&EPA
                United States .
                Environmental Protection
                Agency
                Office of Health and
                Environmental Assessment
                Washington DC 2O46O
EPA/600/6-88/007 Aa
June 1988
Externaf Review Draft
                Research and Development
A  Cancer
Risk-Specific
Dose  Estimate for
2,3,7,8-TCDD
Review
Draft
(Do Not
Cite or Quote)
                                NOTICE
                This document is a preliminary draft. It has not been formally
                released by EPA and should not at this stage be construed to
                represent Agency policy. It is being circulated for comment on its
                technical accuracy and policy implications.

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DRAFT                                                 EPA-600/6-88/007Aa
DO NOT QUOTE OR  CITE                                  June  1988
                                                      Review  Draft
                                    A CANCER

                          RISK-SPECIFIC DOSE ESTIMATE

                                FOR 2,3,7,8-TCDD
                                    NOTICE

THIS DOCUMENT IS A PRELIMINARY DRAFT.  It has not been formally released by the
U.S.  Environmental  Protection  Agency  and  should  not  at  this  stage  be  construed
to represent Agency policy.  It is being circulated for comments on its
technical accuracy and policy implications.
                    U.S. Environmental  Protection Agency
                               Washington, D.C.

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                                DISCLAIMER
     This document is an external draft for review purposes only and does
not constitute Agency policy.  Mention of trade names or commercial products
does not constitute endorsement or recommendation for use.
                                      11

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                                    CONTENTS

Tables and Figures	vi
Preface	vii
Authors and Reviewers	ix
 I.  OVERVIEW		1
     A.  NEW DATA AND METHODS	1
     B.  COMPARISON OF POTENCY ESTIMATES	5
     C.  ORGANIZATION.	7
II.  ANALYSIS.  .	8
     A.  BACKGROUND	8
          1.   Exposure Considerations	 . .9
          2.   Carcinogenicity of 2,3,7,8-TCDD	 . .9
              a.   Chronic Animal Studies 	 10
              b.   Epidemic logic Studies	12
          3.   Altered Cell Function	15
          4.   Weight-of-Evidence Conclusion	17
     B.  MECHANISMS OF CARCINOGENISIS	17
         1.   Hypothesis 1:  2,3,7,8-TCDD as a Direct-Acting,
             Complete Carcinogen 	  	 19
             a.   Qualitative Considerations	19
             b.   Quantitative Considerations  	 21
         2.   Hypothesis 2:  2,3,7,8-TCDD as a "Pure"  Promoter	22
             a.   Qualitative Considerations	22
             b.   Quantitative Considerations  	 26
                                     iii

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                             CONTENTS (continued)
         3.   Hypothesis 3:   Secondary or Indirect  Mechanisms  of
             Carcinogenesis	26
             a.
General Considerations	26
             b.   Qualitative Evaluation of 2,3,7,8-TCDD as an
                 Indirect or Secondary!Carcinogen.  .  	  28
             c.   Quantitative Considerations 	  31
     C.   EVALUATION OF 2,3,7,8-TCDD AS|AN "ANTICARCINOGEN" 	  32
     D.   USE OF PREDICTIVE MODELS.  .	I .  .  32
III.  CONCLUSIONS	 •	38
     A.   QUALITATIVE CONSIDERATIONS. .  	  41
     B.   QUANTITATIVE CONSIDERATIONS 	  42
         1.  Selection of Models	43
             a.   Noel/Uncertainty Factor (Threshold Approaches)	43
             b.   Sielken Approach.  .	44
             c.   M-K-V Model	44
             d.   LMS Model	45
         2.  Selection of the RsD Range	46
             a.   Base Analysis	46
             b.   Modification of Base Analysis . .  :	  47
REFERENCES	52
APPENDIX A:  QUANTITATIVE IMPLICATIONS OF THE USE OF DIFFERENT
             EXTRAPOLATION PROCEDURES FOR LOW-DOSE CANCER RISK
             ESTIMATES FROM EXPOSURE TjO 2,3,7,8-TCDD
APPENDIX B:  EPIDEMIOLOGIC CANCER STUDIES ON POLYCHLORINATED
             DIBENZO-fi-DIOXINS, PARTICULARLY 2,3,7,8-TCDD
                                      iv

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                              CONTENTS  (continued)
APPENDIX C:  REPRODUCTIVE AND DEVELOPMENTAL TOXICITY OF 2,3,7,8-TCDD

APPENDIX D:  EPIDEMIOLOGIC DATA ON REPRODUCTION AND EXPOSURE TO
             2,3,7,8-TCDD:  ITS USEFULNESS IN QUANTITATIVE RISK
             ASSESSMENT

APPENDIX E:  IMMUNOTOXICITY OF 2,3,7,8-TCDD:  REVIEW, ISSUES,
             AND UNCERTAINTIES

APPENDIX F:  RATIONALE FOR A HORMONE-LIKE MECHANISM OF
           .  2,3,7,8-TCDD FOR USE IN RISK ASSESSMENT

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                                     TABLE
Table 1.  Promoting effect of 2,3,7,8-JTCDD on hepatocarcinogenesis
          in partially hepatectomized female rats by a single dose of
          diethylnitrosamine	\	
                                                                  24
                                    FIGURES
Figure l.;


Figure 2.


Figure 3.
Example of risk specific doses and reference
doses for 2,3,7,8-TCDD. . J 	
Potential secondary mechanisms of carcinogenic activity
of 2,3,7,8-TCDD 	 	
"Multiple mechanism" hypothesis for 2,3,7,8-TCDD carcinogenesis
37
40
                                      vi

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                                   PREFACE
      Responding to both internal  and  external  questions about the U.S.
 Environmental  Protection Agency's (EPA's)  1985 Health Assessment Document for
 Polychlorinated Dibenzo-p.-Dioxins,  the  EPA Administrator asked the Assistant
 Administrator  of the  Office  of Research and Development (ORD) to re-examine the
 data and  methodology  upon which the assessment for 2,3,7,8-tetrachlorodibenzo-
 p_-dioxin  (2,3,7,8-TCDD)  was  based in  light of  new data or alternative
 interpretations of the  older literature that have become available since 1985
 and,  if appropriate,  to  modify EPA's  approach.  This report, entitled "A Cancer
 Risk-Specific  Dose Estimate  for 2,3,7,8-TCDD,"* and its appendices present the
 results of that effort.
     Although  there are  many components  to any risk assessment for
 2,3,7,8-TCDD,  two  factors have been particularly important in recent Agency
 decisions, i.e., estimates of cancer  potency and estimates of human exposure.
 Consequently,  while other issues  were reviewed and are briefly discussed in the
 appendices to  this report, the report itself focuses on cancer potency and
*In EPA terminology, the risk specific dose (RsD) is an estimate of dose, or
 exposure, that would equal the dose estimated to result in an upper-bound
 estimate of incremental lifetime cancer risk, e.g., one in a million.
 The reference dose (RfD)s which is referred to later in this document,  is an
 estimate (uncertainty spanning perhaps an order of magnitude) of a daily
 exposure to the human population (including sensitive subgroups) that  is
 likely to be without an appreciable risk of deleterious effects during  a
 lifetime (U.S. EPA, 1987b).

                                     vii

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exposure issues.  This analysis is thus not a complete risk characterization
for 2,3,7,8-TCDD, but rather a re-examination of the hazard identification and
dose-response assessment for the potential human carcinogenicity of this
chemical.
     An ad hoc inter-office workgroup (hereafter, the "Workgroup") prepared the
report and recommendations.  While scientists outside of this group have
provided useful analyses, review, and!comment, the conclusions and
recommendations are those of the Workgroup alone.  Similarly, although the
       ;    _ .  i*                       |
appendices to the report contain important background information on a broad
range of 2,3,7,8-TCDD issues discussed in the report, the special focus of the
report precluded use of many of these analyses in the final document.  Other
major related  sources include:
     •  the report of an EPA Workshop! on  the Development of Risk Assessment
        Methodologies for Tumor Promoters,
     •  the report on Estimating Exposure to 2,3,7,8-TCDD
     -  the report of the "Dioxin" Update Committee
     This report, its appendices, and| the reports listed above represent  an
effort by many people within EPA to grapple with the difficult scientific
issues presented by the very large bujt incomplete data  base on 2,3,7,8-TCDD.
Credit for each report or appendix in| this overall effort belongs to the
individual authors.

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                              AUTHORS AND REVIEWERS
 AUTHORS
      This  document,  "A Cancer Risk-Specific Dose  Estimate  for 2,3,7,8-TCDD,"
 was  prepared  by an  interoffice Workgroup.   Members  of the  Workgroup are  as
 follows:

 Peter W. Preuss,  Ph.D.,  Chairman
 Director
 Office of  Technology Transfer and  Regulatory Support

 Donald G.  Barnes, Ph.D.
 Acting Director
 Science Advisory  Board

 William H.  Farland,  Ph.D.
 Director
 Office of  Health  and Environmental Assessment

 Dorothy E.  Patton,  Ph.D., J.D.
 Executive  Director
 Risk Assessment Forum

 Patricia A. Roberts,  J.D.,  B.S.
 Office of  General Counsel

 Hugh L. Spitzer,  B.A.*
 Office of  Technology Transfer and  Regulatory Support

 Deborah A.  Taylor, M.A., B.A.*
 Office of  the Administrator

 *Mr.  Spitzer and  Ms.  Taylor contributed to  the report  before  they  left EPA  in
 February  1988  and August 1987, respectively.


      The Appendices  were prepared  by scientists from ORD's Office  of Health and
 Environmental Assessment and  Office of Health Research, and by outside
 consultants.

 Appendix A:  Steven  P. Bayard,  Ph.D.
             Carcinogen Assessment Group

             Todd W.  Thorslund, Ph.D.
             ICF-Clement Associates
             Fairfax,  VA

Appendix B:  David L.  Bayliss, M.S.
             Carcinogen Assessment Group


                                     ix

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Appendix C:


Appendix D:


Appendix E:
Appendix F:
Gary L. Kimmel, Ph.D.
Reproductive Effects Assessment Group

Sherry G. Selevan, Ph.D.
Reproductive Effects Assessment Group

Babasahab R. Sonawane,!Ph.D.
Reproductive Effects Assessment Group
                      i
Ralph J. Smialowicz, Ph.D.
Office of Health Research

Robert W. Luebke, Ph.D;.
Office of Health Research

Michael A. Gallo, Ph.D.                         .
Department of  Environmental and Community Medicine
University of  Medicine and Dentistry of New Jersey
Robert Wood Johnson Medical School
REVIEWERS
              Dr.  Milt  Clark
              U.S.  Environmental  Protection  Agency
              Region  5               |
              Chicago,  IL  60605     |

              Mr.  David H.  Cleverly i
              Office  of Air Quality Planning and Standards
              U.S.  Environmental  Protection  Agency
              Research  Triangle Park,  NC  27711
              Mr.  Barry Commoner
              Queens College
              Flushing, NY  11367
              Mr. Michael B. Cook
              Office of Drinking Water
              U.S. Environmental Protection Agency
              Washington, DC  20460

              Dr. Diane K. Courtney!
              Health Effects Research Laboratory
              U.S. Environmental Protection Agency
              Research Triangle Park, NC  27711

              Dr. Marilyn Fingerhut[
              National Institute for Occupational
                Safety and Health
              Cincinnati, OH  45226

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 Dr.  Mike Gough
 Resources for the Future
 Washington,  DC  20036

 Mr.  Richard  N. Hill
 Office of Pesticides  and Toxic Substances
 U.S.  Environmental  Protection Agency
 Washington,  DC  20460

 Dr.  David Hoel
 National  Institute  for  Environmental
   Health Sciences
 Research Triangle Park,  NC   27709

 Dr.  Vernon Houck
 Centers  for  Disease Control
 Atlanta,  GA   30323

 Dr.  Renate Kimbrough
 Regional  Operations
 U.S.  Environmental  Protection Agency
 Washington,  DC  20460

 Dr.  Gary Kimmel
 Reproductive Effects Assessment Group
 U.S.  Environmental Protection Agency
 Washington,  DC  20460

 Dr. Richard  J. Kociba
 The Dow  Chemical  Company
 Midland,  MI   48674

 Mr. James  C.  Lamb
 Office of Pesticides and Toxic Substances
 U.S.  Environmental Protection Agency
 Washington,  DC  20460

