6
Noncancer End Points

This chapter reviews the U.S. Environmental Protection Agency (EPA) assessment of the noncancer end points, including immune function, reproduction and development, diabetes, thyroid function, lipid levels, and other effects related to exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, also referred to as dioxin), other dioxins, and dioxin-like compounds (DLCs) in animals and humans. The purpose of this chapter is to critically assess, to the extent possible, whether EPA has met the criteria set forth in the “Statement of Task” with respect to the noncancer effects of TCDD, other dioxins, and DLCs. Toward this end, this chapter focuses on the uncertainties and assumptions made by EPA in determining whether TCDD, other dioxins, and DLCs have noncancer effects in humans; determining the methods and models used for assessing these effects; determining the breadth and robustness of the studies used and the balance with which the studies are presented in the Reassessment,1 and finally, determining whether the conclusions reached by EPA are consistent with the current scientific peer-reviewed literature.

IMMUNE FUNCTION

EPA uses a sizeable immunotoxicology database derived largely from laboratory animal studies and a smaller number of epidemiological studies in

1

The Exposure and Human Health Reassessment of 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and Related Compounds (EPA 2003a, Part I; 2003b, Part II; 2003c, Part III) is collectively referred to as the Reassessment.



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Health Risks from Dioxin and Related Compounds: Evaluation of the EPA Reassessment 6 Noncancer End Points This chapter reviews the U.S. Environmental Protection Agency (EPA) assessment of the noncancer end points, including immune function, reproduction and development, diabetes, thyroid function, lipid levels, and other effects related to exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, also referred to as dioxin), other dioxins, and dioxin-like compounds (DLCs) in animals and humans. The purpose of this chapter is to critically assess, to the extent possible, whether EPA has met the criteria set forth in the “Statement of Task” with respect to the noncancer effects of TCDD, other dioxins, and DLCs. Toward this end, this chapter focuses on the uncertainties and assumptions made by EPA in determining whether TCDD, other dioxins, and DLCs have noncancer effects in humans; determining the methods and models used for assessing these effects; determining the breadth and robustness of the studies used and the balance with which the studies are presented in the Reassessment,1 and finally, determining whether the conclusions reached by EPA are consistent with the current scientific peer-reviewed literature. IMMUNE FUNCTION EPA uses a sizeable immunotoxicology database derived largely from laboratory animal studies and a smaller number of epidemiological studies in 1 The Exposure and Human Health Reassessment of 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and Related Compounds (EPA 2003a, Part I; 2003b, Part II; 2003c, Part III) is collectively referred to as the Reassessment.

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Health Risks from Dioxin and Related Compounds: Evaluation of the EPA Reassessment its assessment of immunotoxicity produced by TCDD, other dioxins, and DLCs. The assessment of changes in immune competence, regardless of the cause, is complex, as the immune system is composed of a large and diverse group of cellular and soluble components. In addition, compensatory and overlapping mechanisms in host immunity can make it difficult to identify subtle or modest changes within the immune system. Because of the many different cell types and soluble factors, which alone or cooperatively mediate a wide variety of evocable immunological responses, there is no one test or assay that can measure all the different elements. Therefore, the approaches used to identify changes in immune status are multifaceted, including pathological examination of lymphoid organs, enumeration of leukocyte subpopulations, quantification of soluble mediators, and measurement of immune function responses, such as susceptibility to infection or reduced immunological responses to vaccines. Standard assays are available for all the above determinations; however, because of the sheer enormity of the task to measure them all, immune competence is typically assessed by using a small number of immunological end points, often quantifying various aspects of innate, humoral, and cell-mediated immunity. Therefore, the EPA review draws on a large but diverse database of studies that are often difficult to compare because different assays, model systems, responses, and animal species were used. EPA draws several important conclusions about the immunotoxicity of TCDD, other dioxins, and DLCs that are summarized in the Reassessment, Part III, Integrated Summary and Risk Characterization. The first is that “there appears to be too little information to suggest definitively that 2,3,7,8-TCDD at levels observed (in the reported studies) causes long-term adverse effects on the immune system in adult humans” (p. 2-34; lines 19 to 21). The second is that “cumulative evidence from a number of studies indicates that the immune system of various animal species is a target for toxicity of TCDD and structurally-related compounds, including PCDDs, PCDFs [polychlorinated dibenzofurans] and PCBs [polychlorinated biphenyls]” (p. 2-34; lines 24 to 26). Third, animal studies show that TCDD suppresses both “cell-mediated and humoral immune responses, suggesting that there are multiple cellular targets within the immune system that are altered by TCDD” (p. 2-34; lines 26 to 28). EPA goes on to state that “it can be inferred from the available data that dioxin-like congeners are immunosuppressive” in animals. Fourth, the weight of evidence from animal studies in vivo and in vitro “supports a role for Ah-mediated immune suppression” by DLCs (p. 2-35, lines 27 to 28); “other in vivo and in vitro data, however, suggest that non-AHR (aromatic hydrocarbon receptor)-mediated mechanisms may also play

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Health Risks from Dioxin and Related Compounds: Evaluation of the EPA Reassessment some role in immunotoxicity” (p. 2-35, lines 28 to 30). Finally, EPA concludes that “there are insufficient clinical data from these studies to fully assess human sensitivity to TCDD exposure. Nevertheless, because of extensive animal work, the database is sufficient to indicate that immune effects could occur in the human population from exposure to TCDD and related compounds at some dose level. At present, it is EPA’s scientific judgment that TCDD and related compounds should be regarded as nonspecific immunosuppressants and immunotoxicants until better data to inform judgment are available” (p. 2-37, lines 4 to 10). The strengths and weaknesses of those conclusions are discussed below. Uncertainties and Assumptions in Determining Whether TCDD Is Immunotoxic in Humans Is the Assumption Correct That the Immune System in Humans and in Animal Models, Primarily Mice, Are Similar? Historically, the mouse has been the animal model of choice for immunologists; it has also been widely embraced as the model of choice for immunotoxicological studies. Hence, most immunotoxicological studies used by EPA in preparing its report are based on studies in mouse models. The human and mouse immune systems are similar in composition and function; therefore, from the standpoint of comparisons based on the composition of this target organ, it is reasonable to assume that studies in mice provide important qualitative insights into the mechanism of action of TCDD, other dioxins, and DLCs on the human immune system. Providing, insights into the mechanism of action is one of the primary strengths of the mouse model for which there are genetically defined AHR high- and low-responding mouse strains, congenic mice at the Ahr locus, and AHR null (Ahr-/-) mice. More reagents, assays, and biological probes are available for the mouse immune system than for any other species except the human immune system. However, as is the case for other toxicological end points, information-derived from animal studies is qualitative in that the pharmacodynamics, pharmacokinetics, half-lives of the compounds, affinity of AHR, linkage of the receptor to signal transduction pathways, and numerous other factors can be significantly different, at least quantitatively, between humans and other animal species. Therefore, direct quantitative extrapolations from animal models to humans can result in a significant underestimation or overestimation of risk. When strong scientific evidence exists concerning specific species differences, these factors should be incorporated into the risk characterization.

