)
1
3
10
30
100
300
1000
3000
Number
6
6
12
6
6
6
6
5
TCDD Concentration
Liver
(ng/g)
0. 0031 ±0. 0009
0.0102±0.0020
0.0406+0.0121
0.162±0.032
0.699±0.130
3.38±0.22
10.7±2.2
27.9*2.4
TCDD Concentration
Adipose Tissue
(ng/g)
ND
0.0139±0.0015
0.0494*0.0084
0.139*0.021
0.335±0.065
0.819*0.075
2.02±0.17
3.66±0.31
Concentration Ratio:
Liver/Adipose Tissue
NA
0.74±0.15
0.82*0.20
1.16*0.07
2.10*0.27
4.14*0.31
5.27*0.96
7.65*0.64
"Source:  Abraham et al., 1988




 Concentrations were measured 7 days after  injection



 ND  = Not detectable; NA = not applicable
                                             1-36
08/11/92

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(Curtis et al.,  1990).   Similarly, a dose-related increase in hepatic uptake of




[^ I]-2-iodo-3,7,8-trichlorodibenzo-p-dioxin was observed after pretreatment of




mice with 2,3,7,8-TCDD  (Poland et al.,  1989; Leung et al., 1990).  Shen and Olson



(1987)  also  observed an  increase  in  the uptake of 2,3,7,8-TCDD  by  isolated



hepatocytes from 2,3,7,8-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 2,3,7,8-TCDD intake of 100 ng for 2 years had an




average 2,3,7,8-TCDD content of  8100  ppt in fat and 24,000  ppt  in the liver.



Rats given 10 ng/kg/day had an average of 1700 ppt 2,3,7,8-TCDD in the fat and



5100 ppt  in the liver.  For both of these exposures the liver/ adipose tissue



concentration ratio  of 2,3,7,8-TCDD was  -3.   At the  lowest  dose  level  of 1



ng/kg/day, both fat  and  liver contained  an  average of  540  ppt  2,3,7,8-TCDD.



Kociba et al.  (1976) presented evidence that steady state had been  reached by <13




weeks of feeding of 2,3,7,8-TCDD.



     Other studies  do  not  support  the  dose-dependent  tissue  distribution of



2,3,7,8-TCDD and related compounds described above.  Rose et al. (1976) reported




a lack of a dose-dependent accumulation of ^C-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



^g/kg/day, Monday through Friday.   The rates  of accumulation  of TCDD-derived



radioactivity were similar in fat, liver and whole body;  however, the concentra-



tion in the liver  was -5-fold greater  than that in fat.  Recently, Clark et al.




(1991)  and Tritscher et  al. (1992) also  reported a lack of a dose-dependent



hepatic disposition  of  TCDD in  female Sprague-Dawley rats exposed biweekly to



TCDD for 30 weeks at doses  equivalent to 3.5,  10.7, 35.7 and 125 ng/kg/day.  A



linear relationship between administered dose and the concentration in the liver



was observed over  the dose range used  in this chronic exposure  study.  Brewster



and Birnbaum  (1987) also observed similar concentrations  (% dose/g) of 2,3,4,7,8-




PeCDF in liver, adipose tissue and other tissues at 3 days after oral exposure




at doses of 0.1, 0.5 or 1.0 /L/mol/kg bw.   These results  conflict with the above



studies which support the  dose-dependent tissue distribution of  these compounds.
                                     1-37                             08/11/92

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While it is not possible at this time to explain these differences, most of the



available data support a dose-dependent relationship.



     The dose-dependent tissue distribution of 2,3,7,8-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  since the  general human population is exposed to much



smaller daily  doses  (possibly  0.3 pg 2,3,7,8-TCDD/kg/day) than  those used in



experimental disposition studies.  Due  at  least partly to the long half-life of



2,3,7,8-TCDD in humans,  however, this exposure results  in concentrations of 3-18



pg/g in human adipose tissue (Leung et al.,  1990).  Similar levels of 2,3,7,8-



TCDD in adipose tissue (14 pg/g) were observed in rats  7  days after subcutaneous



exposure to  3  ng/kg bw  (see Table 1-7)  (Abraham  et al.,  1988).   Under these



experimental conditions, the liver/adipose tissue 2,3, 7,8-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 2,3,7,8-TCDD was more persistent in  adipose tissue



than in  liver  in rats exposed to 300 ng/kg bw (see Figure  1-1  and Table 1-5)



(Abraham et  al.,  1988).  Human  data  on the  liver/adipose tissue concentration



ratio of 2,3,7,8-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.  (1990) observed a geometric mean adipose tissue 2,3,7,8-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 2,3,7,8-TCDD in both tissues were approximately the



same.   More variability between  individuals  was  observed in the  CDD and CDF



concentrations in liver  and adipose tissue from 25  subjects from the Munich area



(Thoma et al.,  1989).  For example, the liver/adipose tissue concentration ratio



for  2,3,7,8-TCDD was >1.0  for 5  of  the 25 individuals (Thoma  et al., 1989).



While the majority of individuals had liver/adipose tissue concentration ratios



<1.0 for CDDs  and CDFs,  ratios >1.0 were observed for HpCDD  (5 of 25), OCDD (2



of 25),  PeCDF  (2 of  25), HxCDF  (1 of  25), HpCDF  (7 of 25) and OCDF (3 of 25).



Considerable variability in CDD and CDF concentrations in  liver  and adipose



tissues was  also observed between individual marmoset monkeys (Neubert et al.,




                                     1-38                             08/11/92

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                         DRAFT — DO  NOT QUOTE OR CITE





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 Mechanise* for the Dose-Dependent Tissue Distribution.  The



observation that exposure to higher  doses of 2,3,7,8-TCDD and related compounds



results in a disproportionally greater hepatic concentration of these compounds



may be explained by a hepatic binding species that is induced by 2,3,7,8-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



cytochrome P-4501A2.



     Poland et al. (1989) reported that TCDD and other Ah agonists (2,3, 7,8-TCDF,



fi-naphthof lavone, 3,3 ' ,4,4' , 5, 5 '-hexabromobiphenyl) act through the Ah receptor



to  increase a  liver binding  species  that  increases the  hepatic  uptake  of


 1 *)e

[   I]-2-iodo-3,7,8-trichlorodibenzo-p-dioxin (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 (IxlCT1




mol/kg) of [ ^I]-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  (1x10




mol/kg), the hepatic  accumulation of radiolabel was 25-30%  of that administered.



A  similar,  though less  dramatic effect,  was  observed  in  vitro,  with  liver


                                                                  19S
homogenate from TCDD-treated mice binding about four  times  more  [ l^JI ]-2-iodo-




3,7,8-trichlorodibenzo-p-dioxin than homogenate from control mice.  The adminis-



tration of TCDD to C57BL/6J  mice produced a dose-related stimulation of in vivo



hepatic  uptake  of  [ ^I]-2-iodo-3,7,8-trichlorodibenzo-p-dioxin,  increased




binding of radioligand to liver homogenate  and  induction of hepatic activity,



with an ED^Q ranging from 1.5 to 4.0x10"" mol/kg.  In congenic C57BL/6J (Ah<*/Ahd)
mice, which express the lower affinity Ah receptor, the ED^Q values for all three




responses were shifted to doses  that  were  about  10-fold higher.  The observed



effects on hepatic disposition were tissue specific,  with no remarkable disposi-



tional changes being observed, in kidney,  lung, spleen, small intestine or muscle.





                                     1-39                             08/11/92

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                         DRAFT—DO NOT QUOTE OR CITE





This is significant in that TCDD and other agonists for the Ah receptor induce



cytochrome P-4501A1 in liver and other tissues,  whereas cytochrome P-4501A2 is



apparently inducible only in liver and nasal olfactory mucosa (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., 1989).



     The  following evidence reported  by  Poland  et  al.  (1989)  supports  the



hypothesis  that  the  TCDD-inducible  hepatic binding  protein  is  cytochrome



P-4501A2:  the TCDD-induced hepatic binding species was found predominantly in



the microsomal fraction  and was inactivated by  heating at  60°C,  trypsin and



mercurials; the TCDD-induced hepatic binding species was specific for the liver,



with a  large  pool  size (Bmax  of 22+5 nmol/g liver);  and  the major microsomal




binding species covalently  labeled with the photo-affinity ligand [^I]-2-iodo-




3-azido-7,8-dibromodibenzo-p-dioxin migrates with that  immunochemically stained



with polyclonal antiserum that binds to cytochrome P-4501A2.