 Dr. Debdas Mukerjee
 Environmental  Criteria and Assessment Office
 U.S.  Environmental Protection Agency
 Cincinnati,  OH  45268

 Dr. Marvin Schneiderman
 National  Research Council
National Academy  of Sciences
Washington,  DC   20418

Dr. Sherry Selevan
Reproductive  Effects Assessment Group
U.S.  Environmental Protection  Agency
Washington, DC  20460

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Dr. Ellen Silbergeldi
Environmental Defense Fund
Washington, DC  20036

Mr. Ralph Smialowiczj
Health Effects Research Laboratory
U.S. Environmental Protection Agency
Research Triangle Park, NC  27711
                     I
                     i
Dr. Bob Sonawane
Reproductive Effects Assessment Group
U.S. Environmental Protection Agency
Washington, DC  20460
                         xii

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                                 I.  OVERVIEW

      This report re-examines the scientific basis and methods used by the U.S.
 Environmental Protection Agency (EPA) for estimating the cancer potency for
 2,3,7,8-tetrachlorodibenzo-p.-dioxin (2,3,7,8-TCDD) (U.S. EPA, 1985).   The
 object is to determine if the 1985 assessment should be modified in light of
 recent data and other plausible risk assessment methods or alternative data
 interpretations.
    ;   The Analysis uses two different approaches.   One examines EPA's earlier
 analysis  in terms of new data and  recent reviews that offer scientific
 information and views for re-assessing 2,3,7,8-TCDD cancer risks.   The other
 involves  comparing EPA's 1985 assessment with that of other regulatory agencies
 in this country and elsewhere.   The Agency Workgroup could not reach  consensus
 on all  issues.    However,  for the  reasons developed below, the Workgroup
 convened  for this task agreed that (1)  the 1985 assessment that associates a
 0.006  pg/kg/day dose with  a plausible upper-bound  increased cancer risk of one
 in a million (10"6)  should be reconsidered,  and (2) a majority of  the group
 agreed that  a change to a  0.1 pg/kg/day dose as a  plausible upper-bound
 associated with  an  increased lifetime risk of one  in a million is  consistent
 with the  available  data and theories,  and represents a reasonable  science
 policy position  for  the Agency.

A. NEW DATA  AND  METHODS
     Although the scientific literature  is  replete with studies on
2,3,7,8-TCDD, which might  be brought  into a  comprehensive  characterization of
cancer risk, most discussions and debate  about  quantitative risk focus on the

                                      1

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interpretation and use of a small  subset  of  animal and human studies.
                                     i
Laboratory studies conclusively establish a  relationship between exposure to
2,3,7,8-TCDD and cancer in test animals.   There  is,  however, considerable
uncertainty and controversy about theimechanism  by which 2,3,7,8-TCDD causes
                                     i
cancer, an uncertainty that can strongly  influence both qualitative  assessments
                                     i
and the mathematical methods used to assess  cancer risk to humans.   Also,
variabilities in conduct and response among  studies  in human populations  that
may have been exposed to 2,3,7,8-TCDD--an assumption that  is  itself
                                     i
uncertain—raise additional questions and obscure the overall  assessment.
     A question often asked is whether 2,3,7,8-TCDD  is  a  "complete  carcinogen,"
a "promoter," or whether it produces cancer by some  unknown mechanism that  may
functionally have elements of initiation, promotion, and  progression.1   The
previous EPA assessment (U.S. EPA, 1985)  analyzed 2,3,7,8-TCDD as a complete
carcinogen.  Recent studies support the assertion put forth a number of years
ago that one of the major mechanisms of action for 2,3,7,8-TCDD involves the
"promotion" of carcinogenesis in cells.  However, despite changes and additions
to the data base, the analysis for risk assessment is neither obvious nor
simple, and important uncertainties remain.
     These considerations give rise to the  issues upon which this analysis is
                                     [
founded.
According to generally  accepted  theory  (OSTP,  1985;  U.S.  EPA,  1986a),  both
 complete carcinogens  and  promoters  are  capable of increasing  cancer  incidence
 in humans.  Thus,  the question of complete  carcinogenicity versus  promotion
 has little effect  on  identifying potential  human  cancer hazard associated
 with exposure  to 2,3,7,8-TCDD.   Differences,  however,  in mechanisms  of
 carcinogenesis may lead to  differences  in  approaches to quantitative risk
 assessment, with resulting  differences  in  numerical  risk estimates.

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      •   Is  the  carcinogenic  mechanism  of  action  better  understood  today than  it
         was for the  previous assessment?
      •   Is  the  linearized multistage  (IMS) model  as  employed  by  the  EPA to
         estimate the risk associated with exposure to carcinogens  appropriate
         for estimating  risk  from  exposure to  2,3,7,8-TCDD?
      •   Are there other appropriate ways  to characterize  the  risk  associated
         with exposure to 2,3,7,8-TCDD  that more  fully incorporate  the
         biological data? Are these approaches more  appropriate  than the IMS
         model {as used  by EPA)  for 2,3,7,8-TCDD  assessments?
      -   Does the choice of approach (and  related  assumptions)  have any  bearing
         on  a discrepancy perceived by  some between the  observed  human cancer
       .  experience and  human risk estimates based on animal studies?
      •   What is currently understood about the mechanism(s) of action of
         2,3,7,8-TCDD?
      •   How significant are  the remaining uncertainties?
      This analysis identifies several  reasonable  approaches to estimating
2,3,7,8-TCDD cancer  risk, but concludes that  there do not appear to  be
compelling  scientific reasons for regarding any one  of  them as a "most
appropriate"  approach.   Indeed, among  the several contributors to  this  report,
there was a diversity of viewpoints.   Preferring  somewhat different  assumptions
and interpretive  criteria, the  individual contributors  brought different
perspectives  to  the  review process.  Therefore, based on  rationales  grounded in
science  and/or  science  policy,  several different  risk assessment approaches
have been considered.
     The Workgroup recognizes that there  is a range of cancer  risk estimates
for 2,.3,7,8-TCDD  (see Figure  1).  Estimates at one end of the  range  are based
on several  different linearized models and those at the other  end are based on

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California

EPA
(LMS)
Relative
Half-life*
0.001-
0.004

0
Netherlands
NRC __.
Canada FHG

CD
007
i
C
PDi
o.oi

0.006 0.03
• * * U I J
I 1 1
0.0001 0.001 0.01

\



M-K-V
Model
1

0 07 0-6
*, '*'
1

N
Sta
3


Y
te
4


Canada

3
•
1
Slelkln
(MLE)
260

2.0 1 10.0
r 1

0.1 1.0
rl 1 	 *. 	 _
1 1
10.0 100.0
pg/kg/day
Figure 1.  Some examples of risk specific doses (10"s ) and reference doses calculated by
Individual scientists, scientific organizations, and regulatory agencies for 2,3,7,8-TCDD.  Solid
lines represent conclusions reached by regulatory agencies or scientific organizations; hatched
lines represent research efforts.  Some values represented as lines with a single point could
be represented as a range. The point shown is generally the lowest RsD or RfD of the range.


* Very preliminary analysis, taking into account the longer half-life of 2,3,7,8-TCDD in humans
  relative to rats.
Abbreviations used in the chart are as follows:
    CDC      Centers for Disease Control, U.S. Public Health Service
    EPA      U.S. Environmental Protection Agency
    FDA      U.S. Food and Drug Administration
    FRG      Ftderal  Republic of Germany  !
    LMS      Linear multistage model
    M-K-V    Moolgavkar, Knudson,  Venzon
    MLE      Maximum likelihood estimate
    NRC      National Research Council, Canada
                                           i

 The Una  Indicated as "Canada" represents both Health and Welfare Canada and the
 Province of Ontario, Canada

 Source: Taken from Appendix A.

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 a traditional  toxicological  approach.2  Based on science policy considerations
 developed in section III.B.2.b.6.,  the Workgroup proposes that the Agency adopt
 a dose of 0.1  pg/kg/day as a plausible upper-bound increased lifetime risk of
 one in a million.   The estimate is  consistent with the available data and
 theories and represents a reasonable science policy position.

 B.   COMPARISON OF  POTENCY ESTIMATES
      Plausible upper-bound cancer potency estimates for 2,3,7,8-TCDD published
 by  EPA,  other  U.S.  agencies,  some state agencies,  foreign governments,  and
 individual  investigators fall  within a range that  spans more than three orders
 of  magnitude.   This range represents the lack of current consensus regarding
 approaches  to  estimating levels  associated with  potential  cancer risk.
 Comparison  of  the various assessments  has two purposes:   (1) to  demonstrate how
 scientists,  using the  same data  but  different assumptions  and/or science
 policies, arriye at different  risk estimates,  and  (2)  to discuss  alternative
 methods  to  EPA's customary approach, which is  based on  the upper-confidence
 limit  (UCL)  of the  IMS model,  for estimating  human cancer risk from exposure to
 2,3,7,8-TCDD.  This  explicit discussion of assumptions  and policy choices
 highlights some of  the uncertainties inherent  in the risk assessment process
generally, and in analyses and assessments specific to the carcinogenicity of
2,3,7,8-TCDD.
2It should be recognized that neither of these methods attempts to estimate the
 "true risk" posed by exposure to a chemical.   In the case of the linearized
 models, because our understanding of the mechanism of carcinogenesis is so
 limited, an upper limit to the risk is calculated.  In its risk assessment,
 using the IMS model, EPA stresses that the true risk is likely to be lower
 than the "plausible upper bound," and may be  zero.  Likewise,  the traditional
 toxicological approach does not attempt to estimate risk; rather, it estimates
 a lifetime daily dose likely to be without significant risk.

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     Alternative approaches to estimating cancer risk are  discussed  in  terms of
existing 2,3,7,8-TCDD risk assessments to give EPA risk assessors  and risk
managers established,points of reference and to distinguish science,  science
policy, and assumptions.  The analysis shows that differences among  the risk
assessments for 2,3,7,8-TCDD developed by various regulatory agencies  are not
due to disagreements about the scientific data base per se, but rather are due
to the judgments, science policy positions, and methods used in estimating
human risk.  Indeed, a major factor accounting for the differences in the
various assessments is the judgment reached on whether or not a threshold
exists for'the carcinogenic activity |of  2,3,7,8-TCDD.  U.S.  agencies,  including
EPA, have  selected the  LMS model, a mathematical model based on a dose-response
function that does not  have a  threshold  (that is,  some non-zero risk can be
                                     I              „•     :
calculated for all dose  levels)  and h|as  a low-dose response characteristic that
is essentially linear.   Some  Canadian and European environmental  agencies, as
well as some  state  agencies  in this Country,  have selected a traditional
toxicological approach  based  on an  experimentally established
no-observed-effect-level (NOEL)3 to estimate a presumed "safe dose."   While
choice of  model  often reflects, in  part, the historical  or philosophical
tradition  of a  particular agency, in  the case of 2,3,7,8-TCDD it also reflects
important  differences in the way different scientists interpret and weigh the
scientific evidence and related uncertainties.
 3Dose in the chronic animal bioassay at which no Increase in tumor  incidence
  was observed.

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C.  ORGANIZATION
     The background information in Chapter II refers to a report on sources and
routes of human exposure to 2,3,7,8-TCDD, summarizes animal and human data on
the potential for human canceri, and surveys existing quantitative cancer risk
assessments for 2,3,7,8-TCDD.  The section on mechanisms of carcinogenesis
evaluates 2,3,7,8-TCDD in light of several different mechanistic hypotheses,
while the remainder of the chapter focuses on mathematical approaches for risk
extrapolation.  Chapter III synthesizes the range of qualitative and
quantitative considerations bearing on the human cancer risk potential of
2,3,7,8-TCDD, and explains the basis for selecting 0.1 pg/kg/day as a cancer
risk-specific dose (10~6) for this chemical.
      While this report draws on EPA's 1985 Health Assessment Document (HAD)
for Polychlorinated Dibenzo-p_-Dioxins (U.S. EPA, 1985) for certain data, it
incorporates new information and alternative interpretations of the scientific
evidence.*  The analysis follows EPA's Guidelines for Carcinogen Risk
Assessment which call for articulation of "major assumptions, scientific
judgments and to the extent possible, estimates of the uncertainties embodied
in the assessment. .  .distinguishing clearly between fact, assumption, and
science policy" (U.S. EPA,  1986a).
40ther sources include a "Dioxin" Update Committee Report of a meeting held
 July 1-2, 1986 (submitted to the Office of Pesticides and Toxic Substances
 August 28, 1986; hereafter called the "Pitot Report") (U.S. EPA, 1986c), the
 "Report of the EPA Workshop on the Development of Risk Assessment
 Methodologies for Tumor Promoters" (hereafter called the U.S. EPA "Promoter
 Workshop") (U.S. EPA, 1987c), and six issue papers developed as background
 information for this reanalysis (Appendices A through F).

