EPA
United States
Environmental Protection
Agency
Office of Health and
Environmental Assessment
Washington DC 20460
EPA/600/8-84/014F
September 1985
Final Report
Research and Development
_ _ ^^ * *3lt«5jv«> --->-.*- • —H •'- lass^
Health Assessment
Document for
Polychlorinated
Dibenzo-p-Dioxins
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EPA/600/8-84/014F
September 1985
Final Report
Health Assessment Document
for
Polychlorinated
Dibenzo-p-Dioxins
U.S. ENVIRONMENTAL PROTECTION AGENCY
Office of Research and Development
Office of Health and Environmental Assessment
Environmental Criteria and Assessment Office
Cincinnati, Ohio 45268
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NOTICE
This document has been reviewed In accordance with U.S.
Environmental Protection Agency policy and approved for
publication. Mention of trade names or commercial products
does not constitute endorsment or recommendation for use.
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PREFACE
The Office of Health and Environmental Assessment has prepared this Health
Assessment Document on polychlorlnated d1benzo-p_-d1ox1ns at the request of the
Office of A1r Quality Planning and Standards.
In the development of this assessment document, the scientific literature
has been Inventoried, key studies have been evaluated, and summary and conclu-
sions have been prepared such that the toxldty of polychlorlnated d1benzo-p_-
dloxlns 1s qualitatively and where possible, quantitatively, Identified.
Observed effect levels and dose-response relationships are discussed where
appropriate 1n order to Identify the critical effect and to place adverse
health responses 1n perspective with observed environmental levels.
This document was reviewed by a panel of expert scientists during the peer
review workshop held at the Cincinnati Convention/Exposition Center, Cincin-
nati, OH, on July 27, 28 and 29, 1983. The Environmental Health Committee and
the Environmental Effects, Fate and Transport Committee of the U.S. EPA's
Science Advisory Board Independentally reviewed the document 1n a public
session.
111
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AUTHORS, CONTRIBUTORS AND REVIEWERS
The EPA Office of Health and Environmental Assessment (QHEA) was
responsible for the preparation of this draft health assessment document. The
OHEA Environmental Criteria and Assessment Office (ECAQ-C1ndnnat1) had
overall responsibility for coordination and direction of the document
preparation and production effort (Dr. Debdas Mukerjee, Project Manager, Dr.
Jerry F. Stara, Director, ECAO-C1nc1nnat1). The following Individuals
contributed substantially to portions of various chapters of the document and
their assistance Is greatly appreciated.
Dr. D1pak K. Basu
Life and Environmental
Sciences Division
Syracuse Research Corporation
Syracuse, NY
Dr. Debdas Hukerjee
Environmental Criteria and
Assessment Office
U.S. Environmental Protection Agency
Cincinnati, OH
Dr. Hlchael W. Neal
Life and Environmental
Sciences Division
Syracuse Research Corporation
Syracuse, NY
Dr. James R. Olson
Department of Pharmacology
and Therapeutics
School of Medicine
State University of New York
at Buffalo
Buffalo, NY
Dr. Shane Que Hee
Ketterlng Laboratories
Department of Environmental Health
University of Cincinnati Medical School
Cincinnati, OH
Dr. Stephen H. Safe
Department of Physiology and
Pharmacology
College of Veterinary Medicine
Texas A&M University
College Station, TX
Dr. Marvin A. Schnelderman
Bethesda, MD
The OHEA Carcinogen Assessment Group (CA6) was responsible for prepara-
tion of the sections on carc1nogen1c1ty. Participating members of CA6 are
listed below and the authors for the Carc1nogen1c1ty Chapter are Indicated by
an asterisk.
Roy E. Albert, M.D. (Chairman)
Elizabeth L. Anderson, Ph.D.
Larry D. Anderson, Ph.D.
Steven Bayard, Ph.D.*
David L. Bayllss, M.S.*
Chao W. Chen, Ph.D.
Herman J. 61bb, B.S., H.P.H.
Bernard H. Haberman, D.V.M., M.S.
Charallngayya B. Hlremath, Ph.D.*
James W. Holder, Ph.D.
Robert E. McGaughy, Ph.D.
Jean C. Parker, Ph.D.
Charles H. R1s, M.S., P.E.
Dharm V. Singh, D.V.M., Ph.D.
Todd W. Thorslund, Sc.D.
1v
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me roi lowing individuals were asKea to review tms aocument ana earner
drafts of this document:
Dr. Bernard H. Haberman U.S. EPA Carcinogen Assessment Group
Dr. Franklin L. M1nk U.S. EPA ECAO-C1nc1nnat1
Dr. Charles H. Nauman U.S. EPA Exposure Assessment Group
Dr. Sheila L. Rosenthal U.S. EPA Reproductive Effects Assessment
Group
Dr. Mil11am E. Pepelko U.S. EPA ECAO-C1nc1nnat1
W. Bruce Pelrano U.S. EPA ECAO-C1ndnnat1
David J. Relsman U.S. EPA ECAO-C1nc1nnat1
John L. Schaum U.S. EPA Exposure Assessment Group
The following members of the ECAO-C1nc1nnat1 Technical Services Staff
were responsible for document production:
Patricia A. Daunt Judith A. Olsen
Erma R. Durden Bette L. Zwayer
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POLYCHLORINATED DIBENZO-p_-DIOXINS PEER REVIEW PANEL MEMBERS
July 27, 28 and 29, 1983 Cincinnati, Ohio
Co-Chairmen:
Dr. Debdas Nukerjee, ECAO-C1nc1nnat1
Dr. Jerry F. Stara, ECAO-C1nc1nnat1
MEMBERS
Dr. Roy Albert
Institute of Environmental Medicine
New York University Medical Center
Dr. Donald G. Barnes
Office of Pesticides and Toxic
Substances
U.S. Environmental Protection Agency
Dr. K. Diane Courtney
Health Effects Research Laboratory
Research Triangle Park
U.S. Environmental Protection Agency
Dr. Frederick Coulston
White Sands Research Center
Dr. David Firestone
Food and Drug Administration
Dr. S. Garatt1n1
Institute d1 Recerche
Farmacologlc "Mario Negrl"
Milan, Italy
Dr. Dolores Graham
Health Effects Research Laboratory
Research Triangle Park
U.S. Environmental Protection Agency
Dr. Richard Grlesemer
Biology Division
Oak Ridge National Laboratory
Dr. Lennart Harden
University Hospital
Umea, Sweden
Dr. Robert Harless
Environmental Monitoring Systems
Laboratory, Research Triangle Park
U.S. Environmental Protection Agency
Dr. Rolf Hartung
University of Michigan
Dr. AUstalr W.M. Hay
University of Leeds
Leeds, United Kingdom
Dr. Otto Hutzlnger
University of Amsterdam
Amsterdam, The Netherlands
Dr. R.D. Klmbrough
Centers for Disease Control
Dr. Richard 3. Koclba
Dow Chemical Company
Dr. Frederick Kopfler
Health Effects Research Laboratory
Cincinnati
U.S. Environmental Protection Agency
Dr. Marvin Legator
University of Texas Medical Branch
Dr. Ruth Lllls
Mt. Sinai School of Medicine
Dr. Prab D. Lotllkar
Temple University School of Medicine
Dr. Fumlo Matsumura
Michigan State University
Dr. E. McConnell
National Institute of Environmental
Health Sciences
Dr. W.P. McNulty
Oregon Regional Primate Research
Center
v1
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Dr. Robert MUler
National Cancer Institute
Dr. James R. Olson
State University of New York
at Buffalo
Dr. Francesco Pocch1ar1
Institute SupeMore d1 Sanlta
Rome, Italy
Dr. Shane Que Hee
University of Cincinnati Medical
Center
Dr. Chrlstoffer Rappe
University of Umea, Sweden
Dr. Stephen H. Safe
Texas A&M University
Dr. Marvin Schnelderman
Environmental Law Institute
Larry SUbart
National Wildlife Federation
Dr. Ellen Sllbergeld
Environmental Defense Fund
Dr. David Stalling
Columbia National Fisheries Research
Laboratory
Dr. Lewis Thlbodeaux
University of Arkansas
Dr. Thomas Tlernan
Wright State University
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SCIENCE ADVISORY BOARD
Environmental Health Committee
Chairmen: Dr. Herschel E. Griffin, San Diego State University, Sart Diego, CA
MEMBERS
Dr. Seymour Abrahamson
University of Wisconsin
Madison, WI
Dr. Morton Corn
The John Hopkins University
Baltimore,_HD
Dr. Ronald D. Hood
University of Alabama
Tuscaloosa, AL
Dr. John Doull
University of Kansas Medical Center
Kansas City, KS
Dr. Marvin Kuschner
State University of New York
Stony Brook, NY
Dr. D. Warner North
Decision Focus Inc.
Los Altos, CA
Dr. Bernard Weiss
University of Rochester
Rochester, NY
Dr. Ronald Wyzga
Electric Power Institute
Palo Alto, CA
COUNSULTANT
Dr. Stephen H.Safe
Texas A&M University
College Station, TK
Dr. William Lowrance
Rockefeller University
New York, NY
Environmental Effects, Transport and Fate Committee, D1ox1n Subcommittee
Dr. Robert Huggett
Institute of Marine Science
College of William and Mary
Gloucester Point, VA
Dr. John Laseter
Envlro-Health Systems, Inc.
New Orleans, LA
V111
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TABLE OF CONTENTS
Page
1. INTRODUCTION 1-1
2. SUMMARY AND CONCLUSIONS 2-1
2.1. SUMMARY 2-1
2.2. CONCLUSIONS ..... ..... 2-7
2.3. NEEDS FOR FUTURE RESEARCH 2-8
3. PHYSICAL AND CHEMICAL PROPERTIES/ANALYTICAL METHODOLOGY 3-1
3.1. INTRODUCTION 3-1
3.2. PHYSICAL AND CHEMICAL PROPERTIES 3-1
3.2.1. Chemical Formula and Synonyms . . 3-1
3.2.2. Physical Properties 3-3
3.2.3. Chemical Properties . 3-5
3.3. ANALYTICAL METHODOLOGY 3-5
3.3.1. General Procedure for the Analysis of PCDDs .... 3-7
3.3.2. Analysis of PCDDs 1n Specific Environmental Media . 3-20
3.3.3. B1oanalys1s of PCDDs 3-30
3.3.4. Critique of Sampling and Chemical Analysis 3-30
3.4. SUMMARY 3-34
4. PRODUCTION, USE, SYNTHESIS, ENVIRONMENTAL SOURCES AND
ENVIRONMENTAL LEVELS 4-1
4.1. PRODUCTION AND USE 4-1
4.2. SYNTHESIS 4-1
4.2.1. Reaction of Dlchlorocatechol Salts with
1,2,4,5-Tetrachlorobenzenes 1n DMSO . 4-1
4.2.2. Substitution Reaction 4-2
4.2.3. Photoproductlon 4-2
4.2.4. Ullmann Condensation Reactions 4-2
4.2.5. Pyrolysls of Chlorophenates 4-4
4.2.6. Conversion Through Nitration 4-4
4.3. ENVIRONMENTAL SOURCES ..... ...... 4-5
4.3.1. Manufacturing Processes 4-5
4.3.2. Municipal Incinerators 4-14
4.3.3. Other Combustion Processes 4-15
4.3.4. Chemical Dump Sites 4-17
4.3.5. Photochemical Process . . 4-17
4.4. RELATIONSHIP BETWEEN SOURCES AND CONTAMINATION IN
ENVIRONMENTAL MATRICES 4-18
1x
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TABLE OF CONTENTS (cent.)
Page
4.5. ENVIRONMENTAL LEVELS 4-18
4.5.1. Water 4-20
4.5.2. A1r 4-21
4.5.3. Soil 4-25
4.5.4. Foods and Biological Samples. ........... 4-28
4.6. EXPOSURE 4-32
4.7. SUMMARY 4-38
5. ENVIRONMENTAL FATE AND TRANSPORT PROCESSES 5-1
5.1. FATE 5-1
5.1.1. Water 5-1
5.1.2. A1r 5-6
5.1.3. Soil 5-7
5.1.4. Food 5-11
5.2. TRANSPORT 5-12
5.2.1. Water 5-12
5.2.2. A1r 5-13
5.2.3. Soil 5-14
5.3. BIOACCUMULATION/BIOCONCENTRATION 5-15
5.4. SUMMARY 5-18
6. ECOLOGICAL EFFECTS 6-1
6.1. EFFECTS ON ORGANISMS 6-1
6.1.1. Aquatic Life Toxicology 6-1
6.2. TISSUE RESIDUES 6-8
6.3. ECOSYSTEM EFFECTS 6-14
6.4. SUMMARY 6-19
7. COMPOUND DISPOSITION AND RELEVANT PHARMACQKINETICS. 7-1
7.1. ABSORPTION. 7-1
7.1.1. Absorption from the Gastrointestinal Tract 7-1
7.1.2. Absorption Through the Skin 7-6
7.2. DISTRIBUTION 7-7
7.3. METABOLISM 7-12
7.4. ELIMINATION 7-16
7.5. SUMMARY 7-20
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TABLE OF CONTENTS (cont.)
Page
8. TOXICOLOGY: ACUTE, SUBCHRONIC AND CHRONIC 8-1
8.1. EXPERIMENTAL ANIMALS 8-1
8.1.1. Acute 8-1
8.1.2. Subchronlc. ... 8-44
8.1.3. Chronic 8-50
8.2. HUMAN . . 8-60
8.2.1. Acute Exposure 8-60
8.2.2. Chronic Studies . 8-65
8.3. MECHANISM OF TOXICITY 8-69
8.3.1. Receptor-Mediated Tox1c1ty 8-70
8.3.2. Metabolism 8-78
8.3.3. Vitamin A Depletion 8-80
8.3.4. L1p1d Perox1dat1on 8-81
8.3.5. Endocrine Imbalance 8-82
8.4. SUMMARY . 8-84
8.4.1. Experimental Animal Data 8-84
8.4.2. Human Data 8-88
8.4.3. Mechanisms of Tox1c1ty 8-88
9. TERATOGENICITY AND OTHER REPRODUCTIVE EFFECTS 9-1
9.1. STUDIES ON EXPERIMENTAL MAMMALS ....... 9-1
9.1.1. 2,3,7,8-TCDD Administered as a Contaminant of
Other Chemicals 9-1
9.1.2. 2,3,7,8-TCDD Studies In Mice 9-6
9.1.3. 2,3,7,8-TCDD Studies 1n Rats 9-13
9.1.4. 2,3,7,8-TCDD Studies 1n Rabbits and Ferrets .... 9-18
9.1.5. 2,3,7,8-TCDD Studies 1n Nonhuman Primates 9-20
9.1.6. Studies 1n Chickens 9-22
9.1.7. Studies of the Teratogenlc and Reproductive
Effects of HxCDD 9-23
9.2. STUDIES ON HUMAN POPULATIONS 9-23
9.3. OTHER REPRODUCTIVE EFFECTS 9-34
9.1. SUMMARY 9-35
x1
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TABLE OF CONTENTS (cont.)
Page
10. MUTAGENICITY AND OTHER INDICATIONS OF GENOTOXICITY 10-1
10.1. RELEVANT STUDIES 10-1
10.1.1. Assays In Microorganisms 10-1
10.1.2. Interactions with Nucleic Adds 10-7
10.1.3. Cytogenetlc Effects of 2,3,7,8-TCDD 10-8
10.2. SUMMARY 10-13
11. CARCINOGENICITY 11-1
11.1. ANIMAL STUDIES 11-1
11.1.1. Studies Using 2,3,7,8-TCDD 11-1
11.1.2. Studies Using HxCDD 11-39
11.1.3. Summary of Animal Carc1nogen1c1ty 11-51
11.2. CASE REPORTS AND EPIDEMIOLOGICAL STUDIES 11-60
11.2.1. Case Reports 11-60
11.2.2. Ep1dem1olog1c Studies 11-64
11.2.3. Summary of Case Reports and Ep1dem1olog1c Studies . 11-108
11.3. QUANTITATIVE ESTIMATION OF RISKS OF EXPOSURE TO
2,3,7,8-TCDD and HxCDDs 11-109
11.3.1. Introduction 11-109
11.3.2. Procedures for the Determination of Incremental
Unit from Animal Data and Description of the
Low-Dose Animal Extrapolation Model 11-110
11.3.3. Selection of Data 11-112
11.3.4. Calculation of Human Equivalent Dosages for
An1mal-to-Man Extrapolation 11-112
11.3.5. Alternative Methodological Approaches 11-113
11.3.6. Interpretation of Quantitative Estimates 11-114
11.3.7. Incremental Unit Risk Estimates for 2,3,7,8-TCDD
via the Oral and Inhalation Routes 11-115
11.3.8. Incremental Unit Risk Estimates for HxCDDs
(1,2,3,6,7,8 and 1,2,3,7,8,9) via the Oral
and Inhalation Routes 11-119
11.3.9. Relative Potency 11-125
11.4. SUMMARY AND CONCLUSIONS 11-131
11.4.1. Summary 11-131
11.4.2. Conclusions 11-133
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TABLE OF CONTENTS (cent.)
Page
12. SYNERGISM AND ANTAGONISH 12-1
12.1. CHEMICAL CARCINOGENS 12-1
12.2. NON-CARCINOGENIC CHEMICALS 12-1
12.3. SUMMARY 12-2
13. REGULATIONS AND STANDARDS . 13-1
13.1. WATER 13-1
13.2. AIR 13-1
13.3. FOOD 13-1
13.4. SUMMARY 13-2
14. EFFECTS OF MAJOR CONCERN AND HEALTH HAZARD ASSESSMENT 14-1
14.1. PRINCIPAL EFFECTS 14-2
14.1.1. Toxldty 14-2
14.1.2. Mutagenlcity 14-7
14.2. SENSITIVE POPULATIONS 14-7
14.3. FACTORS INFLUENCING HEALTH HAZARD ASSESSMENT 14-8
14.4. QUALITATIVE HEALTH HAZARD ASSESSMENT 14-9
14.4.1. Animal ToxIcHy Data 14-10
14.4.2. Animal Carc1nogen1c1ty 14-11
15. REFERENCES. 15-1
APPENDIX A A-l
APPENDIX B B-l
APPENDIX C C-l
APPENDIX D D-l
X111
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LIST OF TABLES
No. Title Page
3-1 Physical Properties of a Few Selected Polychlorlnated
Dloxlns 3-4
3-2 A Few Estimated Physical Parameters of Chlorinated
D1benzo-p_-d1ox1ns 3-6
3-3 Potential Interferences 1n the Determination of TCDOs
at m/e Values of 319.8966 and 321.8936 3-12
3-4 Some Packed and Capillary Columns Used for the Analysis
of PCDDs. 3-14
3-5 The Detection Limit, Resolution and Ions Monitored by a Few
Mass Spectrometrlc Systems for the Determination of TCODs . . 3-17
3-6 Some Published Method Validation Data for 2,3,7,8-TCDD
Recovered from Fortified Matrices and Determined by GC/MS . . 3-31
4-1 Levels of Tetra-, Penta- and Hexa-chlorod1benzo-p_-d1ox1ns
Reported 1n Chlorophenols and a Few Pesticides
Originating from Chlorophenols 4-7
4-2 Locations of Companies that have been Major Producers and
Formulators of Chlorophenols and Their Derivatives. ..... 4-10
4-3 Levels of TCDD 1n Soils and Sediments from Different
Locations 4-26
4-4 Predicted BCFs from Calculated and Measured Values of Kow . . 4-36
4-5 Measured B1oaccumulat1on Factor for 2,3,7,8-TCDD 1n
Freshwater Aquatic Organisms 4-37
5-1 Bloconcentratlon Factor of TCDD for Several Aquatic
Organisms 5-16
6-1 Effect of Acute Exposure to 2,3,7,8-TCDD on Aquatic Animals . 6-2
6-2 Effects of Chronic or Subchronlc Exposure to 2,3,7,8-TCDD
on Aquatic Animals 6-5
6-3 Levels of 2,3,7,8-TCDDs 1n F1sh and Shellfish ... 6-10
6-4 TCDD Levels 1n Wildlife 6-15
7-1 Gastrointestinal Absorption of 2,3,7,8-TCDD 7-2
7-2 Liver Accumulation of 2,3,7,8-TCDD In Guinea P1gs 30 Days
after a Single Intragastrlc Exposure to 2,3,7,8-TCDD 7-4
xlv
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LIST OF TABLES (cont.)
No. Title Page
7-3 Distribution of 2,3,7,8-TCDD 7-8
7-4 Elimination of 2,3,7,8-TCDD 7-17
8-1 Lethal Doses of 2,3,7,8-TCDD Following Acute Exposure .... 8-2
8-2 Toxic Responses Following Exposure to 2,3,7,8-TCDD:
Species Differences 8-11
8-3 Estimated Single Oral LD50 - 30 Values for PCDDs 8-12
8-4 Immunologlcal Effects of 2,3,7,8-TCDD 1n Animals 8-28
8-5 Effects of Chronic Exposure to 2,3,7,8-TCDD on Laboratory
Rodents 8-52
9-1 Studies on the Potential Teratogenlc Effects of 2,3,7,8-TCDD
Contaminated 2,4,5-T 9-2
9-2 Studies on the Potential Teratogenlc Effect of 2,3,7,8-TCDD . 9-7
10-1 The Results of Mutagenldty Assays for 2,3,7,8-TCDD 1n
Salmonella typh1mur1um 10-2
11-1 2,3,7,8-TCDD Intake and Mortality 1n Hale Sprague-Dawley
Rats 11-3
11-2 Benign and Malignant Tumors 1n Rats Ingesting 2,3,7,8-TCDD. . 11-5
11-3 Liver Tumors 1n Rats Ingesting 2,3,7,8-TCDD 11-6
11-4 Hepatocellular Carcinomas and Hepatocellular Hyperplastlc
Nodules 1n Female Sprague-Dawley Rats Maintained on Diets
Containing 2,3,7,8-TCDD 11-9
11-5 Tumor Incidence 1n Female Rats Fed Diets Containing
2,3,7,8-TCDD 11-10
11-6 Tumor Incidence 1n Male Rats Fed Diets Containing
2,3,7,8-TCDD 11-11
11-7 Dow 2,3,7,8-TCDD Oral Rat Study by Dr. Koclba, WHh
Dr. Squire's Review (8/15/80) Sprague-Dawley Female Rats -
Spartan Substraln (2 years) 11-13
11-8 Dow 2,3,7,8-TCDD Oral Rat Study by Dr. Koclba, WHh
Dr. Squire's Review (8/15/80) Sprague-Dawley Male Rats -
Spartan Substraln (2 years) 11-14
xv
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LIST OF TABLES (cont.)
No. Title
11-9 Incidence of Primary Tumors 1n Hale Rats Administered
2,3,7,8-TCDO by Bavage 11-16
11-10 Incidence of Primary Tumors In Female Rats Administered
2,3,7,8-TCDD by Gavage N 11-17
11-11 Cumulative Data on Tumor Incidence , 11-18
11-12 Incidence of Primary Tumors 1n Male Mice Administered
2,3,7,8-TCDD by Gavage 11-22
11-13 Incidence of Primary Tumors 1n Female Mice Administered
2,3,7,8-TCDD by Savage 11-23
11-14 Promoting Effect of 2,3,7,8-TCDD on Hepatocardnogenesls
by a Single Dose of D1ethyln1trosam1ne (DEN) and
Partial Hepatectomy (PH). . 11-24
11-15 Incidence of Primary Tumors 1n Mice Administered
2,3,7,8-TCDD or 2,3,7,8-TCDD Following DMBA by Dermal
Application . 11-28
11-16 Effects of Intraperltoneal Administration of 2,3,7,8-TCDD
on 3-MC-In1t1ated Subcutaneous Tumors 11-31
11-17 Effect of Intraperltoneal or Subcutaneous Administration
of 2,3,7,8-TCDD Given 2 Days Before or Simultaneous With
Subcutaneous Administration of 3-MC on Tumorlgenesls 1n
D2 Mice 11-32
11-18 Incidence of Tumors 1n Mice Treated With 3-MC and WHh
3-MC and 2,3,7,8-TCDD 11-34
11-19 Liver Tumor Incidences 1n Male and Female Osborne-Mendel
Rats Administered HxCDD for 104 Weeks 11-41
11-20 Liver Tumor Incidences 1n Female Osborne-Mendel Rats
Administered HxCDD by Gavage for 104 Weeks 11-43
11-21 Liver Tumor Incidences 1n Male and Female B6C3F1 Mice
Administered HxCDD by Gavage for 104 Weeks 11-44
11-22 Liver Tumor Response for HxCDD (Observed) and TCDD
Contaminant (Calculated). . , 11-46
11-23 Carc1nogen1c1ty Bloassays of 2,3,7,8-TCDD and HxCDD
by Dermal Application to Mice ..... 11-49.
xv1
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LIST OF TABLES (cent.)
No. Title Page
11-24 Carc1nogen1c1ty Bloassays of PCDD Administration by the
Oral and Dermal Route 11-52
11-25 Distribution of Tumor Types 1n Two Case-Controls Studies
of Soft-Tissue Sarcoma * 11-67
11-26 Exposure Frequencies 1n Two Case-Control Studies of
Soft-Tissue Sarcoma 11-68
11-27 Relative Risks of Soft-Tissue Sarcoma 1n Relation to
Exposure to Phenoxyacetlc Adds and Chlorophenols In
Two Case-Control Studies. . . . ........ 11-70
11-28 Distribution of H1stolog1cal Types of Soft-Tissue
Sarcomas. . . 11-75
11-29 Midland County Soft and Connective Tissue Cancer
Deaths 1960-1981 11-84
11-30 Other Occupations (Minus Forestry/Agriculture). ....... 11-95
11-31 Other Occupations (Minus Forestry/Agrlculture/Woodworkers . . 11-96
11-32 Analysis of Stomach Cancer Mortality 1n a Group of West
German Factory Workers Exposed to 2,3,7,8-TCDD. ....... 11-101
11-33 Reanalysls of Stomach Cancer Mortality 1n a Group of
West German Factory Workers Exposed to 2,3,7,8-TCDD ..... 11-103
11-34 Stomach Cancer Mortality 1n Three Studies of Workers
Exposed to Phenoxyacetlc Acid Herbicides and/or
2,3,7,8-TCDD 11-105
11-35 NTP HxCDD (Gavage) Bloassay. Osborne-Mendel Rats
(2 years) Incidences of Neoplastlc Nodules and Hepato-
cellular Carcinomas . 11-120
11-36 NTP HxCDD (Gavage) Bloassay. B6C3F1 Mice (104 weeks)
Incidences of Adenomas Nodules and Hepatocellular
Carcinomas 11-123
11-37 Relative Carcinogenic Potencies Among 55 Chemicals
Evaluated by the Carcinogen Assessment Group as Suspect
Human Carcinogens . 11-128
14-1 No-Observed-Effect Levels and Low-Observed-Effect Levels
Obtained from Subchronlc and Chronic Oral Tox1c1ty Studies
of 2,3,7,8-TCDD 14-3
XV11
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LIST OF TABLES (cont.)