     One observation of Poland et al.  (1989) does not support the  hypothesis that



the TCDD-inducible hepatic  protein is cytochrome P-4501A2.  These investigators



found that dietary  administration of isosafrole did not stimulate hepatic uptake


    19S
of [i'"I]-2-iodo-3,7,8-trichlorodibenzo-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 cytochrome P-4501A2 (Ryan et al.,  1980).  Poland et



al. (1989) suggest  that this may be attributable to the high affinity binding of



an  isosafrole metabolite to the protein,  which  might  inhibit  the binding of


 19S
[1ZJI]-iodo-3,7,8-trichlorodibenzo-p-dioxin to cytochrome P-4501A2 at or near the




active site of the enzyme.   This does not  explain why  TCDD, which also has high



affinity  for  cytochrome P-4501A2,  cannot displace   some  of  the isosafrole



metabolite from the protein, which should produce enhanced hepatic disposition



of TCDD.



     Voorman and Aust  (1987, 1989) support  further the hypothesis that cytochrome



P-4501A2  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 cytochrome P-4501A2  through the immunoprecipitation of cytochromes P-4501A1




                                     1-40                             08/11/92

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                          DRAFT—DO  NOT QUOTE OR  CITE






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 cytochrome P-4501A2.  A similar association of PAHs with immunoprecipi-



tated cytochrome P-4501A2 was observed for other agonists for the Ah receptor,



including 2, 3,7,8-TCDD,  3,3',4,4'-TCB, 3, 3',4,4',5-PeCB and 3, 3 ' ,4, 4 ' , 5,5'-HxCB.



The  association of 2,3,7,8-TCDD  with cytochrome  P-4501A2  occurred  within 2



minutes,  with  maximum  inhibition  of estradiol  2-hydroxylase occurring  at a



concentration comparable to the concentration of the enzyme (50 nm).  Cytochrome



P-4501A2 was inhibited (complexed) by 2,3,7,8-TCDD with nearly  1:1 stoichiometry




and the Kj for 2,3,7,8-TCDD  was calculated to be 8 nM.  Therefore, 2,3,7,8-TCDD




can be considered a higher binding inhibitor of cytochrome P-4501A2.



     The TCDD-induced binding species was found to have an apparent equilibrium


                           19^
dissociation constant, KJ-J, [ 1^Ji ]-2-iodo-3,7,8-trichlorodibenzo-p-dioxin of 56±16




nM and a  pool size, B^^, of  22±5 nmol/g of  liver in C57BL/6J mice (Poland et




al., 1989).  The induced microsomal binding  species has an affinity about  10



                                                              o

times less than the Ah  receptor but a pool size that is -2xlOJ greater.  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 cytochrome P-4501A2.



     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 2,3,7,8-TCDD in that they  are



slowly  metabolized and  that  metabolism  is required  for urinary  and biliary



elimination.   2,2',4,4',5,5'-HxCB  and -HxBB  distribute primarily to adipose



tissue with partition coefficients (tissue/blood  ratio) ranging from 300-500 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 2,3,7,8-TCDD  and



related compounds.  Partition coefficients in the  liver range from 10-30 in these



species.  2,2 ' ,4,4',5,5'-HxCB and -HxBB do not induce hepatic  cytochrome P-4501A1



or 1A2 and do not exhibit dioxin-like activity.  The lack of induction of hepatic



cytochrome P-4501A2 may  explain the  lack of a dose-dependent  hepatic disposition



of these  compounds.




                                      1-41                             08/11/92

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                         DRAFT—DO NOT QUOTE OR CITE






     Kedderis et al.  (1991b)  assessed the dose-response relationship  for the




induction of hepatic cytochrome P-4501A1 and P-4501A2 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 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 (Birnbaum,  1992).  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  deposition  observed  at  higher  exposures of 2,3,7,8-TBDD




(Kedderis et al., 1991a).



     Other factors may also regulate  the  intracellular distribution of 2,3,7,8-




TCDD  and  related  compounds.   The  possible role of hepatic  lipoproteins as



intracellular carriers in the transport of 2,3,7,8-TCDD has been  assessed by in



vitro and in vivo studies (Soues et al., 1989a,b).   2,3,7,8-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 2, 3,  7,8-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 2,3,7,8-TCDD and related compounds.



1.3.   METABOLISM AND EXCRETION



     Although early in vivo and in vitro investigations were unable to detect the




metabolism  of 2,3,78-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 2,3,7,8-TCDD to polar metabolites (Ramsey et al., 1979,  1982;




Poiger and  Schlatter, 1979; Olson et  al., 1980; Olson, 1986; Gasiewicz et  al.,



1983; Poiger  et al.,  1982;  Sijm  et  al.,  1990; Kleeman et al., 1986a,b, 1988).



Although metabolites of 2,3,7,8-TCDD have not been directly identified in humans,









                                      1-42                              08/11/92

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                          DRAFT—DO NOT QUOTE OR  CITE






recent data  regarding feces samples  from humans in a  self-dosing experiment



suggests that humans can metabolize 2,3,7,8-TCDD (Wendling et al., 1990).



     Table 1-8 summarizes data on the metabolism and excretion of 2,3,7,8-TCDD



and  related  compounds  after  exposure  to  a  single  radiolabeled  congener.



Investigations of 2,3,7,8-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



1-8), with the exception of OCDD;  however, although 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 1-8, essentially all of the  CDD, BDD, CDF or PCB-derived radioactivity in



liver, adipose tissue and other tissues represented parent compound, suggesting



that the metabolites of these compounds were readily excreted.  Thus,  with the



exception  of  OCDD,   the  metabolism of 2,3,7,8-TCDD  and related  compounds is



required for 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



2,3,7,8-TCDD and related compoundst that is not regulated by metabolism.



1.3.1.   Structure of Metabolites.   Several metabolites  of 2,3,7,8-TCDD have



recently  been  identified.   Sawahata  et  al.  (1982)  investigated  the  in vitro



metabolism of 2,3,7,8-TCDD in isolated rat hepatocytes.   The major product was



deconjugated with fl-glucuronidase, derivatized with diazomethane and separated



into two compounds by HPLC.  These metabolites were subsequently identified as



l-hydroxy-2,3,7,8-TCDD and 8-hydroxy-2,3,7-trichlorodibenzo-p-dioxin.  Poiger et



al.  (1982b) identified six  metabolites  in the bile of dogs that  were given a



lethal dose of [^H]-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



l,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 differ-



ences in the relative amounts of various 2,3,7,8-TCDD metabolites in dog and rat







                                     1-43                             OB/11/92

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DRAFT—DO NOT QUOTE OR CITE











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                1-48
                                          08/11/92
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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 2,3,7,8-TCDD in the dog.  This is



in good  agreement  with the unique  ability of the dog to  readily metabolize




persistent PCBs such as 2,4,5,2'4'B'-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  GC/MS.  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.  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 2,3,7,8-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 1-5 and 1-8 indicate that  this reaction occurs at



a faster rate for the furan, since the rate of urinary and biliary elimination



and resulting persistence of these compounds depends on metabolism.



     Data  summarized in Tables 1-5  and  1-8 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 > 1,2,3,7,8-PeCDF > 2,3,7,8-TCDF  (Masuda et al.,  1985).



     Pluess et al.  (1987) investigated the structure of  1,2,3,7,8-PeCDF metabo-



lites in rat bile.  A dihydroxy-tetra-CDF was identified as the major metabolite.




                                     1-49                             08/11/92
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                         DRAFT—DO NOT QUOTE OR CITE






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 a  dihydroxy-tri-CDF,  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 a dihydroxy-penta-CB



and a 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 metabo-




lites  of  2,3,4,7,8-PeCDF  include  a  hydroxy-tetra-CDF,   dihydroxy-tri-CDF,



dihydroxy-tetra-CDF and  a 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  sulfur-containing 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 that  were  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',5-TCB 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 while 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, while  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




a  methylsulphonyl-TCB  as  major metabolites  in  the mouse  fetus.   Sulphur-



containing metabolites of noncoplanar 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




                                     1-50                             08/11/92
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                         DRAFT—DO NOT QUOTE OR CITE



al., 1986,  1989).  PCB methyl sulphones are stable lipophilic metabolites formed


by  the  mercapturic  acid  pathway.   The  toxicological  significance  of  these


metabolites remains generally unknown.