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                                 II.  I ANALYSIS

     In this chapter data from laboratory and human  studies  are  evaluated  in
terms of the factors that influence risk assessment  methodology  generally  and
that raise specific questions about existing cancer  risk assessments  for
2,3,7,8-TCDD.  In place of models that appear to be  based upon a dichotomous
view of carcinogens as either complete carcinogens or pure promoters,
researchers are exploring more sophisticated models  which span a variety of
direct and indirect mechanisms, including a combination of several  modes of
action.  Qualitative answers to the questions regarding mechanism(s)  by which
2,3,7,8-TCDD exerts its carcinogenicity could have a significant effect on the
quantitative estimates of risk.
     This chapter addresses these questions.  Section A identifies relevant
data from laboratory and human studies.  Section B briefly reviews current
                                     [
theories on mechanisms of carcinogenesis as they apply to this chemical.   In
section C, several risk assessment models are reviewed in light of current data
on 2,3,7,8-TCDD and mechanistic considerations.  The special question of body
burden data, particularly its meaning for the epidemiologic studies, is
reviewed in section D.  The implications of these several factors for human
cancer risk from exposure to 2,3,7,8-TCDD is discussed  in section E.
                                     i
A.  BACKGROUND                       ;
     This section summarizes basic data  on  the  effects  of 2,3,7,8-TCDD in
animal, human, microbial, and  in vitro studies.   Section  1  outlines  data  on
sources and routes of exposure;  section  2 summarizes the  animal  and  human
studies that provide the foundation  for  most risk assessments for  2,3,7,8-TCDD;

                                       8

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 and section 3 presents relevant ancillary information.   These  summaries
 abstract data reviewed and analyzed in the issue papers  prepared  for  this
 report (Appendices A through F) and other sources cited.
 1.  Exposure Considerations
      A comprehensive review and analysis of human exposure  to  2,3,7,8-TCDD
 appears in a draft document entitled "Estimating Exposures  to  2,3,7,8-TCDD"
 (U.S.  EPA, I987a).  This report should be consulted for  information on  sources
 of 2,3,7,8-TCDD,  its movement through the environment, routes  of  human
 exposure,  and possible human doses  resulting from those  exposures.     v
      The report was prepared by scientists and  engineers  from  the Exposure
 Assessment Group,  Office of Health  and Environmental  Assessment.   The primary
 purpose of the report is to provide a review and update  of  information  related
 to exposure to 2,3,7,8-TCDD that  has come to light since  1984.  In addition,
 this report provides an  illustration of the application  of  this information  in
 performing exposure assessments for 2,3,7,8-TCDD.   This  is  accomplished by
 using  the  information to construct  several  scenarios  where  contaminated
 material may result in exposure to  2,3,7,8-TCDD,  and  estimating what  the
 exposure would  be  for various  pathways  from source to humans exposed.   Sources
 used as examples in this report include contaminated  soil,  various land
disposal situations,  and municipal  waste incinerators.   It  must be emphasized
that these  scenarios  are not to be  interpreted  as  an  exposure/risk assessment
for all sources of these types.   This  report should,  however,  provide a sound
starting point  for many  exposure  assessments of 2,3,7,8-TCDD contamination.
2-  Carcinogenlcltv of 2.3.7.8-TCDD
     The carcinogenic  potential of  2,3,7,8-TCDD for humans  has been the focus
of intensive study  arid debate  for almost a  decade,  and the  issue  is still not

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resolved.  While chronic exposure studies  in  laboratory  animals demonstrate
that 2,3,7,8-TCDD is a potent carcinogen in rodents,  epidemiologic  data  have
been much more difficult to interpret and  more controversial.  These  issues are
addressed in this section and described more fully in the issue papers prepared
by Bayard (Appendix A) and Bayliss (Appendix B).
     a.  Chronic Animal Studies
     There is general agreement that 2,3,7,8-TCDD is carcinogenic in  laboratory
animals.  The critical studies are from two independent laboratories  and show
effects in both rats and mice.  These and other experimental animal studies are
fully discussed in EPA's HAD (U.S. EPA, 1985).
     In a chronic toxicity and oncogenicity study by Kociba et al. (1978),
dietary doses of approximately 0, 1,000, 10,000, and 100,000 pg/kg/day
2,3,7,8-TCDD were fed to rats for up to 2 years (1 pg =  10'12 gram, so that
100,000 pg/kg/day is equal to 0.1 ug/kg/day).  In the high-dose group, both
male and female animals had significant site-specific increases in tumors.  The
target organs and tumor types in male animals  were squamous cell carcinomas of
the tongue, hard palate, and nasal turbinates, and adenomas of the adrenal
cortex; in female animals, the target organs  and tumor  types were
                                    i
hepatocellular carcinomas and squamous  cell carcinomas  of the tongue, nasal
turbinates, and lung.  Most investigators  interpret  this study as  demonstrating
                                    I
that dietary exposure to 2,3,7,8-TCDD at  100,000 pg/kg/day  or greater results
                                    i
in increased tumor Incidences in both male and female rats.   If  neoplastic
                                      10

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nodules are combined with hepatocellular carcinomas,  a statistically
significant response is also seen at 10,000 pg/kg/day in female rats.^
     The National Toxicology Program (NTP) has also tested 2,3,7,8-TCDD for
carcinogenicity in rats and mice following administration by gavage (NTP,
1982).  Rats were exposed to weekly doses of 0.0, 10,000, 50,000,  and 500,000
pg/kg.  The only tumors that appeared to be treatment-related were follicular
cell adenomas or carcinomas of the thyroid in male animals, and neoplastic
nodules or hepatocellular carcinomas of the liver in female animals.   The
incidence of these tumors was significantly greater in the high-dose groups
than in controls, and the incidence of tumors at both sites showed a positive
dose-related trend.  Under the conditions of this bioassay, the NTP concluded
that 2,3,7,8-TCDD was carcinogenic in both male and female rats.
     In the NTP mouse study, male mice were exposed to weekly doses of 0.0,
10,000, 50,000, and 500,000 pg/kg, while female mice were exposed to weekly
doses of 0.0, 40,000, 200,000, and 2,000,000 pg/kg.  An increased incidence of
liver tumors was also observed in the NTP study in the high-dose male mice and
in the high-dose female mice.  Female mice also had an increased incidence of
follicular-cell adenomas of the thyroid.  In this study, 2,3,7,8-TCDD was
carcinogenic to mice, with effective doses ranging between 500,000 and
2,000,000  pg/kg/week (0.5 and 2.0 ug/kg/week) depending on sex.
5The EPA Guidelines for Carcinogen Risk Assessment (U.S. EPA, 1986a) address
 the question of combining benign and malignant lesions of identical histogenic
 origin.  In addition, the Agency's Risk Assessment Forum has published
 guidance on the use of various pro!iferative hepatocellular lesions of the
 rat in risk assessment (U.S. EPA, 1986b).  In the case of 2,3,7,8-TCDD, both
 would recommend combining benign and malignant lesions; however, both suggest
 that when such lesions are combined, the impact of the benign lesions on the
 quantitative response should be presented explicitly.
                                      11

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     On the basis of information contained  in  these  two  studies, and other

animal data described in the HAD (U.S.  EPA,  1985), the Agency concluded that
                                     i
the evidence from animal studies for a  carcinogenic  response induced by
                                     L

exposure to 2,3,7,8-TCDD is "sufficient"  under the weight-of-evidence  system  in

EPA's then Proposed Guidelines for Carcinogen  Risk Assessment  (U.S.  EPA,  1984).

     b.  Epidemiologic Studies       j

     Several epidemiologic studies designed to evaluate  cancer  in  humans

potentially exposed to 2,3,7,8-TCDD or  substances presumed to  contain

2,3,7,8-TCDD are subjects of sharp debate.   Results  of these studies,  including

both cohort and case-control designs, iare arguably conflicting, with some

studies reporting high risks and others reporting low or no detectable risks.
                                     i
     Although most of the studies have followed standard epidemiologic
                                     I
procedures, the conclusions of all are subject to specific limitations.  Of

particular concern are uncertainties about the nature and extent of actual

exposure to 2,3,7,8-TCDD.6  In every instance, because  of a lack of empirical
                                     i
exposure data, some surrogate basis for estimating  exposure has been  used.
                                     t
Studies in which human populations have been  examined for carcinogenic

responses to exposure to substances containing 2,3,7,8-TCDD are reviewed  in  the

issue paper by Bayliss  (Appendix  B).

     Two epidemiologic  studies  from Sweden  (Hardell  and Sandstrom,  1979;
                                     i

Eriksson et a!., 1981)  are  considered  by many to be critical studies  in

assessing potential human cancer  risk.  They  suggest a  high cancer  risk

associated with exposure to chemicals!  contaminated  with 2,3,7,8-TCDD.  These
6Recent analyses of tissue  from  subjects  in  some  of  these  studies  suggest that
 "control" and "exposed" have  roughly  the same  levels  of 2,3,7,8-TCDD in their
 tissues (Hardell, 1987).   The impact!  of  this finding  on the interpretation of
 the studies has not been fully  assessed.

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 studies  have  reported  statistically  significant,  five-  to  sevenfold  elevated
 risks  of soft tissue sarcoma  (STS)7  related  to occupational exposure to phenoxy
 herbicides  and/or  chlorophenols,  some of which were  assumed to contain
 2,3,7,8-TCDD.   While some methodological questions have been raised  about these
 studies,  it^appears that the  elevated risks  (at some level of exposure) are
 real and,,,,should be considered in  hazard evaluation.
     In  addition to the two Swedish  studies  and certain case reports, other
 studies  (including studies in certain U.S. populations) may give  some support
 to the association between exposure  to 2,3,7,8-TCDD  and STS (see  Appendix B);
 however,  such  an assessment is widely debated.  These and  other studies have
 also reported  associations between other forms of cancer and exposure to
 2,3,7,8-TCDD  or chemicals likely  to  be contaminated  with chlorinated dibenzo-fi-
 dioxins  (CDDs).  For example, Hardell et al.  (1981)  reported a statistically
 significant risk of non-Hodgkin's lymphoma (NHL)  in  agricultural, forestry, and
 woodworking employees  exposed to  phenoxy herbicides, chlorophenols,  or both.
 The relative  risk  ratio ranged from  4.3 to 6.0 for both classes of compounds
 together  as well as separately.   In  addition to NHL, results from these studies
 have raised questions  about increased risks of stomach  cancer, prostate cancer,
 Hodgkin's disease,  and kidney cancer.  Since reports of increased risks for
 cancers other than STS and NHL occur sporadically throughout these studies,
they are generally considered inconclusive.
7"Soft tissue sarcomas constitute a category of rare cancers with a total
 mortality rate of 1 STS per 100,000 persons per year in the United States.
 The soft tissue sarcomas are malignant neoplasms of diverse histologic
 subtypes, which occur throughout the body in mesenchymal connective tissue
 other than bone.  These malignancies are often not reported accurately on
 death certificates and may not be recognized accurately by general
 pathologists" (Fingerhut,  1986).  The effect of such misdiagnoses could either
 under- or overreport the number of STS cases in a study.
                                      13