No. Title Page
14-2 No-Observed-Effect Levels and Low-Observed-Effect Levels
Obtained from Subchronlc and Chronic Oral Toxldty
Studies of HxCDD 14-5
14-3 Carc1nogen1c1ty Bloassays of 2,3,7,8-TCDD 14-12
14-4 Carc1nogen1c1ty Bloassays of a 1:2 Mixture of 1,2,3,6,7,8-
and 1,2,3,7,8,9-HxCDD 14-16
XV111
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LIST OF FIGURES
No. Title Page
4-1 unmann Condensation Reactions 4-3
4-2 Possible Potential Relationship Between Various Sources
of PCDDs and the Environmental Matrices Where PCDDs have
been Detected 4-19
11-1 Time-Dependent Inhibition by 2,3,7,8-TCDD of Tumor
Initiation. 11-37
11-2 Histogram Representing the Frequency Distribution of the
Potency Indices of 55 Suspect Carcinogens Evaluated by
the Carcinogen Assessment Group 11-127
x1x
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LIST OF ABBREVIATIONS
ADI
AHH
bw
BCF
BromoPeCOD
DCDD
DHSO
DMA
EC/GC
ED50
PEL
GC/MS
SC/SIM/MS
HPLC
HRGC
HRHS
HxCDDs
LC50
LD50
LOAEL
LRHS
HFO
NICI
NOAEL
NOEL
Acceptable dally Intake
Aryl hydroxycarbon hydroxylase
Body weight
B1oeoncentrat1on factor
Bromopentachlorod1benzo-£-d1ox1n
D1chlorod1benzo~£-d1ox1n
Dlmethylsulfoxlde
Deoxyr1bonucle1c add
Electron capture/gas chromatography
Median effective dose
Frank effect level
Gas chromatography/mass spectrometry
Gas chromatography/spec1f1c 1on monitoring/mass spectrom-
etry
High performance liquid chromatography
High resolution gas chromatography
High resolution mass spectrometry
Hexachloro derivatives of d1ben2Q-j»-d1ox1ns
Concentration lethal to 50% of recipients
Dose lethal to 50% of recipients
Lowest-observed-adverse-effect level
Low resolution mass spectrometry
Mixed function oxldase
Negative Ion chemical 1on1zat1on
No-observed-adverse-effect level
No-observed-effect level
xx
-------
LIST OF ABBREVIATIONS (cont.)
OCDD
PCDDs
PCP
PeCDDs
ppb
ppm
ppt
RBC
RNA
SA
TCODs
TMCDD
2.4,5-T
TWA
UV
WCOT
OctachloMnated d1benzo-£-d1ox1ns
All polychlorlnated d1benzo-£-d1ox1ns
Pentachlorophenol
Pentachloro derivatives of d1benzo-p_-d1ox1ns
Parts per billion
Parts per million
Parts per trillion
Red blood cells
Rlbonuclelc acid
Satellite association
Tetrachloro derivatives of d1benzo-|)-d1ox1ns
Tr1chlorod1benzo-£-d1ox1n
2,4,5-Tr1chlorophenoxyacet1c ac1d
Time-weighted average
Ultraviolet
Wall-coated open tubular
xxl
-------
1. INTRODUCTION
Dloxlns are a class of compounds that contain the d1benzo-p_-d1ox1n
nucleus. In chlorinated dloxlns, the d1benzo-£-d1ox1n nucleus Is substi-
tuted with chlorine at different positions of the fused benzene rings.
Depending on the number and position of chlorine substitution, 75 congeners
are possible for the chlorinated dloxlns. This document deals with the most
toxic chlorinated dloxlns, namely, 2,3,7,8-tetrachloro-, 1,2,3,7,8-penta-
chloro-, 1,2,3,6,7,8-hexachloro- and l,2,3,7,8,9-hexachlorod1benzo-£-d1ox1n.
Of these four congeners, the 2,3,7,8-tetrachlorod1benzo-j)-d1ox1n has been
studied extensively and Is often described In both popular and technical
literature as "TCDD" or simply "dloxln."
A few documents exists at the present time that deal with selected
aspects of polychlorlnated d1benzo-p_-d1ox1ns 1n the environmental media.
This document, however, has been prepared to provide a comprehensive multi-
media assessment of the analytical methodologies, environmental levels and
ecological and health effects of the four chlorinated dloxlns. The follow-
ing acronyms will hereafter be used when^dlscusslng the polychlorlnated
d1benzo-£-d1ox1ns:
PCDDs Polychlorlnated d1benzo-p_-d1ox1ns
2,3,7,8-TCDD 2,3,7,8-Tetrachlorod1benzo-£-d1ox1n
1,2,3,7,8-PeCDD 1,2,3,7,8-Pentachlorod1benzo-j>-d1ox1n
1,2,3,6,7,8-HxCDD 1,2,3,6,7,8-Hexachlorod1benzo-p_-d1ox1n
1,2,3,7,8,9-HxCDD 1,2,3,7,8,9-Hexachlorod1benzo-p_-d1ox1n
1-1
-------
2. SUMMARY AND CONCLUSIONS
2.1. SUHMARY
Polychlorlnated d1benzo-jj-d1ox1ns are a class of chlorinated trlcycllc
aromatic hydrocarbons consisting of two benzene rings connected by a pair of
oxygen atoms. According to the position and number of chlorine atoms 1t 1s
possible to form 75 different congeners of chlorinated dloxlns. The word
"dloxlns" 1s often used to refer to this class of compounds, especially with
respect to the highly toxic and environmentally widely distributed 2,3,7,8-
tetraehlorod1benzo-fi-d1ox1n (TCDD). This class of compounds 1s rather
stable toward heat, adds and alkalis. The solubility of 2,3,7,8-TCDD 1n
water 1s 0.2 vg/i. This Isomer and the three other PCDDs discussed 1n
this document are soluble 1n certain aromatic and aliphatic solvents. The
PCDDs are chemically relatively stable and start to decompose at tempera-
tures >500°C; the percent of decomposition depends upon the residence time
1n the high temperature zone and the proportion of oxygen 1n the heated zone.
The commonly used method for the determination of these compounds 1n
different samples consists of solvent extraction, followed by sulfurlc add
and base washes to remove I1p1ds and other Impurities from the solvent
extract. The extract 1s then subjected to two liquid chromatographlc
clean-up procedures. The cleaned-up extract 1s finally analyzed for the
PCDDs by the gas chromatographlc-mass spectrometrlc methods. Despite the
specialized methods used for the determination of PCDDs, the results of
analysis at very low levels (possibly <9 ppt 1n biological matrices) can be
questionable unless special precautions, Including addition of Internal
standard, are made.
None of the PCDDs are either commercially manufactured or have any known
use. They are produced as unwanted contaminants primarily during the
2-1
-------
manufacture of chlorophenols and their derivatives. The primary sources of
PCDD contamination 1n the environment result from the Industrial manufacture
of chlorophenols and their derivatives and the subsequent disposal of wastes
from these Industries. Hun1c1pal Incineration may also produce some envi-
ronmental emission of PCDDs. From the available data, 1t 1s difficult to
ascertain the comparative Importance of these three sources 1n contributing
to environmental emissions. The 1,2,3,7,8-PeCDD found 1n environmental
samples has only been reported 1n emissions from Incinerators.
The monitoring data to date Indicate that the maximum level of PCDDs Is
likely to be found In soil and drainage sediment samples near chlorophenol
manufacturing Industries and chemical waste disposal sites. With the excep-
tion of air near certain contaminated sites, only very limited attempts have
been made to determine the level of PCDDs 1n air samples. In the United
States, the highest levels are reported at certain hazardous waste sites and
1n fish and wildlife tissue from areas contaminated with 2,3,7,8-TCDD.
The environmental fates of the four PCDDs are not known with certainty.
Host of the Investigations 1n this field have been conducted with 2,3,7,8-
TCDD, and the conclusions regarding the environmental fate of the other
three PCDDs have been drawn by analogy. Few data exist In the literature
that would Indicate significant chemical and biological transformation of
these compounds 1n atmospheric, aquatic or soil media. The role of photo-
chemical transformation 1n determining the fates of these chemicals 1n var-
ious ambient media Is not known with certainty, but the PCDDs are suscepti-
ble to photochemical reactions 1n the presence of hydrogen donors. In the
aquatic media, a substantial proportion of the PCDDs may be present In the
sedlment-sorbed state or 1n the biota. In the atmosphere, the PCDDs are
expected to be present 1n the vapor-phase and partlculate-sorbed states.
2-2
-------
The atmospheric transport of these compounds can be predicted from disper-
sion modeling equations. In the case of the accidental release of 2,3,7,8-
TCDD at Seveso, Italy, 1t has been estimated from laboratory experiments
that 2,3,7,8-TCDD deposition from air to soil follows an exponential decay
pattern along the downward wind direction. The most probable transport
mechanisms of the PCDDs from soils are transport to the atmosphere by con-
taminated dust particles, direct volatilization from the surface or near
surface zones (<5 cm), and transport to surface water by eroded soil.
Both the calculated and the experimental results show that the PCDDs
will concentrate 1n sediments and biota present 1n aquatic media. It has
been shown by static test procedures that, depending on the species, the
bloconcentratlon factor (BCF) for 2,3,7,8-TCDD 1n fish ranges from
-2000-30,000. The U.S. EPA's best estimate of the BCF for 2,3,7,8-TCDD 1s
5000 (U.S. EPA, 1984).
In mammals, 2,3,7,8-TCDD 1s readily absorbed through the gastrointesti-
nal tract, and absorption through Intact skin has also been reported.
Absorption may decrease dramatically 1f 2,3,7,8-TCDD 1s adsorbed to partlcu-
late matter such as activated carbon or soil. After absorption, 2,3,7,8-
TCDD 1s distributed to tissues high 1n I1p1d content; however, 1n many
species, the liver 1s a major storage site. Metabolism of 2,3,7,8-TCDD
occurs slowly, with the polar metabolites excreted 1n the urine and feces.
Unmetabollzed 2,3,7,8-TCDD can be eliminated 1n the feces and 1n the milk.
It 1s metabolized by the P-450 monooxygenase system through a reactive
epoxlde Intermediate. The metabolism of 2,3,7,8-TCDD seems to be a detoxi-
fication process resulting 1n the production of metabolites that are less
toxic than the parent compound. Available scientific data supports the
contention that the toxic response to 2,3,7,8-TCDD exposure 1s mediation
through cytosollc Ah-receptor site binding.
2-3
-------
The PCDDs discussed 1n this document are among some of the most toxic
compounds known, with the lowest LD5« level for male guinea pigs, the most
sensitive species, being 0.6 vg/kg for 2,3,7,8-TCDD. The other congeners
are somewhat less toxic; however, the LD5Q values are stm 1n the jig/kg
range. Although 2,3,7,8-TCDD 1s highly toxic 1n all species tested, there
are large species differences 1n sensitivity, with the LDgo for hamsters
being 1157-5051 vg/kg. The characteristic signs and symptoms of lethal
poisoning are severe weight loss and thymlc atrophy. Death usually occurs
many days after the exposure. In rats, rabbits and mice, 2,3,7,8-TCDD pro-
duces an acute Hver Injury that 1s not observed 1n either monkeys, hamsters
or guinea pigs. In mice, the Immune response 1s also suppressed. After
subchronlc or chronic exposure to 2,3,7,8-TCDD In rats or mice, the liver
appears to be the most severely affected organ, although systemic hemor-
rhage, edema and suppressed thymlc activity are also observed. The limited
data available for the other PCDDs Indicate that these chemicals produce the
same symptoms as 2,3,7,8-TCDD In a given species; however, the doses
required are higher.
Humans have been exposed to herbicides and other chlorinated chemicals
containing 2,3,7,8-TCDD as a contaminant. The symptoms of toxldty In many
cases are similar to those observed 1n animals, with exposure leading to
altered liver function and llpld metabolism, porphyMa cutanea tarda, neuro-
toxlclty and pathologic changes 1n hematologlc parameters. In addition,
exposure of humans to 2,3,7,8-TCDD produces skin lesions such as chloracne
and hyperplgmentatlon. Although some signs such as chloracne are attributed
to the PCDDs, the other signs of toxldty may arise, at least 1n part, from
the other chemical of which PCDDs are a minor contaminant.
Animal studies have demonstrated that 2,3,7,8-TCDD 1s teratogenlc and
fetotoxlc 1n rats, mice, rabbits and ferrets; and fetotoxlc 1n monkeys.
2-4
-------
exposure 10 t,j,i,o-\^uu in mice pruuuces rat ta i €ieib:>, wuiie
rats results 1n edema, hemorrhage and kidney anomalies; rabbits have a
higher Incidence of extra ribs. In rats a reduction In the gestation Index,
decreased fetal weight, Increased I1ver-to-body weight ratio and Increased
Incidence of dilated renal pelvis 1n the offspring has been observed.
Certain human epidemiology studies have shown positive associations with
exposure to chemicals contaminated with 2,3,7,8-TCDD and birth defects and
abortions, while others have not.
There 1s a limited data base with conflicting evidence for 2,3,7,8-
TCDD's mutagenlc potential; therefore, the available evidence 1s Judged to
be Inconclusive. There are no studies 1n the published literature regarding
the mutagenldty of HxCDD or any other congeners of PCDD.
There 1s evidence from chronic animal cancer bloassay studies that
2,3,7,8-TCDD and HxCDO are probable human carcinogens. There are no chronic
cancer bloassay studies available that evaluate the carcinogenic potential
for other PCDDs. The available data for 2,3,7,8-TCOD and HxCDD come from
gavage and feeding studies, there being no studies available for Inhalation
exposure. The epidemlologic evidence for the carclnogenlclty of 2,3,7,8-
TCDD alone 1s Inadequate, while the evidence for phenoxyacetlc herbicides
and/or chlorophenols with 2,3,7,8-TCDD as an Impurity 1s limited. There
have been no epidemlologic evaluations, as yet, for HxCDD as the sole
compound of concern.
A number of chronic animal cancer bloassays show that 2,3,7,8-TCDD 1s an
animal carcinogen. In rats, oral exposure to 2,3,7,8-TCDD resulted 1n an
Increased Incidence of hepatocellular carcinomas, squamous cell carcinomas
of the tongue and hard palate/nasal turblnates, and squamous cell carcinomas
of the lung. In both male and female mice, Increased Incidences of liver
2-5
-------
lumors were ooservea. A mixture or tne two isomers or HXUUU, discussed in
this document has been tested for cardnogenldty and shows Increased Inci-
dences of liver tumors 1n rats and mice. Also, 2,3,7,8-TCDD has produced
flbrosarcomas at the site of application after dermal administration,
although there was no significant Increase In dermal tumors when the mixture
of HxCDDs was tested. Since both compounds produce statistically signifi-
cant Increased Incidences of tumors 1n two species of animals, there 1s
sufficient evidence, according to the Interim EPA we1ght-of-ev1dence classi-
fication criteria, to conclude that both 2,3,7,8-TCDD and HxCDD are animal
carcinogens. 2,3,7,8-TCDD has been shown to be a promoter as well as an
Initiator 1n rodent test systems. Evidence 1s available from epldemlologlc
studies that Implicate exposure to herbicides contaminated with 2,3,7,8-TCDD
with a significantly elevated risk of soft tissue sarcomas and to a lesser
extent non-Hodgk1ns lymphomas; however, the exposures to 2,3,7,8-TCDD were
always compounded with exposures to the herbicide chemicals.
Assuming that 2,3,7,8-TCDD and HxCDD are carcinogenic In humans, upper
bound Incremental unit cancer risks have been estimated for both 1ngest1on
and Inhalation exposure. The unit risks have been estimated using a multi-
stage extrapolation model that 1s linear at low doses. Available metabo-
lism and pharmacoklnetlc data are Insufficient to alter typically used
assumptions for estimating the human equivalent dose. Since Incidence data
exist only for oral studies 1n animal test systems, the Inhalation risk
estimates are based upon the cancer potency derived from the oral studies
along with appropriate conversion assumptions.
Using data from a feeding study with female rats the upper limit
Incremental cancer risk for 2,3,7,8-TCDD 1s estimated to be 1.56x10"* per
ng/kg/day. The upper limit estimate of Incremental cancer risk Is
2-6
-------
4.5xlO~3 for a continuous lifetime exposure to 1 ng/l of 2,3,7,8-TCQQ In
drinking water and 3.3xlO~s for a continuous lifetime exposure to 1
pg/m3 of 2,3,7,8-TCDD In ambient air.
Using data from an Ingestlon study with female rats and male mice, the
cancer potency for HxCDD 1s estimated to be 6.2xlQ~3 per ng/kg/day. The
upper limit estimate of Incremental cancer risk 1s 1.8xlO~4 for a contin-
uous lifetime exposure to 1 ng/l of HxCDD 1n drinking water and 1.3xlQ~6
for a continuous lifetime exposure to 1 pg/m3 of HxCDD 1n ambient air.
2.2. CONCLUSIONS
The PCDDs, 2,3,7,8-TCDD, 1,2,3,7,8-PeCDD, 1,2,3,6,7,8- and 1,2,3,7,8,9-
HxCDD, are highly toxic following acute exposure. All animal species
administered high levels of these compounds developed weight loss and thymlc
atrophy. In some species liver damage, edema, hair loss and Immunosuppres-
slon were also observed. Chronic toxldty studies have been conducted only
on 2,3,7,8-TCDD and a mixture of the two Isomers of HxCDD. In these
studies, the primary nonneoplastlc lesion was fatty and necrotlc change 1n
the liver.
In the species studied, the fetus has been shown to be highly sensitive
to the toxic effects of 2,3,7,8-TCDD. In rats the fetotoxlclty observed
Included hemorrhage, edema and kidney anomalies, while 1n mice the predomi-
nant lesions were cleft palate and kidney anomalies. The lowest reported
exposure 1n rats, 1 ng/kg, produced a significant (by some analyses but not
others) effect on the fetus, and was similar to the LOAEL observed 1n
chronic studies.
Evidence from oral animal cancer bloassays 1s "sufficient" (according to
EPA and IARC criteria) to conclude that 2,3,7,8-TCDD and a mixture of the
two Isomers of HxCDD are animal carcinogens. 2,3,7,8-TCDD has Increased the
2-7
-------
Incidence of a variety of tumors, Including hepatocellular tumors 1n rats
and mice, while the mixture of HxCDD tested Increased the Incidence of
hepatocellular tumors 1n both sexes of rats and mice. The available
ep1dem1olog1c evidence for the cardnogenlcity of 2,3,7,8-TCOD alone Is
Inadequate and there have been no ep1dem1olog1c evaluations, as yet, for
HxCDD as the sole compound of concern. Considering the animal evidence
together with the ep1dem1olog1c data, the overall we1ght-of-ev1dence classi-
fication for 2,3,7,8-TCDD using EPA's Interim classification scheme 1s cate-
gory B2 meaning that 2,3,7,8-TCDD should be regarded as a "probable" human
carcinogen. The overall we1ght-of-ev1dence classification for HxCDD 1s also
category B2 meaning that 1t should be regarded as a "probable" human car-
cinogen. In terms of low dose potency, 2,3,7,8-TCDD and the HxCDD mixture
are the two most potent carcinogens evaluated by the EPA's Carcinogen
Assessment Group. Ep1dem1olog1c studies of workers exposed to chemicals
contaminated with 2,3,7,8-TCDD such as 2,4,5-trlchlorophenoxyacetlc add and
2,4,5-trlchlorophenol have produced positive findings that are suggestive of
an elevated risk of cancer 1n humans. These ep1dem1olog1c findings are not
Inconsistent with the premise that 2,3,7,8-TCDD Is probably carcinogenic for
humans. There are no chronic studies available regarding the carclnogenlc-
1ty of 1,2,3,7,8-PeCDD.
2.3. NEEDS FOR FUTURE RESEARCH
The basic physical properties such as water solubilities and
vapor pressures of the PeCDDs and HxCDDs need to be deter-
mined. These parameters are Important In predicting the envi-
ronmental fate of these compounds.
New analytical methodologies must be established to determine
the low levels of these compounds 1n environmental matrices
without ambiguity.
More monitoring data, particularly 1n air and aquatic media as
well as 1n vegetables grown near urban Incinerators, should be
developed by a diversity of research groups.
2-8
-------
Isotoptcally labeled Internal standard compounds (37C1 or
13C) should be prepared for PeCDDs and HxCDDs.
More research efforts should be directed to determining the
environmental fate of the PeCDDs and HxCDDs. The determina-
tion of the fate of these chemicals with respect to the possi-
bility of photochemical transformations 1n different environ-
mental matrices needs special attention.
Pharmacoklnetlc studies should be conducted to demonstrate
more clearly the degree of absorption of the PCDDs by all
routes. In particular, studies are needed on respiratory
absorption and on PCDDs adsorbed to environmental media.
Although a number of studies demonstrate that 2,3,7,8-TCDD 1s
a teratogen, the other congeners should be tested for terato-
genlc potential.
There 1s no Information on the effects of chronic exposure to
1,2,3,7,8-PeCDD, and studies should be conducted to determine
both the toxic effects of this compound and Its carcinogenic
potential.
Further epidemiology data on the effects 1n human populations
exposed to PCDDs might assist 1n determining which effects
observed 1n animals are also present 1n humans. In these
studies, careful quantHatlon of PCDD levels 1n humans and
Industrial hygiene samples might provide dose-response data
necessary for health assessment.
B1oava1lab111ty studies from contaminated soil, fly ash, etc.,
are needed.
Hechan1sm-of-act1on studies should be conducted to determine
the fundamental mode of action of the PCDDs.
New destruction methods should be Investigated 1n order to
provide feasible methods for decontaminating environmental
sites where PCDDs have been detected.
Determination of BCF for all these most toxic PCDDs 1n state-
of-the-art test systems.
2-9
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3. PHYSICAL AND CHEMICAL PROPERTIES/ANALYTICAL METHODOLOGY
3.1. INTRODUCTION
D1benzo-§-d1ox1n Is a derivative of the basic chemical structure
p_-d1oxane. The structure of d1benzo-p_-d1ox1n and the conventional numbering
system used for defining substltuent positions are shown below:
9 1
A number of substHuents Including nltro, amlno, alkyl, alkoxy and
halogen can be Introduced at the different positions of the two benzene
rings. Most environmental Interest 1n substituted d1benzo-£-d1ox1ns and
most studies of this family of compounds have centered on chlorinated
d1benzo-p_-d1ox1ns that are loosely referred to as "dloxlns." Theoretically,
there are 75 different congeners of chlorinated d1benzo~p_-d1ox1ns. In this
document, only four polychlorlnated d1benzo-p_-d1ox1ns, namely 2,3,7,8-tetra-
chlorod1benzo-£-d1ox1n (2,3,7,8-TCDD), If2,3,7,8~pentaehlorod1benzo-j5-d1ox1n
(1,2,3,7,8-PeCDD), 1,2,3,&,7,8-hexachlorod1benzo-:p_-d1ox1n (1,2,3,6,7,8-
HxCDD) and l,2,3,7,8,9-hexachlorod1benzo-£-d1ox1n (1,2,3,7,8,9-HxCDD) will
be discussed.
3.2. PHYSICAL AND CHEMICAL PROPERTIES
3.2.1. Chemical Formula and Synonyms.
2,3,7,8-Tetrach1orod1benzo-£-d1ox1n {2,3,7,8-TCDD)
3-1
-------
Chem. Abstr. Name: 2»3,7,8-tetrachlorod1benzo[b,e]{l,4)-d1ox1n
Synonyms: Dloxln; TCDBD; TCDD; 2,3,7,8-tetrachlorodlbenzodloxln, 2,3,7,8-
tetrachlorod1benzo-l,4-d1ox1n.
l,2,3,7t8-Pentach1orod1benzo-£-d1ox1n (l,2»3,7,8-PeCDO>
Chem. Abstr. Name: l»2,3,7»8-Pentachlorod1benzo[b»e](1t4)d1ox1n
Synonym: !,2,3,7,8-Pentachlorod1benzod1ox1n
1,2,3,6,7,8-Hexachlorod1benzo-jp.-d1ox1n (1,2,3,6,7,8-HxCDD)
Chem. Abstr. Name: It2,3,6,7,8-Hexachlorod1benzo[b,e](l,4)d1ox1n
Synonym: 1,2,3,6,7,8-Hexachlorod1benzod1ox1n
l»2,3,7t8f9-Hexachlorod1benzo-fi-d1ox1n {1,2,3,7,8,9-HxCOO)
Cl Cl
CL ^k ^O^ ^^ XI
Chem. Abstr. Name: It2,3,7,8t9-Hexachlorod1benzo[b»e](l,4)d1ox1n
Synonym: 1,2,3,7,8,9-Hexachlorod1benzod1ox1n
3-2
-------
3.2.2. Physical Properties. The physical properties of the four poly-
chlorinated dloxlns are given 1n Table 3-1. Although the physical proper-
ties of 1,2,3,7,8-PeGDD, 1,2,3,6,7,8-HxCDD and 1,2,3,7,8,9-HxCDD have not
been well studied, these properties have been more Intensively studied for
2,3,7,8-TCDD. 2,3,7,8-TCDD 1s UpophlUc, exhibiting a higher degree of
solubility 1n fats and oils than 1n water. The solubility of 2,3,7,8-TCDD
1n various solvents (at unspecified temperatures) 1s as follows (Crummett
and Stehl, 1973):
Solvent Solubility (ppm)
water 2 x 10~4
lard oil 44
benzene 570
o-d1chlorobenzene 1400
chloroform 370
acetone 110
n-octanol 50
methanol 10
The solubilities of HxCDD (Isomer unspecified) In benzene and toluene
are 1600 and 1800 ppm, respectively (U.S. EPA, 1978). The known solubility
data (NRCC, 1981a) suggest that while the lower congeners (e.g., d1-CDD and
tr1-CDD) are more soluble 1n aliphatic solvents (e.g., acetone, methanol),
the higher homologues are more soluble 1n aromatic hydrocarbon solvents.