1.3.2.   Toxicity of Metabolites.   The   above  discussion indicates  that  the


metabolism of 2,3,7,8-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  2,3,7,8-TCDD  suggests that reactive epoxide


intermediates may be formed.  Poland and Glover  (1979) have investigated the in

                       •5
vivo  binding of  [1,2- H]-2,3,7,8-TCDD  derived radioactivity  to rat hepatic


macromolecules,  and  found maximum levels equivalent to  60 pmol of  2,3,7,8-


TCDD/mol of nucleotide in RNA, and 6  pmol of 2,3,7,8-TCDD/mol of nucleotide in


DNA.  This corresponds to  one  2,3,7,8-TCDD-DNA adduct/35 cells.  These investi-


gators  suggest  that  it is  unlikely  that  2,3,7,8-TCDD-induced  oncogenesis  is


through a mechanism  of covalent binding to DNA  and  somatic mutation.   Further


studies of 2,3,7,8-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.  (1982) investigated the toxicity of 2,3,7,8-TCDD metabolites


by administering extracts  of bile  from 2,3,7,8-TCDD-treated dogs to male guinea


pigs in single oral dgses equivalent to 0.6,  6.0 and 60 pg/kg of parent compound.


Other groups  of guinea pigs  were given  bile  extract  form untreated  dogs  or


2,3,7,8-TCDD itself.   A comparison of the mortality data at 5 weeks after dosing


indicated that the acute toxicity of 2,3,7,8-TCDD to guinea pigs was at  least 100


times higher thai} was the acute toxicity of its metabolites.


     Mason  and  Safe  (1986)  synthesized  2-hydroxy-3,7,8-TCDD  and  2-hydroxy-


1,3,7,8-TCDD, which are metabolites  of 2,3,7,8-TCDD,  and assessed the toxicity


of these compounds in male Wistar rats.   The compounds produced no significant


effect on body weight gain,  thymus, liver  or spleen weights after exposure to a


dose of <5000 ^g/kg  bw,.   2-Hydroxy-3,7,8-TCDD  induced  hepatic microsomal AHH,


EROD  and  4-chlorobiphenylhydroxylase  activity at  an  exposure  of  1000  and




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5000 jjg/kg bw, while 2-hydroxy-l,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 less than that of 2,3,7,8-TCDD.   Furthermore,  results are consistent



with the expected rapid conjugation and excretion of these 2,3,7,8-TCDD metabo-



lites (Weber et al., 1982).



     Metabolism of coplanar 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  coplanar 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 2,3,7,8-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




2,3,7,8-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 2,3,7,8-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/or  unidentified




metabolites may contribute to one or more of the toxic responses of 2,3,7,8-TCDD








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and related compounds.  Further studies on the nature of the blotransformation




products of these compounds will help to 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




2,3,7,8-TCDD and related compounds.  Metabolism plays a major role in regulating



the excretion and relative persistence of these compounds, since metabolism is




required for  urinary and  biliary  excretion.   Although the relative  rate of



metabolism of 2,3,7,8-TCDD and'related compounds can be estimated from tissue and



excretion half-life data (see Tables 1-5 and 1-8), 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 2,3,7,8-



TCDD and related compounds.  Therefore, in vivo disposition data  (see Tables 1-5




and 1-8) 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 cytochromes P-4501A1



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 which  may occur  at  exposures  which are  associated with enzyme




induction.  Characterization of the dose dependent disposition of 2,3,7,8-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 2,3,7,8-TCDD and related compounds into bile



represents  a direct means  for estimating the rate of metabolism, since 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  rate 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) 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








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male Fischer 344 rats.  All biliary  radioactivity  was attributable to metabo-


lites.  This rate of elimination is similar to the fecal excretion (-8% of the


dose) 24 hours after intravenous administration of 1 nmol/kg [%]-2,3,7,8-TBDD


(Kedderis et al., 1991a) and reflects  the effect of intravenous bolus versus oral


administration on distribution and elimination.  The large % 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., 1992a).   To assess  the ability of


2,3,7,8-TCDD and 2,3,7,8-TBDD to induce their own metabolism (biliary elimina-


tion), rats were pretreated with 100 nmol/kg,  per  os,  of each  compound 3 days


prior to intravenous injection  of  1  nmol/kg of the respective [ H] congeners.


Biliary excretion of the radiolabeled dose was  quantitatively and qualitatively


unaffected by pretreatment, despite a 2-fold increase in hepatic  levels of  [3H]


in the pretreated animals and significant induction of cytochrome P-4501A1 and


1A2  (Kedderis et al., 1991b).  Therefore, under these conditions, autoinduction


of 2,3,7,8-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  [  C]-2,3,7,8-TCDD,  despite significantly


enhanced levels of 2,3,7,8-TCDD in the livers of pretreated mice.

     While the above studies suggest  that autoinduction of  2,3,7,8-TCDD metabo-


lism 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 period, with pretreated rats  (10


pg/kg, intraperitoneal) excreting 9.7±1.9% of  the radiolabeled dose of 2,3,7,8-


TCDD (200-300 ^ig/kg,  per os)  compared to 7.0±0.9% excreted by naive animals.  In


addition to being  small changes,  these results were  obtained using a dose of


2,3,7,8-TCDD  in  excess of the  LD^Q  in  the rat.   Poiger and Schlatter  (1985)


examined the influence  of pretreatment with phenobarbital and 2,3,7,8-TCDD on the

                      ^
biliary excretion of [ H]-2,3,7,8-TCDD metabolites in a dog given a single oral


dose of the [ H] congener (31 or 33.8  ng/kg).  Without pretreatment,  24.5% of the




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absorbed dose was excreted  in the bile within 110 hours.  Phenobarbital did not



alter this rate, whereas pretreatment  with 2,3,7,8-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 investiga-




tion,  the  results  suggest  that  significant  autoinduction of  2,3,7,8-TCDD



metabolism and biliary excretion may occur in the dog.   Nonetheless, the small



increase in  metabolism and biliary excretion of  2,3,7,8-TCDD in the rat observed



by Poiger and Buser (1984)  and the negative results of Kedderis et al. (1991b)



and Curtis et al. (1990)  suggest that autoinduction of 2,3,7,8-TCDD metabolism




and biliary excretion in the rat may not occur, or occurs to an extent that is



not biologically relevant.




     Limited data suggest that autoinduction of  metabolism and biliary excretion



does occur  for CDFs  in contrast to CDDs and HDDs.   Pretreatment of rats with




2,3,7,8-TCDF (1.0 ^mol/kg,  3 days earlier) significantly increased the biliary



excretion of a subsequent dose  of  [ ^C]-2,3,7,8-TCDF  (McKinley  et al.,  1991).




The naive rats excreted 5.69±2.35% of the dose over the initial 8 hours, while




the pretreated rats excreted 13.18±3.15%  of the  [14C]-2,3,7,8-TCDF.  Similarly,




pretreatment of rats with 2,3,4,7,8-PeCDF  (500 pg/kg, per os,  3 days earlier)



resulted in  a 2-fold increase  in the biliary elimination  of a subsequent dose of




[* C]-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-methylchol-



anthrene-inducible form of  hepatic cytochrome P-450 (1A1 and/or 1A2), which is



also induced by 3,3',4,4'-TCB (Shimada and Sawabe, 1983;  Mills  et al.,  1985).