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Health Risks from Dioxin and Related Compounds: Evaluation of the EPA Reassessment Is the Toxic Equivalency Factor/Toxic Equivalent Quotient Approach for Estimating the Immunotoxicity of Mixtures Scientifically Justified? The toxic equivalency factor/toxic equivalent quotient (TEF/TEQ) approach is based on a well-defined structure-activity relationship for persistent dioxins, other than TCDD, and DLCs for which there is a positive correlation between AHR affinity and toxic potency. There is good general agreement in comparisons of results across studies, primarily in mice, where the acute immunotoxic effects for individual dioxinlike congeners were examined, suggesting that the immunotoxic potency for various congeners and AHR activation exhibit the same qualitative rank order. Few immunotoxicological studies investigated structure-activity relationships for immunotoxic potency. Of those studies, with only a few exceptions, the rank order immunotoxic potency correlated positively with AHR activation. Several exceptions to this relationship are found in the current literature, and some are discussed in the Reassessment, Chapter 4, Part II. For example, 2,7-dichlorodibenzo-p-dioxin, a congener that would be predicted to exhibit low binding affinity for AHR, was found to be equipotent in suppressing the anti-sheep red-blood-cell (anti-SRBC IgM) antibody-forming response to TCDD. For this example, the TEF/TEQ approach would not provide a reasonable estimate of immunotoxicity. Because in vivo immunotoxicity data for 2,7-dibenzo-p-dioxin are available only in the mouse, it is unclear whether the unexpected potency of this congener occurs in other animal species and humans. A second example pertains to certain halogenated aromatic hydrocarbons (HAHs), including several diortho PCB congeners, which exhibit antagonist activity when administered in a mixture with AHR agonists. The latter point may not be trivial, as several of the diortho PCB congeners (e.g., PCB153) are abundant environmental cocontaminants with dioxins, other than TCDD, and DLCs. The aforementioned examples of exceptions for which the TEF/TEQ approach might not predict toxic potency accurately are presented and discussed in a balanced manner (Part II, pp. 4-6 to 4-7, lines 19 and 20). In spite of the aforementioned caveats, based on what is known about the cell biology of AHR and the mechanisms of immunotoxicity for TCDD and related compouds, the TEF/TEQ approach for assessing the immunotoxic potency of mixtures of persistent dioxins, other than TCDD, and DLCs is scientifically justified. Having said that, the effective application of the TEF/TEQ approach for assessing immunotoxic potential ultimately would critically depend on the TEF values assigned to individual congeners for a given immunological response. What is unclear is which immune responses should be used in risk management, as all are not equally sensitive to modulation by HAHs.

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Health Risks from Dioxin and Related Compounds: Evaluation of the EPA Reassessment What Is the Profile of Immune Toxicity in Humans Exposed to DLCs? Well-documented human exposure to TCDD, other dioxins, and DLCs has occurred in the occupational setting and within the general population from industrial accidents and through consumption of contaminated food. Even though immune function and status have been examined in exposed individuals, only a small number of studies have been conducted with appropriate controls and accurate measures of exposure. Likewise, a small number of epidemiological investigations evaluating immunologically related outcomes from chronic exposures have been reported. The results from these human studies for the most part have yielded inconclusive results. In several studies, modest changes in immune status were observed; yet in other studies, the findings were not reproduced or were even contradicted. In animal studies, it is clear that TCDD, other dioxins, and DLCs markedly suppress both humoral and cell-mediated immune responses. This profile of activity has not been unequivocally demonstrated in humans. The most obvious reason for the inconclusive findings in human studies is that, in many cases, a very small number of subjects were evaluated, their primary exposures were often long before measurement of immune end points and concentrations of TCDD and related compounds, and the nature of those exposures were often unclear. Furthermore, obtaining an accurate estimate of the level of exposure through back-extrapolation from current body levels may be more complex than simply using a single half-life throughout. Another contributing factor to the inconsistent results is that immune responses to defined stimuli are highly variable among humans. This variability can be attributed to genetic variability, age, environmental history, and other still undefined causes. This variability in individuals substantially limits the ability to identify subtle and even moderate alterations of immune function after exposure to agents, especially in human populations. Therefore, in the absence of more comprehensive immunotoxicological human data, it is reasonable to assume that TCDD, other dioxins, and DLCs will exert a profile of immunotoxicity comparable to that observed in animal models, such as mice. For risk analysis, the more critical issue is whether the immunotoxic potency observed in certain animal models is significantly greater (by orders of magnitude) than in humans. What Is the Sensitivity of the Human Immune System to DLCs? For many of the same reasons as discussed in the previous section, the sensitivity of humans to immune suppression by TCDD, other dioxins, and DLCs is also currently unclear. There are four separate reports of a longitudinal study in a cohort of Dutch children suggesting that the developing

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Health Risks from Dioxin and Related Compounds: Evaluation of the EPA Reassessment human immune system may be susceptible to immunotoxic alterations from exposure to Western European environmental levels of TCDD, other dioxins, and DLCs (Weisglas-Kuperus et al. 1995, 2000, 2004; ten Tusscher et al. 2003). Exposures before 1990 in the Netherlands resulted in breast milk concentrations of TEQ (at the start of the study) at 30 to 60 parts per trillion (ppt) (lipid), whereas concentrations in the United States at approximately the same time period were in the 15- to 24-ppt range. Three industrial areas were compared with a rural area with about 20% less PCBs in maternal plasma (Koopman-Esseboom et al. 1995). The Dutch study measured the major PCB congeners in plasma in the mother and the newborn and all TCDD and related compounds in maternal breast milk 10 days and 3 months postpartum (Feeley 1995). About half the study subjects were fed a formula that had TCDD and related compounds at less than 2 ppt, and the other half of the test subjects were breast-fed and could be divided into groups breast-fed less than 4 months and those breast-fed more than 4 months. Therefore, individual calculations of total exposure could be correlated with plasma PCB concentrations in children at 3 months, 18 months, 42 months, and 9 years. Three more recent studies (Weisglas-Kuperus et al. 2000, 2004; ten Tusscher et al. 2003) are important because some of the same findings observed in these later studies were also reported in the 1995 studies, indicating persistence of alterations. Weisglas-Kuperus et al. (2000) reported on 207 healthy mother-infant pairs with increased prenatal exposure to TCDD, other dioxins, and PCBs; the results showed an association between exposure and immunological changes, which included an increase in number of lymphocytes, γ-δ T cells, CD3+HLA-DR+ (activated) T cells, CD8+ cells, CD4+CD45RO+ (memory T cells), and lower antibody levels after mumps and measles vaccination at preschool age. In addition, an association was found between prenatal exposure and decreased shortness of breath with wheeze, and current PCB burden was associated with a higher prevalence of recurrent middle-ear infections and chicken pox and a lower prevalence of allergic reactions. Although an association between TCDD and PCB exposure and changes in immune status was observed, all infants were found to be in the normal range. In a second study, ten Tusscher et al. (2003) reported modest but persistent changes in immune status in children with perinatal exposure to dioxin as evidenced by a decrease in allergy, persistently decreased thrombocytes, increased thrombopoietin, increased CD4+ T cells, and increased CD45RA+ cell counts in a longitudinal subcohort of 27 healthy 8-year-old children with documented perinatal exposure to TCDD. The original cohort at 42 months demonstrated an association between reduced vaccine titers, increased incidence of chicken pox, and increased incidence of otitis media with higher TEQ. However, by 8 years of age, the more frequent recurrent ear infections were still apparent