However, the solubilities of both lower and higher homologues of polychlorl-
nated dloxlns may be comparable In chlorinated aliphatic hydrocarbons,
namely chloroform.
Because of the ir -» ir* transitions the polychlorlnated dloxlns have two
absorption maxima 1n the near UV region. The absorption coefficients
resulting from this transition at longer wavelengths are presented In Table
3-1. The partition coefficient of 2,3,7,8-TCDD 1n a hexane water system was
estimated to be 1000 (temperature unspecified) (Matsumura and Benezet,
3-3
-------
TABLE 3-1
Physical Properties of a Few Selected PolychloMnated Dloxlns
w
1
**
Compound
2,3.7,8-TCDD
1,2,3,7,8-PeCDD
1,2,3,6,7,8-HxCOO
1.2,3,7,8,9-HxCOD
CAS
Reg. No.
1746-01-6
40321-76-4
57653-85-7
19408-74-3
Rolecular
Formula
C12H4Cl402
Ci2H3Cl502
C12H2C1602
C12H2C1602
Molecular
Height
321.9
356.5
390.9
390.9
Description
colorless
needles
NA
NA
NA
Melting
Point
CC)
305-306
240-241
285-286
243-244
fcfsaxa
(chloroform)
(ran}
310
308
316
317
Elb
1
173.6
171.4
152
104
Reference
Pohland and
Vang, 1972
Gray et al.,
1976
Gray et al.,
1975
Gray et al.,
1975
aTh1s Is the wavelength of maximum absorption.
''This Is the absorption coefficient for a IX chloroform solution of substrate In 1 cm cell at the
absorption coefficient (IT* cm"1), multiply by one-tenth of the molecular weight.
NA - Not available
To convert this to the molar
-------
7973). Values for other physical properties for these compounds have been
estimated from various correlation equations and are given In Table 3-2.
The Infrared, mass, phosphorescence, and nuclear magnetic spectra of
2,3,7,8-TCDD are available from various sources (Mahle and Shadoff, 1982;
Pohland and Yang, 1972; Chen, 1973; Kende and Wade, 1973). The mass spectra
of the three other PCDOs are also available (Mahle and Shadoff, 1982; Gray
et a!., 1975, 1976). The response ratios of electron Impact (El) and nega-
tive chemical 1on1zat1on (NCI) and fragmentation of 11 of the TCDD Isomers
have been reported by Rappe et al. (1983a). These spectra, particularly the
mass spectra, are very useful 1n Identifying the various homologues/lsomers
of the PCDDs, but they give limited Information for the Identification of
particular Isomers.
3.2.3. Chemical Properties. All four PCDDs are rather stable toward
heat, adds and alkalies, although heat treatment with alkali (under condi-
tions similar to alkaline extraction of tissue) completely destroys octa-CDD
(Albro, 1979). These compounds begin to decompose at 500°C, and at a
temperature of 800°C, virtually complete degradation of 2,3,7,8-TCDD occurs
within 21 seconds (Stehl et al., 1973). The PCDDs are susceptible to photo-
degradation 1n the presence of UV light. They also undergo photoreductlve
dechlorlnatlon 1n the presence of an effective hydrogen donor. Gamma
radiation degrades 2,3,7,8-TCDD 1n organic solvents (FanelH et al., 1978).
3.3. ANALYTICAL METHODOLOGY
Several publications on the analytical methods for the determination of
PCDD levels 1n different media are available. The analytical methodologies
for the separation of the different Isomers of PCDDs are difficult and
expensive. Many Investigators, particularly the earlier ones, failed to
characterize the Individual Isomers and 1t Is not always clear whether a
3-5
-------
TABLE 3-2
A Few Estimated Physical Parameters of Chlorinated D1benzo-p_-D1ox1nsa
Parameter
Vapor pressure (mm of Hg)
at 25°C and 1 atmosphere
Octanol/water partition
coefficient at 25°C
Sorptlon partition
coefficient (Koc)
Water solubility (ppb)
at 25°C
2,3,7,8-TCDD
1.7 x 10~*
1 x 10~«c
1.4 x 10*
6.9 x 10*c
1.9 x IQ'd
1.4 x 10*e
9.9 x 10s
3.3 x 106C
0.2f
PeCDDb
NA
7 x 10«
5 x 10*
0.04
HxCDDb
NA
4.2 x 10'
3 x 10'
0.008
aSource: NRCC, 1981a (unless otherwise stated), based on vapor pressure
data (Firestone, 1977a) and the octanol/water partition coefficient value
{Kenaga, 1980}
are estimated values for nonspecific Isomers
cHabey et al., 1981
dU.S. EPA, 1984
eTh1s 1s a measured value (Neely, 1979)
f"Th1s 1s the experimental value (Crummett and Stehl, 1973}
NA = Not available
3-6
-------
isomer or a mixture of Isomers was responsible for the observed
effect(s). However, analytical methods for detecting specific Isomers at
low ppt levels are now available for human samples (Crummett, 1983). In the
case of TCDDs, the specific Isomer 2,3,7,8-TCDD has been more thoroughly
studied than any of Its other isomers because of Its high toxldty. It 1s
not the purpose of this section to review the various analytical methodolo-
gies available for PCDDs. Such reviews of recent analytical methods have
been done 1n a Canadian document (NRCC, 1981b), a U.S. EPA (1980a) report
and by Tlernan (1983). Instead, this section will attempt to point out the
various problems that may be encountered 1n the analysis of these compounds
and provide a critique of a few typical analytical methods available for
PCODs.
3.3.1. General Procedure for the Analysis of PCODs, The analysis of
PCDDs can be broadly divided Into three basic steps (sample preparation,
sample cleanup and sample analysis). The description of each of these steps
with the associated difficulties that may be encountered are discussed below.
3.3.1.1. SAMPLE PREPARATION — In this step, the sample 1s homoge-
nized or digested and extracted with a suitable solvent or a solvent mixture
to remove the bulk of the sample matrix and to transfer the PCDD residue
Into the solvent(s). Both the selection of the proper solvent(s) and the
method of extraction can be critical 1n obtaining a satisfactory recovery of
PCDDs from the sample matrix. A number of solvents Including hexane,
hexane-acetone, benzene, toluene, chloroform and methylene chloride gener-
ally have been used for extracting PCDDs from sample matrix (Kooke et a!.,
1981; Harless et al., 1980; Van Ness et a!., 1980). If the sample does not
contain water, as 1s the case with fly ash and atmospheric partlculate
samples, either benzene or toluene appears to be the desirable solvent
3-7
-------
(Kooke et al., 1981). Toluene should be preferred over benzene, however,
because of Us lower toxldty. For the extraction of PCODs from aquatic
media, a solvent leading to high partition coefficient should be selected.
No systematic study, however, has been done on the extractablllty of these
compounds from aquatic media by different solvents.
The I1p1d content of different tissues may also Influence the amount and
the nature of extraction solvent. For example, chloroform-methanol 1s
effective for serum and plasma, but H produces emulsion with milk contain-
ing higher I1p1d (Albro, 1979).
In other sample matrices that contain high amounts of water, such as
tissues and food samples, the water may alter the extractablHty of a
solvent. For example, although acetone may be a good solvent for soil
extraction, the admixture of a small amount of water decreases the solubil-
ity of the substrate so that 1t cannot be used directly for animal tissues.
Mixtures of polar and nonpolar solvents such as benzene-methanol may
separate Into two phases In the presence of 2% water, resulting In non-
reproduc11ble extraction (Albro, 1979).
Samples that may contain PCODs bound to the matrices, such as tissue,
food, soil and sediment, may require add/base digestion procedures to
release the bound substrate Into the extraction media. The add/base
extraction 1s normally done with concentrated add or an alcoholic base
(Toslne, 1981; Harless et al., 1980). Kooke et al. (1981) reported highest
extraction efficiencies by add treatment of fly ash before extraction. The
Increase 1n efficiency was hypothesized to be due to opening of some of the
pores 1n the fly ash structure, thus making the solvent more accessible to
the sorbed PCOOs. Refluxlng with alkaline potassium hydroxide, however, may
cause decomposition of the higher polychlorlnated dloxlns and oxidation
3-8
-------
of some products (Hass and Frlesen, 1979; Albro, 1979). A neutral extrac-
tion system 1s reported to circumvent the possibility of this loss and has
been used by several authors (O'Keefe et al., 1978; Harless et al.t 1980),
The extraction efficiency may also depend on the method of extraction.
The extraction efficiencies of PCDDs by simple shaking, ultrasonlcatlon and
soxhlet extraction were studied by a few Investigators (Kooke et al., 1981;
Chess and Gross, 1980). While Chess and Gross (1980) reported no signif-
icant Improvement In extraction efficiencies of PCDDs from fly ash by
sonlcatlon or soxhlet extraction, Kooke et al. (1981) found soxhlet extrac-
tion to be a better procedure than the other two methods. Similarly, Albro
(1979) reported that the nature of the sample matrix Influences the effec-
tiveness of extraction. Thus, while It may be possible to extract liver In
a Teflon-glass homogenlzer, brain tissues may require a blender, and skin a
powerful disintegrator such as the Polytron for the extraction of residues.
3.3.1.2. SAMPLE CLEANUP — The sample cleanup procedure normally
consists of three essential steps. A fourth step 1s usually required 1f an
Isomer specific Identification and quantification 1s required. The first
step 1n the cleanup procedure consists of the removal of llplds from the
extracted sample matrix. The llpld cleanup can be achieved by two routes,
namely, solvent extraction or reaction with an add or a base. The use of
solvents such as hexane, hexane-acetone, chloroform, chloroform-methanol and
petroleum ether (NRCC, 1981b) Is common. The use of nonpolar solvents
(hexane or CC1.) gives excellent results when I1p1ds consist primarily of
trlglycerldes and/or phosphoHplds. When the I1p1d consists of cholesterol
esters, however, sulfurlc add treatment gives a better result than non-
polar solvent extraction (Albro, 1979). Similarly, base wash of the organic
phase may remove Interfering I1p1ds and other materials through saponlflca-
tlon, hydrolysis or degradation. However, add wash 1s more commonly used
3-9
-------
than base wash presumably because of the probability of decomposition
{Albro, 1979} and oxidation (Hass and Frlesen, 1979) of sample components as
a result of base wash. The possibility of decomposition of higher PCDDs by
the base may be the reason for Its less frequent use. It should be men-
tioned that some Investigators used chromatographlc columns such as silica
gel containing sulfurlc add for the acid/base cleanup step Instead of wash-
Ing off the I1p1ds by simple shaking (Lamparskl et al., 1979; FanelH et
al.f 1980a; Langhorst and Shadoff, 1980; Buser, 1978; DIDomenlco et al.,
1980a).
The second step In the cleanup procedure consists of removal of common
Impurities such as pesticide residues from the PCDDs. Liquid chromatog-
raphy with alumina, Flor1s1l, silica, foam charcoal or carbon dispersed on
glass fibers has been used for this purpose (Harless et al.t 1980; Hltchum
et al., 1980; Chess and Gross, 1980; Buser, 1978; Tlernan et al., 1980;
Stalling et al., 1983; Buser and Rappe, 1983). A few Investigators have
used AgNO_-1mpregnated silica gel columns (Lamparskl et al., 1979; Toslne,
1981; Langhorst and Shadoff, 1980). The AgNOg/slHca column system 1s
claimed to be effective 1n the removal of DDE, chlorinated allphatlcs and
sulfldes.
There 1s a difference between the various alumina columns (Lamparskl et
al., 1979; Harless et al., 1980). The separation of PCDDs from PCBs may be
accomplished with acidic, neutral and basic alumina; most authors have
provided no reason for choosing one over the other. However, 1t has been
shown by Albro (1979) that acidic alumina may be better than basic alumina,
which 1n turn may be better than neutral alumina for the separation of
residual Uplds from the PCDDs 1n the sample extracts.
3-10
-------
The third step 1n the cleanup procedure Is used solely as an additional
cleanup of contaminants and has been used by a few Investigators (Langhorst
and Shadoff, 1980; Lamparskl et al., 1979; Hltchum et a!., 1980). The
removal of these additional Impurities has been obtained by using HPLC with
both normal and reversed phase packing materials. Recently, PhllUpson and
Puma (1980) reported that chlorinated methoxyblphenyls In fish extract could
coelute with TCDDs through an alum1na-Flor1s1l cleanup sequence and Inter-
fere with the determination of TCDDs. A few compounds that may Interfere
with the determination of TCDD at m/e values of 319.8966 and 321.8936 are
given In Table 3-3.
The additional cleanup step using the HPLC separation procedure may be
essential for the unequivocal separation of Impurities that may Interfere
with the MS analysis of PCDDs.
The fourth and final cleanup step consists of the separation of PCDDs
Into several different fractions by means of chromatographlc techniques.
Both liquid chromatography with alumina columns (Hass et al., 1978; Albro
and Corbett, 1977) and HPLC with normal and reverse phases have been used
(Toslne, 1981; Ryan and PHon, 1980; Langhorst and Shadoff, 1980; Mltchum et
al., 1980). The separation of PCDDs using HR6C 1s necessary for the unequi-
vocal separation of 2,3,7,8-TCDD, 1,2,3,7,8-PeCDD and 1,2,3,7,8,9-HxCDD from
the other congeners. Buser and Rappe (1983) have shown that this separation
can be achieved using a 55 m SHar column. The unequivocal separation of
2,3,7,8-TCDD from other Isomers has been accomplished by a combination of
reverse phase and normal phase HPLC, and packed column GLC by Langhorst and
Shadoff (1980). The cleanup procedure used by most of the other Investi-
gators has failed to demonstrate this unequivocal separation of all the TCDD
Isomers. The various cleanup and analysis procedures have been compared by
Brumley et al. (1981).
3-11
-------
TABIh 3-3
Potential Interferences 1n the Determination of TCDDs at m/e Values of 319.8966 and 321.8936*
Compound
Heptachlorobiphenyls
Nonachlorobiphenyls
Tetrachloroniethoxy
blphenyls
Tetrachlorobenzyl-
phenyl ethers
Pentachlorobenzyl-
phenyl ethers
DDT (4 Isomers)
DDE (4 Isomers)
Hydroxytetrachloro-
dlbenzofurans
Tetrachlorophenyl-
benzoqulnones
Tetrachloroxanthenes
Molecular Formula
C12H3«C17
C-|2H 3SClg
C]2H «C!8 »'C'I
C13H8 «C140
C"|3H8 3SCl3 37C10
C13H8 «C140
Cl3H8 35Cl3 37C10
Ci3H7 35Cl4 37C10
C14H9 3SCl3 37C12
C~)4Hg 3SC12 37Cl3
Ci4H8 3SC12 37C12
C14H8 35C1 37C13
C12H4C1402
C12H4C1402
C13H60 35C13 37C1
CisH&O 35C12 37C12
Interfering Ion m/e
H* -23SC1 a? 1.8678
Hf -43SC1 319.8521
H^ _3:iH(;| 37d 321.8491
H+ 321 ^9299
Hf 319.93?9
H* 321 .9300
ft* -H8SC1 319.9143
Hf _H35ci 321.91138
H* -H8SC1 319.9321
H* -H35C1 321.92917
H* 319.93?!
«f 321.92916
321.8936
H* 319.8966
321.8936
H^ 319.9143
H* 321.9114
Resolution for
Separation
12476
7189
7233
8805
8848
8813
8843
18043
18104
9006
9050
9011
9052
NR
NR
NR
NR
18043
18104
*Source: NRCC, ISSlb
NR = Not resolved by MS
-------
The cleanup of the samples through liquid chromatography with subsequent
quantification of PCODs requires concentration of the sample solution.
Evaporation to dryness by an Inert gas stream appears to be an accepted
procedure for concentrating the TCOD solutions. If the concentration proce-
dure 1s not properly controlled, 1t can Introduce error 1n two different
ways. It has been shown by Lamparskl et al. (1979) that concentration of
sample solution with prepurlfled nitrogen can Introduce severe contamina-
tion. Therefore, further purification of the gas stream with a series of
traps containing 10% Aplezon L plus 10% each mlcronlzed Carbopack B and
Amoco PX-21 on 60/80 Chromosorb W-AW, 13 x molecular sieve, 20% H2S04 on
B1o-S1l A, and Carbosleve 8S were required. Secondly, O'Keefe et al. (1982)
have demonstrated that significant losses of 2,3,7,8-TCDD occur when
nitrogen evaporation to dryness 1s done at temperatures >50°C»
3.3.1.3. SAMPLE ANALYSIS — The final analysis of PCDDs 1s almost
exclusively performed by iC/MS. Although some of the earlier Investigators
(Laraparskl et al., 1978; Firestone, 1977b) used 6C with electron capture
detection, 1t does not have the sensitivity for complex samples containing
low levels (<10 ng kg"1) of PCODs (Hass and Frlesen, 1979).
The final separation procedure for PCDD analysis uses GC with packed or
capillary columns. A typical 11st of packed and capillary columns used for
the analysis of PCDDs 1s given 1n Table 3-4. Capillary columns are prefer-
able over packed columns because they provide better separation of compon-
ents 1n a complex mixture than packed columns. There are other advantages
of capillary columns, namely, that the narrow band width of the separated
components enhances MS sensitivity, and the capillary columns with their low
bleed rates enhance MS sensitivity by keeping the background contamination
low. A disadvantage of the capillary columns relative to the packed columns
3-13
-------
TABLE 3-4
Some Packed and Capillary Columns Used for the Analysis of PCDDs
PACKED COLUMNS
1.8 m x 2 mm 1.d.t 3% Dexsll 300
0.6-2 m x 2.5 mm 1.d., 3H OV-1, 3% OV-17,
3% QV-61, 2% OV-101
1.8 m x 2 mm 1.d., 354 OV-7
2 m x 2 mm 1.d., 354 OV-210
2 m x 2 mm 1.d. specially packed 0.2% carbon
wax 20 M (Aue packing)
2 m x 2 mm l.d., 0.6% OV-17/0.4% Poly S179
2 m x 4 mm 1.d., 1.2H SHar IOC
1.8 m x 2 mm 1.d., 5% SE-30
CAPILLARY COLUMNS
18 m x 0.3 mm 1.d., OV-61 WCOT
22 m x 0.3 mm 1.d., OV-17, 101, S1lar IOC
50 m x 0.36 mm 1.d., OV-17 WCOT
30 m x (1.d. not given), SE-30 WCOT
30 m x 0.25 mm 1.d., OV-101 WCOT
30 m x 0,25 mm 1.d., SE-30 WCOT
20 m, SP-2100 SCOT
25 m x 0.2 mm 1.d., quartz, methyl slUcone
WCOT
30 m x 0.5 mm 1.d.t glass, 60/40 w/w OV-17/
Poly S-179
50 m x 0.25 mm 1.d., glass SHar IOC
55 m x 0.37 mm 1.d., glas OV-17
55 m x 0,40 mm 1.d., glass OV-101
60 m x 0.26 mm 1.d., Supelco SP-2330
50 m x 0.4 mm 1.d., OV-101 fused silica
60 m OV-101 WCOT (l.d. unspecified)
Van Ness et a!., 1980
D1Domen1co et al., 1980a
Tlernan et al., 1980
Parker et al., 1980
Elceman et al., 1981
Langhorst and Shadoff, 1980
Firestone et al., 1979
Baughman and Meselson, 1973
Buser, 1975
Buser, 1976
Buser and Rappe, 1978
Harless and Oswald, 1978
Harless and Lewis, 1980a
Harless et al., 1980
Hltchum et al., 1980
Norstrom et al., 1982
Nestrlck et al., 1980
Buser and Rappe, 1980
Buser and Rappe, 1980
Buser and Rappe, 1980
Rappe et al., 1983b
Tlernan, 1983
Van Ness et al., 1980
3-14
-------
1s the problem of easy overload In the presence of other coextracted
Impurities. One group of researchers (Langhorst and Shadoff, 1980) has used
a packed column for the unequivocal determination of 2,3,7,8-TCDD In the
presence of 21 other Isomers. However, this determination was possible
because of the prior separation of components through fractlonatlon by HPLC
with a combination of a reverse phase Zorbax ODS column and a normal phase
silica column. D1Domen1co et al. (198Qa) also found low resolution SC
suitable for the analysis of ppt levels of TCDDs 1n environmental samples,
provided the samples are adequately precleaned. Although the analysis of
environmental samples from the Seveso accident by 01Domen1co et al. (198Qa)
may not have required HRGC column because no other Isomers were expected to
have been formed (Buser, 1978), a packed column may not be satisfactory for
the unequivocal determination of 2,3,7,8-TCDD 1n the presence of Interfer-
ence from other TCDD Isomers (Hummel and Shadoff, 1980).
The separation of 2,3,7,8-TCDD from all the other 21 Isomers 1s
difficult even with capillary columns. A combination of OV-101 and OV-17
glass capillary columns of 20-30 m length and 0.35-0.37 mm 1.d. was required
for unequivocal separation of 2,3,7,8-TCDD from the other 21 Isomers of TCDD
(Buser, 1978). However, a Sllar IOC glass capillary column of 55 m length
and 0.25 mm 1.d., and with a theoretical plate number of 192,000, provided
almost unambiguous separation of 2,3,7,8-TCDD from Us other Isomers (Buser
and Rappe, 1980). Other capillary columns known to separate 2,3,7,8-TCDD
from the other TCDD Isomers Include SP-2340, SP-2330 and SHov (Tlernan,
1983). A 50 m length of a Sllar IOC capillary column has been recommended
by the U.S. EPA (1982a) for the determination of 2,3,7,8-TCDD 1n municipal
and Industrial wastewaters. The same column can also be used for the
unequivocal separation of 1,2,3,7,8-PeCDD and 1,2,3,6,7,8- and 1,2,3,7,8,9-
HxCDD from the less toxic congeners.
3-15
-------
As previously mentioned, MS Is used almost exclusively for the detection
and quantification of PCDDs. Basically, three MS techniques (LRMS, HRMS and
NICI) have been used. A few different MS systems used for the determination
of TCDDs are shown In Table 3-5. It 1s obvious from Table 3-5 that electron
Impact lonlzatlon In the low resolution mode (resolution <8000, 10% valley)
has been the most widely applied MS method used for the determination of
TCDDs.
The electron-Impact mass spectra of PCDDs show strong molecular Ions
(M*). Fragmentation occurs through the loss of CO and Cl radicals. Major
Ions are at M*-63 (M*-COG1) and M*-126 (M*-2COC1). Doubly charged
molecular Ions (M *) and minor fragmentation Ions occur at M*-35
(M*-ei)» M*-70 {M*"-2ei) and M*-98 {M+-COC1-C1). The usual charac-
teristic 1on clusterings caused by the chlorine Isotopes are also observed.
Based on molecular Ions and fragmentation pattern, PCDDs can be distin-
guished from other chlorinated pollutants. However, this requires monitor-
Ing multiple Ions. The Ions that are commonly monitored for 2,3,7,8-TCDD
are M* and Us chlorine Isotope clusters, that 1s, 320 (3SC14 CDD),
322 • (*SC1 "Cl COD) and 324 ("Cl^Cl. CDD). In some
O £ t
Instances, fragment Ions at 257 (320-C035C1), 259 (322-C085Cl) and 194
(320-2COSSC1) are also monitored. The Intensity ratios 1n the mass spec-
trometrlc peaks that are due to chlorine Isotope proportions 1n native TCDD
can be used for assessing the degree of Interference and confirming the
Identity of the TCDDs. Thus, the relative peak Intensities of pure
2,3,7,8-TCDD at 320:322:324 are expected to be 77:100:49 (NRCC, 1981a). The
response for the 1on at 257 Is -30% of the response for the 1on at 322
(Glaser et a!., 1981). Sometimes Internal standards containing
(C12H4a7Cl402) or ("CizV^V used for TCDO
analysis give prominent 1on peaks at 328 and 332, respectively. The primary
3-16
-------
lAbLt 3-t>
The Detection L1mH, Resolution and Ions Monitored by a Few Mass Spectrometrlc Systems
for the Determination of TCDDs3
w
Ion1zat1on Method and
Reference
ELECTRON IMPACT
Baughman and Neselson, 1973
Crummett and Stehl, 1973
Hummel, 1977
Hummel, 1977
Mahle et al., 1977
Adamoll et al., 1978
Adamoll et al., 1978
O'Keefe et al., 1978
DIDomenlco et al., 1980a
Buser and Rappe, 1980
Cavallaro et al., 1980a
Chess and Gross, 1980
Fanelll et al., 1980a
Harless et al., 1980
Langhorst and Shadoff, 1980
Lamparskl and Nestrlck, 1980
TCDD Limit
of Detection
(pg)
5
6
5-10
5-10
5
50
50
NR
20
40-80
50
250
5-10
5
40-60
-
10,000
600
400
3,000
NR
unit
unit
10,000
unit
unit
unit
2,000
400
9,000
1,000
unit
m/e Values Monitored for TCDD
320 322 324 326 328 332 259 257 194
4-4- 4- 4-
4-4-4-
4-4- 4-
4-4- 4-
4-4- 4-
4-4-4-
+ 4-4-
4-4- 4-
4-4- 4-
4-4-4-
4- 4- 4- 4-
4- + 4-4-
4- 4-
4-4- 4-4- 4-4-
4-4- f
4- 4- 4- 4-
-------
TABLE 3-5 {cont.)