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 2,3,7,8-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 2,3,7,8-TCDD



in guinea pig, rat,  C57BL/6J mouse,  DBA/2J mouse and hamster  hepatocytes was






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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 2,3,7,8-TCDD is metabolized by the guinea pig liver at a rate -5-fold less



than that observed for the rat,  mouse and hamster.   The limited ability of the



guinea pig  to metabolize  2,3,7,8-TCDD  can explain  the limited  excretion of



2,3,7,8-TCDD metabolites in feces, which represents the major route for 2,3,7,8-



TCDD excretion (Olson, 1986).  In addition,  the  limited metabolism in the guinea




pig may partly explain the  relatively  long excretion half-life for 2,3,7,8-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 2,3,7,8-TCDD and related



compounds.    Wroblewski  and Olson   (1988)  investigated  the  metabolism  of




[14C]-2,3,7,8-TCDD (2.2 juM) in hepatocytes isolated from untreated 2,3,7,8-TCDD-,




3-MC-, isosafrole-  and  phenobarbital-pretreated rats  and  hamsters.   In both



species, 2,3,7,8-TCDD and 3-MC pretreatments elevated the rate of 2,3,7,8-TCDD




metabolism  by 5- to 6-fold, 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 pM) indicate that 2,3,7,8-TCDD can



induce its own rate of metabolism  in the rat and hamster.  In contrast, 2,3,7,8-




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 2,3,7,8-TCDD is  metabolized in the liver  by a 2,3,7,8-TCDD



inducible enzyme,  which is expressed in the  rat and hamster, but  not in the



guinea pig and mouse.  More recently, the kinetics of  2,3,7,8-TCDD metabolism was



investigated  in  isolated rat hepatocytes  incubated with [H]-2,3,7,8-TCDD at




concentrations of 0.01,  0.1 and 1.0 pM (Olson et al., 1991).  Lower 2,3,7,8-TCDD



concentrations in the media  result  in concentrations in hepatocytes which are




more similar to the levels  in the liver after in vivo exposure.   For example, the



concentration of 2,3,7,8-TCDD in hepatocytes incubated at 0.01 ^M are similar to



hepatic levels after in vivo exposure of rats at a dose of -10 pg/kg.  At  0.01
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and 0.1 pM, the rate of metabolism of [3H]-2,3,7,8-TCDD was similar in hepato-


cytes isolated from control and 2,3,7,8-TCDD pretreated rats, while at 1.0 /jM,


2,3,7,8-TCDD metabolism was greater  in hepatocytes  isolated from 2,3,7,8-TCDD


pretreated rats.  The results indicate that 2,3,7,8-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.   A dose-dependent autoinduction


of  2,3,7,8-TCDD  metabolism is  consistent with the  lack of  autoinduction of


2,3,7,8-TCDD metabolism and biliary excretion in the rat (Kedderis et al., 1991b;


Curtis et al., 1990).

                        o
     The metabolism of  [JH]-2,3,7,8-TCDF was also investigated in isolated rat


hepatocytes incubated at concentrations of 0.01,  0.1 and 1.0 pM  (Olson et al.,


1991).   At all concentrations,  hepatocytes from  2,3,7,8-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 2,3,7,8-TCDF is


metabolized in rat  liver by a 2,3,7,8-TCDD inducible enzyme,  possibly cytochrome


P-4501A1 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., 1991).


     There is in vivo and in  vitro data suggesting that autoinduction of 2,3,7,8-


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


1-8) support the assumption of  a first-order elimination process consisting of


one or more components.  These studies show that 2,3,7,8-TCDD was excreted slowly


from all species tested, with half-lives  ranging  from 11  days in  the hamster to


2120 days in humans.  2,3,7,8-TCDD is exceptionally persistent in humans relative


to other animal models.   Elimination data in  tissues (see Tables  1-5 and 1-6)


also indicate that 2,3,7,8-TCDD and related compounds are exceptionally persis-


tent  in  nonhuman primates (Bowman  et al., 1989;  Neubert et al., 1990).  These




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differences may also be in part related to the dose-dependency of the excretion



of these  compounds.    In general,  the congener and  species specific  rate of



elimination of 2,3,7,8-TCDD  and related compounds from major tissue depots  (see




Table 1-5) is similar to the excretion data summarized in Table 1-8.



     In the Syrian Golden hamster, the mammalian species least sensitive to the



acute toxicity of 2,3,7,8-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.,




1980b).  A similar excretion pattern was observed  in  mice,  although there was




significant strain variability (Gasiewicz et al., 1983; Birnbaum, 1986).  In all



the other species, excretion occurred mainly through the feces, with relatively



minor amounts of 2,3,7,8-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 1-8  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 [  C]-




2,3,4,7-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 2,3,7,8-TCDD-derived radioactivity  excreted in the urine



and  bile corresponds  to  metabolites of  2,3,7,8-TCDD (see Table  1-8).   The



apparent absence of 2,3,7,8-TCDD metabolites in liver and  fat suggests that  once



formed, the metabolites of 2,3,7,8-TCDD are excreted readily.  Thus,  urinary and



biliary elimination of  2,3,7,8-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 1-8).




     Although urine and bile  appear to  be  free of unmetabolized 2,3,7,8-TCDD,




data indicate that 2,3,7,8-TCDD and its metabolites are excreted in the  feces of




guinea pigs, rats, mice and hamsters treated with [  H]- and/or [^C]-2,3,7,8-TCDD








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(see Table 1-8).  While 15-35% of the  2,3,7,8-TCDD-derived radioactivity in rat,




mouse and hamster feces represents unchanged 2,3,7,8-TCDD,  81%  of the radioa-



ctivity in guinea pig feces represents unmetabolized 2,3,7,8-TCDD  (Olson, 1986;



Neal et  al.,  1982;  Gasiewicz et  al.,  1983; Olson et  al.,  1980).   The daily




presence of unchanged 2,3,7,8-TCDD in feces and its absence in bile  suggests that



direct intestinal elimination may be  the source  for  the  fecal  excretion of



2,3,7,8-TCDD,  Data  also suggest  that  direct  intestinal elimination of parent



compound contributes to the fecal excretion for 2,3,7,8-TBDD (Kedderis et al.,



1991). While the direct intestinal elimination of  parent compound  may occur for



other congeners (see Table 1-8), this conclusion cannot be made at  this time due



to the  lack of  experimental  data.    Nonetheless, the  species-specific  fecal




excretion of  2,3,7,8-TCDF is very similar to that observed for 2,3,7,8-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 CDFa was detected in  rat feces after subcutaneous exposure



to a defined mixture of congeners  (Abraham et al., 1989).  Studies in lactating



rats have also found that unchanged 2,3,7,8-TCDD may be excreted in the milk of




lactating animals (Moor© et al., 1976; Lucier et al.,  1975;  Nau et al., 1986).



Lactation, direct integtinal elimination,  and perhaps sebum  may serve as routes




for excretion of 2,3,7,8-TCDD, which do not depend on metabolism of the toxin.



These data suggest that the in vivo half-life for elimination  of 2,3,7,8-TCDD and



related compounds only provides an approximation  of the rate  of metabolism of



these compounds  in  $ given  animal.   The results  in Table 1-8  do 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 that 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 2,3,7,8-TCDD and related  compounds  is species- and



congener-specific (see Table  1-8).  2,3,7,8-TCDD is most persistent in human and



nonhuman primates.   In  the  hamster,  the least sensitive species  to the acute




toxicity of 2,3,7,8-TCDD, the mean tw was 10.8 days (Olson et al.,  1980a,b), and




in the guinea pig, the most  sensitive species to the acute toxicity of 2,3,7,8-
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TCDD, the mean ti^ was 94 days (Olson,  1986). 2,3,7,8-TCDF was also most persis-




tent in the guinea pig, with a ti^ of  20-40 days  (Decad et  al.,  1981a; loannou




et al.,  1983).  Furthermore,  results indicate that the relatively limited ability



of the  guinea pig to metabolize 2,3,7,8-TCDD  and -TCDF may  contribute to the



greater persistence and greater  acute  toxicity of these congeners in the guinea



pig.



     The time distribution,  metabolism and excretion of 2,3,7,8-TCDD were also



investigated in Han/Wistar and Long-Evans rats,  which were, respectively, more




resistent (LD5Q>3000  jjg/kg) versus more susceptible  (LD^Q -10 /jg/kg)  to  the acute




toxicity of 2,3,7,8-TCDD  (Pohjanvirta et al.,  1990).  The results suggest that




the  metabolism  and disposition of 2,3,7,8-TCDD  do not  have a major role in



explaining the strain differences in toxicity.



     The  intraspecies  differences in the  ti^ of  2,3,7,8-TCDD in  three mouse




strains may  be  due  to the  finding that  the  DBA/2J strain  possesses -2-fold




greater adipose tissue stores than the C57BL/6J and  B6D2F^/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 2,3,7,8-



TCDD in this strain.   Birnbaum (1986)  examined the effect of genetic background



on the distribution and excretion of 2,3,7,8-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 2,3,7,8-TCDD.   Thus, the



distribution and excretion of 2,3,7,8-TCDD were primarily governed  by  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  2,3,7,8-TCDD do 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 2,3,7,8-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








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                         DRAFT—DO NOT QUOTE OR CITE






elimination of [^C]-2,3,7,8-TCDD in rats given repeated oral doses of 0.01, 0.1




or 1.0 pg/kg/day Monday through Friday for 7 weeks or a single dose of 1.0 /ug/kg.