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Health Risks from Dioxin and Related Compounds: Evaluation of the EPA Reassessment (overall), although the chicken pox frequency showed an inverse correlation with PCB and TCDD concentrations. The subcohort in the ten Tusscher study was small, and therefore the results are not as robust as those in the Weisglas-Kuperus et al. (2004) study, which included almost 91% of the original 2000 study cohort. Animal studies also suggest that the developing immune system is sensitive to persistent changes in immune function or status, especially when exposure occurs in the perinatal and neonatal stage and especially in T-cell-mediated immunity. Less compelling studies exist from which to estimate the sensitivity of the adult human immune system. EPA concludes that there is insufficient clinical data from these studies to fully assess human sensitivity to TCDD exposure. Nevertheless, based on the results of the extensive animal work, the database is sufficient to indicate that the immune effects could occur in the human population from exposure to TCDD and related compounds at some dose level. At present, it is EPA’s scientific judgment that TCDD and related compounds should be regarded as nonspecific immunosuppressants and immunotoxicants until better data to inform judgment are available. (Reassessment, Part III, p. 2-37, lines 4 to 10) Indeed, based on the extensive animal data, it is reasonable and prudent for EPA to regard TCDD as an immunotoxicant. Furthermore, the Dutch study provides some suggestive evidence for this conclusion. However, it is unclear in EPA’s conclusion what is meant by TCDD and related compounds being regarded as “nonspecific immunosuppressants and immunotoxicants” and this should be clarified. How Persistent Are the Immunotoxic Effects of TCDD on the Human Immune System (Reversible Versus Irreversible Changes)? Because it has not yet been unequivocally established that TCDD induces immune suppression in humans, it is not possible at this time to delineate the persistence of TCDD-mediated immunotoxicity in humans. The lowered total white-blood-cell numbers reported in the studies of Dutch children (Ilsen et al. 1996) were no longer evident 2 years after birth (Weisglas-Kuperus et al. 2000). The elevated T-cell subpopulations in Dutch children at 42 months of age did not appear to persist at 8 to 10 years of age (ten Tusscher et al. 2003). However, Weisglas-Kuperus et al. (2004) also reported a positive association in children (3 to 7 years of age) between increased postnatal PCB exposure and increased prevalence in recurrent middle ear infections. In addition, there was a positive association between increased prenatal PCB exposure and decreased chicken pox frequency as well as allergy and asthma. A second recent report (Van den Heuvel et al.

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Health Risks from Dioxin and Related Compounds: Evaluation of the EPA Reassessment 2002) suggested that TCDD, other dioxins, and DLCs may produce subtle but persistent changes in immune status as evidenced by a reduction in allergy and asthma. In this study of 200 Flemish adolescents, a negative correlation was observed between exposure to TCDD, other dioxins, and DLCs with TCDD TEQ measured and allergic responses in airways. In addition, serum immunoglobulin G levels were also negatively correlated with PCB exposure. Relevance of Rodent Models to Human Quantitative Risk Assessment for Immunotoxicity In the Reassessment, Part III, Appendix A, Table A-1, EPA centers its risk characterization for adult immunological effects on four studies conducted in mice (Vecchi et al. 1983; Narasimhan et al. 1994; Smialowicz et al. 1994; Burleson et al. 1996) and its developmental immunotoxicological risk characterization on a single rat study (Gehrs and Smialowicz 1999) (see Appendix A, Table A-1; for immune end points; see Figures 12 to 15, pp. A-7 to A-9; same studies identified in Table 5-6, p. 5-39, in the Reassessment, Part III). Based on these studies, lowest-observed-adverse-effect levels (LOAELs) and no-observed-adverse-effect levels (NOAELs) are used to derive ED01 (1% effective dose) and human equivalent intake values. Because of the importance that these studies have to the Reassessment and the potential importance that the derived values may have for risk management, some additional comments are provided here. The study of Burleson et al. (1996) showed the lowest LOAEL (6 ng/ kg), and NOAEL (3 ng/kg) values, which result in calculated “human equivalent intakes” of 1 pg/kg/day and 2 pg/kg/day, respectively. The study showed that the sensitivity to alteration by TCDD of host resistance of mice to H3N2 influenza A (Hong Kong/8/68) virus is strikingly sensitive compared with other LOAELs for immunotoxicological end points in adult rodents given in Table A-1, the LOAEL ranging from 100 to 1,200 ng/kg. In fact, the sensitivity to TCDD in the Burleson et al. study is striking even when compared with similar and more recently published host-resistance studies using influenza virus. For example, Nohara et al. (2002) showed that TCDD doses up to 500 ng/kg did not increase mortality in a number of different strains of mice, including B6C3F1, C57Bl/6, Balb/c, and DBA/2 mice infected with influenza A virus (A/PR/34/8, H1N1). More mice per group were used in the Nohara study than in the Burleson study, thus providing even greater statistical power. In a study using influenza A/ HKx31, Warren et al. (2000) reported that in certain experiments, TCDD treatment (1 to 10 µg/kg) increased mortality, whereas in other experiments no mortality was observed. Furthermore, Warren and coworkers stated that in some experiments TCDD doses as high as 10 µg/kg produced no