O3
lonlzatlon Method and
Reference
Norstrom et al., 1982
Toslne, 1981
Ryan and Pllon, 1980
Tlernan et al., 1980
Tlernan et al., 1980
CHEHICAL IONIZATION
Hass et al., 1978
Mltchum et al., 1980
TCDD Limit
of Detection
(pg)
5-10C
10C
10C
ld
100C
50-500
10
-
unit
unit
1,000
350
12,500
unit
NR
m/e Values Honltored for TCDD
320 322 324 326 328 332 259 257 194
+ 4- +• f +•
4- + * f
+
+ +
4- * f +
323 for HNCIe, 252 and 276 for HONCIf, 176 for
ONCI9
-176 from 320, -182 from 332 by ONIAPCIh
Source: NRCC, 1981a
resolution of mass
ng.kg~1
methane negative chemical lonlzatlon
methane-oxygen negative Ion chemical lonlzatlon
%xygen negative Ion chemical lonlzatlon
oxygen negative In atmospheric pressure chemical lonlzatlon
NR = Not reported
-------
M Ions for PeCDDs and HxCDDs are 356 and 390. If exact masses are used,
the normal 1on masses at 320, 322, 328, 257 and 259 will correspond to
319.8965, 321.8936, 327.8847, 256.9327 and 258.9298, respectively. Thus,
HRHS with appropriate resolution 1n most cases may positively Identify
2,3,7,8-TCDO when the sample cleanup 1s not specific (Hummel and Shadoff,
1980). However, an unequivocal Identification and quantification of
2,3,7,8-TCOD 1n the presence of Us Isomers will still require HPLC frac-
tlonatlon or HRSC separation as described earlier.
The following criteria have been outlined by Harless et al. (1980) for
confirmation of 2,3,7,8-TCDD residues:
1. Correct GC retention time for 2,3,7,8-TCDD.
2. Correct Isotope ratio for the molecular Ions 320 and 322.
3. Correct simultaneous response for the molecular Ions 320, 322
and 328.
4. Correct responses for the co-Injection of sample fortified
with *'C1-TCOO and 2,3,7,8-TCOO standard.
5. Intensity of molecular Ions 320 and 322 must be >2.5 times the
noise level.
Supplemental criteria that Harless et al. (1980) suggested for highly
contaminated extracts are:
1. COC1 loss Indicative of TCDO structure.
2. GC/HS peak-matching analysis of molecular Ions 320 and 322 1n
real time to confirm the 2,3,7,8-TCOD elemental composition.
Although the limit of detection for TCOO Is about the same on both HRMS
and LRMS (Crummett, 1983), the advantage of HRMS over LRHS for PCOO analysis
1s that the former technique requires far less time-consuming cleanup steps
than those required for LRMS although this 1s dependent on the nature of the
3-19
-------
sample. With the use of properly selected analytical techniques, the PCDOs
can be determined down to sub ppt levels (Crummett, 1983).
The use of chemical 1on1zat1on techniques has received limited applica-
tion for the Individual TCOO Isomers. Other methods not requiring coupling
GC with MS have also been used for PCDDs. For example, the method of direct
probe and specific 1on monitoring (M* •» Mt + COC1) based on the
concept of MS-MS was used for the analysis of TCDD (Chess and Gross, 1980).
Although the method had comparable specificity to GC-HRMS, the precision of
the method was not as good.
3.3.2. Analysis of PCODs 1n Specific Environmental Media. Although the
general procedure for the analysis of PCOOs levels has been discussed 1n
Section 3.3.1., the detailed analytical procedures depend on the type of
medium. For this document, the environmental media have been divided Into
four classes, namely, water, air, soil and biological media, and the
techniques used for the sampling and analysis of PCDDs 1n each medium have
been discussed Individually.
3.3.2.1. HATER —
3.3.2.1.1. Sampling Method — Two types of sampling methods can be
used for collecting aqueous samples for PCDDs. In the first method, no
preconcentratlon of the samples during collection 1s made. Grab samples are
collected 1n clean (detergent washed, rinsed with acetone or methylene
chloride, and dried) amber glass bottles of 1 a or 1 quart capacity fitted
with screw caps lined with Teflon or aluminum foil (U.S. EPA, 1982a). If
aluminum foil 1s used as a liner, 1t should be washed with acetone and the
dull side should face the sample to avoid sample contamination (Albro,
1979). Automatic samplers can also be used for collecting flow proportional
composite samples In amber glass bottles (U.S. EPA, 1982a). The sample
3-20
-------
containers roust be kept refrigerated at 4°C and protected from light during
compositing. The grab or the composite-samples should be protected from
light and be kept at 4°C during shipment. All samples must be extracted
within 7 days and completely analyzed within 40 days of extraction (U.S.
EPA, 1982a).
The preconcentratlve method of sample collection was used by D1Domen1co
et al. (1980a). In this method, 2-20 2. of water was allowed to pass
through a 12 cm x 1.5 cm i.d. XAD-2 column at a rate of 60 ma/minute. The
XAD-2 columns containing the PCDDs should be protected from light and kept
at 4°C during transportation and storage.
3.3.2.1.2. Analysis — Most of the methods found 1n the literature
described 2,3,7,8-TCDD analysis Instead of other PCDD analyses 1n aqueous
samples. The methods used for the analysis of 2,3,7,8-TCDD can be used also
for the analysis of the other PCDDs. However, the recovery of the Indi-
vidual PCDDs should be established with added Internal standards.
An appropriate volume of water (depending on the desired detection
limit) with added Internal standard of either 13C12 or 37C14
2,3,7,8-TCDD 1n the amount of 2.5-25 ng (Harless et al., 1980; U.S. EPA,
1982a) can be extracted with hexane (D1Domen1co et al., 1980a), methylene
chlorine (U.S. EPA, 1982a; Harless et al., 1980) or petroleum ether (Van
Ness et al., 1980). Judging from the recovery data (U.S. EPA, 1982a;
DIDomenlco et al., 1980a; Harless et al., 1980) methylene chloride appears
to be a better solvent.
The extract containing 2,3,7,8-TCDD was cleaned by add and base wash
(Harless et al., 1980; U.S. EPA, 1980b; Van Ness et al,, 1980) and further
cleaned by liquid chromatography with alumina column (Harless et al., 1980;
Van Ness et al., 1980). However, U.S. EPA (1982a) recommends another
3-21
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cleanup step using silica gel liquid chromatography, which may be necessary
for wastewater but may be unnecessary for drinking water and clean surface
water samples. The final separation and analysis was performed by low
resolution GC-HRHS (Van Ness et al., 1980; Harless et a!., 1980) or high
resolution GC-HRHS or LRMS (U.S. EPA, 1982a). If an unequivocal Identifica-
tion of 2,3,7,8-TCDO 1s required, the U.S. EPA (1982a) method seems to be
most appropriate since 1t recommends using a 50 m SHar IOC capillary column
and multiple 1on monitoring MS mode that 1s known to unequivocally Identify
and quantify 2,3,7,8-TCDD 1n the presence of Its other Isomers (Buser and
Rappe, 1980). Harless et al. (1980) reported that TCDO 1n water can be
accurately determined to as low a concentration as 0.03 ppt.
3.3.2.2. AIR --
3.3.2.2.1. Sampling Method — Monitoring of PCDDs from point sources
of emission and ambient atmospheric level requires development of sample
collection methods from both sources. The available published work suggests
that the PCDDs are associated primarily with partlculate matters (NRCC,
1981b).
For the collection of air samples from hot point sources, namely exhaust
from an Incinerator, a number of commercially available sampling probe and
sampling trains are available. Most Incorporate filters to Isolate the
particles and a subsequent device to trap gaseous organlcs from the exhaust.
For PCDDs, glass fiber filters of proper pore size are generally used (NRCC,
19815). The filter should be maintained at a temperature of >100°C to
prevent condensation of water. PCDDs that may escape the glass filters may
be collected 1n a polyurethane foam or XAD-2 trap maintained at room
temperature. The sampling must be performed 1n an 1sok1net1c manner to
ensure representative sampling. To permit evaluation, the efficiency of the
3-22
-------
collection method must be documented. The sampling methodology for point
sources 1s 1n a developmental stage (NRCC, 1981b) and more work 1s needed 1n
this area. The recommendations for sample collection procedure given above
follow the general U.S. EPA procedure for collection of air samples from hot
point sources. A modified U.S. EPA Method 5 sampling train (Federal
Register, 1971} consisting of a filtering unit, a condenser unit, a resin
cartridge unit and a series of 1mp1ngers have been used by Stanley et al.
(1982) to collect PCDOs 1n flue gas samples from utility boilers.
The collection of PCDDs 1n ambient atmospheric samples has been achieved
by both dustfall jars and high volume samplers (D1Domen1co et al., 1980b).
Dustfall jars were constructed from 10 a. glass vessels topped with metal
grldded funnels with a collecting cross section of about 0.11 m2. The top
of the funnels were about the human breathing level from the ground. The
grid allowed particles <500 ym to be collected. Samples were collected
for 1 month or the time required for the vessel to be filled with meterolc
water and dust. At the end of the sampling time, the liquid phase was
separated from the particles by filtration and the two phases were analyzed
separately.
The high volume sampling was performed with high volume samplers
equipped with A and E glass fiber filters at a flow rate of 1.5 m3/minute
(DIDomenlco et al., 1980b). The sampling duration was about 160 hours. The
whole sampling unit was assembled Into a protective container. The effi-
ciency of sample collection by either of the above methods was not estab-
lished. The high volume sampling can lead to stripping of PCDDs from the
filter. A backup filter consisting of polyurethane foam plug may be used to
prevent this anticipated loss. Partlculate and vapor phase TCDD was also
3-23
-------
collected by polyurethane foam filters (U.S. EPA, 1982b; Nash and Beall,
1980). The collection efficiency with this system was determined to be 86%
by Nash and Beall (1980).
3.3.2.2.2. Analysis — The analysis of PCDDs 1n the participate
matter begins with an extraction process. As has been shown 1n Section
3.3.1.1., the best extraction efficiency Is obtained with dilute HC'I
pretreated particles, followed by soxhlet extraction with benzene or
toluene. Ubertl and Brocco (1981) found that xylene was a better solvent
than toluene, while Cutle (1981) found that o-d1chlorobenzene may be better
than any of the other solvents. Various extraction procedures for combus-
tion effluent samples have been described by Taylor et al. (1983).
Several methods are available for sample cleanup before analysis,
[Basically, the methods used for the analysis of fly ash can be used for
partlculate matter (L1bert1 and Brocco, 1981; Elceman et al., 1980; Tlernan,
1983; Buser et al., 1978)]. In one analytical procedure, Lamparskl and
NestMck (1980) added Internal standards of «C-2,3,7,8-TCDD, «C-
1,2,3,4,7,8-HxCDD and 13C-OCDD to the partlculate extract. The extract
was cleaned with add and base washes. Next, the extract was cleaned by
liquid chromatography with AgNO^/slUca column and basic alumina column,
0
followed by cleanup and sample fractlonatlon with an RP-HPLC (Zorbax ODS)
and a normal phase HPLC (silica) method. The final analysis was performed
with low resolution GC-LRHS. This method provided an unequivocal Identifi-
cation of Isomers and permitted analysis of a minimum concentration of 110
ppt of 2,3,7,8-TCDD 1n electrostatically precipitated fly ash from a munici-
pal burner.
3-24
-------
In another method (Rappe et al,» 1983b; Buser and Rappe, 1983), the
sample (soot or Kleenex tissue from wipe tests) was spiked with 1-5 ng of
2,3,7,8-i3C10-TCDD, 2,3,7,8-37Cl,,-TCDF (tetrachlorodlbenzofuran) and
Ic. 4
37C1_-OCDD and treated with 1 M hydrochloric add. The PCDDs and PCDFs
o
1n the washed and dried sample were extracted with toluene 1n a soxhlet
extractor and the extract was subjected to column chromatography on silica
gel and basic alumina column. The methylene chlor1de-n-hexane (1:1)
fraction from the second column containing PCODs and PCDFs was subjected to
HRGC/MS analysis. A 55 m x 0.26 mm 1.d. SHar column was found to be suit-
able for the 1somer1c separation of all 22 Isomers of TCDD.
3.3.2.3. SOIL —
3.3.2.3.1. Sampling Method — Since similar analytical methods are
used for both soil and sediments, this subsection describes the sampling and
analytical methods for these two sample types.
Whenever possible, the sites for soil samples should be chosen 1n open
areas away from physical obstacles. If the soil 1s suspected to be contami-
nated because of fallout from a point source, sampling sites should be
established 1n a grid over a topographical map of the suspected area. Son
samples may be collected by Inserting a 0.5 m long and 7 cm l.d. steel
cylinder Into the soil to a depth of 7 cm and then retracting the soil and
the cylinder system. The earth core should be removed and stored 1n sealed
plastic bags (D1Domen1co et al., 1980c). The bags should be cooled to 4°C
during transportation.
To determine the distribution of PCDDs 1n soil, samples can be taken
from the vertical faces of dug trenches of a maximum depth of 2 m. Suitable
steel core cylinders can be Inserted horizontally Into the trench face from
bottom to top. The Individual samples collected 1n this fashion should be
3-25
-------
stored 1n plastic bags at 4°C during transportation them. The details of
the soil sampling procedure have been described by DIDomenlco et al.
(1980a,c).
Although no sampling procedure for the collection of sediment samples
for PCDD analysis 1s available, the accepted method {U.S. EPA, 1979a) for
the collection of bottom sediments should be adequate 1n this case.
Clam-type or similar dredge samplers, such as Peterson, Shlpek or Hopper
samplers, can be used to collect sediment sample. Core samplers can also be
used for collecting bottom sediments. The collected samples should be
stored In glass containers with teflon-Hned screw caps, and stored at 4°C
during transportation.
3.3.2.3.2. Analysis — Several methods are available for the analysis
of PCDDs 1n soil samples (Chess and Gross, 1980; Van Ness et al., 1980;
Buser, 1978; Buser and Rappe, 1980; Harless et al., 1980). Although most of
these methods have been used for the analysis of 2,3,7,8-TCDD, they are
applicable for other PCDDs. The methods used for the analysis of soil can
also be used with very Uttle modifications for the analysis of sediments.
The first step 1n the analysis 1s the extraction of PCDDs from the soil
with a suitable solvent or a solvent mixture. A number of solvents Includ-
ing hexane-acetone (1:1), methylene chloride (Buser and Rappe, 1980),
aqueous KOH/ethanol (Harless et al., 1980), benzene (Chess and Gross, 1980),
petroleum ether (Van Ness et al., 1980), and a number of extraction methods
Including simple shaking (Van Ness et al., 1980; Buser and Rappe, 1980),
refluxlng (Harless et al., 1980), sonlcatlon and soxhlet extraction (Chess
and Gross, 1980), have been used. However, Chess and Gross (1980) demon-
strated that, 1n soil, the results obtained by simple stirring with 1:1
hexane/acetone and the more extensive sonlcatlon or soxhlet extraction with
benzene are consistent.
3-26
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The cleanup procedure for the extract generally consists of an acid and
base wash, liquid chromatography on silica and alumina columns or two
alumina columns, and final analysis by HRGC-LRMS or HR6C-HRMS (Harless et
a!., 1980; Buser and Rappe, 1980). If an unequivocal Identification and
quantification of 2,3,7,8-TCDD 1s required, the 55 m SHar IOC capillary
column used by Buser and Rappe (1980) or the 60 m SP-2330 fused silica
column (Rappe et a!., 1983b) 1s preferable to the 30 m SE-30 capillary
column used by Harless et al. (1980). The HRMS technique used by Harless et
al. (1980) Is expected to provide a better resolution of components than the
LRMS. The method of Harless et al. (1980) was suitable for the determina-
tion of ppt levels of TCDD 1n soils.
3.3.2.4. BIOLOGICAL MEDIA — In this section, the sampling and
analysis of PCDDs 1n a number of media, namely, blood, urine, fish, egg,
gelatin, liver, milk, cream, lean and adipose tissue, grain, grass, leaves,
vegetables and sawdust, will be discussed 1n general.
3.3.2.4.1. Sampling Methods — Only a limited systematic study has
been performed on the methods of sample collection for the different biolog-
ical media. A review of available literature reveals certain facts that
should be considered during sample collection. The concentration of PCDDs
1n blood 1s ~2-3 orders of magnitude lower than their concentrations 1n
adipose tissue (Firestone et al., 1979). There Is also evidence 1n several
species that the accumulation of TCDD 1n liver tissue 1s higher than 1n
adipose tissue (Section 7.2.). Liver 1s also preferable because Its I1p1d
content 1s lower than adipose tissue (samples with high I1p1d content are
more difficult to extract and clean up). One of the most convenient
sampling media that does not require sacrificing or surgically removing the
tissue 1s milk. Because of the high I1p1d content of milk, PCDDs are
expected to be accumulated 1n this medium (Langhorst and Shadoff, 1980).
3-27
-------
The dry solid samples, such as rice grain, grass, vegetables and sawdust
can be collected 1n polyethylene bags. Samples should be frozen 1n dry Ice
during transportation and should be stored 1n a freezer (-18°C) until
analyzed (Jensen et al., 1983). However, 1t has been reported that tissue
samples stored 1n linear polyethylene bottles sorbed ~2% of added 14C-ODT
overnight and the sorbed DDT could not be washed out from the bottle (Albro,
1979)» Similar absorption of 2,3,7,8-TCDD on polyethylene bags or bottles
may take place. The collection of samples 1n clean glass jars sealed with
screw caps lined with Teflon or acetone-washed aluminum foil {dull side
down) 1s preferable (Brumley et al., 1981). The sample should be trans-
ported at 4°C and frozen until analysis.
3.3.2.4.2. Analysis — Numerous analytical methods are available for
the analysis of samples In this category (NRCC, 1981a; Crummett, 1983; Rappe
et al., 1984; Smith et al., 1984). The acid/base and neutral extractions
procedures are available. Neutral extraction procedures are preferred over
add/base procedures since the latter may decompose the higher PCDDs. The
analytical methods for the determination of PCDDs 1n three typical media,
namely, fish and lean tissue, adipose tissue, and milk, will be discussed
here. In choosing the analytical methods, the results of the study of
Brumley et al. (1981) have been given due consldera- tlon.
F1sh and other lean tissue samples should be ground to obtain a homo-
geneous sample. The homogenized sample should be blended with anhydrous
sodium sulfate until a free-flowing powder 1s obtained. The mixture should
be packed Into a glass column and extracted with methylene chloride. The
extract should be first cleaned through a dual-column system of silica,
concentrated sulfurlc add 1n silica, and sodium hydroxide 1n silica,
followed by a second dual-column system of silver nitrate on silica and
3-28
-------
basic alumina. The PCDD fractions should then be cleaned up by normal phase
silica HPLC, followed by reverse-phase (Zorbax-ODS) HPLC. This extraction
and clean-up method 1s a combination of procedures employed by Hucklns et
al. (1978) and Lamparskl et al. (1979), and 1s expected to provide a better
method for the analysis of PCDDs 1n lean tissue samples.
Recently, an Interlaboratory round robin study to estimate the reliabil-
ity of data on the determination of 2,3,7,8-TCDD levels 1n fish and other
aquatic species was conducted (Ryan et al., 1983). No significant differ-
ences 1n the determined concentration of 2,3,7,8-TCDD 1n these species
occurred from methods differing 1n the use of digestion or extraction
technique, HRMS or LRMS, and Isomer specific or nonspecific separation. The
relative standard deviations 1n three fish samples analyzed by seven labora-
tories varied between 14 and 25%. This study Indicated the necessity for
the use of an Internal standard to obtain precise results.
Thawed adipose tissue samples should be ground with anhydrous sodium
sulfate (8 g Na2$04/g fat) 1n a mortar and pestle to remove excess
moisture. The homogenized sample should be extracted with chloroform-
methanol (2:1) 1n a blender. The methanol should be removed from the
extract by adding aqueous KC1. The chloroform layer should then be sub-
jected to the clean-up procedures. For the cleanup of the chloroform
extract, the method described for lean tissue should be followed. The
extraction and clean-up method described 1s a combination of procedures
employed by Hass et al. (1978) and Lamparskl et al. (1979). However,
hexane-acetone (1:2) was used by Ryan and Williams (1983) 1n extracting
2,3,7,8-TCDD from human adipose tissue.
The milk samples should be mixed with sodium oxalate and ethanol and the
^solution extracted with ethyl ether-hexane (1:1.4). The ether-hexane
extract should be dissolved 1n hexane and the clean-up procedure described
3-29
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for lean tissue should be followed. For the extraction and clean-up method,
a combination of procedures employed by O'Keefe et al. (1978) and Latnparskl
et al. (1979) may be employed.
3.3.3. B1oanalys1s of PCDDs. There are currently three methods for the
bloanalysls of PCDDs, namely, rad1o1mmunoassay (Albro et al., 1979; McKlnney
et al., 1981), AHH Induction assay (Bradlaw and Casterllne, 1979) and a
cytosol receptor assay (Hutzlnger et al., 1981; Sawyer et al., 1983). All
of these methods are In the developmental stage and are neither specific for
PCDDs nor are sensitive enough at low levels. The advantages of these
methods are that they are Inexpensive and quick compared with chemical
analytical methods. Therefore, these methods have some potential for high
volume screening of samples for the presence of PCDDs, but should not be
used as substitutes for chemical analysis.
3.3.4. Critique of Sampling and Chemical Analysis. The greatest weak-
nesses that persist 1n the determination of PCDD levels 1n environmental
samples are the lack of data for validating the accuracy of sample collec-
tion, transportation and storage procedures. The lack of representativeness
of samples during collection, loss of sample by sorptlon on container walls
or photodecomposltlon during transportation and storage, and contaml- nation
of the sample by collection equipment or sample containers can all cause
errors, particularly In samples with very low residue levels. However, no
comprehensive study has been done to provide enough guidance In the sampling
procedures.
There are several possible points of weakness 1n the analytical methods
as well. Although some validation data are available for the overall
recovery of 2,3,7,8-TCDD 1n fortified matrices, these data, as shown In
Table 3-6, may not represent the true recoveries, since 1t 1s difficult 1f
3-30
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TABLE 3-6
Some Published Method Validation Data for 2.3,7,8-TCDD Recovered from Fortified Matrices and Determined by GC/RS
m/e Values
to
i
w
320,
320,
320,
320,
335
320,
320,
320,
320,
329
320.
320,
320,
329
320,
322,
322,
322,
322,
322,
322,
322,
322,
322,
322,
322,
322,
335
324
324
324,
328
328
328
328.
328
328
328.
328
ftatrlx
human milk
soil
soil
soil
fish, liver
human milk
water,
sediment
water .
sediment
water,
sediment
water,
sediment
bovine feed
liver
TCOD Level of Fortification. nq/kg'»
Native Isotope
"C, {»'C1)
2.6 166
NA 100a
10 M
50 »»>
0-125 1000*
0-5 2503
0.01-1000 250'
0.7-65 66
2 M
NA 6?5a
13-200 390-1000
20 1000
Mean % Recovery with S.D,
Number of Native Isotopes
Replicates
8 25 * 7 37 i 19
6 NA 87 t IS
28 87 f 17 NA
8 99.2 i 5 59.8
17 i!5c 86 * 15
13 *38C 68
14 +16C 87
12 85-100 71-87
(±8-»17| (±12-*21J
3 83.3 NA
4 NA 64
16 80-100 77-105
(iS-OB) (i'-i18)
9 34 £ 7 27 i 5
Reference
langhorst and
Shadoff, 1980
Humel, 1977
DIDonenlco
et al., 19BOa
Laaparskl and
Nestrlck, 1980
Mr less et al.,
1980
Harless et al.,
1980
Harless et al.,
1980
O'Keefe et al.,
1978
Hahle et al..
1977
Hahle et al.,
1977
O'Keefe et al.,
1978
Baughman and
Heselson, 1973
-------
TABLE 3-6 (cont.J
rvs
m/e Values
320, 322, 324
320, 322, 324
320, 322, 324
Matrix
carrots
beets
spinach
TCDD Level of
Native
0.5-1.0
0.5-1.0
0.5-1.0
Fortification, nq/ka"1
Isotope
«C, |»»C1)
NA
NA
NA
Number of
Replicates
20
20
20
Nean X Recovery
Native
64.5-66.6
(i18.9-t25.5l11
60.8-79.8
46.6-67.7
(i14.2-*24.7)«1
with S.O.
Isotopes
NA
NA
m
Reference
Cavallaro
et al., 1980b
Cavallaro
et al.. 1980b
Cavallaro
et al., 1980b
'Indicates publishing author's recovery data Mas converted from ng to ppt or from ppt to X.
bPlus Indicates fortified with Isotope but amount not specified clearly.
cfhese data Indicate the mean X accuracy for TCOD obtained with quality assurance samples,
^Number In the bracket represents the X variation experienced; unclear as to how calculations were made.
NA - Not added; SO > Standard deviation
-------
not tropossJ&le to Incorporate the Internal standard 1n the same physical/
chemical form 1n the sample matrix as the PCDDs. This situation weakens the
reliability of much of the analytical data on PCDO levels 1n various
matrices.
The recovery of the overall analytical procedures 1s normally done by
measuring the recovery of Internal standards such as 37C1.-TCDD and
13C-TCDD. Methods that used Internal standards that exceeded the native
TCOO by 50-2500 times are at best questionable. Also, the recovery data
based on one Internal standard to correct for another congener or another
Isomer, such as 1,2,3,4-TCDD for OCDD or 2,3,7,8-TCDD, may be questionable
1n view of the fact that recovery and response factors may vary between
congeners and Isomers. This could cause serious problems with the deter-
mined detection limits.
Despite some rigorous criteria (Harless et al., 1980) that may be used
for positive Identification of 2,3,7,8-TCDD (assuming that the SC column
resolves 2,3,7,8-TCDD from other TCDDs), false positive results have been
obtained under certain conditions. A collaborative study conducted by the
U.S. EPA exemplifies this point. Of the total of 20 unsplked samples 1n
this study, 10 gave false positive results (Crummett, 1980). In a recent
method validation study by the U.S. EPA (Gross et al., 1981), 2,3,7,8-TCOD
levels <9 ppt could not be detected with accuracy. Clearly, there 1s a need
for more exhaustive examination for potential Interferences that may cause
false positive results.