In the single-dose study, no * C  was excreted in the urine or expired air; in the




repeated-dose study, however, 3-18% 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 2,3,7,8-



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.,  1991).    These  results  are  consistent with  the in  vivo and  in vitro



evidence suggesting that autoinduction of 2,3,7,8-TCDD  and  2,3,7,8-TBDD metabo-




lism  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., 1991).  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.,   1991; Olson et al.,  1991).   Autoinduction of metabolism would



suggest 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 2,3,7,8-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 Table  1-8).  The half-life




for elimination was estimated to be 2120  days.  Table 1-9 summarizes additional



half-life estimates for 2,3,7,8-TCDD and related compounds  in humans, based on



serum  and/or  adipose tissue  concentrations  at two  or more time points.   In



another study, the  half-life  of 2,3,7,8-TCDD  in  humans was estimated to be ~7



years on the basis of 2,3,7,8-TCDD levels in serum  samples taken in 1982 and  1987



from 36 of the Ranch Hand personnel who  had 2,3,7,8-TCDD levels >10 ppt  in  1987



(Pirkle et al.,  1989).  These studies indicate that 2,3,7,8-TCDD  is exceedingly




persistent  in humans.   Estimated half-lives for  other congeners in Table 1-9



range from 0.8-10 years.  The half-life  values  in  Table 1-9 are rough estimates



based on  a  small  number of  individuals  and based on analysis at as few as two






                                     1-61                             08/11/92
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time points.  Phillips  (1989) discusses  this issue.   Estimates also  assume a



simple, single compartment, first-order elimination process. Recent data suggest



a biphasic elimination  of  2,3,7,8-TCDD,  with a half-life >7  years  (Birnbaum,



1992).



     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 pg/kg, while



Yusho patients had  levels of 0.1-5 pg/kg.   In  the  Yu-Cheng  individuals, half-



lives for three CDFs were 2-3 years, while elimination from Yusho individuals was




more  variable and slower,  with  half-lives  >5  years  (see  Table 1-9)  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. (1990)



and Ryan (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.



     Due  to  the  lipophilic nature  of  milk,  milk  secretion  can provide  a



relatively efficient mechanism for decreasing the body burden of 2,3,7,8-TCDD in



females.  As discussed by Graham et al. (1986), this elimination of 2,3,7,8-TCDD



through mother's milk can result  in high exposure levels in the infant.  Since



both  milk and the  fatty tissues  of  fish are  essentially  providing  an oily



vehicle,  it would be likely that these sources would provide 2,3,7,8-TCDD in  a



form that is  readily bioavailable.



     Several  investigators have quantified the levels of 2,3,7,8-TCDD in human



milk samples. Many of the milk samples were pooled (Jensen, 1987).  Rappe et  al.



 (1984) reported levels of 1-3 ppt  2,3,7,8-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  2,3,7,8-TCDD  in milk  fat  from four



volunteers in northern Sweden.  Furst et al.  (1986)  reported an average  level of



9.7 ppt 2,3,7,8-TCDD  in milk fat  from three individuals  in the Netherlands  and



<1.0 ppt 2,3,7,8-TCDD in milk fat  from two individuals in Yugoslavia.  Nygren et




al.  (1986)  reported average levels of 2,3,7,8-TCDD in human milk samples from



 four  subjects in  Sweden to be  0.6 ppt in milk  fat,  in five subjects from West




                                      1-64                             08/11/92
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                          DRAFT—DO  NOT QUOTE OR  CITE






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 2,3,7,8-TCDD have  been detected  in  the milk  of mothers



exposed to  high levels of 2,3,7,8-TCDD in  the  environment.  Reggiani  et al.



(1980) reported levels between 2.3 and 28.0  ppt 2,3,7,8-TCDD in whole milk from



mothers in  Seveso.   Baughman  (1975)  reported levels  between 40.0 and 50.0 ppt




2,3,7,8-TCDD in whole milk from mothers in South Vietnam.  Schecter et al. (1987)



also  found  high ppt levels of  2,3,7,8-TCDD in  human milk  samples  from South



Vietnam.  These authors found that samples taken in 1985 from South Vietnamese




mothers were comparable to the  level of 2,3,7,8-TCDD presently  found in North



American human milk samples (5 ppt).



1.4.   PHYSIOLOGICALLY BASED PHARMACOKINETIC MODELS



     PB-PK  models have  been  developed for 2,3,7,8-TCDD in C57BL/6J and DBA/2J



mice  (Leung et al., 1988), rats  (Leung et  al.,  1990) and  humans (Kissel and



Robarge,  1988).   PB-Pk  models incorporate  known  or estimated anatomical,



physiological  and physicochemical parameters to describe  quantitatively the



disposition of  a  chemical  in  a  given species.  PB-Pk models can assist in the




extrapolation of high-to-low dose kinetics within a species, estimating exposures



by different routes of administration, calculating effective doses and extrapo-



lating  these values  across  species  (Scheuplein et  al.,  1990).   Table 1-10



summarizes  the  pharmacokinetic  parameters  for 2,3,7,8-TCDD that  were used in



developing these PB-Pk models.  In many cases,  these parameters were estimated




from in vivo experimental data.



     A five-compartment (blood,  liver, fat,  muscle/skin, viscera), flow-limited



PB-Pk model for 2,3,7,8-TCDD  was developed for the Ah-responsive C57BL/6J mouse



and the  Ah-nonresponsive DBA/2J mouse  (Leung et al., 1988).   The  model also



included binding in the hepatic cytosol and hepatic microsomes and first-order



hepatic metabolism.  There was general agreement  between the simulated descrip-



tion generated by the model and the  experimental disposition data of Gasiewicz



et al.  (1983).   The greater accumulation of 2,3,7,8-TCDD  in  the liver of the



C57BL/6J mouse, compared to the  DBA/2J mouse, was not attributable to the 2-fold



greater total  fat content in the DBA/2J  strain.  The  authors  suggested that



strain  difference  in  hepatic  disposition  was  due  to differing  affinity of




                                     1-65                             08/11/92
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2,3,7,8-TCDD for the microsomal binding protein in  the two strains, and proposed




using a different microsomal dissociation  constant  for  each strain.   Alterna-



tively, the strain difference in hepatic disposition may be  due to the different



doses of 2,3,7,8-TCDD needed to induce cytochrome  P-4501A2 in the two strains.



In  contrast to  the  high  capacity/low  affinity   hepatic  microsomal  binding



proteins,  the low capacity/high affinity hepatic cytosolic binding protein (Ah



receptor)  did not play a major role  in determining the overall tissue distribu-




tion pattern of 2,3,7,8-TCDD in this model.



     A similar five-compartment PB-Pk model was developed to describe the tissue




disposition of 2,3,7,8-TCDD in the Sprague-Dawley rat (Leung et al.,  1990).  This



description included  blood,  liver  (cytosolic receptor  and microsomal  binding



protein),  fat,  muscle/skin  and visceral tissue compartments.  The authors found



generally good agreement between the PB-Pk  model simulated  data and the experi-




mental data  for  the single-dose  study  of  Rose et  al.  (1976)  and the  7- and



13-week multiple-dose  studies  of Kociba et al.  (1978).    The  model was not



satisfactory for the  2-year  feeding study of Kociba et al.  (1978),  underpre-



dicting the 2,3,7,8-TCDD  concentration  in  the fat  at the  low  dose  (0.001



pg/kg/day) and overestimating the concentration achieved at the high dose (0.1



^g/kg/day).   The model  found  the  hepatic  disposition  of 2,3,7,8-TCDD  to  be



dependent on the high capacity/low affinity hepatic microsomal binding protein



with a dissociation  constant of 7 nM  and a basal and induced  concentration in the




liver of 25 and 175 nmol/liver,  respectively. As discussed  earlier, Voorman and