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     In contrast, several  studies involving populations  believed to have been
exposed to 2,3,7,8-TCDD or 2,3,7,8-TCDD-containing  chemicals  have  not  shown any
significant increased incidence of cancer (Appendix B).
     Studies of Vietnam veterans have been the subject of particular  interest
because it is thought that some of these veterans were exposed  to  2,3,7,8-TCDD
as a result of exposure to Agent Orange in Vietnam.8  Based on  Agent  Orange use
and potential for exposure under the conditions in  Vietnam, it  has been  assumed
                                       i
some subsets of the population may have been exposed to relatively high  levels
of 2,3,7,8-TCDD.  To date, most of these studies have shown no  statistically
        <• . •                             j
significant correlation between Vietnam service (and, therefore,  possible
exposure to 2,3,7,8-TCDD)  and an increased risk of  cancer.  For example, the
Ranch Hand study has so far reported only a limited number of deaths  (six)  from
cancer among the exposed group, none of which was from STS or lymphoma
(Fingerhut et al., 1984).   Studies of the mortality patterns among New York
service men with and without Vietnam experience found no significant
association between cancer and serviced in Vietnam  (Greenwald et al.,  1984),
                                       i
although it should be noted that a study of Massachusetts Vietnam veterans
reports a significant excess of connective tissue  sarcomas compared to
non-Vietnam veterans (Kogan and Clapp, 1985).
     Although the epidemiologic data afe not  persuasive  regarding one
interpretation over another, the high  relative  risks  seen  in the  Swedish
studies are noteworthy.  While an association may  exist  between exposure to
8Agent Orange, an herbicide widely use'd  in Vietnam, was  composed  of equal  parts
 of butylesters of 2,4,5-trichlorophenpxyacetic  acid  (2,4,5-T)  and  2,4-
 dichlorophenoxyacetic acid (2,4-D).   2,3,7,8-TCDD, a contaminant in Agent
 Orange, has been shown to originate  from the  2,4,5-T component with levels
 ranging from 0.1 to 47 ug/g  (U.S. EPA,  1985).
                                      14

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 chemicals contaminated  with  2,3,7,8-TCDD  (e.g., phenoxy herbicides) and
 increased incidences  of cancer,  the data  are  still too uncertain to attribute
 the effects seen  to 2,3,7,8-TCDD.9
      In light of  the  above considerations, and  in accordance with the Agency's
 Guidelines for Carcinogen Risk Assessment  (U.S. EPA, 1986a), the human evidence
 supporting an association between exposure to 2,3,7,8-TCDD and cancer is
 considered inadequate.
 3.   Altered Cell  Function
     Animals  given  2,3,7,8-TCDD  as a single dose of 300,000 to 3,000,000 pg/kg
 exhibited  a significant  increase in a variety of enzymes responsible for the
 toxification/detoxification of foreign chemicals in the liver (increased
 enzymatic  activity  has also been found in other organs) (Poland and Glover,
 1975).  Sloop and Lucier (1987) have shown a statistically significant increase
 in arylhydrocarbon hydroxylase (AHH) in animals exposed to 15,000 pg/kg of
2,3,7,8-TCDD in corn oil.10  In other systems it has been shown that the
toxification/detoxification process can,  in some cases, yield genotoxic
intermediates when metabolizing ingested  chemicals to harmless substances
 One current investigation involves a National  Institute for Occupational
 Safety and Health (NIOSH) registry of U.S.  workers who have
 been employed in industries that manufactured  chemicals thought to be
 contaminated with 2,3,7,8-TCDD,  as well  as  other chlorinated dibenzo-p.-
 dioxins (CDDs)  and chlorinated dibenzofurans (CDFs).   A study of this
 group of 7,000  workers,  scheduled for completion in 1989,  could provide
 substantial,  valuable,  and additional information on  the question of the
 carcinogenic potential  of 2,3,7,8-TCDD in humans.
 AHH is  a cytochrome P45G-mediated microsomal mono-oxygenase that metabolizes
 numerous chemicals.   AHH is induced by a sequence of  events starting with
 the binding of  2,3,7,8-TCDD to the Ah receptor located at the cellular mem-
 brane.   It is the translocation  of the Ah receptor/2,3,7,8-TCDD complex to
 5e«[]ucleus and b1nd™9  to. the DNA that  leads  to the  transcription/induction
 of  AHH.
                                     15

-------
suitable for excretion.   2,3,7,8-TCDDjis  poorly metabolized, and therefore the
                                     I
increased activity of these enzymes i?  likely  to  have little effect on the
genotoxic potential of 2,3,7,8-TCDD itself.  Available  data suggest that
2,3,7,8-TCDD has little or no ability to  act as  a direct  genotoxin.   However,
the increase in toxification/detoxification  enzymes does  increase  the
possibility that other exogenous chemicals will  be activated  to genotoxic
substances.  Thus, it is possible to postulate a process  whereby 2,3,7,8-TCDD,
                                     I
through a secondary route, causes cancer through a mechanism involving
genotoxicity.
     The available information on enzyme induction indicates that 2,3,7,8-TCDD
                                     i
is one of the most potent inducers studied.   Poland and Glover (1984) found
that 0.85 nmoles/kg of 2,3,7,8-TCDD ejlicited the ED50 level of AHH hydrocarbon
hydroxylase in rat liver compared to J25,500 nmoles/kg of 3-methylcholan-
threne.ll
     In addition, the increased enzymatic activity elicited by 2,3,7,8-TCDD
remains elevated, near peak level, for over 30 days while the enzymatic
activity elicited by 3-methylcholanth|rene returned to normal in 8 days,
reflecting the influence of 2,3,7,8-TCDD's long  half-life  (see Appendix A).
Limited data using human cells  in  culture indicate that  enzyme  induction is
likely to take place  in humans  exposed to 2,3,7,8-TCDD (Jaiswal et al., 1985).
Additional cellular data suggest an  impact  of 2,3,7,8-TCDD on  such diverse
                                     I
responses as increased proliferation,!  antagonism of hormone-mediated responses,
cytotoxicity, and  in  vitro  transformation (see  section B).
 n3-Methylcholanthrene is used routinely by investigators as an inducer of
  AHH.
                                       16

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     While this information 1s of interest and does signal  qualitative  concern
for carcinogenicity in humans exposed to 2,3,7,8-TCDD, it is not possible at
present to factor these observations into a quantitative risk assessment.
4-  Weight-of-Evidence Conclusion
     Based on sufficient evidence in animal studies, inadequate human evidence,
and consideration of ancillary or supportive information, 2,3,7,8-TCDD is
classified as a B2--probable human carcinogen in EPA's weight-of-evidence
scheme.  The Agency reached the same conclusion in the 1985 HAD without
   •:      -,•'••
invoking the contribution of the ancillary data.
     This classification represents a qualitative judgment as to the likelihood
that 2,3,7,8-TCDD may be a human carcinogen at some dose.  It does not reflect
a  calculation of potency nor does it resolve the issue of threshold versus
nonthreshold approaches to describe the carcinogenic dose-response.  In  its
simplest terms, this classification represents the consensus of scientific
opinion that ". . .in the absence of adequate data on humans, it is
biologically plausible and prudent to regard agents for which there is
sufficient evidence. . .of carcinogenicity in experimental animals as  if they
presented a carcinogenic risk to humans."  (IARC, 1987).

B.  MECHANISMS OF CARCINOGENESIS
     Qualitative evidence for designating a chemical as a potential human
carcinogen comes from a variety of observations—human epidemiologic studies,
chronic animal  bioassays, in vitro studies, metabolic studies, and mechanism
studies.  Thus, conclusions depend not on a single piece of  information, but
rather, a weight-of-evidence assessment of all data bearing  on whether the
chemical is or is not carcinogenic.

                                      17

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     At present, carcinogenesis,  as a process,  is  hypothesized to be a
continuum of events characterized by a number of steps — some  irreversible and
others reversible—that result in the [transformation  of a  normal cell to a
malignant one.  While some of these steps have been defined by responses
observed in laboratory experiments, the actual  sequence of cellular events
leading to (1) initiation of a cell, (2) clonal expansion  of  the population  of
                                     I
initiated cells, and (3) progression leading to malignant  transformation  is
                                     i
still unknown for even a single chemical.*2  Thus, chemicals  are often
                                     i
categorized by responses seen in vivo!and in vitro studies and/or  where in  the
carcinogenic process they act.  For example, simply stated,  a "complete
carcinogen" is one that can lead to tumor formation in the absence of any other
known exogenous factor and a "promoted" is a substance that can  affect  the
growth and clonal expansion of a population of initiated cells,  and can alter
gene expression (U.S. EPA, 1987c).
     While such categories are theoretically easy to describe,  in  practice it
is often difficult (if not impossible! to separate carcinogens into discrete
categories based on mechanism of actibn.  This process is further complicated
by the possibility of multiple carcinogenic mechanisms, direct and indirect,
occurring as a result of exposure to a single  compound.   In fact,  especially at
low doses it is not clear that any carcinogen  affects  all  stages;  the
hypothesis of "linearity-at-low-doses" does not require this.
12It should be noted, that initiation!, promotion, and transformation may
  also be made up of a series of steps.
                                      18

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 1-   Hypothesis 1:   2.3,7,8-TCDD as a Direct-Acting.  Complete Carcinogen
      a.   Qualitative Consideration
      At  present,  carcinogens are defined operationally because of a lack of
 understanding of  the mechanisms by which chemicals cause  cancer.   Thus,  obser-
 vations  of an increased  number of rare  tumors,  a  decrease in the  time-to-tumor,
 or  a statistically significant increase of  site-specific  tumor incidence in
 multiple species  and strains in animal  bioassays  have  generally been  taken  to
 support  the conclusion that  the test chemical  is  a complete  carcinogen.
 Unfortunately,  the bioassay  gives us no information  about the process leading
 to  the tumorigenic response.   To give further  support  to  the characterization
 of  a chemical  as a direct-acting,  complete  carcinogen,  i.e.,  one  that acts
 through  a  mechanism that  involves DNA damage,  the weight-of-evidence  analysis
 includes information from a  variety of  other observations.   These  include
 short-term mutagenicity studies,  irreversible  binding  to  DNA,  clastogenicity,
 and  other  indications of  genotoxicity.   Positive results  in  some  or all  of
 these types  of  tests are  considered  by  some as  necessary  before a  chemical  can
 be considered to be  a complete  carcinogen.  Conversely, negative results in
 these types  of  tests  are  considered  by  some as  an indication  that  a chemical
may  not  be a complete carcinogen.  Although some mechanisms,  such  as  oncogene
activation, may also be considered as "direct" negative results in more
traditional genotoxicity tests, they have been used to argue against
interpretation of positive bioassays results as the consequence of direct,
complete  carcinogenesis.13
13The use of the term "direct" in this context is meant to convey the notion of
  a direct impact of a chemical  or its metabolites on the carcinogenic process
  and should not be confused with the use of the term direct (parent compound)
  versus indirect (metabolite) carcinogenicity as described by some in the
  scientific literature.
                                     19

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     2,3,7,8-TCDD produces tumors inlanimals in lifetime studies  (Kociba  et
a!., 1978; NTP, 1982).  Tumors were seen at multiple sites in rats and mice  of
both sexes.  However, attempts to demonstrate that 2,3,7,8-TCDD is either
mutagenic or genotoxic have been mostly negative.   Recent reviews by Fishbein
(1987), Giri (1987), and Shu et al. (1987) of over 20 studies have concluded
that 2,3,7,8-TCDD is probably not gehotoxic.  In addition, Randerath et al.
                                    [
(1987) demonstrated that 2,3,7,8-TCDD exposure does not result in measurable
                                    j
DNA adducts, and, therefore, probably does not bind irreversibly to DNA.
Furthermore, Lim et al. (1987) published data that show a lack of      ;
clastogenicity in animals exposed to!2,3,7,8-TCDD.  Thus, the kind of
mechanistic data that would support (classification of 2,3,7,8-TCDD as a
direct-acting, complete carcinogen on the basis of genotoxicity are lacking.
                                    i
     A recent review comparing the results from short-term tests and long-term
chronic bioassays for 76 chemicals (Tehnant et al.» 1987) showed that the
                                    i
concordance between short-term tests! and a response in a chronic bioassay was
only 60%.  While highly predictive for certain classes of compounds and for
those substances that are routinely positive, this review indicates that the
lack of positive results in short-term tests is often not a  predictor  of the
outcome of a chronic bioassay, and, 'therefore, may not be a  reliable  predictor
of direct, complete carcinogenicity.
Also, Reynolds et al. (1987) have shown
that at least two chemicals that tes|t negative  in mutagenicity tests conducted
by the NTP can activate oncogenes wh|ich may result  in  a  specific  irreversible
                                    i
cellular change.  This is considered by many  scientists  to  affect control  of
cell proliferation and, perhaps, to !be an  important step in the carcinogenic
process.  These kinds of observations suggest that  as  new approaches to
investigating molecular events potentially involved in the  carcinogenic process

                                    1  20

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 become available, our understanding of how particular  chemicals exert their
 carcinogenic effect may change.
      It is importan^ to note that while the promoting  capability  of
 2,3,7,8-TCDD has been clearly demonstrated in  the  liver,  the  tumors  observed  in
 one animal bioassay in the lung,  soft palate,  and  nasal  turbinates can  be  taken
 as evidence supporting complete  carcinogenicity; although,  even in these
 tissues,"promotion of pre-existing initiation  (or  complete  carcinogenesjs  by
 unknown mechanisms that are not  representative of  response  at the administered
 dose),  based on  locally high levels of 2,3,7,8-TCDD  found in  food particles,
 cannot  be  ruled  out (Kociba,  1984).   In addition,  studies that use a liver
                                                   .           '             '\
 system  to  show promoting effects  of 2,3,7,8-TCDD yield data suggesting  that
 some  initiating  potential  cannot  be totally ruled  out.   In  sum, it is not
 possible,  at this time,  to conclude definitively whether  or not 2,3,7,8-TCDD  is
 acting  as  a  complete  carcinogen  in some tissues.
      b.  Quantitative Considerations
      For complete carcinogens  that act  by  directly causing  an irreversible
 initiating event  and  then  by  fostering  promotion and progression  of  the cells
 to  a  frank tumor,  no  threshold would be expected on  the basis of  current
 theory.  In  addition,  if a carcinogen  caused its effect by  adding irreversibly
 to  a  background process  already underway,  linearity  at low  doses  would  be
 expected.  The EPA has  integrated  the  two  concepts of  irreversibility and
 additivity in deriving the use of  a  plausible  upper  bound from the IMS  model
 for carcinogens as  a  matter of science  policy.  While  a number of models,  in
 addition to  the IMS model,  incorporate  the concept of  low-dose linearity,  there
 is currently no biological  basis for the choice of one of these alternatives
over the IMS model.