Another major factor limiting the research 1n the field 1s the shortage
or lack of availability of Individual Isomers. Unless the authentic com-
pounds are available, analytical data developed for one Isomer on the basis
of the response factor of another Isomer will remain largely questionable.
3-33
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3.4. SUMMARY
The solubility of 2,3,7,8-TCDD 1n water 1s 0.2 yg/fi,. This congener
and the other three PCDDs are more soluble 1n aromatic solvents than
aliphatic solvents. The PCODs are relatively stable 1n the environment and
they start to decompose at temperatures >500°C.
The general method for the determination of these compounds 1n different
sample matrices consists of a solvent extraction procedure to transfer the
PCDD residue Into the solvent(s), followed by H^SO, and base washes to
remove the excess I1p1d and other Impurities from the solvent extract. The
extract 1s then subjected to two liquid chromatographlc clean-up procedures,
The cleaned up extract Is finally analyzed for the PCDDs by a SC/MS method.
All the possible GC/MS combinations, namely, HRGC-LRMS, LRGC-LRMS, LRGC-HRM8
and HRGC-HRM6, have been used. However, 1f an unequivocal Identification
and quantification of several specific Isomers 1s required, two methods are
suitable. One Involves a 55 m SHar IOC glass capillary or a 60 m SP-2330
fused silica column In combination with LRMS. Another method using RP-HPLC
and normal phase HPLC separation In combination with LRMS has been found to
be satisfactory.
3-34
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4. PRODUCTION, USE, SYNTHESIS, ENVIRONMENTAL SOURCES
AND ENVIRONMENTAL LEVELS
4.1. PRODUCTION AND USE
PCDDs Including the four compounds discussed 1n this document are not
commercially produced. Rather, these compounds are formed as trace amounts
of unwanted Impurities 1n the manufacture of other chemicals, primarily
chlorophenols and their derivatives. There 1s no known technical use for
the PCDDs (Rappe et al., 1979). The amount of total PCDDs entering the
Canadian environment/year has been speculated to be ~3300 pounds and 75# of
this amount has been estimated to be due to OCDD alone {NRCC, 1981a).
4.2. SYNTHESIS
Although the PCDDs are not commercially produced, some of these com-
pounds have been synthesized according to reactions discussed below (U.S.
EPA, 1980a).
4.2.1. Reaction of Dlchlorocatechol Salts with l,2,4,S-Tetrachlorobenzenes
1n DHSO. This general reaction has been used to synthesize 2,3.7.8-TCDD
according to the reaction scheme shown below:
K CL ^^ JCI
OK
KOH.DMSO
r»flm
The yield of 2,3,7,8-TCDD by this reaction 1s low (Kende et al., 1974).
A better method 1s the reaction of o-dlchlorocatechol with 3-n1tro-2,5,6-
trlchlorobenzene as shown below (Gray et al., 1976):
DMSO
4-1
-------
4.2.2. Substitution Reaction. The following substitution reactions have
been used for the synthesis of 2,3,7,8-TCDD:
CI2 •«- Fed,
—"* p*nta-CDD +
trl-CDD
The yield of 2,3,7,8-TCDD by this reaction has been reported to be low (U.S.
EPA, 1980a). However, when the chlorlnatlon of the unsubstltuted d1benzo-p_-
dloxln was conducted without the Fed,, the yield of 2,3,7,8-TCDD was
reported to be 40-50% (U.S. EPA, 1980a). The substitution of dlbenzo-fi-
dloxln with 2,3-d1chlorod1benzo-p_-d1ox1n 1n the presence of FeClQ and
*j
Iodine, on the other hand, reportedly also produced a high yield (4154) of
2,3,7,8-TCDD (Kende et al., 1974).
4.2.3. Photoproductlon. Small amounts of mixtures of lower PCDDs have
been produced by the UV Irradiation of OCDD (Buser, 1979). For example, a
mixture of tr1-, penta-, hexa- and hepta-CDD has been produced by this
method.
4.2.4. Ullmann Condensation Reactions. The condensation reactions as
shown 1n Figure 4-1 have been used for the synthesis of tetra- and hexa-CDO.
The yield of the desired products by the condensation reactions are not
always satisfactory because of other competing reactions. Examples of some
of these competing reactions are condensation with Cl atoms meta to a
hydroxyl group, condensation of Cl atoms para to the hydroxyl group,
dechlorlnatlon reactions, and Smiles rearrangement (U.S. EPA, 1980a).
Although the best conditions for dloxln formation are unknown, 1t has been
speculated that a temperature of 180-400°C, a pressure of >1 atmosphere
4-2
-------
OK
.o.
cr ^^ ci
c
ct
aao c
1-4 hr.
CI
CI
ci
.o.
OK
c
280 C
1-4 hir.
CI
CI
CI
CI
major
minor
OK
OK
C
CI
280* C
1-4 hr.
cr ^v" "ci
CI
CI CI
CI
C
Cl
CI
FIGURE 4-1
Ullmann Condensation Reactions
4-3
-------
(necessary to retain some precursor compounds 1n the liquid state to permit
dloxln formation), and the presence of some catalyst provide the most suit-
able conditions for dloxln formation (U.S. EPA, 1980a). However, some of
the catalysts, namely, Cu, Fe, Al-salts and !„, may encourage competing
reactions, thereby reducing the yield of the desired product(s) (U.S. EPA,
1980a).
4.2.5. Pyrolysls of Chlorophenates. All 22 TCDD Isomers have been syn-
thetically prepared from different Chlorophenates (d1-, tr1- and tetra-J
using a simple pyrolysls procedure (Buser and Rappe, 1980). Pyrolyses of
these Chlorophenates were conducted by placing 1 mg of the Chlorophenates 1n
a glass reaction tube plugged with glass wool and alumina. They were heated
for 30-60 minutes at 300°C. The yields of the TCDDs have been reported to
be 1n the ng range (Buser and Rappe, 1980).
4.2.6. Conversion Through Nitration. It has recently been shown by
Oliver and Ruth (1983) that l,2,3,6,7,8-hexaehlorod1benzo-£-d1ox1n can be
selectively prepared from two synthetic routes each consisting of dlnltra-
tlon of a tetrachlorod1benzo-j)-d1ox1n, followed by reduction and a Sandmeyer
reaction as shown below:
ci
.« . ^*^ JCt
HHJH03
trlflijoro-
ffit&ld. CI
CI
The recovery of 1,2,3,6,7,8-HxCDD was excellent by this method.
4-4
-------
4.3. ENVIRONMENTAL SOURCES
The sources of PCDDs and particularly 2,3,7,8-TCDD, 1,2,3,7,8-PeCDD,
1,2,3,6,7,8-HxCDD and 1,2,3,7,8,9-HxCDD In the environment can be broadly
divided Into five categories, namely, manufacturing processes,- municipal
Incinerations, other combustion processes, chemical disposal sites and
photochemical processes. The last source may not significantly contribute
to PCDO contamination 1n the environment. Each of these categories 1s dis-
cussed Individually 1n the following subsections.
4.3.1. Manufacturing Processes. PCDDs are generally produced during the
production of chlorinated phenols, during the production of chemicals uti-
lizing the chlorophenols (I.e., 2,4,5-T and 2,4-D) and 1n various Industrial
Incinerators where materials containing chlorinated phenol and polychlorl-
nated dlphenyl ethers are Incinerated.
4.3.1.1. PRODUCTION OF CHLOROPHENOLS — PCDDs are formed as
by-products during the manufacture of chlorophenols. Chlorophenols are
produced by two processes, the chlorlnatlon of phenols and the alkaline
hydrolysis of the appropriate chlorobenzenes. Hypothetically, both pro-
cesses can lead to the formation of PCDDs according to the mechanism
depicted below (U.S. EPA, 1980a):
Cl ^ OH(ONa) ClVxXNs ^0— :X^JC1
n
cr^-"" ti
2,4,5-Tr1chlorophenol(phenolate)
NaOH
1,2,4,5-Tetrachlorobenzene
4-5
-------
Similarly, HxCOOs are formed during the manufacture of tetraehlorophenols by
the above reaction process. PCODs are also expected to be formed during the
hydrolytlc production of polychlorlnated benzenes. The amounts of PCDDs 1n
commercial chlorophenols vary according to manufacturing process and condi-
tions. The levels of TCDDs, PeCDDs and HxCDDs found 1n different chloro-
phenols have been shown In Table 4-1, It can be seen from Table 4-1 that
the specific Isomers of the TCDDs, PeCDDs and HxCDDs have not always been
Identified 1n the products. However, 2»3,7,8-TCDD has been Identified 1n
commercial trlchlorophenols (Table 4-1). On the other hand, 2,3,7,8-TCDD 1s
not produced 1n the manufacture of PCP (Buser and Rappe, 1978). The main
HxCDD Isomers produced during the manufacture of PCP are 1,2,4,6,7,9-,
1,2,3,6,8,9- and 1,2,3,6,7,8-HxCDD present 1n a ratio of 1:4:5 (Buser,
1979). However, the composition and quantities of PCDDs 1n PCP may vary
widely from batch to batch and manufacturer to manufacturer, depending on
the manufacturing processes.
The annual world production of chlorophenols 1s estimated to be ~150,000
tons (Rappe et a!., 1979). U.S. production figures for d1- and tetraehloro-
phenols are not available. However, the 1977 estimated figures Indicate
that the annual production capacity for PCP 1n the United States was 53
million-pounds (U.S. EPA, 1980a). Canadians manufacture ~4000 tons of
chlorophenols annually with the total release Inventory to the environment
estimated at >1365 tons/year (Environmental Canada, 1984). The chloro-
phenols are used as fungicides, herbicides, sllmaddes, bacterlcldes and
Intermediates 1n the production of chlorinated phenoxy acid herbicides 1n
agriculture and forestry. The antiseptic, hexachlorophene, 1s also prepared
from 2,4,5-trlchlorophenol (Rappe et al., 1979). Therefore, the use or
presence of contaminated chlorophenols 1n facilities such as chlorophenol
4-6
-------
TABU 4-1
Levels of Tetra-, Penta- and Hexa-chlorod1benzo-p_-d1ox1ns Reported 1n Chlorophcnols
and a Few Pesticides Originating from Chlorophenols
Chlorod1benzo-p-d1ox1n (-CDD) level,
Compound
o-Chlorophenol
2,4-Olchlorophenol
?.6-01ch1oropheno1
2,4,S-TCPC
2.4,6-TCP
2,4,5-TCP (Na salt)
TCP (unspecified)
2,3,4,6-Tetrachlorophenol
Tetrachlorophenol
(unspecified)
pcpe
Tetra-
ND
0.037b
NO
NO
ND-6.2 {2,3,7,8-)*
ND-0.3 (1,3,6.8-)
49 (1,3,6,8-)
1.40 (2,3,7.8-)
ND
NO
0,7
NR
m
ND
NO
NR
NO
NO
NO
Penta-
NO
NR
NO
NO
ND-1.5
ND
NO
NR
ND
5.2
NR
NR
ND
NR
NR
NR
NR
NO
. ppm
Hexa-
ND
NR
NO
ND
ND
ND
ND
ND-<10
NO-29
9.i
6
ND-<100
0.17-39
ND-<100
9
9-27
0.02-42
0.03-10
No. Contain.3
No. Tested
0/1
several
samples
0/1
0/1
3/4
1/1
1/2
4/6
2/3
NA
1/1
3/3
6/6
10/11
1/1
several
samples
2/2
12/13
Reference
Firestone
et al., 1972
Anonymous, 1979
Firestone
et al., 1972
Firestone
et al., 1972
Firestone
tt 81., 1972
Firestone
et al., 1972
Firestone
et al.. 1972
Wool son et al.
1972
Firestone
et al.. 1972
Rappe et al. ,
1978
Buser. 1975
Wool son et al.
1972
Firestone
et al.. 1972
Wool son et al.
1972
Buser, 1975
AWPI, 1977
¥1 1 laneuva
et al., 1973
Buser and
Bosshardt, 19"
-------
TABLF 4-1 (cont.)
00
Chlorodlbenzo-p-dloxln (-COD) level.
Compound
PCP (cont.)
PCP (Na salt)
2,4-0 (-08, -OP)f
2,4-0 and 2,4,5-T mixtures
(formulated products)
2.4-D (acid, esters, and
amines)
2,4-0 (add, esters, and
amines)
2.4,5-Th
2,4,5-T (add, esters, and
formulated products)
S1 1 vex*
Agent Orange (1:1 mixture
of butyl esters of 2,4-0
and 2,4,5-T)
Agent Purple (5:3:2 mixture
of n-butyl 2.4-0. n-butyl
2,4,5-T and Iso-butyl 2,4,5-T)
Tetra-
NR
ND
0.06-0.4
ND
ND
NO-8.739 (1.3.6.8-/
1,3,7,9-)
D (1.3,6,8-)
ND-<100
0.010-0.080
(2,3.7,8-)
ND-<10
1.98'
(2.3.7.8-)
32.8'
(2.3,7,8-)
Penta-
NR
ND
NO-0.08
ND
ND
NR
NR
NR
NR
NR
NR
NR
ppm
Hexa-
ND-2
14-20
ND-6.8
ND-<10
ND
NR
NR
ND-<100
NR
ND
NR
NR
No. Contam.*
No. Tested
several
samples
2/2
6/6
1/2B
0/10
28/58
2/30
23/42
12/30
1/7
490/490
NR
Reference
Dow, 1978
Firestone
et al.. 1972
Buser and
Bosshardt, 1976
Wool son et al.,
1972
Norstrom
et al.. 1979
Cochrane
et al.. 1981
Thomas. l?80a;
Harless. 1981
Wool son et al.,
1972
ACP, 1980
Wool son et al.,
1972
Young, 1983
Young, 1983
aThese are the ratios of the number of samples contaminated with any chlorodloxlns to the number of samples tested.
D2,3,7,8-1somer detected but not quantified
CTCP: trlchlorophenol
"These Indicate specific dloxln concentrations.
ePCP: pentachlorophenol
'These are dlchlorophenoxy-acetlc, -butyric acid and -proplonlc acid.
9The Isomers could not be separated.
"This Is 2,4,5-trlchlorophenosy acetic acid.
'This Is an average value.
ND « Not detected; NR -- Not reported; D - detected; NA = Not available
-------
and pesticide/herbicide plants, cooling towers, pulp and paper Industry,
Incinerators and disposal sites are potential exposure areas for PCDDs
(Josephson, 1983).
The locations of current and former producers and formulators of
chlorophenols are presented 1n Table 4-2. The Inclusion of the locations of
the former producers has been judged necessary for the Identification of
past sources of contamination that may present an environmental hazard 1n
the future (I.e., airborne contaminated dust particles) because of the
environmental persistence of 2,3,7,8-TCDD (Chapter 5).
4.3.1.2. PRODUCTION OF CHLOROPHENOL DERIVATIVES — PCDDs have been
detected also as contaminants produced during the manufacture of commonly
used chlorophenol derivatives, such as 2,4-D, 2,4,5-T and hexachlorophene by
mechanisms hypothesized to be similar to those discussed In the case of
chlorophenols. The amounts of 1,3,6,8- and 1,3,7,9-TCDD 1n commercial 1so-
octyl-, mixed butyl- and propylene glycol butyl ether ester of 2,4,-D varied
from nondetectable to 8.7 rag/kg (Cochrane et a!., 1981). Agent Orange,
which 1s a 1:1 mixture of the butyl esters of 2,4-D and 2,4,5-T, has been
shown to contain 2,3,7,8-TCDD 1n quantities 1n the range of 0.1-47 jig/g
(Rappe et a!., 1979). The 2,3,7,8-TCDD Impurity 1n Agent Orange has been
shown to originate from 2,4,5-T. The mean levels of 2,3,7,8-TCDD In Agent
Orange and Agent Purple (5054 n-butyl 2,4-D, 30% n-butyl 2,4,5-T and 20%
Isobutyl 2,4,5-T) preparations used 1n the 1960s were shown to be 1.98 and
32.8 ppm, respectively (Young, 1983). Efforts were made during the 1970s to
control and minimize the formation of 2,3,7,8-TCDD and, at the present time,
all the producers claim that their products contain <0.1 ng/g of 2,3,7,8-
TCDD (Rappe et a!., 1979).
4-9
-------
TABLE 4-2
Locations of Companies that have been Major Producers and Formulators
of Chlorophenols and Their Derivatives3
Chemical Producer
2,4-D Add and Esters Alco Chemical Corp., Philadelphia, PA
*Amvac-Chem1cal Corp., Los Angeles, CAb
Chempar, Portland, OR
*D1amond Shamrock Corp., Tuscaloosa, AL
Cleveland, OH
Diamond Alkali, Newark, NJ
*Dow Chemical, U.S.A., Midland, MI
Fallek-Lankro Corp., Tuscaloosa, AL
GAF, Linden, NJ
*Guth Corp., Hillside, IL
Hercules, Inc., Jacksonville, AR
Imperial, Inc., Shenandoah, IA
MUler Chemical, WhHeford, MD
Monsanto, Co., Sauget, IL
North American Phillips Corp., Kansas City, KS
*PBI-Gordon Corp., Kansas City, KS
Rhodla, Inc., Portland, OR
St. Paul, MN
St. Joseph, MO
*Rhone-Poulenc, Inc., Portland, OR
*R1verdale Chemical Co., Chicago Heights, IL
Rorer-Amchem, Fremont, CA
St. Joseph, MO
Thompson Chemical, St. Louis, MO
Union Carbide Corp., Ambler, PA
*Vels1col Chemical Corp., Beaumont, TX
Bayport, TX
Vertac, Inc., Jacksonville, AR
Woodbury, Orlando, FL
2,4,5-T Chempar, Portland, OR
Diamond Shamrock, Cleveland, OH
Dow Chemical, U.S.A., Midland, MI
Hoffman-Taft, Inc., Springfield, MO
Monsanto Co., Sauget, IL
North American Phillips Corp., Kansas CHy, KS
PBI-Gordon Corp., Kansas CHy, KS
Rhodla Inc., Portland, OR
St. Joseph, MO
*R1verdale Chemical Co., Chicago Heights, IL
Rorer-Amchem, Ambler, PA
Fremont, CA
4-10
-------
TABLE 4-2 (cont.)
Chemical
Producer
2,4,5-T (cont.)
2,4,5-T derivatives
Sllvex esters and salts
Ronnel
Erbon
Hexachlorophene
2,4,5-TCP and salts
2,3,4,6-Tetrachlorophenol
PCP and salts
Rorer-Amchem, St. Joseph, HO
Jacksonville, AR
Thompson Chemical, St. Louis, MO
Union Carbide Corp., Fremont, CA
St. Joseph, HO
Ambler, PA
Vertac, Inc., Jacksonville, AR
Dow Chemical U.S.A., Midland, HI
Hercules, Inc., Jacksonville, AR
North American Phillips Corp., Kansas CHy, KS
*R1verdale Chemical Co., Chicago Hts., IL
Vertac, Inc., Jacksonville, AR
*Dow Chemical U.S.A., Midland, MI
*Dow Chemical U.S.A., Midland, MI
Glvaudan Corp., Clifton, NJ
Diamond Shamrock Corp., Cleveland, OH
Dow Chemical, U.S.A., Midland, HI
GAF Corp., Linden, NJ
Hercules, Inc., Jacksonville, AR
Hooker Chemical, Niagara Falls, NY
Merck and Co., Inc., Rahway, NJ
Nalco Chemical Co., Chicago, IL
North Eastern Pharmaceuticals, Verona, MO
Roberts Chemical, Inc., N1tro, WV
Rhodla, Inc., Monmouth Junction, NJ
Vertac, Inc., Jacksonville, AR
Dow Chemical U.S.A., Midland, MI
Sanford Chemical, Port Neches, TX
J.H. Baxter and Co., San Hateo, CA
Dow Chemical U.S.A., Midland, MI
ICC Industries, Inc., Dover, OH
Monsanto Co., Sauget, IL
Nalco Chemical Co., Chicago, IL
*Re1chhold Chemical, Inc., Tacoma, WA
Sanford Chemical, Port Neches, TX
*Vulcan Materials Co., Wichita, KS
Sources: U.S. EPA, 1980a; SRI, 1982; USITC, 1982
^Company names Indicated with an asterisk are the major producers of
chlorophenols and their derivatives at the present time.
4-11
-------
As can be seen from Table 4-1,, 2,4-D, 2,4,5-T and their formulated
products may contain other PCDDs 1n addition to TCDDs. It has also been
reported that Agent Orange and 2,4,5-T samples used during the Vietnam con-
flict contain other PCDDs at levels similar to that of 2,3,7,8-TCDD. Agent
Orange and European 2,4,5-T formulations from the 1960s, on the other hand,
may contain primarily 2,3,7,8-TCDD and only minor amounts of other PCDDs
(Rappe et al., 1979). The average 2,3,7,8-TCDD contents 1n Agent Orange and
Agent Purple given 1n Table 4-1 refer to these materials manufactured 1n the
1960s.
Hexachlorophene 1s prepared from the same starting material as 2,4,5-T,
namely, 1,2,4,5-tetrachlorobenzene. Because of additional purification,
however, the level of 2,3,7,8-TCDD 1n this product has been reported to be
<0.03 yg/g (Rappe et al., 1979).
The locations of current and former producers of chlorophenol deriva-
tives have been shown 1n Table 4-2.
4.3.1.3. CONTAHINATED MANUFACTURING EQUIPMENT — Production trains
are often used for the production of chemicals whose manufacture necessi-
tates the use of similar process equipment. In the manufacture of chemicals
on a production train previously contaminated with PCDDs, both the products
and waste generated can be contaminated with PCDDs. Thus, the manufacture
of 2,4-D, which otherwise was not expected to be contaminated with 2,3,7,8-
TCDD, did Indeed contain 2,3,7,8-TCDD because the equipment used had been
employed previously to produce 2,4,5-T, and the equipment remained contami-
nated with 2,3,7,8-TCDD (Federal Register, 1980a).
4-12
-------
4.3.1.4. DIPHENYL ETHER HERBICIDES ~ The presence of TCDDs, PeCDDs
and HxCDDs as contaminants 1n dlphenyl ether herbicides was reported by
Yamag1sh1 et al. (1981). The source of PCDDs 1n these herbicides was specu-
lated to be the trlchlorophenol used 1n their production. The concentra-
tions of the two major Impurities, TCODs and PeCDDs, 1n commercial formula-
tions were -150 and 30 ppm, respectively. The 1somer1c distribution of
TCDDs showed that the major components were 1,3,6,8- and 1,3,7,9-lsomers.
The Isomer 2,3,7,8-TCDD was not detected 1n the commercial products.
4.3.1.5. INCINERATION OF SELECTED INDUSTRIAL WASTES — The combus-
tion of a variety of chlorinated hydrocarbons has been shown to produce
PCDDs (Tlernan et al., 1982a). The formation of PCDDs would likely occur 1n
Incinerators operating at 750-900°C; chlorophenols are probably the precur-
sors of PCDD formation. At temperatures >1200-1400°C and residence time of
<1 second, PCDDs are Hkely to decompose and these compounds are not
expected to form (dunk and Richard, 1981). From kinetic and thermodynamlcal
considerations, Shaub and Tsang (1983) estimated that 99.99K gas phase dis-
sociation of tetrachlorod1benzo-p_-d1ox1ns at 727*C may require -15 minutes,
while the same decomposition at 977°C may require <1 second.
In an Industrial boiler 1n the United States where PCP was known to have
been burned, Rappe et al. (1983b) reported ~5 ppm PCDDs 1n the bottom and
baghouse ash. More than 90H of the PCDDs were lower chlorinated congeners
than OCDD and only a small amount, of 2,3,7,8-TCDD was detected. Soot
analysis of a recent transformer fire In Blnghamton, NY, 1n February, 1981,
revealed that 2,3,7,8-TCDD (0.6 ppm) and 1,2,3,7,8-PeCDD (2.5 ppm) were the
dominating Isomers of the PCDDs formed (Buser and Rappe, 1983; Rappe et al.,
1983b). The origin of the PCDDs was probably the chlorobenzenes 1n the
transformer oil (Buser, 1979). The analysis of wipe tests from a garage
4-13
-------
adjacent to this site did reveal the presence of PCDDs before cleaning the
garage. Following the cleanup, no contamination was found (Tlernan et a!.,
1982b; Tlernan, 1983). Therefore, 1t 1s Important to recognize the possi-
bility of production of PCDDs and PCDFs 1n fires Involving PCB and chloro-
benzene transformers.
4.3.2. Municipal Incinerators. PCDDs have been detected both 1n the fly
ash and air partlculate matter from municipal Incinerators by several
Investigators 1n Canada, Europe and the United States. The partlculate
matter forming the emissions (air partlculates) has a 10-fold greater con-
centration of PCODs than the precipitated material (fly ash) (Lustenhouwer
et a!., 1980). The concentration of total TCODs, PeCDDs and HxCDDs In the
fly ash from a variety of municipal Incinerators 1n Canada, Europe and the
United States have been studied by several authors (Elceman et a!., 1979,
1980; Nestrlck et a!., 1982; Karasek et a!., 1982; Bumb et al., 1980; Buser
and Bosshardt, 1978; Tlernan et al., 1982a; Taylor et al., 1983). The TCDD
Isomer known to be the most toxic (I.e., 2,3,7,8-TCDD) was either not
detected or detected at a low level. The quantities emitted 1n Incinerators
vary, probably because of differing efficiencies, and since few municipal
Incinerators have been reliably characterized for PCDD/PCDF emissions over
extended time Intervals, the data base 1s still Inadequate. Whereas Bumb et
al. (1980) and Buser and Rappe (1980) detected 0.4 ng/g of 2,3,7,8-TCDD In
the fly ash from a United States municipal Incinerator, the U.S. EPA con-
cluded that emissions from five municipal waste combustors did not present a
public health hazard for residents living 1n the Immediate vicinity (CEQ,
1981). Evaluation of stack emissions of PCDDs have to be based on the
amount of dloxlns 1n both the flue gas condensate followed by an effective
absorption or adsorption step (Ballschmlter et al., 1984). PCDDs have been
4-14
-------
detected 1n the emissions of some municipal waste Incinerators 1n Europe
(G1zz1 et al.. 1982; Benfenatl et al., 1983; Taylor et al., 1983; OHe et
al., 1982, 1983; Lustenhouwer et al.. 1980; Barnes, 1983). Observations on
PCDD emissions from an Industrial boiler have been discussed 1n Section
4.3.1.5. (Rappe et al., 1983b).