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Health Risks from Dioxin and Related Compounds: Evaluation of the EPA Reassessment mortality, whereas 80% mortality was observed at the same dose in other experiments. Such data emphasize the variability typically observed in host-resistance studies. The reason for the significantly greater sensitivity to TCDD in the Burleson et al. (1996) study is unclear but strongly suggests that further studies are needed before using results from this study for risk characterization. The LOAEL, NOAEL, ED01, and human equivalent intake values for immunotoxicity are based on suppression by TCDD of the anti-SRBC IgM antibody-forming cell response in studies by Vecchi et al. (1983), Narasimhan et al. (1994), and Smialowicz et al. (1994) (the other three studies identified in Table A-1 and used for risk characterization of the adult immune system). Numerous laboratories have demonstrated suppression of the antibody-forming cell response by TCDD, and in general, there is good concordance in the ED50 doses (600 to 770 ng/kg) derived from these studies (see Table 4-1, p. 4-38) (Vecchi et al. 1980; Davis and Safe 1988; Kerkvliet and Brauner 1990; Kerkvliet et al. 1990). In contrast, some variability in the LOAEL values identified in Table A-1 were observed in three other studies: 100 ng/kg (Narasimhan et al. 1994), 300 ng/kg (Smialowicz et al. 1994), and 1,200 ng/kg (Vecchi et al. 1983). The variation is due primarily to dose selection in each of the studies. It is clear how the LOAEL and, in the case of the Narasimhan study, the NOAEL were identified from the data presented in each of the published reports. It is not clear, however, how the ED01 values were calculated. Several concerns also exist about using the Gehrs and Smialowicz (1999) study for characterizing developmental immunotoxicological risk by TCDD. The Gehrs and Smialowicz study gives no indication as to the number of rat offspring studied; therefore, it is unclear whether the results are robust. In addition, both males and females were found to be more sensitive to immune suppression by TCDD after 14 months of age than at 4 months of age, as measured by a delayed type hypersensitivity response, which is somewhat puzzling. Although hypotheses could be advanced to explain these unexpected findings, it would be valuable and prudent to repeat that study before using those results for characterizing developmental immunotoxicological risk by TCDD. Congruence with Full Document In Part II, Chapter 4 of the Reassessment, a comprehensive and balanced synthesis of the immunotoxicology literature on TCDD, other dioxins, and DLCs is presented. Results from more than 200 published studies are discussed in an organized and logical manner. Moreover, there is good congruence between Chapter 4 and section 2.2.3 on immunotoxicity in the Executive Summary.

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Health Risks from Dioxin and Related Compounds: Evaluation of the EPA Reassessment CONCLUSIONS AND RECOMMENDATIONS ON THE IMMUNOTOXICITY OF TCDD, OTHER DIOXINS, AND DLCS Present clinical findings are inconclusive about whether or in what way TCDD, other dioxins, and DLCs are immunotoxic in humans, a conclusion that EPA acknowledges, and human data are also sparse. Perhaps the most compelling data that these compounds are human immunotoxicants, at possibly relevant environmental levels, come from the studies of the Dutch children’s cohort. These studies show an association between prenatal exposure and changes in immune status. However, the effects are modest and do not lie significantly outside the full range of normal. The correlation of increased otitis media in the very young with perinatal TEQ is the only statistically significant immunological clinical finding. Some of the same findings were made in acutely exposed Taiwanese and Japanese cohorts (Yu et al. 1995). Concordant with findings in Dutch children are a number of animal studies that also suggest that the developing immune system is especially sensitive to modulation by TCDD, other dioxins, and DLCs. Collectively, in light of the large database showing that these compounds are immunotoxic in laboratory animal studies together with sparse human data, EPA is being prudent in judging TCDD, other dioxins, and DLCs to be potential human immunotoxicants in the absence of more definitive human data. EPA’s conclusion that TCDD, other dioxins, and DLCs are immunotoxic at “some dose level” by itself is inadequate. At a minimum, a section or paragraph should be added that discusses the immunotoxicology of these compounds in the context of current AHR biology. Specifically, there is evidence showing that the affinity of TCDD for the human AHR is at least an order of magnitude lower than that in high-responding Ahrb-1 mouse strains (Ramadoss and Perdew 2004), which has been the most commonly used animal model for investigations of immunotoxicity of TCDD, other dioxins, and DLCs. Other properties of AHR, in addition to binding affinity, such as specificity for target genes and transactivation potential, will contribute to the toxicity produced by AHR ligands. Nevertheless, EPA supports a TEF/TEQ approach for estimating the immunotoxic potency of mixtures of dioxins, other than TCDD, and DLCs. The Reassessment assumes that immunotoxicity is therefore primarily mediated through an AHR-dependent mechanism, so some discussion should be included acknowledging the possibility that rodents, especially certain mouse strains expressing Ahrb-1 might be significantly more sensitive to the immunotoxic effects of TCDD, other dioxins, and DLCs than humans. Some discussion should also be included on the strengths and weaknesses of using genetically homogeneous inbred mice to characterize immunotoxicological risk in the genetically variable human population. Expanding the

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Health Risks from Dioxin and Related Compounds: Evaluation of the EPA Reassessment discussion to include the above crucial points would provide additional balance to Part III of the Reassessment. EPA centers its risk characterization for adult immunological effects on four studies conducted in mice (Burleson et al. 1996; Smialowicz et al. 1994; Narasimhan et al. 1994; Vecchi et al. 1983), and its developmental immunotoxicological risk characterization on a single rat study (Gehrs and Smialowicz 1999) (see Part III, Appendix A, Table A-1; for immune end points; see Figures 12 to 15, pp. A-7 to A-9). Concerns about Table A-1 are the following: The calculations of ED01 values and the scientific assumptions made in deriving those values need further clarification. Likewise, EPA should provide a clear scientific rationale for selecting ED01 as a benchmark dose. Considerations of the Burleson et al. (1996) study with no consideration of two similar studies—Nohara et al. (2002) and Warren et al. (2000)—that yield very different results requires justification. On the basis of concerns discussed earlier, it would be prudent to replicate the Gehrs and Smialowicz (1999) study before using its results for characterizing developmental immunotoxicological risk of TCDD. An important animal study by Oughton et al. (1995) was not included in either Part II or the tables in the Executive Summary of the Reassessment. The importance of the study is that it is the only low-level chronic exposure investigation published (TCDD at 200 ng/kg/week once a week to Bb mice at 2 to 16 months of age) in which immunotoxicological parameters have been assessed—specifically, a phenotypic analysis by flow cytometry of major cell subpopulations in the mouse spleen, thymus, and peripheral blood. The study showed only subtle alterations in the immune system as demonstrated by a modest increase in γ-δ T cells, which the authors considered “questionable biological relevance,” and a small decrease in the frequency of memory CD4 cells (by phenotype). However, these changes, although of questionable biological relevance, have also been observed in humans and in high-exposure animal studies. REPRODUCTION AND DEVELOPMENT Animal Data EPA provided an overview of the effects of TCDD, other dioxins, and DLCs on development and reproduction based on published animal studies and accidental human exposures. Determination of the alterations in development and reproduction is a highly complex process because hormonal as well as intracellular processes and compensatory mechanisms, including hormonal feedback mechanisms, are affected. The Reassessment compre-