                                      21

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     Consequently, if the Agency considers 2,3,7,8-TCDD  to  be  a direct-acting,

complete carcinogen, then, the use by IEPA of a plausible upper bound derived
                                      i        - '•                        '
from the IMS model is appropriate and Iconsistent with Agency science policy.
                                      i         '              • ,  -       •
To the extent that the mechanism of action of 2,3,7,8-TCDD  is  not in accord

with the derivation of the model, the |use of the IMS model  may be less

appropriate.                          ,

2.  Hypothesis 2:  2,3,7,8-TCDD as a "Pure" Promoter
                                      f
                                      f              '          .
     a.  Qualitative Considerations   |
                                      i                      .    '        .  .
     Like complete carcinogens, promoters are defined operationally.   Promoters

are defined as providing a certain pattern of results in initiation/promotion

tests.  In theory, therefore, because "pure" promoters do not cause initiation,

one might expect a promoter to give negative results in an animal

carcinogenicity bioassay.  In practice, however, a promoter could yield

positive results in such a test because of initiated cells present as a result

of "background" events that can be promoted to yield a tumorigenic response.

For most promoters, the sensitivity of the bioassay would be expected to be too

low to yield a statistically significant response based only on background
                                      i                       •          ,
events.  Thus, under these terms, bioassay studies would allow certain

promoters to be distinguished from complete carcinogens.

     In order to characterize a chemical as having promoting potential only,
                                      I .           .
additional information from other tests  is generally considered  to be

necessary, such as:  a lack of  initialing potential  as  measured  by a lack of
                                    -  i                        .
genotoxicity, reversibility of  the promotion  response,  inhibition of

cell-to-cell communication14 and/or demonstration of a  dose-response having
^Several investigators  (e.g., Troskoi,  1983)  have  suggested  that  many  promoters
  may act by inhibiting  cell-to-cell Communication as measured  in metabolic
  cooperation or dye-transfer  studies!.

                                .     '22 '     ,        .

-------
 threshold characteristics.  It should be noted, however, that promoters do not
 necessarily have to exhibit all of these characteristics, but that positive
 results in these kinds of tests can help to support the categorization of a
 chemical as having only promotion potential.
      Currently, two in vivo systems are commonly used to study the promotional
 phase of the carcinogenic process—rat liver and mouse skin.   In both systems
 the animal is given a very small  amount of a known potent initiator followed by
 a promoter; in the case of one of the controls, the order is  reversed.
 Assuming the test compound is a promoter only,  the results of such experiments
 can be expected to yield results  as follows:
          Test Protocol                             Result
          initiator only                           no tumors
          initiator followed                       tumors
           by a promoter
          promoter followed                        no tumors
           by an initiator
          promoter only                             no tumors
      In  both  rat  liver  and in  some  mouse  skin studies,  2,3,7,8-TCDD  is  a potent
 promoter when  tested with  a known initiator  (U.S.  EPA  1986c;  1987c).   In both
 systems, however,  a low incidence of tumors  in  animals  given  only 2,3,7,8-TCDD
 suggests that  2,3,7,8-TCDD may have some  initiating  potential  or that the
 observed tumors are the result of potent promotion of background events or
 unidentified initiators found in the animals' environment (e.g., substances in
 food).  The results of  such a study involving diethylnitrosamine (DEN) and
 2,3,7,8-TCDD in partially hepatectomized rats are illustrated  in Table 1.
     In addition to the in vivo studies for promotional activity, a number of
laboratories have investigated the promotion potential of 2,3,7,8-TCDD in
                                     23

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TABLE 1   PROMOTING EFFECT OF 2,3,7,8-TCDD (TCDD) ON HEPATOCARCINOGENESIS IN PARTIALLY
TABLE 1.                 FEMALE RATS BY A SINGLE DOSE OF DIETHYLNITROSAMINE (DEN)
No. of enzyme-
altered foci per
No. of cubic centimeter
Group Treatment animals of liver
1 DENa 4 . ' i 309 ± 98b
2 TCDD (low dose)c 4 34 ± 17
3 TCDD (high dose)d 5 25 ± 7
4 Phenobarbital (control) 4 .56 ± 13
5 DEN + TCDD (low dose) 5 1068 ± 166
6 DEN + TCDD (high dose) 7 871 ± 66
7 DEN + phenobarbital 10 533 ± 103
a DEN given at dose of 10 mg/kg.
b Mean ± SD.
c 0.14 ug/kg/2 weeks.
d 1.40 ug/kg/2 weeks.
e Three rats exhibited neoplastic nodules in the liver.
f One rat exhibited neoplastic nodules in the liver.
Source: Pi tot et a!., 1980.
24
Mean volume No. of
of enzyme- % liver rats
altered volume with
foci (cubic occupied car-
millimeter) by foci cinoma
0.02 0.7 0
0.05 0.2 0
0.04 0.1 0
0.01 0.1 0
0.08 9.0 Oe
0.49 43.0 5f
0.15 6.0 8









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 isolated  cell  systems  (see,  for example, Abernethy et al.,  1985).  The results
 of these  studies clearly demonstrate that 2,3,7,8-TCDD affects  isolated cells
 as a promoter  but does not alter the cells in a manner which would suggest that
 an initiating  event has occurred.  Hence, under the most stringent conditions
 (in vitro), 2,3,7,8-TCDD may act as a "pure" promoter, but  under in vivo
 conditions the results are not as clear cut.
     The  reversibility of clonal expansion after the removal of a promoting
 substance has  been considered a key effect in characterizing promotional
 activity.  For example, Pitot et al.. (1987) showed that the size of liver foci
 in rats increased in the presence of phenobarbital (a chemical generally
 regarded  as a  "pure" promoter) and then returned to normal when phenobarbital
 was removed from the diet.15  Investigation of 2,3,7,8-TCDD for reversibility
 of foci formation in similar experiments has not been attempted because of its
 long half-life in tissues, making results less easily interpretable.
     It can also be argued that 2,3,7,8-TCDD does not meet the above criteria
 for a "pure" promoter.  Some tumors observed in the lung, nasal turbinate, and
 hard palate may be the result of "complete carcinogenicity," although, as
 discussed previously, this is not uniformly accepted.  Similarly, the
 reversibility of the promoter step has not been demonstrated for 2,3,7,8-TCDD.
 Finally, some evidence of clastogenicity exists although the results have not
 been duplicated (Green et al., 1977).
15It should be noted that re-administration of phenobarbital in this experiment
  resulted in a greater than anticipated clonal expansion which could be
  interpreted to mean that in addition to reversible effects, some irreversible
  step(s)  had occurred.

                                     25

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     b.  Quantitative Considerations
     A "pure" promoter that acts by facilitating clonal expansion of a
population of initiated cells without! any capacity to induce irreversible
changes in DNA may be expected to dempnstrate a threshold, theoretically,
although no quantitative framework exists which demonstrates that thresholds
must exist under all circumstances.  h>r example, the Moolgavkar, Knudson, and
                                     i
Venson (M-V-K) model, now being investigated by a number of scientists for
modeling the carcinogenicity of promoters, does not require an assumption of a
                                     I
threshold.  The theoretical basis for| the threshold response would be the
likelihood of a dose at which the netj response of clonal expansion and normal
cell death (or control of cell proliferation) would be zero.  Consequently, for
such a situation an appropriate method to estimate a level of exposure that
would be unlikely to pose a significant risk may be the traditional
toxicological approach for organ-specific toxicity via the derivation of  a
reference dose (RfD).  This RfD is calculated from the no-observed-adverse-
effect-level  (NOAEL) based on carcino|genicity noted in the bioassay, divided by
                                     i
several uncertainty factors.
                                     i
     To the  extent that the data demonstrate that 2,3,7,8-TCDD  is  a  "pure"
                                     [
promoter, the traditional toxicologicjal approach is arguably appropriate.
Conversely,  data that  indicate that  2,3,7,8-TCDD has the  ability to  initiate
                                     i
the carcinogenic process or that 2,3,!7,8-TCDD may be a complete carcinogen
would make the application of the  abpve method  less appropriate.
3.  Hypothesis 3:  Secondary or  Indirect  Mechanisms of Carcinoaenesis
     a.  General Considerations
     As stated earlier in this chapter, a carcinogenic response in animals  may
be the result of direct interaction  of the  chemical  (or  its metabolites)  under
                                      26

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 test with sensitive cellular targets.   On the other hand,  this response may be
 the result of indirect effects or even a combination of the two.
      While it is not possible to distinguish  between direct and indirect
 responses in animal  bioassays,  a variety of test  data may  be used  to  draw  a
 conclusion that  indirect  carcinogenicity may  be responsible for an observed
 effect.   The nature  of such  indirect effects  may  be such that  the  chemical
 affects  the pool  of  "initiated  cells"  either  qualitatively by  making  cells  more
 sensitive to initiating agents  or quantitatively  by causing increased numbers
         • -   '                                              •              t
 of  initiated cells.   On the  other hand,  chemicals may indirectly affect  the
 establishment or progression of preneoplastic  lesions by making the cellular
 environment more conducive to such events  or  by inhibiting  the cellular
 processes which  keep tumor growth in check.   It is  quite plausible that
 induction or inhibition of enzymes, competitive inhibition  of  normal  feedback
 mechanisms regulating cell proliferation,  cytotoxicity, or  effects on the
 immune system could  be responsible for an  indirect  increase  in carcinogenic
 response.
     A wide variety  of experimental data may be used  to evaluate the  inference
 of  indirect carcinogenicity.  These may be  studies  focused  on  molecular
 mechanisms  or they may be at  the  level of  altered organ function.  The
 following  section discusses  such  experimental  data  on 2,3,7,8-TCDD.   It  should
 be  understood  that this discussion is included to establish the plausibility of
 this approach, and does not represent an indepth review of potential
mechanistic data.  A number of lines of evidence require further development
before their  impact on earcinogenicity can be  assessed.  A more detailed
discussion of several aspects of this approach can be found in Appendix F.
                                     27