Aust (1987,  1989) and Poland et al. (1989a,b) provided evidence that this binding



species is cytochrome  P-4501A2.  Induction of the microsomal binding protein was



necessary in order to  account for  the differences in hepatic disposition at low



(0.01 p
-------
                         DRAFT—DO NOT QUOTE OR CITE





                                                                 19S
     A PB-Pk model was also developed  for female C57BL/6J mice for  [ *•"!]-2-iodo-




3,7,8-TCDD, an analog of 2,3,7,8-TCDD  (Leung et al., 1990).  Mice were pretreated



with 0.1 ^mol/kg of  2,3,7,8-TCDD or the vehicle only, followed by 2-iodo-3,7,8-



TCDD (0.1  nmol/kg)  3  days  later.   Naive mice had liver/fat 2-iodo-3,7,8-TCDD



concentration ratios of 0.17-0.38, while the 2,3,7,8-TCDD  pretreated mice had



ratios of 2.0-6.1.  This is  in agreement with the dose-dependent tissue distribu-



tion of  2,3,7,8-TCDD  described earlier  (Abraham et al., 1988; Poiger  et al.,



1989).   As  with 2,3,7,8-TCDD,  the  model found  that the  2-iodo-3,7,8-TCDD



concentration in the  liver was most  sensitive  to the  binding  capacity of the



hepatic  microsomal   protein.    Whole-body elimination  of  2-iodo-3,7,8-TCDD



approximated first-order kinetics, and induction by pretreatment with an inducing



dose of  2,3,7,8-TCDD almost doubled the rate of excretion  (ti^ of 14.2 days in




naive versus  8.0 days in  induced mice)  (see Table  1-8).   The distribution in



naive and  pretreated mice  was described by  a  PB-Pk model  in  which induction



(2,3,7,8-TCDD pretreatment) increased the amount of hepatic microsomal binding



protein  from 1.75-20 nmol/liver and increased the rate constant for metabolism



of  free  2-iodo-3,7,8-TCDD  from 1-3  hours/kg liver.   Although the more rapid



elimination of 2-iodo-3,7,8-TCDD in 2,3,7,8-TCDD pretreated mice suggests that



the rate of metabolism of 2-iodo-3,7,8-TCDD was  induced by pretreatment, no data



were provided on the effect of this pretreatment of body weight and composition,



which may in turn alter the rate of elimination.   2,3,7,8-TCDD pretreatment may



also  alter deiodinase activity.    Furthermore,  in  vivo and in  vitro studies



suggest  that  the autoinduction of  2,3,7,8-TCDD  metabolism may not occur under



these conditions.   Kedderis et  al.   (1991b) and Curtis et  al.  (1990) found no



autoinduction of 2,3,7,8-TCDD metabolism and biliary excretion in the rat.  In



addition,  Shen  and  Olson  (1987)  found  that while 2,3,7,8-TCDD pretreatment of



C57BL/6J mice increased the uptake of 2,3,7,8-TCDD by hepatocytes in  suspension



culture, pretreatment did not  increase the rate of metabolism of 2,3,7,8-TCDD by



hepatocytes.   Therefore,  this  PB-Pk model  may  not   accurately  describe the



metabolism of 2,3,7,8-TCDD for exposures  which result in  varying degrees of



induction  of  the   hepatic monooxygenase  system.   While  the dose-dependent



pharmacokinetics of 2,3,7,8-TCDD may not include  autoinduction of  2,3,7,8-TCDD






                                     1-68                             08/11/92
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metabolism, this may not be the  case  for other CDDs, BDDs, CDFs, BDFs, PCBs and




PBBs.  For example, the autoinduction of metabolism has been reported for CDFs



(Brewster and Birnbaum, 1987; McKinley et al.,  1991; Olson et al., 1991).



     Andersen et al.  (1992) recently derived a receptor-mediated PB-PK model for



the tissue distribution and enzyme inducing properties of 2,3,7,8-TCDD. The data



used for this analysis were  from  two previously published studies with Wistar



rats (Abraham et al.,  1988; Krowke et al., 1989).   The model was  used to examine



the tissue disposition of 2,3,7,8-TCDD and the induction of both a dioxin-binding



protein (presumably cytochrome P-4501A2)  and cytochrome P-4501A1.




     Kohn et al. (1992) recently developed a mechanistic model of the effects of



dioxin on glue expression in the rat liver (referred to as the NIEHS model).  The



model includes the tissue distribution of 2,3,7,8-TCDD in the  rat and its effect



on the concentrations of CYP1A1 and CYP1A2,  and the effects of 2,3,7,8-TCDD on




the  Ah,  estrogen  and  EGF receptors  over   a  wide  2,3,7,8-TCDD dose  range.



Experimental data  from Tritscher  et  al.   (1992) and Sewall et  al. (1992) were




incorporated into the  NIEHS model.  Female Sprague-Dawley rats were  injected with



an  initiating  dose of  diethylnitrosamine,  and after  20 days, the  rats were



exposed biweekly to 2,3,7,8-TCDD  in  corn  oil by gavage at doses equivalent to



3.5-125 ng/kg/day for 30 weeks.  The  NIEHS model predicts  a linear relationship



between administered  dose  and the concentration  in the  liver  over  this dose



range, which  is in agreement with the data of Tritscher et al.  (1992).   The




biochemical response  curves for all these proteins were hyperbolic, indicating



a proportional relationship between target tissue dose and protein concentration



at low administered doses of 2,3,7,8-TDCDD.



     A fugacity-based PB-Pk model for  the elimination of 2,3,7,8-TCDD from humans



was  developed  by Kissel and  Robarge (1988).  Transport within  the  body was



assumed to be perfusion-limited  (flow-limited).  2,3,7,8-TCDD was  assumed to be



uniformly distributed within each  tissue  or  fluid  phase,  and  tissue levels were



considered  to be  in equilibrium with  exiting fluids  (blood,  bile,  urine).




2,3,7,8-TCDD  appears  to be  poorly  metabolized in  humans,   thus  reducing the



necessity  of  modeling the  fate of metabolites.   2,3,7,8-TCDD also  seems to




exhibit fugacity-based partitioning behavior in  humans as evidenced by relatively



constant lipid-based tissue distribution (Leung et  al.,  1990; Ryan et al., 1987),




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although this is not the case  in  rodents (Leung et al., 1988, 1990).   With a



daily human background  intake of  2,3,7,8-TCDD  in North America of  -50 pg/day



(Travis and Hattemer-Frey,  1987), the steady state adipose tissue concentration



predicted by the model,  assuming no metabolism,  was 7.7  ppt.  This is similar to



the lipid-based blood tissue levels reported in the general population with no



known unusual exposure.   The model was also used to predict the elimination of



2,3,7,8-TCDD from Ranch Hand Vietnam veterans.   The model simulation assumed a



background exposure of  50  pg/day  and no metabolism.   Under these conditions,



apparent half-lives of 4.4, 5.2,  5.9, 7.2,  9.1  and 20 years were estimated for



individuals with adipose tissue concentrations of 100, 50, 30, 20,  15  and 10 ppt,



respectively.  The model predicted half-lives  are  similar  to the experimental



value of 7.1 years, based on analysis of 2,3,7,8-TCDD in blood lipids of veterans



with adipose burdens greater than  10  ppt  (Pirkle et al., 1989) (see Table 1-9).



The apparent half-lives derived from the model  increased as the adipose tissue



concentrations  approached  the steady-state  level  associated with  background



exposure.  Ryan and Masuda  (1991) also reported  a similar relationship for CDFs,



with experimentally derived half-lives increasing in individuals with lower body



burdens of the compounds.   Finally, the model was also  found to approximate the



elimination  of 2,3,7,8-TCDD  from one  volunteer  as   reported  by  Poiger  and



Schlatter (1986).   Taken together,  the comparisons described above suggest that



a fugacity-based PB-Pk model for 2,3,7,8-TCDD in humans can provide one method



for describing the elimination of 2,3,7,8-TCDD from humans.