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Health Risks from Dioxin and Related Compounds: Evaluation of the EPA Reassessment largely on committee-based, consensus evaluation of the available data rather than on specifically commissioned, rigorous analyses constructed according to established criteria that both formally evaluate the strengths of the available evidence and integrate, by quantitative systematic review, the data across available studies (Sackett et al. 2000; NCI 2002; CEBM 2005; Guzelian et al. 2005). On the whole, the potential for increased risk of noncancer end points after exposure to TCDD at or near background levels is cautiously presented in the Reassessment. However, the Reassessment explicitly characterizes TCDD as “developmental, reproductive, immunological, endocrinological, and carcinogenic hazards” (Part III, p. 6-3, lines 10 and 11), although the formal criteria for defining human hazard in the context of these noncancer end points are not defined precisely in the Reassessment. Further, although the Reassessment acknowledges that “some have argued that in the absence of better human data, deducing that a spectrum of noncancer effects will occur in humans overstates the science” (Part III, p. 6-3, lines 33 and 34), the EPA position is that an inference of human effects “is reasonable given the weight of evidence from available data” (Part III, p. 6-4, lines 1 and 2). Nonetheless, as EPA concedes, available human data currently do not permit resolution of these divergent evaluations. Human Reproductive and Developmental Outcomes The available human reproductive and developmental studies available at the time of the Reassessment draft are presented in detail, although a number of the more recent follow-up studies are obviously not reported, as mentioned below. EPA provides an overall conclusion that “subtle effects, such as the impacts on … developmental outcomes … or the changes in circulating reproductive hormones in men exposed to TCDD, illustrate the types of responses that support the finding of subtle yet arguably adverse effects at or near background body burdens” (Part III, p. 6-2, lines 6 to 11). The committee agrees that the results are subtle but disagrees that the reported effects are truly clinically adverse, especially when confidence in the observations is low and the reported changes could be non-significant at the biological level and clinical outcome. In this context, the Reassessment also notes that “there is no reason to expect, in general, that humans would not be similarly affected [as animals] at some dose, and a growing body of data supports this assumption. On the basis of the animal data, current margins of exposure are lower than generally considered acceptable, especially for more highly exposed human populations. The human database supporting this concern for potential effects near background body burdens is less certain” (Part III, p. 6-32, lines 19 to 23).

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Health Risks from Dioxin and Related Compounds: Evaluation of the EPA Reassessment Male Reproductive Hormones The Reassessment’s description of the National Institute of Occupational Safety and Health (NIOSH) study report (Egeland et al. 1994) showing a significant positive correlation of serum LH and FSH levels with serum TCDD does not discuss the weak nature of this correlation, the wide confidence intervals (CIs) around the regression, or the hormone values within the normal range (Part II, section 7.13.5.1). Similarly, the text further describes a two to four times higher prevalence of low testosterone levels among workers exposed to TCDD but does not report that the CIs around the risk ratios at the higher serum TCDD levels not only are very broad but also cross 1.0, indicating limited confidence in the significance of the relationships (Part II, section 7.13.5.1). Nor does the EPA Reassessment report that no dose-response effect was observed (odds ratio = 3.9 at lowest range of TCCD levels and 2.1 at highest levels), although the 95% CIs of the odds ratios themselves are so broad as to raise significant uncertainty about whether there is indeed a dose response relationship indicated by these studies (Part II, section 7.13.5.1). Similarly, the Reassessment states that the Ranch Hand study (Roegner et al. 1991) (Part II, section 7.13.5.1) reported lower serum testosterone levels in Ranch Hand veterans with current serum TCDD levels exceeding 33.3 pg/g, although the reported difference (10.2 ng/dL) was “statistically nonsignificant” and unlikely to have a measurable physiological effect. The EPA Reassessment also describes three additional negative studies (CDC 1988; Grubbs et al. 1995; Henriksen et al. 1997), concluding that “the human data offer some evidence of alterations in male reproductive hormone levels associated with substantial occupational exposure to 2,3,7,8-TCDD” (Part II, p. 7B-38). Thus, although “some evidence” has been reported, the bulk of the reported evidence is either negative or uncertain to a degree. The Department of Defense (DOD) released the latest report of the Ranch Hand study in 2005. The committee did not have the opportunity to review the report in detail because its release coincided with the end of the committee’s deliberations. However, the document reports that “the difference in adjusted free testosterone means in Ranch Hand versus Comparisons was 10.95 versus 10.47, respectively. The LH means for Ranch Hand and Comparison officers were 4.49 mIU/mL versus 4.09 mIU/mL, respectively. Both were well within one standard deviation of normal-age matched populations. No evidence of a dose-response effect was seen based on categorized dioxin or 1987 dioxin levels” (DOD 2005, p. 18-156). The report concludes that “the association of dioxin with … gonadal abnormalities appeared weak at best and unlikely to be clinically significant” (DOD 2005, p. 18-156) and “associations between dioxin level and …

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Health Risks from Dioxin and Related Compounds: Evaluation of the EPA Reassessment gonadal hormone abnormalities were unlikely to be clinically important” at these levels (DOD 2005, p. 21-8). Female Reproductive System In the Reassessment’s discussion of potential effects of TCDD exposure on endometriosis, the more recent Seveso data (Eskenazi et al. 2002a) are not included. Compared with women with TCDD concentrations of ≤20 ppt, the relative risk of endometriosis is 2.1 in women with TCDD concentrations >100 ppt, but the 90% CI ranges from 0.5 to 8.0, indicating little confidence in the true magnitude of the rate ratio or the significance of the reported average relative risk of 2.1. One conclusion from these data might be that women whose serum TCDD levels were >20 ppt had no more endometriosis than those whose serum TCDD concentrations were ≤20 ppt. Another defensible conclusion might be that the study did not have the power to come to any convincing conclusion on this issue. The authors of the study, Eskenazi et al. (2002a), chose to describe their findings as a “doubled, nonsignificant risk for endometriosis among women with serum TCDD levels of 100 ppt or higher, but no clear dose response.” Finally, in a recent review of the nonhuman primate and the human data assessing the relationship between TCDD exposure and endometriosis Guo concluded that “there are no solid, credible data available at this moment to support the hypothesis that dioxin exposure may lead to the development of endometriosis” (Guo 2004). Data published within the past 2 years on effects of exposure to TCDD, other dioxins, and DLCs on the menstrual cycle in women are obviously not referenced in the 2000 Reassessment. Thus, data from the Seveso incident surveying women who were exposed to TCDD postnatally, but while they were prepubertal, found “no change in the risk of onset of menarche with a 10-fold increase in TCDD,” and there was “no evidence of a dose-response trend” (Warner et al. 2004). Likewise, postmenarchal women exposed in Seveso showed no association of TCDD exposure with menstrual cycle length, but, in women exposed before menarche who had a 10-fold increase in serum TCDD concentrations, the menstrual cycle was lengthened by 0.93 day, although the 95% CI ranged from −0.01 to 1.86, and the strength of the relationship between menstrual cycle length and serum TCDD concentration shown in Figure 1A of the report is not convincing, with widely scattered data points (Eskenazi et al. 2002b). An observational study of wives and sisters of Swedish fishermen found a 0.49-day shorter menstrual cycle (95% CI 0.03 to 0.89) in those with a high dietary exposure to polychlorinated organochlorine compounds, including TCDD, but found no association with early life exposure (Axmon et al. 2004).