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     b.  Qualitative Evaluation of 2,3,7,8-TCDD as an Indirect
         or Secondary Carcinogen
     (1)  Enhancement of Initiation
     One way in which 2,3,7,8-TCDD could affect the pool of initiated cells is
by inducing enzymes that activate other endogenous and exogenous chemicals to
proximate carcinogens.  The effects, of 2,3,7,8-TCDD on the induction of AHH
                                   I
activity is well established in a number of in vitro and in vivo systems
(Poland and Knutson, 1982).  This ehzyme induction could result in increased
levels of reactive intermediates frfDm other xenobiotic chemicals, and
                                   i
ultimately increased numbers of initiated cells and increased potential
                                   I               .        ,     .   .
carcinogenicity.  Further enhancement of carcinogenicity could result from the
potent promotional activity of 2,3,7,8-TCDD acting on indirectly initiated
cells.  Studies have shown increased genotoxicity of chemicals such as
benzo(a)pyrene that require metaboljic activation when they are administered or
incubated with 2,3,7,8-TCDD in systems capable of responding to enzyme inducers
(Pahlman and Pelkonen, 1987).      :
                                   i
     The integrity of the DNA molecjjle is generally thought to be important for
normal cellular function.  Specific types of DNA damage alter DNA molecular
weight and may increase the probability of initiation.  One measure of both
                                   i
normal metabolic processing and increases in DNA damage is quantification of
single strand breaks in DNA.  Such jstrand breaks may be caused by increased
levels of free radicals in actively metabolizing tissues.  Direct measurements
of strand breaks have indicated increased breakage with exposure to
2,3,7,8-TCDD (Randerath et al., 198J7).  Studies that examined changes  in DNA
                                   i
molecular weight after 2,3,7,8-TCDD treatment have provided conflicting
results.  Molecular weight increases were observed in treated animals
                                      28

-------
 indicating  fewer rather than  Increased  numbers  of single  strand  breaks.   Strand
 breaks  may  also  be  associated with  increases  in DNA  adducts  either  directly  or
 indirectly  to  chemical  exposures.   Randerath  et al.  (1987) found no DNA  adducts
 within  the  limits of detection of his assay in  the livers of rats chronically
 exposed to  low doses of 2,3,7,8-TCDD, and  Romkes and Safe (1987)  did not find
 2,3,7,8-TCDD enhancement of DNA adduct  formation from endogenous steroids.
      Changes in  DNA repair  capacity could  also  have  an impact on the pool of
 initiated cells.  While few studies have measured repair  parameters after
 2,3,7,8-TCDD treatment,  several  observations  can be  noted.   Treatment with
 2,3,7,8-TCDD causes increases in 0-6-methylguanine content in DNA.   This
 observation may  be  consistent with  an indirect  effect on  methylating capacity
 or on a failure  to  rapidly  repair these lesions.   Studies to date have not
 demonstrated that 2,3,7,8-TCDD causes increases  in unscheduled DNA  synthesis
 (UDS).   This observation  suggests that, under the conditions of  the study, DNA
 damage  has not increased  to a level  that can be  measured  with this  type  of DNA
 repair.  Based on recent  studies by Busser and  Lutz  (1987) and Den  Engelse et
 al. (1986), effects  on  the repair system itself  cannot be ruled out.
     Another way  that 2,3,7,8-TCDD  could impact  the pool  of  initiated cells
 indirectly is through its ability to cause organ-specific cell proliferation,
thereby  increasing the number  of pre-existing initiated cells, which may
represent an increase of potential   tumors.   The  ability of 2,3,7,8-TCDD to
cause hypertrophy and hyperplasia in several  tissues where tumors arise has
been noted in chronic-toxicity studies (Kociba et al., 1978).  The  question of
stimulation of DNA synthesis caused by exposure to 2,3,7,8-TCDD is  still  an
open one (Busser and Lutz, 1987).
                                      29

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     (2)  Enhancement of Carcinogenic! Progression
     Currently accepted theory suggests that an aspect of the carcinogenic
                                     i
process is the ability of chemical carcinogens to affect the progression of
preneoplastic cells or foci towards the malignant state.  It is generally held
that this part of the process shows ah irreversible dedifferentiation based on
permanent genetic change.  In the cas|e of 2,3,7,8-TCDD, the long half-life of
the molecule in human and animal systems allows for a constant impact over a
prolonged period of time, somewhat akin to the constitutive presence of a
regulator of cellular differentiation.  While there is no evidence of permanent
genetic changes associated with 2,3,7,8-TCDD exposure, there are data to
suggest that the chemical can affect terminal differentiation and carcinogenic
transformation in vitro.  Increases in "flat cell"/"XB cell" (Gierthy and
Crane,  1985) transformation support the notion of the potential to cause
dedifferentiation, and studies  in  "10T1/2 cells" (Abernethy et al.,  1985) show
increased cellular transformation.   ;
                                     [
     These activities represent a  release from growth control which  is
characteristic of the carcinogenic process and might  allow for the appearance
of increased carcinogenic response in  an indirect manner.  Further support for
this role of 2,3,7,8-TCDD comes from  its apparent ability to act as  a hormone
agonist or antagonist  (Umbreit  and Gallo, 1988), without competing for  sites on
certain hormone receptors.  The suggestion that  2,3,7-8 TCDD may be  acting
                                     i
through cellular receptors as opposed  to acting  through the disruption  of
membranes, as many "classical promoters" do,  is  further supported by evidence
that 2,3,7,8-TCDD does not disrupt cell-to-cell  communication  in vitro  (Lincoln
et al., 1987).  Disruption of cell-tb-cell communication has been suggested  by
some investigators (e.g., Trosko,  1983) to be  an attribute of  many promoting

                                     ! 30

-------
compounds.  This  activity,  or  perhaps  lack of activity,  is found  in cells with
and Without an  inducible AHH system, so the characteristic is  independent of
that event, again  suggesting the necessary but not sufficient  role that enzyme
induction might have  in the carcinogenic process.  This  issue  is  described more
fully in the  issue paper prepared by Gallo (Appendix F).
     c.  Quantitative Considerations
     The suggestion,  in qualitative terms, that 2,3,7,8-TCDD may  be acting as
an indirect or  secondary complete carcinogen through multiple  mechanisms
affects the way that  this chemical is  viewed in quantitative terms.  This
hypothesis is also consistent  with the observed potency  of 2,3,7,8-TCDD in
producing a carcinogenic response in vivo, in that the observed tumors could be
postulated to result  from mechanisms such that 2,3,,7,8-TCDD:   may increase the
pool of initiated  cells, either directly or indirectly;  is a potent promoter
based on experimental evidence in specific test systems; may be actively
involved in stimulating progression of tumors from benign to malignant; and may
be indirectly influencing both repair  and surveillance capabilities in vivo.
Such activities,  if viewed  as  adding to events that are  already underway and
contributing to the background or "spontaneous" incidence rate of tumors, have
been argued as reasons for  considering a nonthreshold, low-dose linear type of
dose-response curve as being applicable (OSTPj 1985; Crump and Howe, 1984).
This would imply an approach,  under the Agency's current guidance, that might
be identical  to that generally used for carcinogens, namely the use of the IMS
model to describe a; plausible  upper bound on the risk.   It would  not be likely  .
that such indirect effects would result in a greater response  than would direct
effects,  although this cannot  be ruled out.   These issues are  discussed further
in section II:B.l.b.  of this document  and in Appendix F.

    •  ".'     .•'  -.'.-.'.  "  ".'  •   : : ..•.  si   '  "  ••   •..'.•  . :     •

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C.  EVALUATION OF 2,3,7,8-TCDD AS AN "ANTICARCINOGEN"
     Another attribute of chemical carcinogens that should be taken Into
                                    i
account when attempting to understand their behavior is the activity termed
                                    i
"anticarcinogenesis."  In some cases, chemicals have been shown to overcome
background processes or their own effects at some doses to produce a net
negative effect on the carcinogenic
outcome of animal bioassays.  Such
responses could have a profound effect on expectations of the dose-response if
anticarcinogenic effects were cancelling out carcinogenic effects at a given
dose in some tissues.  The reproducible dip in response (at low doses) to below
                                                                            '
background tumor levels in female rat liver (Kociba et al., 1978; NTP, 1982)
has suggested to some observers that* 2,3,7,8-TCDD may show such a phenomenon.
These observations are supported by ,more recent data which showed that
induction of liver foci were reduced at low doses of 2,3,7,8-TCDD in an
                                    i
initiation/promotion assay (Pitot et al., 1987).  Responses in other rat
tissues (i.e., uterine and breast tissue), also show decreases in background or
spontaneous tumor incidence which again support the observation that there is
an overall decrease in the carcinogenic response with some doses of
2,3,7,8-TCDD (Kociba et al., 1978).
     The implications for an analysis of dose-response and the applicability
of certain models based on the observation of a potentially "anticarcinogenic"
response for 2,3,7,8-TCDD is discussed in the next section.
D.  USE OF PREDICTIVE MODELS        i
     Scientific interest in interspecies and high- to low-dose extrapolation
has led to the development of a variety of predictive models, including models
for estimating the likelihood of hunian cancer based on data from animal
                                     32

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 studies.   Selection of the appropriate model  in a given set of circumstances is
 difficult because of a lack of knowledge of concordant events across species
 and at low doses.  Selection is further complicated by the possibility of
 multiple  effects affecting the carcinogenic process.
       Pleiotropism, i.e.,  action through multiple pathways,  is not an uncommon
 finding with molecules such as 2,3,7,8-TCDD.   One has  only to review the
 earlier experiments on multistage mouse skin  carcinogenesis  of 2,3,7,8-TCDD  to
 see that,  in some cases,  it inhibited  tumor formation  by PAH initiators
   '*'.'•<,': f
 (DiGiovanni-et  al.,  1977;  1980;  Berry  et al.,  1978).   It must be  emphasized
 that the  responses in  multistage models are dependent  on time,  sequence  of
 administration,  dose,  and  species.  Hence,  inhibition  under  some  conditions
 might have  been  predictable.   This  is  contrasted  with  the two-stage  liver model
 (Pitot et al.,  1980) in which  it has been shown that orally  administered
 2,3,7,8-TCDD enhances  the  tumorigenic  action  of DEN.   In subsequent  experiments
 at  lower doses of 2,3,7,8-TCDD,  a parabolic dose-response curve has  been
 reported  in  the  DEN/TCDD initiation-promotion  protocol  (Pitot et  al.,  1987).
 These results are not  well  understood,  but  they do  not  appear to  be  solely the
 function of  enhanced metabolism  or Ah  receptor binding  (Safe  et al.,  1987).
 Perhaps it  is the result of alteration  of epidermal growth factor (EGF)
 receptors at  low doses  (Madhukar et al., 1984) which displays  a commonality
 with  several  steroid hormone receptors.
      These findings are of  importance to the approximation of human  and  animal
 health risks from exposure  to  2,3,7,8-TCDD  and related molecules.  Mathematical
modeling of physiological phenomena, especially those related  to  receptor
 function,  is often conducted using the Michaelis-Menton equation  (1913)  as
modified by Clark  (1933) for the  "classical" receptor model.  The weight of
                                     33

-------
evidence for the most prevalent 2,3,7J,8-TCDD effects falls into the category of
the receptor model (Poland and Knutsoh, 1982).  According to recent findings,
hepatocarcinogenesis observed after exposure of animals to 2,3,7,8-TCDD is
related to estrogen levels or to the presence of functional ovaries (Goldstein
et a!., 1987), and di ethyl nitrosamine; hepatocarcinogenesis in partially
hepatectomized rats is first inhibited and then promoted by 2,3,7,8-TCDD (Pitot
et al., 1980; 1987).  These findings indicate that 2,3,7,8-TCDD is not causing
its myriad of effects in liver by a simple one-step event such as binding to
the Ah receptor and subsequent induction of cytochrome PI-450.
     Risk modeling for carcinogenic xenobiotics can be segregated into three
classes or types of models:  (1) physiologically based pharmacokinetic (PBPK)
models in which the body is considered to be a small group of physiological
compartments (Hoel et al., 1983; Krewski et al., 1986; Bischoff, 1987); (2)
biologically motivated models of carcinogenesis (BMMC) in which the
carcinogenic process is considered to occur through a series of linked
reactions that result from two or more molecular events followed by a cellular
                                     i
amplification by  "promoter" molecule^  (Moolgavkar et al.,  1987; Thorslund et
al., 1987; Krewski et al., 1987; U.S.  EPA,  1987a); and (3) the IMS model of
Armitage-Doll as modified by Crump and Howe (1984) in which it is assumed that
a sequence of events occur within a single  cell, some of which are
irreversible, leading to the neoplastic change  (Armitage,  1985).
                                     j
     A model that appears to accommodate most of the critical  components from
the biological data  base on 2,3,7,8-jCDD is the BMMC model, which  is generally
                                     I
referred to  as the M-V-K model  (Moolgavkar .and  Venzon, 1979; Moolgavkar and
                                     L
Knudson, 1981).   This model allows for several  of the concepts of
initiation-promotion-progression, along with  the growth-stimulating role  of