     Kedderis et al.  (1992a,b) recently developed a PB-PK model for 2,3,7,8-TBDD



in the  rat.   The model  is  based  on previously  developed physiologically-based



models  for 2,3,7,8-TCDD (Leung et al., 1990; Poland et al., 1989) and utilizes



published data on the disposition of a single exposure to 2,3,7,8-TBDD at a dose



of  1  or  100  nmol/kg,  intravenous   (Kedderis et  al.,  1991a,b)   and dermal



disposition  data  (Jackson  et al., 1991).   In  the model,  the  dose- and time-



dependent  accumulation in  the  liver was  attributed  to specific  binding of



2,3,7,8-TBDD  with the  inducible  protein,   CYP1A2.    The model  also   includes



diffusion-limited tissue uptake of 2,3,7,8-TBDD, transluminal excretion of parent



compound via the gut into the feces, growth of tissue compartments  and a  separate



skin compartment.   This  model provides further validation of the model structure




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originally  developed to  describe  important  dispositional  determinants  for




2,3,7,8-TCDD.



     A five-compartment  (blood, liver, fat, skin, muscle)  flow-limited physiolog-



ical model was developed  to  describe the  tissue  distribution and excretion of



2,3,7,8-TCDF-derived material in  rats, mice and monkeys  (King  et  al.,  1983),



based on experimental data reported earlier  (Birnbaum et al., 1980, 1981; Decad



et al., 1981b).  Partition coefficients (tissue/blood distribution ratios) and




metabolic clearances  were estimated  from in  vivo  experimental data  and are



summarized in Table 1-11.   All pharmacokinetic  parameters  for 2,3,7,8-TCDF were




based on  in vivo data  after a  single intravenous  exposure  at a  dose  of 0.1



pmol/kg (30.6 pg/kg).   Therefore, the model  is limited  in not considering the



potential dose-related  distribution  and  excretion of  2,3,7,8-TCDF.   Recent



studies indicate that 2,3,7,8-TCDF is able to induce its own rate of metabolism



and biliary  excretion  at  higher  doses  (McKinley  et al.,  1991; Olson  et al.,



1991).  This model will need to be  revised as additional data on the dose-related




distribution and excretion of 2,3,7,8-TCDF become available.



     PB-Pk models  are  primarily  limited  by the availability of  congener and




species-specific data  that  accurately describe  the dose- and  time-dependent



disposition  of  2,3,7,8-TCDD and related  compounds.    The  pharmacokinetic



parameters summarized in Tables 1-10 and 1-11 were derived from available  in vivo



and in vitro experimental  data.   As  additional data become available,  partic-



ularly on the dose-dependent disposition of these compounds, more  accurate models



can be  developed.   In  developing a  suitable  model  in  the human, it  is also



important to consider that the half-life estimate of 7.1 years for 2,3,7,8-TCDD



was based on two serum values taken 5 years  apart, with the assumption of a



single compartment, first-order elimination process (Pirkle et al., 1989).  It



is likely that the excretion of 2,3,7,8-TCDD in humans is  more complex, involving



several compartment, tissue-specific bonding proteins and a  continuous daily



background exposure.  Furthermore, changes in body weight and body composition




should also  be considered in developing PB-Pk models for 2,3,7,8-TCDD and related



compounds in humans.




     An empirical model  of dioxin  (toxic equivalents) disposition in animals and



humans has  also  been recently developed  by Carrier and  Brodeur  (1991).   The




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TABLE 1-11
Pharmacokinetic Parameters for 2,3,7,8-TCDF Used in the PB-Pk Model
Described by King et al. (1983)

C57BL/6J
Mouse
DBA/2J
Mouse
Fischer 344
Rat
Rhesus
Monkey
PARTITION COEFFICIENTS
Liver
Fat
Skin
Muscle
130
25
8
2
100
40
12
4
100
35
4
2
30
30
7
2
CLEARANCES
Metabolism
Km (mL/minute/kg)
Metabolism excretion ratio K^/KL*
0.07
2.8
0.14
0.06
2.4
0.27
1.0
4.0
0.03
2.25
0.45
0.19
aUrinary clearance/biliary clearance
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kinetic analysis begins with  the  observation that the  tissue distribution of




dioxin-like HPAH in humans  and  in animals  is  dose-dependent  (or, alternately,



body-burden dependent).  As  total body burden of TCDD equivalents  increases, the



proportion of  the  body burden  associated with  the  liver  increases  toward a



maximum value.   The data were then analyzed with an empirical,  saturable binding



isotherm equation:
                   liver fraction (fH) = fwsaiCbody/(Kd
If the body burden-Cj^   (^g/kg)— is considered a surrogate for liver concentra-




tion, this  equation can  be  loosely interpreted  as  the induction  of binding



species in the liver as dose increases.   In the analyses, Kd was found to be very



similar for people and experimental animals, indicating similar protein induction



dynamics in various  animal species. This model, however, is not physiologically-



based and the  terms,  C^jy and f^x, are difficult to  interpret in biological




terms.   In  working  with different isomers,  fjj^  and K^ values vary somewhat,




presumably due to binding affinities in the liver.



     This empirical model is successful in providing a description that "fits"



the observed data in various species.  It still is  largely a  fitting exercise to



a particular equation, not an examination of biology by computer modeling.  In



addition, there  are at  least  two assertions that seem incorrect.   First, the



assumption that the  limit of the hepatic fraction at very low doses  is  zero.  It



seems more  likely that  the  limit is some  finite value, determined  by liver



partitioning of  dioxin and the binding  parameters of the Ah receptor, and the



dioxin binding species in the liver in the linear, low-dose region.  Secondly,



that metabolism of dioxin becomes  saturated with the maximum induction of liver



sequestration  of dioxin.   There  is no  justification  for this  at  present.



Nevertheless,  the model  indicates clearly that with respect to dosimetry and



induction of  hepatic  binding  species for dioxin,  people and rodents  are very



similar.   Furthermore,  the empirical model  of Carrier and Brodeur (1991)  is



generally consistent with the PB-PK models.






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1.5.   PHARMACOKINETICS IN SPECIAL POPULATIONS



1.5.1.   Pregnancy and Lactation (Prenatal and Postnatal Exposure of Offspring).



The distribution  and excretion  of  [14C]-2,3,7,8-TCDD  (30 pg/kg)  and  [14C]-




2,3,7,8-TCDF  (800  /jg/kg)  were studied  in pregnant  C57BL/6N mice  after oral



exposure on gestation day 11 (Weber and Birnbaum,  1985).   The distribution and



excretion of 2,3,7,8-TCDD and 2,3,7,8-TCDF in pregnant mice were similar to that



of males of the same strain (Gasiewicz et al., 1983; Decad et al., 1981b) (see



Tables 1-5 and 1-8), although elimination rates were higher in the pregnant mice



for both congeners.  For 2,3,7,8-TCDD, liver,  urinary and fecal elimination were



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 were 1.3, 1.8 and 1.8  times faster than



observed for males.  Elimination data from pregnant mice was based on only three



time  points (gestation days  12,  13  and  14)  and  thus  represents  only  rough



estimates.   In addition,  the greater fecal  excretion could have been  due to



incomplete  absorption of 2,3,7,8-TCDD  after  oral  exposure.   Although  these



results  need further substantiation, it  is conceivable  that  the  sex  of the



animal, pregnancy and/or the route of exposure could have a significant impact



on the pharmacokinetics of these compounds.



      In a related study,  Krowke  (1986) compared the 2,3,7,8-TCDD concentrations



in the liver 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 2,3,7,8-TCDD concentrations were 7 and 32 ng/g in pregnant and



nonpregnant  mice,  respectively.   At the  higher exposure,  5.5 times  lower



concentrations of 2,3,7,8-TCDD were found in the livers of pregnant animals on



gestation day 18.  A similar effect on hepatic  2,3,7,8-TCDD levels was observed



also  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 2,3,7,8-TCDD in pregnant mice are



consistent with the Weber and Birnbaum (1985) observation of more rapid elimina-



tion  of 2,3,7,8-TCDD in pregnant mice.  Further investigations are necessary to



better  characterize the  apparently  significant effects  of pregnancy  on the



disposition of 2,3,7,8-TCDD and related compounds.
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     Weber and  Birnbaum  (1985)  also  investigated the  distribution of  [14C]-




2,3,7,8-TCDD  (30 /jg/kg)  and [ 14C]-2,3,7,8-TCDF  (800 M9/k9) 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 approxi-



mately  11-fold  higher concentrations of 2,3,7,8-TCDD  than  2,3,7,8-TCDF when



exposed on a 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  to 2,3,7,8-TCDD.