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Health Risks from Dioxin and Related Compounds: Evaluation of the EPA Reassessment The discussion on spontaneous abortions briefly mentions the study on the NIOSH cohort as “in press” (Part II, section 7.15.3.4.5). This study has now appeared (Schnorr et al. 2001) and found no effect on the incidence of spontaneous abortion or on the sex ratio of offspring. The authors concluded that the study provided “additional evidence that paternal TCDD exposure does not increase the risk of spontaneous abortions at levels above those observed in the general population.” Likewise, recent data from the Seveso cohort (Eskenazi et al. 2003) showed no association of TCDD with spontaneous abortions. Other recent relevant studies include birth-weight results reported for the NIOSH cohort (Lawson et al. 2004) and the Seveso cohort (Eskenazi et. al. 2003). The recent NIOSH report (Lawson et al. 2004) found that paternal TCDD exposure had no effect on birth weight for term infants, and a “somewhat protective” association of preterm delivery with paternal TCDD (odds ratio = 0.8), although the 95% CI ranged from 0.6 to 1.1. There was no obvious increase in birth defects, although the results were descriptive only. The authors concluded that “because the estimated TCDD concentrations in this population were much higher than in other studies, the results indicate that TCDD is unlikely to increase the risk of low birth weight or preterm delivery through a paternal mechanism.” The recent Seveso follow-up (Eskenazi et al. 2003) also showed no association of TCDD concentration with offspring birth weight or with the birth of infants small for gestational age. Finally, the Reassessment does not discuss the female Vietnam veterans study reported by Kang et al. (2000), which also reported no increase in spontaneous abortions, stillbirths, low-birth-weight infants, or infant deaths among women veterans who had served in Vietnam (and possibly exposed) compared with those who had served in the United States, although there are no body burden measurements made. The data on birth-weight effects were described adequately (Part II, section 7.13.12.8), and the summary comment (Part II, section 7.13.12.9) reflected appropriately the uncertainty of whether there were any birth-weight effects of exposure to TCDD at the time of the Reassessment. However, the likelihood of TCDD exposure having a measurable effect on birth weight has been substantially reduced by the recently reported studies of Kang et al. (2000), Eskenazi et al. (2003), and Lawson et al. (2004). To reflect and appropriately weigh this new information, EPA should correspondingly modify the summary comment (Part II, section 7.13.12.9). For the state of available information in 2000, the Reassessment describes adequately the observed effects of TCDD exposure on offspring sex ratio (Part II, sections 7.13.12.7, 7.15.3.4.8). As noted in the report, increased female births were observed after the Seveso accident (Mocarelli et al. 1996, 2000). They were also observed in a study of offspring of Russian pesticide producers exposed to TCDD (Ryan et al. 2002). However, the

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Health Risks from Dioxin and Related Compounds: Evaluation of the EPA Reassessment Schnorr study mentioned in the Reassessment as “in press” has now been published (Schnorr et al. 2001) and found no effect of TCDD exposure on sex ratio of offspring in the NIOSH cohort. In the United States, both exposure to TCDD and the male-to-female sex ratio at birth have declined since the early 1970s (Matthews and Hamilton 2005). This parallel decline is opposite that postulated from TCDD poisoning incidents. Thus, because sex ratios at birth not only undergo temporal trends but also show racial and nationality differences and are affected by both maternal age and infant birth order (Matthews and Hamilton 2005), EPA should also acknowledge the uncertainties inherent in evaluating sex ratios at birth without properly controlling for the aforementioned variables. The committee recognizes, however, that the TCDD exposure studies that showed altered gender ratios at birth have reported ratio values that were greater than the changes that might normally be expected to be caused by the variables mentioned above. Childhood Growth and Postnatal Development The Reassessment text describes appropriately the cited growth data and conveys adequately the uncertainty of whether TCDD exposure has effects on postnatal growth in humans (Part III, section 7.13.12.9). The issue would not be further clarified by including the omitted Swedish fish exposure study (Rylander et al. 1995) that reported diminished height, but not weight, at age 18, because this report includes neither TCDD nor TEQ data. Two of the longest-term studies of chlorinated toxicant effects on growth published subsequently (Blanck et al. 2002; Gladen et al. 2000) deal with PCB exposure and thus do not contribute to resolving the debate about the effects of TCDD on childhood growth. Similarly, the longest neurodevelopmental follow-up studies (Jacobson and Jacobson 1996; Gray et al. 2005) are reports on PCB exposure and do not directly contribute to the current TCDD issues since no TEQ is derived. However, the ongoing Dutch follow-up study referenced repeatedly in the Reassessment has now published its findings in 6.5-year-old children (Vreugdenhil et al. 2002a). At that age, there were no cognitive or motor differences between breast-fed infants (primarily postnatally exposed) and formula-fed infants (primarily exposed in utero with background postnatal exposure), including no overall differences in global cognitive index, memory, or motor performance, except when children from “less optimal homes” were analyzed separately. This observation suggested to the authors that less optimal home environment may allow the effects of TCDD on neurodevelopment to become manifest more readily while, in more optimal home environments, the additional beneficial environmental influences overcome the detrimental effects of exposure to TCDD. This is clearly