                                      34

-------
endogenous substrates such as hormones (Moolgavkar, 1987).  Incorporation of
some of the factors necessary for the PBPK model can also be done using the
M-V-K model as expanded by Thorslund et al. (1987).  These expansions of the
M-V-K model give the risk assessor a powerful tool for looking at cancer risk
mechanistically.  This approach requires the use of assumptions for several
critical parameters and, thus, is testable but as yet unvalidated.  This
approach is not available with the IMS model as originally proposed.
     The IMS model might be accommodated if one hypothesizes that:  (1) the
initiating event is the result of an indirect action of 2,3,7,8-TCDD through
modification of exogenous or endogenous compounds; (2) a population of
initiated cells exist; or (3) 2,3,7,8-TCDD acts through a variety of
mechanisms, some of which are, at low doses, additive to other processes
related to carcinogenicity arid already underway.
     Recent reports have shown that 2,3,7,8-TCDD and other promoters in liver
enhance stimulation of DNA synthesis in situ, and stimulate repair of
0-6-methylguanine in liver DNA (Busser and Lutz, 1987; Den Englese et al.,
1986).  Lutz et al. (1984) presented a scheme for promoter potency based on
stimulation of DNA synthesis and the assumption that cell  division is a
prerequisite for several stages in the carcinogenesis process.   These reports
indicate that 2,3,7,8-TCDD may act as a complete, indirect carcinogen,
including promoter activity, despite the lack of DNA binding or direct
mutagenesis.  The sum of all these findings, along with the myriad of other
toxic responses, suggests a complex hypothesis for 2,3,7,8-TCDD carcinogenesis
in rodent liver, which is illustrated schematically in Figure 2.   This
hypothesis can account for the dose-response data in the bioassays and the
multistage promotion experiments,  as well  as allow for incorporation into
                                     35

-------
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-------
existing risk models, and the scheme is not inconsistent with the reports of
decreased tumor formation in some tissues.  If the pathway through AHH activity
can be verified by demonstration of a net increase of reactive intermediates
after 2,3,7,8-TCDD treatment, then the IMS model can be used.  The scheme also
presents several testable hypotheses that should be examined.
                                    37

-------
                               III.
CONCLUSIONS
     This re-examination of the carcinogen risk assessment for 2,3,7,8-TCDD
identifies several approaches to describing the possible carcinogenic
                                    I
mechanisms for this chemical and reviews several different mathematical models
for estimating its carcinogenic potency.  However, the enormously rich data
base on 2,3,7,8-TCDD is incomplete when it comes to answering the questions
                                    j
posed at the beginning of the analysis.  The available data permit
consideration of a broad array of possible scientific theories and science
policy approaches when reconsidering EPA's 1985 carcinogen risk assessment for
2,3,7,8-TCDD (U.S. EPA, 1985), but they do not provide a clear basis for
confidently choosing among them.  The evaluation of each possible theory
results in varying degrees of confidence as to their plausibility, as discussed
                                    i
in more detail below.
     The literature review regarding 2,3,7,8-TCDD leads the Workgroup to
                                    i
several conclusions:
     •  2,3,7,8-TCDD is a potent promoter of carcinogenesis;
     •  the possibility that 2,3,7,8-TCDD acts as a direct-acting, complete
        carcinogen cannot be eliminated;
     •  2,3,7,8-TCDD may act through a secondary mechanism(s) which may affect
        the carcinogenic process at|different stages; and
     •  2,3,7,8-TCDD may act through a number of different mechanisms so that
        the observed effects represent an integrated composite of several
        mechanisms in operation.    !
     After considering all of the data, the Workgroup has concluded that
                                    i
thinking about 2,3,7,8-TCDD either solely as a "promoter" or as a "complete"
carcinogen is an oversimplification!  Rather, while it is clear that 2,3,7,8-
TCDD acts as a potent promoter, it may also affect other important carcinogenic
                                      38

-------
processes, some of which may result In a linear carcinogenic response in the
low-dose region.
     Schematically, this multiple mechanism hypothesis for 2,3,7,8-TCDD
carcinogenicity is graphically displayed in Figure 3.  The upper curve is the
UCL of the IMS model, as applied in U.S. EPA (1985), in which the chemical is
treated as a complete carcinogen with no distinction made for the specific
contributions of initiation, promotion, or progression to the response.  The
low-dose behavior that is appropriately modeled by the IMS approach for the
multiple mechanism hypothesis is depicted by the dotted line for the composite
effect, represented here by an arbitrarily placed line.  However, the magnitude
of any, difference between the slopes of these two lines is uncertain.
Particularly from the available information, it is impossible to determine
whether there is any difference at all, or if the "true" difference is
negligible or substantial.  The fact that the promoting behavior is taken as a
major factor in the tumor response for 2,3,7,8-TCDD could argue for a greater
rather than a lesser difference between the two slopes.  On the other hand,
because the animal tumor response observed is the net result of many processes,
2,3,7,8-TCDD potency at low doses could be characterized by the LMS upper-bound
estimate, even if promotion is the predominant activity, because the linear
portion of the curve would be the composite of a number of different activities
acting in concert.
     Quantitatively, it is not. easy to fit such a hypothesis into currently
available and well-accepted risk assessment models.  Accordingly, the Workgroup
recommends that, until either a more appropriate model is developed or
additional data demonstrate that a currently available model is correct, as
assessment approach that recognizes the possibility of linearity at low doses

                                     39

-------
R

I

S

K
                                                 UCL-LMS (1985)
                                                      - "Composite (1988)
                        D   OS   E
   Figure 3.  "Multiple mechanism" hypothesis for 2,3,7,8-TCDD
               carcinogenesis.   j
                                  40

-------
and presents estimates in terms of plausible upper bounds would be preferable
as a interim approach.
     The basis for these conclusions is set forth below.  Section A summarizes
qualitative factors relating to choosing a mathematical model, while section B
reviews related quantitative considerations.  Section C sets forth a rationale
for a recommended cancer risk-specific dose (RsD) for 2,3,7,8-TCDD.

A. -QUALITATIVE CONSIDERATIONS
     Qualitatively, at least three classes of mechanisms of carcinogenic action
for 2,3,7,8-TCDD have been considered in this document.  First, tumors found in
long-term bioassays in which 2,3,7,8-TCDD is the only known agent suggest that
this agent is, at least operationally speaking, a complete carcinogen.  Second,
data from in vivo studies for promoter activity demonstrate that 2,3,7,8-TCDD
is a potent promoter of carcinogenesis with little or no demonstrated ability
to act as a direct genotoxin.  Finally,  an indirect impact on carcinogenic
processes is suggested by studies linking 2,3,7,8-TCDD to responses such as
enhancement of initiation,  increased cell  proliferation, antagonism of
hormone-mediated responses, cytotoxicity,  and in vitro transformation activity.
     Despite evidence supporting each potential  mechanism,  2,3,7,8-TCDD does
not exhibit certain properties generally expected for each of the first two
mechanisms.  For example, if 2,3,7,8-TCDD directly initiated a "complete"
carcinogenic response, genotoxicity or binding to DNA would be expected.   The
absence of these effects in studies involving this chemical  does not rule out
the possibility that 2,3,7,8-TCDD is a direct initiator, but it suggests that
2,3,7,8-TCDD is not a "typical"  complete carcinogen.   Unlike many promoting
agents,  2,3,7,8-TCDD acts at low doses.   (2,3,7,8-TCDD is active at doses 1000

                                     41

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times less than other known promoters.)  Furthermore,  reversibility,  an
                                   j
oft-cited characteristic of promotion, has not and cannot easily be
demonstrated because of the long half-life of 2,3,7,8-TCDD in biological
systems.
     In short, it appears that 2,3,M-TCDD has characteristics of a potent
promoter, and, operationally, of a complete carcinogen.  As observed
previously, the Workgroup has concluded that thinking about 2,3,7,8-TCDD either
solely as a "promoter" or as a "complete" carcinogen is an oversimplification.
Rather, 2,3,7,8-TCDD produces a broad  spectrum of biological responses that
allows many hypotheses regarding the mechanism of 2,3,7,8-TCDD toxicity and
carcinogenicity.  Because the available data are not adequate to absolutely
confirm or refute one or more of thjese approaches, each is considered  in
evaluating potential quantitative methods for estimating the risk  associated
with exposure  to this chemical.

B.  QUANTITATIVE CONSIDERATIONS    !
                                   I
     The  range of potential  mechanisms suggests a range of different
quantitative  approaches.   If 2,3,7,8-TCDD is treated as a direct-acting,
complete  carcinogen, a model incorporating  linearity at low  doses  would be
appropriate for dose-response assessment, for the reasons mentioned  in the  EPA
guidelines and the  OSTP  Cancer  Principles  (U.S. EPA,  1986a;  OSTP,  1985).  At
the other end of the spectrum,  if  2,3,7,8-TCDD  is regarded  solely  as  a
promoter, a threshold  approach  to  quantitative  assessment may  be more
appropriate.   And,  finally,  if  2,3J7,8-TCDD is  regarded  as  an  indirect
carcinogen, possibly acting  by  multiple  mechanisms  in  the  carcinogenic process,
some of which may  display linear behavior at low  doses,  then a linearized model
                                       42

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could be appropriate  although the slope of the response will be uncertain.  It
should be noted here  that the 1985 estimate of the potency of 2,3,7,8-TCDD
(U.S. EPA,  1985) has  always been characterized as a plausible upper bound to
the risk; the extent  of this "overestimate" is unknown, but it may be higher
than previously thought,
     An assessment of several potential approaches for estimating the
carcinogenic potency  of 2,3,7,8-TCDD and related risk-specific doses is
summarized  in the following section.
1.  Selection of Models
     a.  NOEL/Uncertainty Factor (Threshold Approaches)
     If 2,3,7,8-TCDD  acts solely as a promoter, the traditional toxicological
approach based on a NOEL or a lowest-observed-effect-level (LOEL) may be an
appropriate risk estimation approach under certain circumstances.  However,
while there is evidence that 2,3,7,8-TCDD acts as a promoter, the data suggest
that this chemical may also act through multiple mechanisms, direct or
indirect.   If some of these component mechanisms were linear at low doses, then
the composite dose-response curve would be expected to be linear at low doses,
and a threshold approach would not be appropriate.  Although 2,3,7,8-TCDD is a
strong promoter, biological data and statistical limitations to the power of
bioassays suggest that a threshold cannot be adequately demonstrated and may
not exist.  This assessment is tempered somewhat by observations both in vivo
and in vitro suggesting "anticarcinogen" effects at low doses which could
offset the  other effects of 2,3,7,8-TCDD and possibly produce a threshold.  An
anticarcinogenic effect working in conjunction with a carcinogenic effect in
the same tissue might result in a net response of zero over background and
might be described as a threshold effect,  in summary.