In a study of municipal fly ash conducted between a single Incinerator
1n the United States and one In Europe, Lamparskl and Nestrlck (1980)
detected at least 14 of the 22 possible TCDD Isomers. Although the ratio of
Isomers to the total present were similar 1n both fly ashes, their absolute
amounts varied by a factor >10. It has been demonstrated by Rappe et al.
(1979) that minor amounts of the highly toxic PCDD congeners, 1,2,3,7,8-
PeCDD, 1,2,3,6,7,8-HxCDD and 1,2,3,7,8,9-HxCDD, are also formed 1n municipal
Incinerators.
4.3.3. Other Combustion Processes. Scientists from Dow Chemical Co.
(Dow, 1978) reported the detection of PCDDs In partlculate matter from most
combustion sources. These findings led to a hypothesis which suggested that
PCDDs may be formed 1n trace amounts from chemical reactions during the com-
bustion of many chlorinated hydrocarbons (Bumb et al., 1980; Crummett et
al., 1981). These Investigators detected PCDDs Including TCDDs and HxCDDs
1n partlculate matter from municipal and Industrial Incinerators, 1n
mufflers from dlesel truck and passenger vehicles, from home wood-burning
fireplaces and from soot and cigarette smoke. Since the trace chemistries
of fire hypothesis was presented, several Investigators have attempted to
test 1t. Tlernan (1982) reported the detection of 0.65 ppb TCDD 1n soot
from a wood-burning fireplace. Although there Is general agreement regard-
Ing the production of PCDDs from the burning of wood with additional HC1 and
from Incinerators burning chlorinated products or wastes (Tlernan et al.,
4-15
-------
1982a, Tlernan, 1983), production from the combustion of coal and hydro-
carbons (such as occurs 1n gas burners, and auto and truck engines) has not
been confirmed (NRCC, 1981a). For example, Rappe et al. (1979) concluded
from their pyrolysls experiments that PCDDs are produced by the burning of
very specific chemicals, such as chlorinated phenols, polychlorlnated
benzenes and polychlorlnated dlphenyl ethers. Wood pregnated with these
compounds might produce PCDDs during Incineration and the history of wood to
be burned In fireplaces 1s often unknown. Junk and Richard (1981) and
Klmble and Gross (1980) failed to measure TCDD above the detection limits of
1 or 1.2 ppt, respectively, from their analysis of one fly ash sample from
stack emissions of a low sulfur and h1gh-ash coal burning power plant.
Recent Investigations (Halley et al., 1983; Stanley et al., 1982) also
failed to detect (detection limit: flue gas, 100-700 pg/m3; fly ash,
10-70 pg/g) PCDD homologues 1n any sample from four coal-fired power plants.
Independent confirmation of "trace chemistries of fire" as proposed by Dow,
U.S.A., 1s not yet available.
Czuczwa and HHes (1984) and Czuczwa et al. (1984, 1985) analyzed for
the PCDDs and PCOFs In sediments from the Great Lakes Including the sediment
core from Slsklwlt Lake of Isle Royale 1n northern Lake Superior. The
sediment that came from Slsklwlt Lake was used because 1t received only
atmospheric Inputs. In all cases the authors detected the flux of PCDDs and
PCDFs, which began at about 1940. When this "1940 horizon" was compared
with combustion trends 1n the last century, the authors found evidence that
the combustion of synthetic chlorinated organic chemicals Is the primary
source of PCDDs and PCDFs. Furthermore, the authors responded that the flux
of PCDDs and PCDFs to three Swiss lakes, where combustion has been extensive
during the last century, Increased only after the development of the
4-16
-------
chlorinated organic chemical Industry. The authors also addressed the
debate regarding 2,3,7,8-TCDD In coal fly ash. Reaffirming similar find-
Ings, no 2,3,7,8-TCDD was found above a detection limit of 100 ppt. These
results strongly suggest that coal combustion 1s not a significant source of
2,3,7,8-TCDD contamination to the environment.
4.3,4. Chemical Dump Sites. At present, other potential sources of PCDDs
are chemicals known to be contaminated with PCDDs but withdrawn from use and
awaiting disposal, and disposal sites where chemical wastes containing PCDDs
have been dumped. It has been estimated that -11,600 metric ton/year of
hazardous wastes are produced 1n the manufacture of chlorophenols and
-79,000 metric ton/year are produced 1n the manufacture of phenoxy compounds
(Jett, 1982). Process wastes from the manufacture of chlorophenols and
phenoxy compounds are landfUled, or Injected Into deep-well. Treatment
wastes are frequently subjected to on-s1te Impoundment (Jett, 1982). Recent
Canadian environmental data Indicate that 2,3,7,8-TCDD may be leaking Into
the Great Lakes from toxic dump sites (Hallett, 1984).
4.3.5. Photochemical Process. Photochemical processes can also lead to
formation of PCDDs. For example, the d1mer1zat1on of chlorophenols to OCDD
has been studied by Crosby and Wong (1976). Lamparskl et al. (I960) also
reported that photolysis of PCP-treated woods may lead to the formation of
PCDDs. Similarly, photochemical cycHzatlon of predloxlns (chlorinated
2-phenoxyphenols, precursors of PCDDs) can also produce PCDDs. Since pre-
dloxlns are common Impurities (1-5%) 1n commercial chlorophenols, exposure
of chlorophenols containing those Impurities to light may produce PCDDs
(NHsson et al., 1974).
Another photochemical process of potential environmental Importance 1s
the formation of highly toxic TCDD and PeCDD congeners from the dechlorl-
4-17
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nation of higher PCDDs. However, photolysis of 1,2,3,6,7,8-HxCDD and
l,2,3,7,8,9HxCDD produced only 13% of the toxic 1,2,3,7,8-PeCDD and no
2,3,7,8-TCDD (K1m et al., 1975), while the photolysis of octa-CDD was shown
to produce mainly 1,4,6,9-TCDD, 1,2,4,6,9-PeCDD and 1,2,4,6,7,8-HxCDD. Con-
sequently, 1t was concluded that the most toxic Isomers are not likely to be
formed from the photolysis of the higher PCOOs (Buser and Rappe, 1978).
Formation of tetra- and pentachlorodlbenzo-p-dloxlns has been observed
by the photolysis of 1,2,3,6,7,8- and 1,2,3,7,8,9-HxCDOs (Buser, 1979).
There seems to be a preferential dechlorlnatlon of the HxCDDs occurring at
the lateral positions flanked on both sides by adjacent chlorines (Choudhry
and Hutzlnger, 1984). However, formation of trace amounts of 2,3,7,8-TCDD
were also observed from the photolysis of the above two Isomers of HxCDDs
(Buser, 1979).
4.4. RELATIONSHIP BETWEEN SOURCES AND CONTAMINATION IN ENVIRONMENTAL
HATRICES
The potential relationship between various sources of PCDDs and the
environmental matrices where these compounds have been detected (NRCC,
1981a) 1s depicted 1n Figure 4-2, which has been modified from the original
reference to Indicate the possible Inhalation exposures from these sources.
4.5. ENVIRONMENTAL LEVELS
The detection of PCDD residues, particularly the residue of the four
toxic PCDDs under discussion, 1n various environmental matrices 1s Indica-
tive of the potential Impact that the various sources could have on the
environment. However, the monitoring efforts for the determination of the
levels of these compounds In the environment are extremely limited for sev-
eral reasons. The primary reasons are the nonavailability of standardized
sampling methods and the specialized analytical techniques that must be used
for the determination of traces of these difficult to separate compounds 1n
4-18
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mammals
wood preservation plants effluents
receiving waters -biota
chlorophenol-treated woods
food stored in CP treated bins
food
wood shaving for bedding
food
Industrial plants"
air • ». mammals
-••receiving waters—•—
biota
alr:
•mammals
municipal incinerators!
precipitated fly ash in receiving waters -biota
fly ash disposal site
air.
mammals
biocide formulated products
receiving waters
biota
kraft pulp mill
•affluents
receiving waters
biota
.skin scrapings
animals
leather tannery
effluents
receiving waters
biota
unidentified
receiving waters
biota.
FIGURE 4-2
Possible Potential Relationship Between Various Sources of PCDDs
and the Environmental Matrices Where PCDDs have been Detected
Source: Modified from NRCC, 1981a
4-19
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the presence of a large number of Interfering compounds. Measurable quanti-
ties of these compounds have been detected 1n the environment under special
circumstances, that 1s, after accidents 1n factories producing chlorophenols
and their derivatives, 1n the environment after certain herbicide use, and
1n the environment near certain dumpsltes. In other words, the current
available data demonstrate that the major sources of PCDDs 1n the environ-
ment are those associated with the production, use and disposal of chloro-
phenols and their derivatives, Choudhary (1983) 1n a review paper provided
a list for some of the potential workplaces where occupational exposure to
PCODs may occur. It should also be recognized that most of the environ-
mental monitoring Investigations measured 2,3,7,8-TCDD levels, whereas mon-
itoring data for other PCDDs are even more limited. WHh these limitations
1n mind, the levels of 2,3,7,8-TCDD, 1,2,3,7,8-PeCDD, 1,2,3,6,7,8-HxCDD and
l»2t3,7»8,9-HxCDD 1n various environmental media have been presented 1n the
following subsections.
4.5.1. Water. NAS (1977) reported that no 2,3,7,8-TCDD has ever been
detected 1n drinking water using methods with limits of detection In the ppt
range. Other PCDDs Including PeCDD and HxCDD have not been detected 1n
drinking water. However, TCDD, Including the 2,3,7,8-lsomer, has been
reported 1n aqueous Industrial effluent samples and leachates from hazardous
waste disposal sites. For example, Van Ness et al. (1980) analyzed eight
effluents from a trlchlorophenol manufacturing plant site and detected TCDD
1n two of these effluents (detection limit 10-30 pg/g). The concentrations
of TCDD 1n the two samples with detectable TCDD concentrations were 17 and
100 pg/g. Although the specific Isomer was not routinely separated, the
authors concluded from their study that a significant portion of the TCDD
was apparently the 2,3,7,8-lsomer.
4-20
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Jhe analysis of leachate samples from two waste disposal sites for the
analysis of TCOD have also been reported. In one study, 23 water samples
analyzed by Wright State University Inside and outside of a waste disposal
site near Jacksonville, AR (containing wastes from 2,4-D and 2,4,5-T manu-
facture) were found to contain 2,3,7,8-TCOO (Thlbodeaux, 1983). The concen-
tration of 2,3,7,8-TCOD 1n these samples averaged 14 ppt with a concentra-
tion range of none detected to 47 ppb. In another study (U.S. EPA, 1982b),
two untreated leachate samples collected from the Love Canal, NY, chemical
dump site showed a concentration of 1.56 ppb (1560 ppt) for 2,3,7,8-TCDO.
The treated leachate (samples taken after remedial steps were Installed to
minimize PCOD-leachlng possibility), on the other hand, showed no detectable
level of 2,3,7,8-TCDD (detection limit 5-10 ppt). 2,3,7,8-TCDD was not
detected 1n any of the groundwater samples analyzed.
Shadoff et al. (1977) analyzed for 2,3,7,8-TCDD 1n two locations exposed
annually to 2,4,5-T. These locations were an Impoundment from the drainage
of a watershed In Texas where 2,4,5-T had been used for several years for
brush control and from a pond 1n Arkansas used as a reservoir for Irrigating
rice fields treated with 2,4,5-T. Two water samples from each location
failed to show any detectable level of 2,3,7,8-TCOD at a detection limit of
0.1-0.2 ppt.
4.5.2. A1r. One possible source of PCDDs 1n the atmosphere 1s the field
spraying of the herbicide 2,4,5-T. The spraying of 2,4,5-T containing
2,3,7,8-TCDD Impurity may lead to a concomitant exposure to 2,3,7,8-TCDD.
However, the measurement of air concentration at any particular time after
spraying may not be a representative sample because of spray drift to non-
target sites and the Intermittent nature of spray application. From mlcro-
agroecosystem chamber and field studies, Nash and Bean (1980) determined
4-21
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the atmospheric concentration of 2,3,7,8-TCDD at various times after the
application of emulsified and granular SHvex (1.3-2.0 kg/ha SHvex) con-
taining 44 ppb to 15 ppm TCDD Impurity. Using trltlated 2,3,7,8-TCDD, these
authors found that atmospheric concentrations of 2,3,7,8-TCDD decreased with
time either at an exponential rate (granular formulation) or at a log log
rate (emuls1f1able formulation) 1n chambers. The emuls1f1able formulation
resulted 1n considerably higher TCDD concentrations (~1000-fold or more) 1n
air than 1n granular formulation Initially, but with time (200 days)
approached the concentrations 1n air similar to the granular formulation (10
fg/ma; fg = 10~xs g). In a small field trial, with a nonshaded plot,
TCDD concentrations 1n air from the application of 2 kg/ha of emulslflable
SHvex containing 15 ppm TCDD were about twice (620 fg/m3) that of a
shaded plot (270 fg/m3) on the treatment day, but only -33% of the amount
from the shaded plot on the second day. Presumably, this was a result of
the lesser quantities (<50%) of TCDD remaining on the grass for volatiliza-
tion during the second day.
A1r filter samples collected from Elizabeth, NJ, after an Industrial
fire on April 22, 1980, were analyzed for TCDD by Harvan et al. (1981).
Col!1s1on-1nduced-d1ssoc1at1on mass-analyzed 1on kinetic energy spectrometry
was used for the confirmation of the presence of TCDD. Of the nine samples
analyzed by these authors, one contained 20 pg of TCDD, four contained <9 pg
of TCDD, and four others probably contained 5-12 pg of TCDD. However, the
concentration of TCDD 1n the air cannot be given for these samples because
the air volumes corresponding to the filters analyzed were not specified by
the Investigators.
The atmospheric concentrations of TCDD near two hazardous waste sites
have been monitored. In one study, U.S. EPA (1982b) failed to detect
4-22
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(detection 7fmft 7-20 ppt) any 2,3,7,8-TCDD 1n the atmosphere at the Love
Canal, NY, area. In another study of a waste disposal site near Jackson-
ville, AR, an average concentration of 1100 ppt of TCOD 1n two air partlcu-
late samples collected near the disposal site was reported (Thlbodeaux,
1983).
The levels of 2,3,7,8-TCOD 1n atmospheric dust were monitored In the
Seveso, Italy, area between 197? and 1979. The concentrations of 2,3,7,8-
TCDD were found to be 1n the range of 0.06-2.1 ng/g of dust with dustfall
jars as sample collection technique and 0.17-0.50 ng/g of dust with high
volume sampler as sample collection technique (DIDomenlco et a!., 1980b).
The accident 1n Seveso released only 2,3,7,8-TCDD, while most other environ-
mental sources may produce a mixture of PCDDs.
Another source of atmospheric emission of PCDDs 1s Incineration (61zz1
et a!., 1982; Benfenatl et a!., 1983; Taylor et a!., 1983; OHe et al.,
1982, 1983; Lustenhouwer et al., 1980; Barnes, 1983). The concentrations of
TCDD, PeCDD and HxCDD 1n fly ash from Canadian municipal Incinerators have
been studied extensively by Elceman et al. (1980, 1981). Elceman et al.
(1979) also determined the TCDD levels 1n fly ash from Incinerators 1n Japan
and the Netherlands. The average concentrations of the PCDDs 1n the
Canadian studies (Elceman et al., 1979, 1980, 1981) were estimated with the
assumption that the SIM response factors for all the PCDDs were the same as
the response factor from 1,2,3,4-TCDD used as a standard. However, the
analytical method used by these authors has been criticized by Nestrlck et
al. (1982). Recently, Karasek et al. (1982) also determined the total TCDD,
PeCDD and HxCDD levels 1n a French municipal Incinerator to be none detect-
ed, 7.8 and 21.8 ng/g, respectively. It was also concluded by these authors
that the PCDDs tend to concentrate 1n particles of lower mean size (30 ym
vs. >850 pm).
4-23
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In another study, Bumb et al. (1980) studied the PCDD level 1n fly ash
from a municipal Incinerator 1n Nashville, TN, several European municipal
Incinerators, and the Industrial Incinerators of the Dow Chemical Co. facil-
ity 1n Midland, HI. The TCDD concentrations were determined to be 7.7 ng/g
(0.4 ng/g of 2,3,7,8-TCDD), 2-20 ng/g and 0-38 ng/g (2,3,7,8-TCDD not
detected), respectively. The corresponding values of HxCDD were reported to
be 14, 30-200 and 1-20 ng/g. However, the analytical method used by these
Investigators has been criticized by other Investigators (Hay, 1979). Buser
and Rappe (1983) and Buser and Bosshardt (1978) also analyzed the fly ash
from Incinerators 1n Switzerland and Canada. In one such study (Buser and
Bosshardt, 1978), the total amount of PCDDs In the fly ash from a Swiss
municipal and Industrial Incinerator were found to be 0.2 and 0.6 ppm,
respectively. The dloxln Isomers known to be most toxic, namely 2,3,7,8-
TCDD, 1,2,3,7,8-PeCDD, 1,2,3,6,7,8-HxCDD and 1,2,3,7,8,9-HxCDD, were only
minor constituents of the total dloxlns found. In another study (Buser and
Rappe, 1983), the presence of TCDDs (3 ppb), PeCDDs (20 ppb) and HxCDDs (50
ppb) was Indicated 1n the fly ash from a municipal Incinerator 1n Zurich,
Switzerland. The TCDD, PeCDD and HxCDD Isomers with substitution at
2,3,7,8- positions, such as 2,3,7,8-TCDD, 1,2,3,7,8-PeCDD and 1,2,3,6,7,8-,
1,2,3,7,8,9- and 1,2,3,4,7,8-HxCDD were present only as 2, 14 and 24% of the
total TCDDs, PeCDDs and HxCDDs. A fly ash sample from Ontario, Canada, was
also found to contain TCDDs (150 ppb), PeCDDs (550 ppb) and HxCDDs (900
ppb). Although the sample was reported to contain significantly higher
levels of PCDDs 1n comparison with the Swiss fly ash sample, 1t showed
similar proportions of 2,3,7,8-substHuted PCDDs (4, 12 and 21% of the total
TCDDs, PeCDDs and HxCDDs, respectively). It Is not yet known whether the
4-24
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higher levels of PCDDs result from different Incinerator operating condi-
tions, different feed stock or different fly ash collection conditions
(Buser and Rappe, 1983). Similarly, the fly ash from a municipal Incinera-
tor 1n the United States showed the presence of at least 11 TCOD Isomers,
but 2,3,7,8-TCOD was found to be a minor product (U.S. EPA, 1980a).
The U.S. EPA evaluated the magnitude and significance of TCOD emissions
from combustion processes. In 1981, the U.S. EPA sampled five municipal
waste combustors and concluded that emissions from these waste combustors do
not present a public health hazard for residents living 1n the Immediate
vicinity (CEQ, 1981). In view of the recent data of Pocch1ar1 et al. (1983)
reporting the presence of 1,3,6,8- and 1,3,7,9-TCDO (0.4-2 ppt) 1n eplgeal
parts of a large number of plants grown 1n the proximity of municipal
Incinerators, and the lexicological evaluation of TCDD 1n ashes from urban
Incinerators (Bronzettl et al., 1983; R1zzard1n1 et al., 1983), the question
of health hazard for residents living 1n the Immediate vicinity of municipal
Incinerators needs further evaluation. Another reason for the presence of
1,3,6,8- and 1,3,7,9-TCOD 1n eplgeal parts of plants may also be due to
contamination by TCDD-conta1n1ng herbicide or pesticide application, as
observed by Yamag1sh1 et al. (1981).
4.5.3. Soil. The levels of PCODs 1n soil, sediment and dust samples are
presented 1n this subsection. In general, the PCODs have been detected 1n
the samples that originated from the areas around certain Industrial sites,
waste disposal sites, and sites Involved 1n accidental or unintentional
spillage of chemicals containing PCDD contaminants. Very few Investigators
determined the levels of other PCOOs besides TCOO. Even 1n the case of
TCOD, the specific Homer Identification was not performed 1n many cases.
The levels of TCDD 1n different son, sediment and dust samples are shown 1n
Table 4-3.
4-25
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TABLE 4-3
Levels of TCDD In Soils and Sediments from Different Locations
1
NJ
Sample Type
Soils
Sediments
Soils
Sediments
Sediments
Soils
Soils
Soils
Sediments
Soils
Soils
Sampling Site
Love Canal , NY
Love Canal, NY
Love Canal, NY
Love Canal, NY
Love Canal, NY
NR
Eastern Missouri,
U.S.A.
Seveso, Italy
canal north of
Amsterdam
Seveso, Italy
Jacksonville, AR
Sample History
waste disposal
site
sediments from
storm sewers and
creeks near water
disposal site
soils collected
away from source
of contamination
sediments from
storm sewers
sediments from
simp
sample originated
from an Industrial
site
sample originated
from contaminated
horse arena
sample originated
from ICMESA plant
accident site
sample originated
from a dump site
sample originated
from ICEMSA plant
accident site
waste disposal
site
Concentration
Total TCDD
<0. 0025-6. 7 ppb
NR
NR
NR
NR
ND (20-2300 ppt)a-
559 ppb
NR
NR
NR
NR
NR
In Sample
2,3,7,8-TCDD
NR
0.9-312 ppb
ND (-1-20 ppt)a
ND {1-20 pptp-
672 ppb
ND {1-20 ppt)*-
9570 ppb
NR
detected0
detected0
55-5062 ppt
<5-20,000 iig/m»
ND-2.9 ppb
Reference
Smith et al.,
1983b
Smith et al.,
1983b
U.S. EPA, 1982b
U.S. EPA, 1982b
U.S. EPA, 1982b
Van Ness
et al., 1980
Buser and
Rappe, 1980
Suser and
Rappe, 1980
Helda. 1983
DIDomenlco
et al., 1980c
Thlbodeaux,
1983
-------
TABLE 4-3 (cont.)
#»
i
Concentration In Sample
Sample Type
Sediments
Soil/sludge
Soils
Soil/dust
Soil/dust
Soils
Soils
Sampling Site
Jacksonville, AR
Love Canal, NT
unspecified
Midwestern
community In
U.S. A
Midland, HI
Urban U.S. areas
Northwest Florida
Eastern Missouri
Sample History
sediments from
pond and creek
near waste
disposal site
waste disposal
site
sample near a
wire reclamation
Incinerator
sample Inside
Industrial site
no obvious source
of contamination
Eglln A1r Force
test site
horse breeding
arena sprayed
with waste oil
Total TCOO
NR
0.3-199 ppb
NO (<3 ppt)«-
0.021 ppb
1-1 20* ppb
1-4d ppb
NO (1-10 pptH1-
0.03 ppbc
NO (1-10 ppt»a-
0.04 ppba
0.010-0.70 ppbf
12.3 ppbfl
31.8-33.0 ppm
Reference
2,3,7,8-TCOD
NO-22.1 ppb Thlbodeaux,
1983
NR Tlernan, 1982
NR Hryhorczuk
et al., 1981
0.3-100= ppb Bumbetal..
0.7-3d ppb 1980
NR* Bumb et al.,
1980
NR Cockerham
et al., 1980
NR Carter et al,,
1975
aNot detected and the detection limit Indicated within parentheses
Value not quantified
c»alue for soil
dValue for dust
eOust sample from St. Louis, MO, area showed 0.12 ppb 2,3,7,8-TCDO.
This Is the soil residue after 10 years of periodic aerial spraying of 2,4-0 and 2,4,5-T.
'This Is the soil residue Immediately after spraying.
NR * Not reported; NO = Not detected
-------
It Is obvious from Table 4-3 that the waste disposal site 1s respon-
sible for the origin of 2,3,7,8-TCDD In the Love Canal, NY, area. This 1s
reflected by the high level of 2,3,7,8-TCDD found 1n sediments from sump and
1n sediments from storm sewers and creeks near waste disposal sites. The
reported levels of 2,3,7,8-TCDD 1n soil and sediment samples near the
Jacksonville, AR, waste disposal site are such that this site requires care-
ful reexamlnatlon. It can also be concluded from Table 4-3 that the envi-
ronment Inside a manufacturing (2,4,5-trlchlorophenols and derivatives) site
are likely to be contaminated with 2,3,7,8-TCDD by levels that may be higher
than the background level (sites with no obvious sources of contamination).
4.5.4. Foods and Biological Samples. The occurrence of PCDDs In foods
could result from the following: 1) spraying of certain grain crops with
PCDD-contam1nated herbicides, such as SHvex and 2,4.5-T; 2) consumption by
livestock of PCDD-contamlnated forage; 3) magnification of residues through
the food chain; or 4) consumption of fruits and vegetables In the proximity
of municipal Incinerators. Besides determining the PCDD levels 1n food
chains, this subsection will discuss the levels of these compounds 1n wild-
life and 1n human tissues (I.e., urine and milk). The detection of these
compounds In wildlife and human tissue collected near Industrial or waste
disposal sites can be taken as an Indication of anthropogenic exposure.
Sometimes the tissue levels can be used to estimate the extent of exposure
and subsequent excretion and/or accumulation of these compounds.