Assuming that all radioactive  material found  in embryos  was parent compound, at



most, 2.6 ng  (8 pmol) of 2,3,7,8-TCDD and 6.4 ng  (21  pmol)  of 2,3,7,8-TCDF/g



tissue were detected under these conditions.




     The transfer of  [ ^C]-2,3,7,8-TCDD to the embryo during early gestation was




assessed in NMRI mice given a dose of 25 /jg/kg by intraperitoneal injection on



either days 7, 8, 9,  10, 11 or 13 of gestation (Nau and Bass, 1981).  The mice



were sacrificed after 48 hours, and 2,3,7,8-TCDD concentrations  were determined



by liquid scintillation counting of solubilized tissue  and  by  GC-ECD and GC/MS.



Similar results  were given by these methods, suggesting that  2,3,7,8-TCDD derived




[  C]  in maternal and embryonic tissue was the parent  compound.  The maternal




liver contained  from 4-8% of the dose/g or  40-80 ng/g. 2,3,7,8-TCDD in embryonic



tissue from gestation days 11-15 ranged from  0.04-0.1%  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 2,3,7,8-TCDD  was relatively low, as



compared to maternal liver; however, 2,3,7,8-TCDD levels  in fetal livers were 2-4



times higher  than levels  in  other  fetal organs.   Nau and  Bass  (1981)  also



attempted to  correlate  2,3,7,8-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  2,3,7,8-TCDD on gestation day 7 or 10 or 5




pg/kg/day, intraperitoneally, on  gestation  days 7-11.   On gestation day 13,



2,3,7,8-TCDD concentrations in maternal  tissues were very similar in the three





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exposure groups.  At day 13, however, the embryo contained  0.038±0.011%  (0.36




ng/g)/ 0.096±0.027% (0.92 ng/g) and 0.12±0.05% (1.1 ng/g) of the dose (mean±SD)



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 2,3,7,8-



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 2,3,7,8-TCDD-



induced cleft palate in C57BL/6N mice; however, tissue  levels were not investi-



gated .



     In the same laboratory,  Abbott et al. (1989) investigated the distribution



of  2,3,7,8-TCDD  in the C57BL/6N mouse fetus  following maternal exposure on



gestation day 11 to 30 pg/kg.  2,3,7,7-TCDD was detected in the gestation day-11



embryo at 3 hours post-exposure and was equally distributed between the embryonic



head and body.  At  72 hours post-exposure, 0.035% of the total dose was  in fetal



tissues, and  1%  of  the 2,3,7,8-TCDD  in the fetus  (1.4-3.5  pg  was  found in the



palatal shelf.




     Krowke  (1986) also measured  the  concentration  of  2,3,7,8-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



2,3,7,8-TCDD were observed in the placenta, amniotic fluid and  fetus  (-0.5 ng/g)



on day 16  of  gestation.  Fetal liver 2,3,7,8-TCDD concentrations were  at least



five  times greater than other fetal tissue.   Krowke (1986) reported  slightly




lower 2,3,7,8-TCDD levels in  the fetal head relative to other extrahepatic fetal



tissue, while Weber and  Birnbaum (1985) found  a  slightly  higher 2,3,7,8-TCDD



concentration in the head relative to other extrahepatic fetal tissue.



     Nau et al.  (1986) investigated the transfer of 2,3,7,8-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




2,3,7,8-TCDD  (Nau  and Bass,  1981) and found that postnatally, 2,3,7,8-TCDD was



transferred efficiently to mouse neonates  and offspring by lactating  mothers.



During the first 2  postnatal weeks, the pups were given doses of 2,3,7,8-TCDD via



the  milk that were, on a  body weight basis,  similar to those that had  been




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administered prenatally to  their mothers.  2,3,7,8-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 2,3,7,8-TCDD and for



the subsequent exposure of pups.



     The disposition of 2,3,7,8-TCDD in rat pups was  assessed  after the prenatal



(via placental  transfer)  and/or 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 2,3,7,8-TCDD from  maternal tissues, with the  half-life of 2,3,7,8-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 2,3,7,8-TCDD



concentrations that were greater than the corresponding levels  in maternal liver.



In cross-fostering experiments,  the concentrations of 2,3,7,8-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 2,3,7,8-TCDD in  rats  and mice is



relatively limited compared with the efficient  transfer via maternal milk.



     Van den Berg  et al.  (1987) 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 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.  2,3,7,8-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 2,3,7,8-




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TCDD,  1,2,3,7,8-PeCDD  and  the  three  2,3,7,8-aubstituted 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.   2,3,7,8-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 3-fold  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




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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  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 2,3,7,8-TCDD in the 33-day-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 2494 pg I-TE/g,  while the offspring  liver contained  2022 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 2,3,7,8-TCDD to the offspring of rhesus



monkeys was  investigated by Bowman et al.  (1989).    Animals were fed  a diet




containing 2,3,7,8-TCDD at concentrations of 5 or 25 ppt for -4 years and were




on  a  2,3,7,8-TCDD-free  diet for  -18  months prior  to parturition.   Maternal



2,3,7,8-TCDD levels (mean±SE) in adipose tissue were 49±11 (n=7) and 173±81 (n=3)




ppt in  the  5 and  25  ppt groups, respectively.   Corresponding levels  in the



adipose tissue of offspring at weaning  (4 months) were 187158 an 847±298 ppt in



the 5 and 25 ppt groups,  respectively.  From these data,  a 2,3,7,8-TCDD BCF of



4.29 was estimated from mother to nursing  infant.  This value  is similar to that



observed for 2,3,7,8-TCDD in the marmoset  monkey (Hagenmaier  et al., 1990).  The



milk of the rhesus monkeys  in the 25 ppt group contained  from 4-14  ppt of



2,3,7,8-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 17-44% of



their 2,3,7,8-TCDD  body  burden by  lactation.   They  also concluded  that the




results are  generally  consistent with overall triglyceride movement as mediating



the excretion of 2,3,7,8-TCDD in milk.




     In a subsequent study, Bowman et al. (1990)  reported the relative persis-



tence of  2,3,7,8-TCDD in  the  offspring  of rhesus  monkeys   that were exposed




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earlier to  5 or 25  ppt of  2,3,7,8-TCDD  in the diet.   The  concentration of



2,3,7,8-TCDD in  adipose  tissue  was measured in offspring  at -4-5,  12  and 24



months of age.   The decrease of 2,3,7,8-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 2,3,7,8-TCDD  from adipose




tissue at a  faster rate than adult rhesus monkeys,  which  had individual half-



lives ranging from 180-550 days (Bowman et al., 1989).



     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 concentra-



tions of CDDs in human milk  (on a fat basis) ranged from 195 ppt for OCDD to 2.9



ppt for  2,3,7,8-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 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 milk fat (ppt) at  1, 5 and 52 weeks  after delivery were  251, 132 and



119  for OCDD,  7.9,  5.9  and  1.4  for 2,3,7,8-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 post-




partum.  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.








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     Although data  are more  limited  for the  coplanar 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).  Therefore,  lactation appears to be an



effective means  for  the excretion of  coplanar  PCBs  from mothers  and a. major



source of postnatal  exposure  of nursing infants.  Since 3,3 ' ,4,4',5-PeCB and



other coplanar PCBs are present in human  milk at  concentrations up to 60-fold




higher than  2,3,7,8-TCDD, it is important to consider the relative toxic potency



of these dioxin-like compounds and their potential  health  impact on nursing




infants.



1.5.2.   Aging.   The  influence of aging on the intestinal absorption of 2,3,7,8-



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 2,3,7,8-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 2,3,7,8-TCDD.



     Banks et al. (1990) studied the effect of age on the dermal absorption and




disposition of 2,3,7,8-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



2,3,7,8-TCDD, at  3 days after exposure,  decreased from 17.7±2.7% (mean±SD) to



5.6±2.5% 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 22.2±0.2 to



14.7±3.8% 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




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in various depots reflected age-related changes in dermal absorption, while 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 conducted



using 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 2,3,7,8-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/cnr)  2,3,7,8-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., 1992).  Dermal  absorption using acetone as vehicle was greatest



in 3-week-old rats  (129 pmol; 64% of the administered dose),  decreasing to -80



pmol  (40%)  in  B-r 8- and 10-week-old rats and  to  45  pmol  (22%)  in 36-week-old



rats.  The  results  indicate  that 2,3,7,8-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.



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