                                     43

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      Until  such  time  that  some  of  th|ese  critical  Issues  are  resolved,  the
 Workgroup  concluded that it would  not  be prudent  to  adopt  a  threshold  approach
                                     i
 for  estimating human  cancer risk for 2,3,7,8-TCDD.
                                     i
                                     I
      b.  Siel ken Approach
      Sielken  has produced  a risk assessment with  several provocative aspects;
 e.g., generating a maximum likelihood  estimate  (MLE, as  opposed to  a UCL),
                                     i
 discarding  the data obtained  from  thje  highest dose,  using  time-to-tumor
 information,  and drawing attention to  the lower-than-background response
                                     I               '                   i. •  .  . .
 observed at the  lowest dose in  both  ^he  Kociba  et al.  (1978)  and the NTP (1982)
 studies.  While  this  type  of  exploratory analysis is interesting and raises
 points that should be examined  more  Closely, the  Workgroup is reluctant to
 adopt this  approach at this time for several reasons.  For example, the use of
 the  MLE has traditionally  been  avoided due to its instability in the face of
 relatively  small changes in the data| (see Appendix A)  and, for this reason, the
 UCL  has generally been preferred.  Cfump (1987) provided a critical review of
                                     i
 the  Sielken analysis  and observed  that even if  "the  true shape of the  dose
 response is a straight line connecting the background  response and  the response
 at the mid-dose," the probability  of an  MLE of  zero  for  the  linear  term in the
 multistage  model is about  1/3.  He concludes that while  the  data are consistent
 with  Sielken's interpretation of a higher RsD,  they  are  also consistent with
 much  lower  RsDs, as displayed in the confidence limits.
      c.  M-K-V model
                                     j
     This model, which is  based on the use of biologically-based models (see
Section II.D.), represents significant progress in carcinogen risk assessment.
 Its use for estimating the carcinogenic  potency of chemicals in general,  and of
2,3,7,8-TCDD in particular, is viewed by  this group as premature for several

                                     44

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reasons.  There are concerns about its biological bases and assumptions, the
statistical derivation and application of the model, the resulting large range
of "best" estimates (as opposed to upper-bound estimates) for which there are
no adequate criteria for selecting any estimate within the range, and the lack
of use and "experience" with other chemicals.  Other concerns include
uncertainties and sensitivities about application of the model.  Although this
model has many interesting features, and the Agency will continue to encourage
its development, the reasons described above and in Appendix A preclude
recommendation of its use to select an RsD at this time.
     d.  IMS Model
     Although there are many uncertainties about the mode of action of
2,3,7,8-TCDD, data from bioassays and information on possible indirect
mechanisms provide some basis for assuming that this chemical might initiate
the carcinogenic response.  If 2,3,7,8-TCDD is both an initiator and a promoter
of carcinogenesis, or if it functions in some other way as a complete
carcinogen, a model that is linear at low doses would be appropriate for
estimating an upper bound on the carcinogenic potency.  If the chemical acts by
a combination of direct and indirect mechanisms, some with the characteristic
of low-dose linearity, the composite dose-response might be expected to exhibit
low-dose linearity as well, but perhaps with a lower slope than previously
estimated.
     The EPA has elected to use the IMS model for cancer risk assessments, in
general, because it has a plausible biological basis, incorporates the
assumptions of nonthreshold linearity at low doses, provides a plausible upper
bound to the risk, and can be used as a "yardstick" to compare the "potency" of
one chemical with another.  The EPA guidelines for the assessment of risk from
                                      45

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carcinogens point out that other modjels can, and should, be used if biological
                                    I
considerations dictate.  In the casei of 2,3,7,8-TCDD, some other models have

been proposed (Longstreth and Hushon, 1983), but the biological basis that

would justify their use is not any stronger than with the IMS model.

Consequently, the Workgroup has concluded that the use of these alternative

models provides no additional advantages to the risk assessment of

2,3,7,8-TCDD.

     In summary, the Workgroup concludes that none of the available models

adequately describe the carcinogenic; behavior of 2,3,7,8-TCDD at low doses.

Specifically:

     •  While there is evidence that! 2,3,7,8-TCDD acts as a promoter, there is
        little evidence on which to conclude that a threshold exists.  Without
        a more scientific basis for such a radical departure from EPA's
        traditional approach to the risk assessment for carcinogens, the
        Workgroup is unwilling to adbpt a threshold approach for 2,3,7,8-TCDD.

     •  The innovative approaches of;Sielken and Moolgavkar, Venson, and
        Knudson are interesting, butj untested.  Therefore, the Workgroup
        concludes that it would be imprudent to use them at this time for
        2,3,7,8-TCDD.

     •  The available evidence suggests that reliance on the IMS model, as
        traditionally used by EPA, may be less appropriate for 2,3,7,8-TCDD
        than for many other chemicalis, and that the Agency's 1985 assessment
        based on the IMS model may overestimate the upper bound on the risk by
        some unknown amount.  Howeveir, a rationale for a possible linear
        behavior at low doses has been developed in this report, and the IMS
        model provides a useful and familiar context which is widely used in
        the Federal government when discussing risk estimates.  Therefore, the
        Workgroup discusses its recommendation using the IMS model as a
        construct, that is, the plausible upper-bound estimate of risk and the
        risk-specific dose.

2.  Selection of the RsD Range

     a.  Base Analysis

     Application of the IMS model to estimate the carcinogenic potency of

2,3,7,8-TCDD results in a range of RsDs (10~6) from 0.001 pg/kg/day to 1.2
                                    i
P9/k§/day depending on the data used as the basis for the analysis and the

                                    I  46

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assumptions made in the assessment.  This range would be even greater if the
RfDs established in Canada, Europe, and some states in the United States were
included.  Moreover, it is not clear where in the range potency estimates,
based on composite direct and indirect mechanisms, would fall.  In fact, it is
possible that such estimates may fall outside of the IMS model range described
previously.  The discussion in the subsequent sections attempts to narrow this
wide range and provide a bound to the RsD selected.
   ,  b.  Modification of Base Analysis
     (1)  Incorporation of Alternative Inferences
     Incorporation of the factors used by the FDA for scaling from animals to
humans, along with use of only the tumors of the lung, hard palate, and nasal
turbinates observed in the Kociba et al. (1978) study results in an RsD (10~6)
of 1.2 pg/kg/day, the highest RsD we have developed based on the IMS model.
This analysis excludes the tumors observed in the liver because of the strong
promoter activity shown by 2,3,7,8-TCDD in this organ.  However, the Workgroup
gives less weight to this point on the range because (1) it excludes 90% of the
response observed in the bioassay, and (2) there is controversy whether the
tumors of the lung, hard palate, and nasal turbinates should be considered at
all (Kociba, 1984), since they may be a localized carcinogenic response to
inhaled microscopic food particles containing 2,3,7,8-TCDD.  This suggestion
gains support from the fact that these tumors were not observed in experiments
using other routes of exposure, i.e., dermal, gavage, and intraperitoneal,
while liver tumors were common to all four routes.  The Workgroup concludes
that there is not, as yet, sufficient evidence to accept or reject this
hypothesis.
                                      47

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     (2)  Incorporation of Relative Half-Lives
     The lowest RsD in the range is based on differences in the relative
half-lives of 2,3,7,8-TCDD in rats and humans.  The Workgroup gives less weight
                                    I
to this point because of numerous uncertainties about the pharmacokinetics of
2,3,7,8-TCDD, particularly the absence of information on species differences
and rates of incorporation and absorption of 2,3,7,8-TCDD in different tissues
and species.  Similarly, the rate ofjrelease of this chemical is likely to be
different from tissue to tissue and species to species.  Furthermore,  it is not
                                    I
clear how 2,3,7,8-TCDD will behave in different species, particularly humans,
                                    i
under conditions of chronic exposure^ incorporation, and release.
     (3)  Alternative Dose-Response Curves
     The suggestion by Hoel (1987) that AHH induction may be useful for
defining the shape of the dose-response curve is interesting, but it is not
clear how the results of such a calculation would be incorporated into a final
                                    f
risk assessment.  Initially, use of AHH data to define the curve is appealing
because many data points are available, AHH induction is closely related to
many of the toxic effects observed in 2,3,7,8-TCDD, and AHH induction appears
                                    i
to be linear at low doses.  However,;there is no demonstrated correlation
between AHH induction and tumorigenicity, and it is not apparent that the shape
of the AHH dose-response curve reflects the shape of the dose-response curve
for 2,3,7,8-TCDD's carcinogenic effects at low doses.
     (4)  Multiple Mechanisms
     Qualitatively, the concept of multiple mechanisms acting in concert, or
even opposition, is given added weight because it allows inclusion of much of
                                    i
the body of scientific data on 2,3,7|,8-TCDD other than the standard bioassay
                                    i
data.  As discussed previously, it ils not unreasonable to assume that a

                                      48

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composite dose-response curve may, under certain conditions, exhibit linearity
at low doses.  However, quantitative application of this approach is limited
because we do not have information on the slopes of the component curves that
would be necessary to define the overall slope of the linear portion of the
curve.  In general, if such multiple mechanisms could be incorporated into the
LMS methodology, the slope of the line would be lower (extent not quantifiable)
rather than higher than that derived as a plausible upper bound for direct-
acting, complete carcinogens.
      (5)  Different Base Assumptions
     Using the same basic LMS model approach, EPA, CDC, and FDA have estimated
carcinogenic RsDs (10~5) for 2,3,7,8-TCDD of 0.006, 0.03, and 0.06 pg/kg/day,
respectively.  Several different policy-based assumptions account for the
differences.  For example, EPA scales from animals to humans on the basis of
relative body surface area, while FDA uses relative body weights.  In addition,
both EPA and FDA used the 2,3,7,8-TCDD concentration in rat food as a surrogate
for dose, while CDC used the concentration of the chemical in rat liver as a
measure of dose.  There is no obvious scientific basis for excluding any of
these values from the range.
     (6)  Choice of RsD
     As noted previously, a majority of the Workgroup has concluded that the
1985 EPA estimate of the upper-bound potency (RsD) generated from the
application of the UCL LMS model  to the Kociba et al. (1978) data is likely to
have led to an overestimate of risk (or underestimate of the risk-specific
dose).  The weight of evidence indicates that a more appropriate upper-bound
estimate would be obtained by a reduction of the potency by some unquantifiable
amount.  Therefore,  in recommending a new RsD and indirectly suggesting a
                                     49

-------
change in potency, the Workgroup was confronted with the question, "How great a
reduction in slope (or increase in RsD) is appropriate?"
     The Workgroup concluded that tjiere is currently no definitive scientific
                                   i
basis for an answer to that questioji.  Given, however, that the question must
be answered for Agency purposes, this answer should be grounded in rational,
prudent science policy.
     While some argument could be mpunted that a threshold for the
carcinogenicity of 2,3,7,8-TCDD may; in fact exist, the evidence for such a
contention is not compelling.  Therefore, prudence dictates against adopting a
simple threshold approach to setting an RsD.  Hence, the Workgroup recommends
against adopting a policy position similar to that of the European countries at
                                   i
this time.
     The Workgroup encourages the type of analysis generated by Si el ken and the
application of the newer M-K-V modeil to 2,3,7,8-TCDD.  These fresh looks at the
problem stimulate discussion and challenge old ways of thinking.  However, as
noted previously, neither of these approaches is sufficiently developed nor has
received sufficient standing in the| critical scientific community that the
Workgroup feels comfortable in recommending a potency (RsD) on this basis at
this time.                         ;
     The Workgroup is not convinced^ that the UCL IMS model is an appropriate
model for estimating upper-bound cancer'risks associated with exposure to
2,3,7,8-TCDD.  However, without a b[etter alternative the Workgroup has used the
UCL IMS model as a construct within: which to discuss the carcinogenic potency
                                   I
or RsD of 2,3,7,8-TCDD.  The scientific evidence is consistent with, and would
support, a recommended science policy position that the RsD (10~6) for
                                   i                        A
2,3,7,8-TCDD be 0.1 pg/kg/day, which is associated with a qj, in the UCL IMS

                                      50

-------
construct of 1 x W^/mg/kg/day, for the following reasons:
     •  the scientific data indicate that the Agency's current upper bound for
        2,3,7,8-TCDD may be an overestimate;
     •  the scientific data do not permit an estimate of the extent of the
        overestimate;
     •  al] of the UCL IMS RsD estimates generated by the Federal agencies are
        arguably of equal scientific merit at this time;
     •  for strictly policy purposes, there is great benefit in Federal
        agencies' adopting consistent positions in the absence of compelling
        scientific information; and
     •  an order of magnitude estimate of the RsD (potency), as opposed to some
        more precise estimate of the risk-specific dose, helps to convey the
        notion that the numerical expression is only a rough estimate (the
        science permits no greater accuracy).
     The available scientific data can give us no clearer guidance.  Some of
the research now underway holds the promise of clarifying the issue but not
resolving it totally.  While a series of considerations, based on science, may
be brought to bear on the selection of the RsD, the Workgroup does not advance
them as compelling scientific support for a recommended RsD (10"^), which is,
in this case, simply a rational and prudent science policy position.  The
Workgroup further recommends that the 2,3,7,8-TCDD risk-specific dose issue be
examined again regularly as new information becomes available.  It is felt that
the rate of research on this chemical is so great and the fundamental questions
relating to multistage carcinogenicity and risk are so important to Agency
decision-makers that a regular re-evaluation of the science and/or science
policy underlying the selection of an RsD is appropriate.
                                      51

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Sloop, T.C.; Lucier,  G.W.  (1987)  Dose-dependent elevation of  Ah receptor
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                                      56

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                                      57

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