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Health Risks from Dioxin and Related Compounds: Evaluation of the EPA Reassessment a hypothesis at this stage, given the availability of only this single study that has addressed the issues. In an additional report, Vreugdenhill et al. (2002b) also described decreased masculinized play in boys and increased masculinized play in girls at age 7.5 years. Although statistically significant, the biological relevance of these conclusions remains uncertain given the wide scatter of the data and the regression coefficients reported. Cardiovascular and Pulmonary Systems The Reassessment discusses in detail the available data on potential human cardiopulmonary consequences of TCDD exposure highlighting the difficulty of supporting firm conclusions about the presence of a relationship (Part II, sections 7.13.9, 7.13.9.1, 7.13.10). Recently, from the latest data on Ranch Hands (DOD 2005), DOD concluded that “no consistent evidence suggested that herbicides or dioxin were associated with ill effects on respiratory health” (p. 21-9). On the other hand, “the presence of heart disease was found to be higher among Ranch Hands than Comparisons in enlisted flyers” (p. 21-6), and “an increased percentage of Ranch Hands in the high dioxin category were found to have abnormally high diastolic blood pressure. Ranch Hands in both the low dioxin category and the low and high dioxin categories combined were found to have a lower mean systolic blood pressure. Similarly, a smaller percentage of Ranch Hands in both the low dioxin category and the low and high dioxin categories combined had an abnormally high systolic blood pressure” (p. 21-6). However, the report notes that “the prevalence of cardiovascular disease was not increased in the Ranch Hand cohort. In only one analysis, that of diastolic blood pressure noted above, was there any evidence of an increased risk with increased body burden of dioxin” (p. 21-7). OTHER NONCANCER END POINTS Diabetes The Reassessment (Part II, sections 7.13.6, 7.13.6.1, 7.15.2.1.2; Part III, section 2.2.5) presents in detail the then available data on the relationship between TCDD exposure and the development of Type 2 diabetes. This relationship was evaluated in greater depth by an IOM committee (IOM 2000), which concluded that “there is limited/suggestive evidence of an association between exposure to the herbicides used in Vietnam or the contaminant dioxin and Type 2 diabetes.” This is an adequate statement of the state of the science concerning this noncancer end point, and the committee recommends that EPA revise the Reassessment to include the analysis provided by the IOM committee. The Reassessment should also incor-

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Health Risks from Dioxin and Related Compounds: Evaluation of the EPA Reassessment porate the data of the recent Ranch Hand study report (DOD 2005) showing that “mean fasting insulin and the risk of diabetes requiring insulin control increased with initial dioxin. C-peptide and time to diabetes onset decreased as initial dioxin increased.” The risk of diabetes requiring insulin control was increased in the Ranch Hand high-dioxin category. An increase in the risk of diabetes requiring oral hypoglycemic or insulin control was observed as 1987 dioxin levels increased. Time to diabetes onset decreased as 1987 dioxin levels increased. The risk of an abnormally high hemoglobin A1c increased with 1987 dioxin levels. Some findings in the DOD (2005) report appeared inconsistent with the results presented above, such as a decrease in the risk of 2-hour postprandial urinary glucose abnormalities with 1987 dioxin levels. The findings appear consistent with the previously noted association between Type 2 diabetes and dioxin in Ranch Hand veterans. Increased risks of diabetes requiring insulin control were found with initial dioxin, in the high-dioxin category, and with 1987 dioxin levels. In contrast, “associations between dioxin level and thyroid and gonadal hormone abnormalities were unlikely to be clinically important” (DOD 2005, p. 21-8). These data led to the conclusion that “the association noted at previous Air Force Health Study examinations between Type 2 diabetes mellitus and dioxin persisted. A higher prevalence of diabetes, as well as severity, as dioxin increased was evident, even after adjustment for such factors as age and body mass index” (DOD 2005, p. 18-156). Thyroid Function The Reassessment acknowledges that despite the fact that “many effects of TCDD exposure in animals resemble signs of thyroid dysfunction or significant alterations of thyroid-related hormones” (Part III, p. 2-41, lines 32 and 33), the results of human studies “are mostly equivocal” (Part III, p. 2-42, line 1). The Reassessment reports that Pavuk et al. (2003) showed “elevated TSH [thyroid stimulating hormone] means among the high TCDD exposure group in the 1985 and 1987 follow-ups, with an increasing trend across the decade 1982-1992, but no association with the occurrence of thyroid disease” (Part III, p. 2-42, lines 2 to 4). The discussion does not address the fact that the TSH differences, although statistically significant, are quantitatively extremely small and well within the normal range of circulating TSH levels. Further, limitations of the study are not described, including 86 exclusions “from the longitudinal analyses because they had undergone thyroidectomy, or had endocrine cancer, or were on thyroid medication” (Pavuk et al. 2003), the exclusion of one Ranch Hand because of an extremely low TSH value that might have indicated the earliest sign of hyperthyroidism and the fact that “over the five examina-

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Health Risks from Dioxin and Related Compounds: Evaluation of the EPA Reassessment tions, three different radioimmunoassays were used to measure TSH” (Pavuk et al. 2003). In addition the Reassessment does not report that no significant differences “with regard to mean levels of total thyroxine (T4), triiodothyronine (T3)% uptake, or free thyroxine index were observed at any examination” (Pavuk et al. 2003). Recently, DOD (2005) reaffirmed that “as for a dioxin effect related to thyroid disease, the 2002 examination data did not support such a relation.” Further, while the most recently published Ranch Hand follow-up report (DOD 2005) found a difference in adjusted mean TSH levels (1.653 microunits/mL versus 1.557 microunits/mL) in Ranch Hands and Comparisons, respectively, this difference was “not considered clinically significant because a 1% difference is difficult to measure. The same was true from the free T4 values in enlisted flyers (mean of 1.115 ng/dL in Ranch Hands versus 1.054 ng/dL in the comparison groups). If a primary thyroid effect were present, one would expect the TSH to move in the opposite direction of the free T4, which was not seen in these data” (DOD 2005, p. 18-156). The draft Reassessment also highlights the higher TSH values reported in human infants by Pluim et al. (1993) and by Koopman-Esseboom et al. (1994) (Part III, 2.2.6.2 and Part II,7.15.2.2.2), but does not discuss the fact that the TSH changes were very small and possibly not of physiological or clinical significance. Follow-up of the Dutch children’s cohort has now been carried out for more than a decade, and changes in thyroid status in this cohort have not been reported, although it is unclear whether they were in fact thoroughly assessed. The text in the Reassessment does include lengthy hypothetical discussions (Part III, 2.2.1.3 and Part III, 2.2.6.2) of plausible mechanisms for perturbations of human thyroid function, although there is limited human data to support or refute such mechanisms. Teeth Because ectodermal abnormalities are common findings in animal studies, including nonhuman primates, as well as in the human Yusho and Yu-Cheng exposures, the enamel hypomineralization found in 6- to 7-year old Finnish children (Alaluusua et al. 1996, 1999) is highlighted twice (Part II, section 7.13.12.6.1; Part III, section 2.2.2.1). Because the enamel mineralization scores were largely subjective, it is imperative that the observers were blinded to the prior breast-feeding status of the children, but this issue is not specifically mentioned in the publications. Additionally, the Reassessment highlights several other limitations of this study and, in the related commentary (Part II, section 7.13.12.6.2) EPA acknowledges that “the presentation of the results is incomplete.”