The detection of 2,3,7,8-TCDD has been reported In locally grown garden
fruit and vegetables following the ICMESA accident 1n Seveso, Italy, In 1976
(FanelH et al., 1982; Cocucd et aU, 1979; Pocch1ar1 et a!., 1983; W1pf et
a!., 1982). Studies with either the seeds or the mature plants of soybeans
or oats showed that 2,3,7,8-TCDD was neither absorbed by the seeds after
4-28
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spraytttg nor taken up from the son Into the mature plants (Isensee and
Jones, 1971; Matsumura and Benezet, 1973). However, young plants accumu-
lated up to 40 ppb of 2,3,7,8-TCDD {Isensee and Jones, 1971). From the
analysis of several parts of fruit trees and kitchen-garden plants such as
carrots, onions, potatoes and narcissuses collected from the contaminated
(400-lOdO yg/m2 of 2,3,7,8-TCDD In soil) Seveso area 1n Italy, Cocucd
et al. (1979) concluded that 2,3,7,8-TCOD 1s translocated from soil to the
aerial parts of the plants, probably through the conductive vessels. This
study further suggested that the plants may eliminate 2,3,7,8-TCDD by an
unknown mechanism within 4-10 months after transplantation In unpolluted
soils. However, the study of Cocucd et al. (1979) contradicts the Investi-
gations of W1pf et al. (1982) 1n which vegetation samples analyzed from the
Seveso area from 1976 through 1979 suggested that the contamination 1n
vegetation was from local dust and not from plant uptake. Unlike the Seveso
Incident where release of 2,3,7,8-TCDD 1n the environment took place, normal
use of herbicides containing 2,3,7,8-TCDD Impurities may not cause detect-
able 2,3,7,8-TCDO contamination of the crop. Jensen et al. (1983) analyzed
rice grain from fields 1n Arkansas, Louisiana and Texas after application of
2,4,5-T (containing 0.4 ppm TCDD) at a maximum rate of 2.25 pounds/acre. No
2,3,7,8-TCDD residues (detection limit 2-10 ppt) were found 1n these rice
grains nor were any TCDDs found 1n 30 samples of rice purchased In retail
stores throughout the United States. Contamination of fruits, vegetables or
grains 1n the United States with TCDD has never been reported.
The contamination of a large number of vegetables grown 1n the proximity
of municipal Incinerators has been reported by Pocch1ar1 et al. (1983).
These Investigators detected 1,3,6,8- and 1,3,7,9-TCDD 1n the concentration
range of 0.4-2 ppt 1n vegetables whose origin of TCDD was not attributable
4-29
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to the ICHESA plant accident. This finding suggests the possibility of
human exposure of TCDD from edible vegetables grown 1n areas close to mu-
nicipal Incinerators.
Different Investigators have reported the presence of PCDDs 1n the fat
of cattle that had grazed on pasture experimentally treated with 2,4,5-T
(Heselson et a!., 1978; Kocher et al.» 1978). The levels of TCDDs 1n these
studies ranged from 3-70 ppt. Kocher et al. (1978) reported that only 13%
of the fat samples collected (3 of 23 samples) gave a positive response for
2,3,7,8-TCDD at low levels (3-4 ppt).
Results of a collaborative program to analyze a selected beef sample by
the U.S. EPA, Dow Chemical Company, Wright State University and Harvard
University showed that TCDD could be detected In the adipose tissue of
cattle with access to 2,4,5-T-treated rangeland (U.S. EPA, 1984). Of the 85
beef fat samples analyzed, one sample contained 60 ppt of 2,3,7,8-TCDD and
two samples appeared to have 2,3,7,8-TCDD levels 1n the range of 5-10 ppt.
No 2,3,7,8-TCDD was determined 1n the rest of the samples. While several
laboratories detected levels 1n this lower range, the values reported were
very near the limits of detection.
Bovine milk collected after the accident 1n Seveso area was analyzed by
FanelH et al. (1980b). The concentration of 2,3,7,8-TCDD was found to vary
from none detected (detection limit <40 ppt) to as high as 7.9 ppb. Other
Investigators have failed to detect either 2,3,7,8-TCDD (detection limit 1
ppt) or HxCDD (detection limit 25 ppt) 1n surveillance (after normal appli-
cation of 2,4,5-T on pasture) samples of milk from the states of Oklahoma,
Arkansas and Missouri, or quarantined milk 1n the state of Michigan
(Lamparskl et al., 1978; Mahle et al., 1977).
4-30
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TCDDs Including 2,3,7,8-TCDD, PeCDDs Including 1,2,3,7,8-PeCDD and
HxCDDs Including 1,2,3,6,7,8-HxCDD have been detected 1n fish from a few
PCDD-contam1nated areas. This 1s discussed 1n detail In Section 6,2.
PCDDs have been detected 1n gelatin samples obtained from supermarkets
and 1n bulk gelatin (Firestone et a!., 1979), Eleven of 15 commercial
gelatins examined contained a combined amount of 1,2,3,6,7,8-HxCDD and
1,2,3,7,8,9-HxCDD ranging from 30-700 ppt. Three bulk gelatins of Mexican
manufacture showed higher levels of PCDDs. 2,3,7,8-TCDD was not detected 1n
any sample. The origin of PCDDs 1n gelatin was speculated to be PCP and
trlchlorophenol that are routinely used 1n the leather-tanning Industry.
The use of by-product fat materials from PCP-treated hides as animal feed
constituents led to widespread outbreaks of chick edema disease 1n the late
1950s (Firestone, 1973).
Bumb et al. (1980) analyzed charcoal-broiled steak under conditions
representing rare, well-done and overdone samples and failed to detect
either TCDD {detection limit 1-10 ppt) or HxCDO (detection limit 10-50 ppt)
1n the cooked meat of selected meat samples.
The analysis of human milk and urine for 2,3,7,8-TCDD has been per-
formed. A study of 103 samples of breast milk from mothers living 1n areas
within the United States that were sprayed with 2,4,S-T/s11vex revealed no
TCDD at a detection limit of 1-4 ppt and 0.1-10 ppt (U.S. EPA, 1980a; Heath
et al.t 1985). The control samples that were also negative for 2,3,7,8-
TCDD were derived from mothers living 1n areas where no records for 2,4,5-T
or sllvex exposure exist (Heath et al., 1985).
In an Interlaboratory collaborative analytical study on adipose tissue
1t was revealed that tissues from three Vietnam veterans heavily exposed to
4-31
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Agent Orange contained 2,3,7,8-TCDD residues with levels ranging from 20-173
ppt {Gross et a!., 1984). A survey of 72 autopsy tissue materials from
across Canada found 2,3,7,8-TCDD averaging between 5 and 10 ppt, OCDD
averaging between 600 and 800 ppt, and Intermediate levels for penta-,
hexa- and hepta-dloxlns (Ryan et a!., 1985). Results on the distribution of
tetra- and octa-chlorlnated dloxlns 1n autopsy tissues from the general
population, supported by the above observations, Indicate that there 1s
substantial contamination of the general population 1n the United States and
Canada with 2,3,7,8-chlorlne substituted tetra- through octa-d1ox1ns
(Sehecter et a!., 198i). Rappe et al. (1984, 1985} also reports the pre-
sence of PCDDs In human adipose tissue. In the same study Rappe et al.
(1985) reported their analytical results on a survey of mothers' milk from
Sweden, Germany, Denmark and Vietnam. All the samples contained different
levels of PeCDD, HxCDD, HpCDD and OCDD residues. The most toxic Isomer,
2,3,7,8-TCDD, was found only 1n the milk samples of mothers from Sweden and
Germany but not Vietnam. This Isomer was not analyzed In milk samples of
mothers from Denmark (Rappe et al., 1985).
The monitoring of urine samples from two people Involved with spray
application (2,4,5-T) showed no detectable level of TCDD at a detection
limit of ~2 ppt (Lavy et al., 1980).
4.6. EXPOSURE
The exposure of the general United States population to the four PCODs
cannot be estimated because the levels of these compounds 1n air, drinking
water and foods have not been established. In fact, no PCDD contamination
of any United States drinking water has ever been reported. Although the
local atmosphere near a few chemical disposal sites and municipal Incinera-
tors has been reported to be contaminated with TCDD and HxCDD, no comprehen-
4-32
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s)ve study Is available to demonstrate the atmospheric levels of these com-
pounds 1n areas farther away from the point sources. Similarly, some of
these compounds have been detected 1n edible aquatic species. Again, these
fish contaminations have been reported 1n areas near a limited source where
effluents contaminated with these compounds may have been discharged Into
surface waters. One of the consumer products that has been found to be
contaminated with HxCDD 1s gelatin. However, 1t 1s difficult to estimate
the contribution of food to human exposure of PCDDs from such limited data.
It seems more prudent to try to estimate the exposure of these compounds to
populations 1n certain localized areas (e.g., dump sites and known sources
of Industrial pollution) and certain special population groups (I.e.,
occupational) when adequate data are available.
The concentrations of 2,3,7,8-TCDD 1n bottom sediments of a drainage
canal passing through a dump area (wastes from 2,4,5-T production) 1n
northern Amsterdam, Holland, were reported by Helda (1983). The concentra-
tions of 2,3,7,8-TCDD 1n sediments within the dump area varied from 844-5062
ppt and outside the dump area from 55-611 ppt. Analysis of the eel revealed
that only two samples originating from shallow ponds adjacent to the main
drainage canal contained between 1.0 and 1.1 ppt of 2,3,7,8-TCDD. 2,3,7,8-
TCDD was not detected 1n other eel samples collected farther away from the
dump site. This study demonstrates the possibility of TCDD contamination
near dump sites.
The results of analysis for 2,3,7,8-TCDD and HxCDD 1n human milk samples
were reported by Langhorst and Shadoff (1980). About 6 of the 9 samples
showed 2,3,7,8-TCDD at levels slightly higher than the detection limits
(0.2-0.7 ppt). All nine samples showed HxCDD at levels slightly higher than
4-33
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the detection limit (0.2-0.5 ppt). However, these results remain uncon-
firmed because of the lack of validation of the precision and accuracy of
data. Investigations of 103 breast milk samples from mothers living 1n
areas 1n the United States sprayed with 2,4,5-T revealed no TCDO at a
detection limit of 1-4 ppt (U.S. EPA, 1980a).
The monitoring of urine samples from two people Involved with spray
application (2,4,5-T) showed no detectable level of TCDO at a detection
limit of ~2 ppt (Lavy et a!., 1980).
In one Polish study (Gorskl, 1981), 1,2,3,6,7,8-HxCDD was detected In
latex nipples at a concentration of 20-400 ppt. However, no TCOD or PeCDO
was detected. The origin of PCDDs 1n the latex was speculated to be the
result of Y-1rrad1at1on of latex (for crossllnking) containing PCP during
Its manufacturing process.
A BCF relates the concentration of a chemical In aquatic species to the
concentration 1n water. The steady-state BCFs for a I1p1d-soluble compound
1n the tissues of various aquatic species seem to be proportional to the
percent I1p1d 1n the tissue. Thus, the per capita 1ngest1on of a I1p1d-
soluble chemical can be estimated from the per capita consumption of fish
and shellfish, and a steady-state BCF for the chemical.
Data from a recent survey on fish and shellfish consumption 1n the
United States were analyzed by SRI International (U.S. EPA, 1980b). These
data were used to estimate that the per capita consumption of freshwater and
estuarlne fish and shellfish 1n the United States 1s 6.5 g/day (Stephan,
1980). In addition, this Information was used with data on the fat content
of the edible portion of the same species to estimate that the weighted
average percent I1p1ds for consumed freshwater and estuarlne fish and shell-
fish Is 3.0%.
4-34
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Several equations have been developed for predicting the steady-state
BCF for an organic compound from Its octanol-water partition coefficient
(Kenaga and Goring, 1980; Velth et a!., 1980; Velth and Koslan, 1983). All
of these depend on the availability of a useful value for the partition
coefficient. Several estimated values (Leo, 1979; Mabey et al., 1981;
Neely, 1983) and one measured value (Neely, 1979; Kenaga, 1980; Neely, 1983)
have been reported for the octanol-water partition coefficient for 2,3,7,8-
TCDD. Use of six equations with four values for the partition coefficient,
K , results 1n the following predicted BCFs (Table 4-4). The predicted
ow
BCFs range from 7000-900,000 using the calculated values of the partition
coefficient and from 3000-68,000 using the one measured value.
Several measured BCFs have been reported for 2,3,7,8-TCDD (Table 4-5),
but none can be considered definitive values. Many were determined 1n model
ecosystems 1n which the concentrations 1n water were not necessarily con-
stant. The measured BCFs, however, range from 2000-9000. A few other BCF
values are given 1n Table 5-1. Until further Information Is available, the
U.S. EPA's best current estimate for the BCF of 2,3,7,8-TCDD 1n aquatic
organisms Is 5000. An adjustment factor of 3.0/7.6=0.39 can be used to
adjust the estimated BCF from the 7.65C I1p1ds on which the equation 1s based
to the 3.0% I1p1ds that 1s the weighted average percent I1p1ds consumed per
capita from fish and shellfish (U.S. EPA, 1980b). The weighted average BCF
for 2,3,7,8-TCDD 1n the edible portion of all freshwater and estuarlne
aquatic organisms consumed by Americans 1s calculated to be 5000x0.395=1975.
Uptake by fish from lower tropic levels may add to uptake from water, so
this BCF may underestimate concentrations 1n wild aquatic organisms.
4-35
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TABLE 4-4
Predicted BCFs from Calculated and Measured Values of Kow a
Equation
log BCF
log BCF
log BCF
log BCF
log BCF
BCF = 0
*= 0
= 0
= 0
= 0
= 0
.048
.542
.76
.79
.635
.85
Kow
log Kow + 0.124
log Kow - 0.23
log Kow - 0.40
log Kow + 0.7285
log Kow - 0.70
6.
6
93
101
118
130
332
84
,780
,000
,000
,000
,000
,000
log
KOW
Calculated
7.
9
157
174
183
234
663
14
,860
,000
,000
,000
,000
,000
7.
11
201
224
225
308
915
28
,700
,000
,000
,000
,000
,000
Measured1*
6
2
27
28
43
33
67
.15
,870
,800
,740
,000
,700
,800
aSources: Kenaga and Goring, 1980; VeHh et a!., 1980; Velth and Koslan,
1983
k?h1s measured value has been reported by Neely, 1979
4-36
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TABLE 4-b
Measured Bloaccumulatlon Factor for 2,3,7,8-TCOD 1n Freshwater Aquatic Organisms
Species
Alga,
Oedogonlum cardlacum
Alga,
Oedogonlum card lac urn
Snail,
Physa sp.
Snail,
Physa sp.
f Cladoceran,
" Daphnla magna
Cladoceran,
Daphnla magna
Catfish,
Ictalurus punctatus
Hosqultoflsh,
Gambusla afflnls
—
—
Tissue
NR
NR
whole body
whole body
whole body
whole body
whole body
whole body
-
-
Percent
Llpld
NR
NR
NR
NR
NR
NR
NR
NR
NR
NR
Duration
(days)
33
32
33
32
30
32
28
14
-
_
Bloconcentratlon
Factor
3094a
2075b
2033
5471 a
2095b
3731
3895a
7070b
7125
2000
4850b
4875
9080C
5400
Reference
Isensee, 1978
Isensee, 1978
Yocklns st al..
Isensee, 1978
Isensee, 1978
Vocklm et al..
Isensee, 1978
Isensee, 1978
Yocklm et al.,
U.S. EPA, 1983a
Thomas, 1983
Isensee, 1978
Yocklm et al.,
Neely, 1979
Kenaga, 1980
1978
1978
1978
*
»
1978
These are arithmetic mean of several values given
These are values at equilibrium tissue concentrations
Calculated as ratio of uptake and clearance rate constants
NR = Not reported
-------
The BCF for 2,3,7,8-TCDD 1n the earthworm, Allobophora caHglnosa or
rosea. from soil with Initial 2,3,7,8-TCDD concentration 1n the range of
0.06-9.2 ppb has been determined to be ~ 10 (FanelH et al., 1982).
The BCFs for other PCDDs cannot be estimated because of the lack of
solubility data.
Finally, the levels of TCDD 1n wildlife have been determined by various
authors and are discussed 1n detail In Section 6.2.
4.7. SUMMARY
None of the PCDDs are commercially manufactured 1n the United States or
anywhere else 1n the world. They are produced as unwanted contaminants
during the manufacture of primarily chlorophenols and their derivatives,
such as the herbicides 2,4,5-T and SHvex. At the present time, there 1s no
known manufacturer of trlchlorophenol 1n the United States. Its derivatives
distributed 1n the market before banning, however, continue to be used as
pesticides 1n the United States. The level of 2,3,7,8-TCDD contaminants In
commercially available 2,4,5-T and similar formulations had been reduced to
<0.1 ppm before these products were banned.
The primary sources of PCDDs 1n the environment probably are Industrial
manufacturers of chlorophenols or their derivatives, and chemical disposal
sites containing the wastes from these Industries. Municipal waste Inciner-
ation also may produce some environmental emission of PCDDs. The signifi-
cance of this source of emission compared with Industrial emission and prob-
able contamination from chemical disposal sites cannot be assessed with the
available data. The 1,2,3,7,8-PeCDD now found 1n environmental samples has
only been reported 1n emissions from Incinerators.
PCDDs, particularly TCDD and Us specific Isomer 2,3,7,8-TCDD, have been
monitored 1n a number of environmental media, Including air, water, soil,
4-38
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food and biological media. The monitoring data to date Indicate that the
maximum level of PCDDs 1s likely to be found 1n son and drainage sediment
samples near chlorophenol manufacturing Industries and chemical waste
disposal sites. PCDDs have rarely been monitored 1n United States air
samples. Small amounts of PCDD contamination have been found 1n fish and
wildlife 1n the United States 1n areas around chlorophenol manufacturing
Industries and chemical waste disposal sites.
4-39
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5. ENVIRONMENTAL FATE AND TRANSPORT PROCESSES
5.1. FATE
5.1.1. Water.
5.1.1.1. BIQDEiRAOATION -- 2,3,7,8-TCDD exhibits relatively strong
resistance to blodegradatlon. Only 5 of -100 microblal strains that have
the ability to degrade persistent pesticides show slight ability to degrade
2,3,7,8-TCDD (Matsumura and Benezet, 1973). Ward and Matsumura (1977)
studied the blodegradatlon of 14C-labeled 2,3,7,8-TCDD by using lake
waters and sediments from Wisconsin. The observed half-life of 2,3,7,8-TCDD
1n sediment-containing lake waters was found to be 550-590 days. In lake
water alone, -70% of the 2,3,7,8-TCDD remained after 589 days. Using an
outdoor pond as a model aquatic ecosystem and dosing 1t with 14C-labeled
2,3,7,8-TCDD, Tsushlmoto et al. (1982) and Matsumura et al. (1983) estimated
the apparent half-life of 2,3,7,8-TCDD to be ~1 year. Although blodegrada-
tlon may have been responsible for part of the degradation, 1t Is almost
Impossible to estimate the blodegradatlon half-life of 2,3,7,8-TCDD 1n
aquatic systems from this experiment. It 1s likely that the apparent blode-
gradatlon loss was due to volatilization through air/water Interface. Other
Investigators (Huetter and Ph1l1pp1, 1982; Camon! et al., 1983) have demon-
strated the virtually complete lack of degradation of 2,3,7,8-TCDD by micro-
organisms. It could be Inferred from these studies that PeCDD and HxCDD,
having more chlorine substitution on benzene rings, would be even more
resistant to blodegradatlon than 2,3,7,8-TCDD.
The blodegradatlon half-life of 2,3,7,8-TCDD can also be estimated from
the theoretical rate constant values based on relative rates of transforma-
tion reported 1n the literature or on structure-activity analogy values
given by Mabey et al. (1981). Assuming the estimated blotransformatlon rate
5-1
-------
constant of lx!0~10 ma cell'1 hour"1 (Mabey et al., 1981) and the
concentration of microorganisms capable of degrading TCDD as 5xlOs cell
msT1 (Burns et a!., 1981), the half-life of blodegradatlon can be esti-
mated to be >1 year. It should be emphasized that the role that blodegrada-
tlon plays 1n the removal of PCDDs from water 1s not clear.
5.1.1.2. PHOTOTRANSFORMATION — 2,3,7,8-TCDD has a UV absorption
maximum at 310 nm with an extinction coefficient of 5590 M""* cm"3- (NRCC,
1981a). 2,3,7,8-TCDD 1n a pure state 1s photochemlcally stable but 1t will
photolyze 1n sunlight 1n the presence of a hydrogen atom donating substrate
{Crosby and Wong, 1977). For example, PUmmer et al. (1973) reported that a
2,3,7,8-TCDD suspension 1n distilled water remained unchanged when Irradi-
ated with a sunlamp. Similarly, a thin dry film of 2,3,7,8-TCDD on a glass
plate or 2,3,7,8-TCDD on dry and wet soils showed negligible photodegrada-
tlon after Irradiation with sunlamps (Crosby et al., 1971). In contrast,
2,3,7,8-TCDD 1n methanol solution or benzene solution of 2,3,7,8-TCDD 1n
water stabilized by surfactant underwent substantial photodegradatlon under
sunlamp or sunlight Irradiation (PUmmer et al., 1973; Crosby et al., 1971).
Botre et al. (1978) demonstrated that catlonlc surfactants, namely 1-hexade-
cylpyr1d1n1um chloride, act as an energy transfer agent 1n facilitating the
photodecomposltlon of TCDD In aqueous solutions. These laboratory studies
may not be applicable to the ambient environments. To explain the longer
half-life of 2,3,7,8-TCDD 1n a model laboratory ecosystem than 1n an outdoor
pond, Hatsumura et al. (1983) and Tsushlmoto et al. (1982) speculated that
photolysis was the most likely cause. In the outdoor environment where the
Intensity of sunlight was higher compared with the laboratory experiments,
algae-mediated photosens1t1zat1on of 2,3,7,8-TCDD may cause some photode-
composltlon of this compound. Nestrlck et al. (1980) estimated the photo-
5-2
-------
lytic half-life of 2,3,7,8-TCDD 1n n-hexadecane under sunlamp Irradiation to
be ~57 minutes. From the available Information, 1t 1s difficult to predict
the fate of 2,3,7,8-TCOD 1n aquatic media under environmental photolytlc
conditions. In the presence of hydrogen atom donating substrate(s) 1n
surface waters, photolysis may be a significant fate process.
An Increase In chlorine substitution 1s expected to decrease the rate of
photodegradatlon (Nestrlek et al., 1980; Helling et a!., 1973). For
example, Crosby et al. (1971) showed that although complete decomposition of
2,3,7,8-TCDD 1n methanol occurred In 24 hours under UV Irradiation, >80%
OCDD 1n methanol remained unreacted during the same period under similar
Irradiation conditions.
Although the degree of photolysis may be related to the extent of
chlorlnatlon, positional 1somer1zat1on also plays a critical and perhaps
dominant part 1n the photolysis of higher PCDDs. In higher PCDDs, there
appears to be preferential loss of chlorine from the 2, 3, 7 and 8 positions
(Nestrlck et al., 1980; Buser and Rappe, 1978; Choudhry and Hutzlnger,
1984). However, Buser (1979) observed the formation of 2,3,7,8-TCDD 1n
trace quantities, and PeCDD form photolysis of 1,2,3,6,7,8-HxCDD and
1,2,3,7,8,9-HxCDD. PCDD compounds with chlorine substitutions 1n positions
2, 3, 7 and 8 are Hkely to photodegrade faster than compounds not having
these positional substitutions. According to such a predicted rule, 1t 1s
not likely that photodegradatlon of OCDD and other higher PCDDs will yield a
high quantity of 2,3,7,8-TCDD as the stable end product. For example, the
photolysis half-life of 1,2,3,7,8-PeCDD has been estimated to be 7.6 hours
1n n-hexadecane solution under sunlamp Irradiation (Nestrlck et al., 1980).
Similarly, the photolytlc half-lives of 1,2,3,7,8-PeCDD, 1,2,3,6,7,9-HxCDD
and 1,2,4,6,7,9-HxCDD 1n hexane solutions under sunlight Irradiation have
been determined to be 5.4, 17 and 47 hours, respectively (Dobbs and Grant,
5-3
-------
1979). Nestrlck et al. (1980) reported a half-life value of 6.8 hours for
1,2,3,6,7,8-HxCDD 1n n-hexadecane under sunlamp Irradiation. The Interme-
diates of the photodegradatlon of higher PCDDs are probably lower chlori-
nated dloxlns, but the pathways of degradation are not known with certainty
{NRCC, 1981a).
From the preceding discussions of the photolysis of PCDDs 1n the
presence of organic hydrogen donating substrates, 1t 1s difficult to predict
the photolytlc fate of these compounds 1n natural aquatic media where suf-
ficient organic hydrogen donating substrate(s) may or may not be available.
The situation 1s complicated further by the fact that, unlike 1n solution, a
predominant amount of PCDDs 1n surface water may remain sorbed on suspended
particles and settled sediments. Moreover, since the penetration of UV
light Into natural water may be very limited, photolytlc degradation of
PCDDs 1s not likely to be of environmental Importance.
5.1.1.3. RADICAL OXIDATION AND HYDROLYSIS -- Although these processes
occur, hydrolysis of 2,3,7,8-TCDD or oxidation with free radicals (R0?«,
RQ», etc.) In aquatic media are not likely to be of environmental signif-
icance (Callahan et al., 1979; Mabey et al., 1981). Likewise, hydrolysis
and oxidation are even less likely to be environmentally significant
processes for PeCDD and HxCDD.
5.1.1.4. VOLATILIZATION — Although several Investigators Implicated
volatilization as one of the major reasons for the observed disappearance of
2,3,7,8-TCDD from aqueous solution during mlcroblal studies, no quantitative
Information regarding the volatilization of 2,3,7,8-TCDD from aquatic media
Is available (Ward and Hatsumura, 1977; Matsumura et al., 1983; Huetter and
Ph1l1pp1, 1982). 2,3,7,8-TCDD may undergo some water-mediated evaporation
1n aquatic media (Matsumura et al., 1983). Using the formulas of L1ss and
Slater (1974), a vapor pressure value of 1.7x10"* torr (0.2 m Pa) and a
5-4
-------
solubility value of 6.2xlO~10 mole/4, the volatilization half-life for
2,3,7,8-TCDD was 6 minutes from water of 1 cm depth and 10 hours from water
of 1 m depth (NRCC, 1981a). The limitations of this theory to predict the
rate of volatilization have been discussed 1n the NRCC (1981a) document.
The L1ss-Slater model does not consider terrestrial matrices {suspended
solids, sediments, biota, etc.} normally encountered 1n natural surface
water and thus Ignores the effects of these parameters on the volatilization
rate. Employing a computerized EXAMS model for two standardized aquatic
ecosystems (lake and pond; see NRCC, 1981a, for definitions) and the Input
parameters for 2,3,7,8-TCDD given 1n NRCC (1981a), volatilization has been
estimated to account for 100% of the fraction lost; blodegradatlon has been
calculated to be Q%. The volatilization half-life for TCDD has been
estimated to be 5.5 and 12 years from pond and lake water, respectively. A
transport model has also been used to estimate the volatilization rate of
2,3,7,8-TCDD from a cooling pond on an Industrial site (Thlbodeaux, 1983).