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Health Risks from Dioxin and Related Compounds: Evaluation of the EPA Reassessment Chloracne The Reassessment adequately summarizes the uniformly agreed upon and well-documented association between TCDD exposure and the development of chloracne. Elevated γ-Glutamyl Transferase The Reassessment adequately summarizes the data showing “a consistent pattern of increased γ-glutamyl transferase (GGT) levels among individuals exposed to TCDD-contaminated chemical.” (Part III, p. 2-40, lines 23 and 24) and notes that “long-term pathological consequences of elevated GGT have not been illustrated by excess mortality from liver disorders or cancer; or in excess morbidity in the available cross-sectional studies” (Part III, p. 2-41, lines 23 and 24). The report also acknowledges that “the consistency of the findings in a number of studies suggests that the finding may reflect a true effect of exposure but for which the clinical significance is unclear” (Part II, p. 7B-116, lines 20 to 22). The most recent report of the Ranch Hand study found no relationship between TCDD exposure and GGT (DOD 2005, pp. 13-51 to 13-57). Lipid Levels The Reassessment accurately notes that “neither adults nor children from Seveso had lipid levels above the referent level” (Part II, p. 7B-118) despite very high exposures and that “the most recent data suggest that high exposure to 2,3,7,8-TCDD contaminated substances are not related significantly to increased lipid concentrations, specifically total cholesterol and triglycerides” (Part II, p. 7B-118). The Reassessment adds that “slight but chronic elevations in serum lipids may put an individual at increased risk for disorders such as atherosclerosis and other conditions affecting the vascular system” (Part II, p. 7B-118) and that “risk factors such as dietary fat intake, familial hypercholesterolemia, alcohol consumption, and exercise” (Part II, p. 7B-118) were not considered in the Seveso study, even though no effects of TCDD exposure were found. The most recent Ranch Hand follow-up showed no relationship between TCDD exposures and total high-density-lipoprotein cholesterol, although ranch hands had an increased percentage of individuals with increased triglyceride values (DOD 2005, pp. 13-78 to 13-103). This report concluded that “based on the analysis of triglycerides, a subtle relation between dioxin and lipid metabolism cannot be excluded” (DOD 2005, p. 13-218).

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Health Risks from Dioxin and Related Compounds: Evaluation of the EPA Reassessment The various remaining noncancer end points, for which there are fewer data and even less suggestive evidence than the associations discussed above, are adequately summarized throughout the Reassessment. CONCLUSIONS AND RECOMMENDATIONS ON THE REPRODUCTIVE, DEVELOPMENTAL, AND OTHER NONCANCER END POINTS OF TCDD, OTHER DIOXINS, AND DLCS Embryonic and fetal development and female and male reproduction are sensitive end points of toxicity from TCDD, other dioxins, and DLCs in rodents because, as discussed earlier, responses occur at lower administered doses than other end points. However, the sensitivity of these end points in humans is less apparent. The fetal rodent is more sensitive than the adult rodent to adverse effects of TCDD. The human equivalent intake (pg/kg-day) for some adverse effects related to reproduction and development based on ED01 is not adequately supported (Chapters 2, 3, and 4). In humans, there is a clear association of TCDD exposure with chloracne and available studies have shown suggestive associations of TCDD exposure with Type 2 diabetes, but the latter data are not yet robust. In humans, the association of TCDD exposure with other reported, detrimental noncancer effects has not been convincingly demonstrated. The available studies have not yet shown clear associations among TCDD exposures and the risks of individual, clinically significant, noncancer end points. In reference to human disease risks, the overall conclusions about noncancer risks due to TCDD exposure are, in general, cautiously stated, and the uncertainty of suspected relationships is acknowledged. Nonetheless, the limitations of specific human studies are not uniformly addressed, and the broad 95% CIs accompanying some reported statistically significant effects are not discussed in the context of the uncertainty that these broad confidence limits imply. Conversely, statistically insignificant effects are sometimes highlighted. The divergent data across the diverse studies assessing human noncancer end points have not been subjected to systematic review according to currently accepted approaches, including meta-analysis when appropriate, nor has there been formal grading of the quality of the evidence according to accepted principles currently applied in other areas of clinical pathophysiology, including one report of the relationship of TCDD exposure to cancer end points (Crump et al. 2003). EPA should discuss how the ED used in the in utero and lactational exposure rat models relates to human reproductive and developmental toxicity and risk information, including TEFs and TEQs.

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Health Risks from Dioxin and Related Compounds: Evaluation of the EPA Reassessment EPA should more clearly describe how and why ED01 values were determined in animals and transferred to human equivalents for the various noncancer end points and address risk estimate calculations using alternative assumptions (e.g., ED05). Whereas ED01 is conceptually a viable POD, the committee has concerns about how the ED01 is computed and whether there are adequate data at the ED01 level to ensure an acceptable level of confidence in the conclusions derived from using the ED01. The dynamic range approach EPA used to compute ED01 for continuous response is flawed in that the change of 1% total range may not identify any meaningful toxic effects, that 1% change may be well within random variation in the absence of exposure, and that the use of total range is less sensitive than use of a control range because total range can be much wider. EPA should provide a discussion of the dose-response effects of TCDD, other dioxins, and DLCs on the adult female reproductive system that result in endocrine disruption in animals. The impact of the dose-response data provided in these studies on human risk assessment should be presented. With respect to human noncancer end points, the Reassessment text should be revised to include the relevant, more recent data and, when appropriate as discussed above, to reflect study quality and data uncertainty of the studies referenced. For available human, clinical, noncancer end point data, EPA should establish formal principles of and a formal mechanism for evidence-based classification and systematic statistical review, including meta-analysis when possible. The application of systematic review, followed by evidence-based classification, leads to a more explicit statement of, and concrete appreciation of, the level of certainty (and, correspondingly, of uncertainty) that can be accorded the answers to specific questions in a particular field. When the mechanism is established, currently available and newly available human clinical studies should be subject to such systematic review and formal evidence-based assessment. The quality of the available evidence should be reported, and the strength or weakness of a presumptive association should be classified according to currently accepted criteria for levels of evidence. Animal studies have shown that TCDD can cause a variety of noncancer effects. These studies support both the EPA position of the plausibility of corresponding human effects and the need to devise adequately designed investigations that will answer the questions in man. In making its final recommendations, EPA should incorporate and integrate the relevant data from both human and animal studies, as appropriate, according to the levels-of-evidence hierarchy devised. EPA is encouraged to review newly available studies on the effects of TCDD on cardiovascular development in its risk assessment for noncancer end points.