The model accounted for movement of 2,3,7,8-TCDD from the bottom sediment to
the water column and then to the air. Based on the measured concentrations
In the pond bottom sediment (22,100 ng/kg) and the pond surface area
(15,050 mz), the calculated volatilization rate was 15-16 mg/year.
Pertinent data regarding the volatilization of PeCDDs and HxCDDs from
aquatic media could not be found 1n the available literature. However,
these compounds with higher molecular weight and more chlorine substitution
are expected to volatilize more slowly than 2,3,7,8-TCDD from aquatic media.
5.1.1.5. SORPTION — Data from microcosm experiments Indicate that
2,3,7,8-TCDD 1s highly sorbed to sediments and biota (Isensee and Jones,
1975; Ward and Hatsumura, 1978). More than 90% of 2,3,7,8-TCDD 1n an,
i
aquatic medium may be present 1n the adsorbed state (Hard and Matsumura,
5-5
-------
1978; Hatsumura et al., 1983). Considering the low water solubility and the
high octanol/water partition coefficient, this 1s not surprising. In fact,
the equation of Karlckhoff et al. (1979) predicts a sorptlon partition co-
efficient value of 104 for 2,3,7,8-TCDD 1n sediments containing 2% organic
carbon. Similarly, the higher PCDDs are likely to be present predominantly
1n the sedlment-sorbed state 1n aquatic media.
5.1.2. A1r. A number of PCDDs, Including TCDDs, PeCDDs and HxCDDs, have
been detected 1n the dust and fly ash from municipal Incinerators (Cavallaro
et al., 1980b; Clement and Karasek, 1982; Elceman et al., 1981). Size
fractlonatlons of fly ash from municipal Incinerators have shown that larger
concentrations of 2,3,7,8-TCDD and PeCDDs occurred on the larger (550 ym)
particles, while the 30 vm particles had greater relative concentrations
of OCDD (Clement and Karasek, 1982). Tiernan et al. (1982b) also reported
higher concentrations of TCDDs on larger particles (3-10 ^m) from a
refuse-fueled municipal Incinerator effluent than on smaller particles
(<1 ym). PCDDs emitted to the atmosphere from combustion processes appear
to be associated with air partlculate matter (Nestrlck et al., 1980).
Atmospheric PCDDs originating from other noncombustlon sources, such as
herbicide-treated soils and vaporized PCDDs from aquatic media (Thlbodeaux,
1983), are also likely to be associated with air partlculate matter. CupHt
(1980) presented mathematical descriptions of physical removal mechanisms
for the fate of toxic and hazardous materials 1n the air environment. For
the adsorption of chemicals on aerosol particles he developed a general
model based on aerosol surface area and chemical saturation vapor pressure.
His results suggest that adsorption will be a reasonable vapor-phase removal
mechanism from air only for materials with saturation vapor pressures of
10~7 torr (mm) or less. 2,3,7,8-TCDD has an estimated vapor pressure of
1.7xlO~6 torr.
5-6
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Photodegradatlon and wet and dry deposition of partlculate-bound PCOOs
are probably the most Important fate-determining processes for the atmos-
pheric PCDDs. The available data relating the photodegradatlon of these
compounds 1n the sorbed phase or as films are conflicting. For example,
experiments of earlier Investigators Involving photoreactlvlty of 2,3,7,8-
TCDD as films or sorbed on solid surfaces and exposed to the atmosphere
yielded negligible photodegradatlon with sunlight (Crosby et a!., 1971).
However, the more recent work of Buser (1979) and the Investigations of the
other researchers (PUmmer, 1978; Crosby and Wong, 1977) have shown that
some photolysis of TCDD 1n the condensed phase (I.e., coated on glass plate
or on silica) may take place. In the condensed phase, photodecompos1t1on of
TCDD In the bottom layers that are shielded from Incident light by the
surface layer 1s prevented.
Gebefuegl et al. (1977) studied 2,3,7,8-TCDD photochemical degradation
under simulated environmental conditions by exposing silica gel-sorbed
2,3,7,8-TCOD to light of wavelength >290 nm and observed 9254 decomposition
1n 7 days. The half-life for photodegradatlon of 2,3,7,8-TCDD film on glass
surfaces has been estimated to be 5.8 days under Irradiation with sunlamps
(NestMck et al., 1980). It Is not known whether a similar photodegradatlon
of particle-bound 2,3,7,8-TCDD will occur 1n the atmosphere since the state
of sorptlon may be different from those obtained under laboratory condi-
tions. The potential for oxidation of PCDDs by free radicals (OH-, 0«,
etc.) and other molecules (0<,, NO , etc.) that may be present 1n the
w X
atmosphere 1s unknown.
5.1.3. Soil.
5.1.3.1. SORPTION — From the empirical correlation of Karlckhoff et
al. (1979), 1t 1s possible to predict a soil/water partition coefficient of
4.8x10* for a son containing 1054 organic matter.
5-7
-------
Because of their high affinity toward soils, particularly those with
significant organic content, and because of their extremely low water
solubilities, 2,3,7,8-TCDD (and presumably other PCDDs) tend to remain on or
near the surface of soils (U.S. EPA, 1984). With time, 2,3,7,8-TCDD bound
to soil becomes more difficult to desorb (Ph1l1pp1 et al., 1981; Huetter and
PhlUppI, 1982).
Several authors have shown that vertical movement of 2,3,7,8-TCDD 1n
soil 1s negligible, although movement of 2,3,7,8-TCDD may occur by hori-
zontal transfer (eroded soil transported by water) and through contaminated
airborne dust particles (U.S. EPA, 1984; Helling et al., 1973). Therefore,
underground water supplies are unlikely to be contaminated with 2,3,7,8-
TCDD. However, as the organic content of soil decreases, the likelihood of
vertical movement of PCDDs 1n soil Increases. In areas of heavy rainfall
and sandy soil, vertical migration of 2,3,7,8-TCDD and Us lateral displace-
ment by soil erosion and runoff would be enhanced (U.S. EPA, 1984). The
downward vertical migration of 2,3,7,8-TCDD up to 30 cm Into soil has been
suggested to have occurred 1n Seveso, Italy (D1Domen1co et al., 1980d,e).
The monitoring of Seveso soil 1 year after the accident showed that the
highest 2,3,7,8-TCDD levels were not present 1n the topmost soil layer (0.5
cm), but very often 1n the second (0.5-1.0 cm) or third (1.0-1.5 cm) layers.
In view of the low water solubility of 2,3,7,8-TCDD, probable explanations
of this vertical distribution could be due to volatilization through the
a1r/so1l Interface or solvatlon of 2,3,7,8-TCDD by organic solvents (NRCC,
1981a), or blotlc mixing by earthworms or other soil Invertebrates. It 1s,
therefore, possible that 2,3,7,8-TCDD may appear 1n the air above and 1n
normal water leachate of soils, particularly after multiple PCDD application
or accidental release of 2,3,7,8-TCDD on son.
5-8
-------
5.1.3.2. PHOTOTRANSFORMATION - The photodecomposltlon of 2,3,7,8-
TCDD on wet or dry soil under artificial and natural sunlight was studied by
Crosby et al. (1971). The photodecomposltlon was found to be negligible 1n
soils. Similarly, PUmmer et al. (1973) determined that photodecomposltlon
of TCDD on soils was too slow to be detected. In a later experiment,
PUmmer (1978) found that although TCDD decomposed significantly from
precoated silica plate ("22%) 1n 8 hours of sunlight Irradiation, practic-
ally no decomposition of TCDD was observed from TCDD sorbed on soil under
similar conditions.
The photodegradatlon of TCDD 1n combination with other pesticide
mixtures was studied by Crosby and Wong (1977). When Agent Orange contain-
ing 15 ppm of TCDD was applied on the surface of glass plates (5 mg/cm2),
rubber plant, Hevea brasH1ens1s (6.7 mg/cm2), and on the surface of
sieved Sacramento loam soil (10 mg/cm2) and exposed to sunlight, TCDD was
found to photodecompose. The loss of TCDD 1n 6 hours was >50# from glass
plate, ~10054 from the surface of leaves and ~10% from the surface of soil.
The rapid photolysis of TCDD from these surfaces Indicates that the herbi-
cide formulation provided a hydrogen donor that probably allowed the
photolysis to occur. The authors attributed the slower photolysis of
2,3,7,8-TCDD 1n soil to a shading effect by lower layers of soil particles.
5.1.3.3. BIODE6RADATION -- Polger and Schlatter (1980) noted that
2,3,7,8-TCDD absorbs strongly onto soil particles, thereby reducing Its
b1oava1labH1ty. Young (1983) also noted that 2,3,7,8-TCDD 1s not likely to
metabolize readily by soil microorganisms. It can be concluded from the
following discussions that the blodegradatlon half-life 1n soil 1s likely to
be >1 year.
5-9
-------
The overall half-life of 2,3,7,8-TCDD 1n soil has been reported to be
1-3 years by Kearney et al. (1972). Studies performed by the U.S. A1r Force
(Young et al., 1976; IARC, 1977) suggested that soil bacteria may blodegrade
TCDD. The half-life of this chemical 1n soils under relatively dry condi-
tions (Utah test area) was found to be ~330 days and 1n more moist soils and
under warm conditions (Florida test area) was found to be ~190 days. This
Is consistent with the blodegradatlon half-life of -0.5 year for TCDD
determined by Commoner and Scott from the soil 1n rural Missouri after the
accidental spraying of TCDD-contam1nated oil (IARC, 1977). However, these
half-life estimates may greatly underestimate the true value, since 1t has
recently been shown that radlolabeled TCDO adsorbed to soil becomes progres-
sively more resistant to extraction (Ph1l1pp1 et al., 1981; Huetter and
Ph1l1pp1, 1982).
The rate of disappearance of 2,3,7,8-TCDD following an accidental
2,3,7,8-TCDD release from a trlchlorophenol manufacturing plant at Seveso,
Italy, was studied by D1Domen1co et al. (1980d, 1982). The disappearance of
2,3,7,8-TCDD from the topmost soil layer after 1 year was speculated to be
due to photodegradatlon, volatilization or vertical movement through the
soil. These Investigators estimated the Initial half-life of 2,3,7,8-TCDD
1n soil at the time of Its release to be 5 months. One month after release,
the rate of disappearance of 2,3,7,8-TCDD slowed down to the equivalent of 1
year 1n apparent half-life. By the 17th month, the rate declined to an
extremely slow level; the apparent half-life figure for this phase was
calculated to be >10 years. More recent data (Young, 1983; W1pf and Schmld,
1983) Indicate that the half-life of 2,3,7,8-TCDD 1n soil 1s about 10-12
years. Since most of the other PCDDs are no more susceptible to transforma-
tion/degradation than TCODs, their half-lives 1n soil are presumed to be
similar to that postulated for TCDDs.
5-10
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5.1.4. Food. Isensee and Jones (1971) conducted experiments to study the
possibility of absorption and translocatlon of 2,3,7,8-TCDD by plants from
polluted soil. Oats and soybean plants grown to maturity 1n soil contami-
nated with 0.06 ppm 2,3,7,8-TCDD showed <1 ppb of 2,3,7,8-TCDD 1n the seeds.
Cocucd et al. (1979) measured the level of contamination 1n kitchen garden
plants (carrot, potato, onion and narcissus) grown In soil from the contami-
nated Seveso area containing 1000-4000 vg/m2 of 2,3,7,8-TCDD. 2,3,7,8-
TCDD was found to be 3-5 times higher In foliage than 1n fruits. The fact
that the highest 2,3,7,8-TCDD content was found adjacent to the conductive
tissue was Interpreted as evidence of translocatlon of 2,3,7,8-TCDD from
roots to the outer parts of the plants. The Investigation of these authors
also suggested that 2,3,7,8-TCDD may be eliminated from the mature plants.
W1pf et al. (1982), however, failed to detect any measurable 2,3,7,8-TCDD 1n
the flesh of fruits and vegetables collected from the contaminated area 1n
Seveso during 1977-1979, although the soil 2,3,7,8-TCDD concentration was
-10 ppb. These authors concluded that 2,3,7,8-TCDD may not be translocated
from soil to the plants. A similar conclusion was reached by Pocchlarl et
al. (1983) from their uptake experiments with plants. It can be concluded
from these studies that 2,3,7,8-TCDD 1s not Hkely to concentrate 1n plants
grown 1n contaminated soils.
With respect to potential 2,3,7,8-TCDD exposure through aerial parts of
plants, when an aqueous suspension of pure 2,3,7,8-TCDD was exposed to
either artificial light or sunlight, photodecomposltlon was negligible.
However, 1n conjunction with other pesticides, 2,3,7,8-TCDD rapidly degraded
when exposed to light (Crosby and Wong, 1977). This 1s consistent with the
observations that TCDD was found not to persist on foliage (Sundstrom et
al., 1979; Crosby and Wong, 1977) after application with other pesticides
5-11
-------
(2,4,5-T, Agent Orange). The half-life of 2,3,7,8-TCDD disappearance from
grass In Texas treated at a high rate (12 pounds/acre) of 2,4,5-T containing
0.4 ppm 2,3,7,8-TCDD was determined to be 5,6 days (Jensen et a!., 1983).
Cattle fed rations fortified with a maximum of 90 ppt TCDO were monitored
for TCDD content 1n the body fat. TCOD from the body fat presumably dis-
appeared with a half-life of -16.5 weeks (Jensen et al., 1981). Similarly,
cattle fed rations fortified with 500 ppt 2,3,7,8-TCDD showed a maximum
level of 90 ppt of 2,3,7,8-TCDD 1n cows' milk. On withdrawal of 2,3,7,8-
TCDD containing feed, 2,3,7,8-TCDD disappeared from the milk with a half-
life of 41 days (Jensen and Hummel, 1982).
5.2. TRANSPORT
5.2.1. Hater. The two likely transport processes for PCDDs 1n aquatic
media are volatilization and sorptlon onto suspended partlculates and subse-
quent sedimentation. No quantitative data regarding volatilization of any
of these compounds from aquatic media are available, although several
Investigators Implicated volatilization as one of the major reasons for the
observed loss of 2,3,7,8-TCDD from aqueous solutions during mlcroblal
studies (Hard and Hatsumura, 1977; Huetter and PhUlppI, 1982). There 1s a
very wide difference 1n the calculated values of half-life of volatilization
for 2,3,7,8-TCDD. For example, calculation based on the L1ss and Slater
(1974) equation gives a half-life for evaporation of 10 hours from water of
1 m depth (see Section 5.1.1.4.). Calculation based on a reaeratlon rate
ratio of 0.373 (Mabey et al., 1981) and an oxygen reaeratlon rate constant
of 0.19 day"1, 0.96 day"1 and 0.24 day"1 (Mabey et al., 1981) for
pond, river and lake water, respectively, gives half-life values of 10, 2
and 8 days for 2,3,7,8-TCDD In pond, river and lake water, respectively.
These wide variations are conceivable when examined with the volatilization
5-12
-------
models for half-life (Thlbodeaux, 1979). Evaporation half-life 1s shown to
be proportional to water depth and Inversely proportional to the mass-
transfer coefficient. A more realistic calculation based on EXAMS predicts
half-life values for TCDD of 5.5 and 12 years from pond and lake water,
respectively (see Section 5.1.1.4.). The EXAMS calculation routine contains
an added element that accounts for the sorptlon of TCOD both on the
suspended and on-bottom sediment. For substances with high sorptlon coef-
ficients such as TCDD, the evaporation rate 1s reduced significantly. A
comparison of calculated transport rates from an Industrial site Indicates
that evaporation of TCDD from a contaminated cooling water pond sediment 1s
negligible In comparison with other contaminated areas on the site (Thlbo-
deaux, 1983). It will also become apparent from the following discussion
that volatilization may be Insignificant compared with sorptlon processes
for the transport of TCDD and presumably other PCDDs from aquatic media.
It has already been shown (see Section 5.1.1.5.) that 2,3,7,8-TCOD 1s
highly sorbed to sediments and biota (Isensee and Jones, 1975} and >90% of
2,3,7,8-TCDD 1n aquatic media may be present 1n the sorbed state (Ward and
Matsumura, 1978). This 1s consistent with the sorptlon partition coeffic-
ient value of this compound. Although the sorptlon effects of the higher
PCDOs have not been studied, based on their expected higher octanol/water
partition coefficient values, these compounds are likely to be present
predominantly 1n the sedlment-sorbed state In aquatic media.
5.2.2. A1r. All the PCDDs are believed to be transported 1n the vapor-
phase and 1n partlculate bound form 1n the atmosphere (see Section 5.1.2.).
The transport of these compounds from stationary point sources (i.e. stack
emission) and area sources (waste disposal sites) can be theoretically
predicted from dispersion modeling (Oosephson, , 1983). Although such
5-13
-------
dispersion modeling has been performed for 2,3,7,8-TCDD (SAI, 1980), the
correlation between the theoretical value and experimental monitoring data
has never been performed. In the case of accidental release of toxic clouds
containing TCDD at Seveso, Italy, Cavallaro et al. (1982) determined the
transport pattern and the ground deposition of the TCDD from the cloud.
They determined that the TCDO deposition from air to soil should follow an
exponential decay pattern along the downwind direction and follow a Gaus-
sian-distribution along the cross-section of the downwind direction. From
regression equations, these Investigators determined that the aerial deposi-
tion Y (vg/ma) should be y - 2900 e x for x<2 km and Y = 45
-0 5x
e * for 2 km
-------
120 and 1200 g/year of TCDD were volatilized from a highly contami-
nated soil surface between 1978 and 1979 before the Implementation of
remedial measures. Over the same period 1t was estimated that 28-37 g/year
left the site by wind-blown particle entrapment, 0.1-1.0 g/year evaporated
from a burial site and 0.98-2.3 g/year 1n water runoff. All these sources
are areas In which the 2,3,7,8-TCDD was found to remain sorbed on the soil.
It appears that volatilization from son and downward migration caused by
son movement, or through blotlc mixing by earthworms or other soil Inverte-
brates are more probable mechanisms by which 2,3,7,8-TCDD may be transported
from soils.
5.3. BIOACCUMULATIQN/BIGCQNCENTRATIQN
The bloconcentratlon of TCDD 1n various aquatic species has been studied
under controlled laboratory conditions using static test chambers. The
results of these Investigations have been discussed In Section 4.6. and are
given 1n Table 5-1. In all these experiments, the total amounts accumulated
were found to be related to the Initial TCDD concentrations 1n aquatic
phase. The Investigation of Ph1l1pp1 et al. (1981) made H clear that
bloaccumulatlon would be significantly affected by the physical form (sorbed
or 1n solution) 1n which TCDD occurs 1n the environment. Isensee (1978)
reported that the concentration 1n the tissues of the tested species reached
equilibrium 1n 7-15 days. In the absence of any experimental BCFs derived
under dynamic test conditions, the values of Isensee (1978) reported 1n
Table 5-1 probably represent the best experimental values available (1n
species other than fish) since these values were derived from equilibrium
concentrations of TCDD 1n the tested tissues. The BCF for 2,3,7,8-TCDD 1n
the earthworm, Allobophora callglnosa or rosea. from soil with Initial
2,3,7,8-TCDD concentration 1n the range of 0.06-9.2 ppb has been determined
to be -10 (FanelH et al., 1982).
5-15
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en
I
TABLE 5-1
Bloconcentratlon Factor of TCDD for Several Aquatic Organisms3
Species
Algae, Oedogonlum cardlacum
Algae. Oedogonlum cardlacum
Algae, Oedogonlum cardlacum
Ostracod
Duckweed. Lemna minor
Snail, Physa sp.
Snail, Physa sp.
Snail, Helosoma sp.
Daphnlds, Daphnla magna
Daphnlds, Daphnla magna
Initial Aquatic
Concentration (ppt)
0.05-1300
0.05-1300
O.lc
2.6
0.05-1300
0.05-1300
0.05-1300
O.lc
0.05-1300
0.05-1300
B1oconcentrat1on
Factor
2,075
9,000b
2,080
110
3,625b
2,095
20,OOQb
2,080
7,070
26,000b
Reference
Isensee, 1978
Isensee and Jones,
1975
Yocklm et al.,
1978
Hatsumura and
Benezet, 1973
Isensee and Jones,
1975
Isensee, 1978
Isensee and Jones,
1975
Yocklm et al.,
1978
Isensee, 1978
Isensee and Jones,
Daphnlds, Daphnla magna
0.4
2,200
1975
Hatsumura and
Benezet, 1973
-------
TABLE S-l (cent.)
Species
Mosquito fish. Gambusla afflnls
Mosquito fish, Gambusla afflnls
Mosquito fish. Gambusla afflnls
Mosquito larvae, Aedes aegyptl
Brine shrimp, Artlmla salina
Wl
_l
*J Catfish, Ictalurus punctatus
Catfish, Ictalurus punctatus
Brook Sllverslde, Laludesthes slcculus
Pond Heed, El odea nut tall and
Ceratophyllon demersum
Initial Aquatic
Concentration (ppt)
0.05-1300
0.05-1300
O.lc
0.45
O.ic
0.05-1300
0.05-1300
1.3
53.7
Bloconcentratlon
Factor
4,850
26,000b
4,875
9,200
1,570
9,000b
4,875
545d
30,300
Reference
Isensee, 1978
Isensee and Jones,
1975
Yocklm et al.,
1978
Hatsumura and
Benezet, 1973
Hatsumura and
Benezet, 1973
Isensee and Jones,
1975
Yocklm et al.,
1978
Hatsumura and
Benezet, 1973
Tsushlmoto et al.,
1982
aBCF values derived by Isensee and Jones (1975) were based on dry weight for all biological and sediment
materials.
Average of several values
cThese are Initial concentrations of TCDD In soil added to water.
Error In the original publication corrected 1n the value reported here.
-------
5.4. SUMMARY
The four transformation processes (photoreactlon, blotransformatlon,
hydrolysis and radical oxidation) that control the fate of a chemical In
aquatic media do not appreciably transform TCDD and possibly other PCDDs In
aquatic media. However, the two former processes may be more Important for
the transformation of 2,3,7,8-TCDD 1n aquatic media. The transport of these
compounds to the atmosphere by volatilization from surface water may take
place through a water-mediated process, particularly 1n the case of 2,3,7,8-
TCDD, but significant transport of these compounds to the atmosphere through
water may not be likely. Therefore, the PCDDs are expected to be very
persistent In aquatic media.
The potential for oxidation of PCDDs by tropospherlc free radicals Is
not known. Although appreciable photolysis of TCDD coated on glass plate or
sorbed onto silica has been observed, 1t 1s not known whether a similar
photodegradatlon of particle-bound TCDD and other PCDDs will occur 1n the
atmosphere. The transport of vapor phase and particle-bound PCDDs may be
theoretically predicted from dispersion modeling equations. In the case of
accidental release of toxic clouds containing TCDD at Seveso, Italy, H has
been demonstrated that the TCDD deposition from air to soil followed an
exponential decay pattern along the downwind direction and a Gaussian
distribution pattern along the cross-section of the downwind direction.
PCDDs are resistant toward photochemical and blodegradatlon reactions 1n
soil. The half-life of 2,3,7,8-TCDD 1n soils may be >10 years. These
compounds are likely to be transported from soil through movement of
partlculate matter containing sorbed PCDDs. The most probable transport
mechanisms are transport of these compounds to the atmosphere by contami-
nated airborne dust particles, evaporation, and transport to surface water
5-18
-------
via eroded soil transported by water. Leaching Is a less likely transport
process for these chemicals except for very sandy soils.
Both the calculated and experimental results show that these compounds
will bloaccumulate 1n aquatic organisms. The experimental BCF varies with
the species and ranges from ~2000-30,000. However, studies with flow-
through systems should be performed to establish the realistic bloaccumula-
tlon factors for these compounds 1n different aquatic species.
5-19
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6. ECOLOGICAL EFFECTS
6.1. EFFECTS ON ORGANISMS
6.1.1. Aquatic Life Toxicology. Almost all of the available Information
concerning the toxldty of PCDOs to wildlife pertains to aquatic species,
and most of the aquatic Information 1s based on acute exposure to calcu-
lated, rather than measured concentrations of 2,3,7,8-TCDD.
6.1.1.1. ACUTE TOXICITY — The effects of acute exposure to 2,3,7,8-
TCDD have been reported for four species of freshwater fish and one species
of amphibians (Table 6-1). In almost all of these studies, toxic effects
were observed only after the acute exposure period ended. Miller et al.
(1973, 1979) exposed juvenile coho salmon, Oncorhynchus klsutch. to a range
of 2,3,7,8-TCDD concentrations for up to 96 hours. Concentrations were
expressed as ng/g wet bw and as ng/l of water, based on the amount of
2,3,7,8-TCDD added to the water In the test containers and the Initial body
weight of fish. Test concentrations were measured during the exposure
period. After exposure, the fish were transferred to clean flowing water
and observed for up to 114 days during which they were fed to satiation 3
times/week. Experiments were conducted with two groups of fish that dif-
fered 1n Initial mean wet weight (3.51 and 6.63 g). Food consumption,
growth and survival of smaller fish were measured until 60 days after expo-
sure and were found to be significantly reduced at 5.4 ug/kg bw (0.0056
vg/l), but not at 0.54 ug/kg bw (0.00056 yg/l) or lower. Growth
and survival of larger fish were measured until 114 days after exposure and
were significantly reduced at 5.4 tig/kg bw (0.0105 jig/i) but not at
0.54 vg/kg bw (0.00105 ug/l) or lower. The actual concentrations 1n
fish and water were undoubtedly lower than the calculated values, because
much of the added 2,3,7,8-TCDD would be adsorbed to all containers.
6-1
-------
TABLE 6-1
Effect of Acute Exposure to 2,3,7,8-TCDO on Aquatic Animals
Species
Coho Salmon,
Oncorhynchus klsutch
Coho Salmon,
Oncorhynchus klsutch
Rainbow Trout,
Salmo qalrdnerl
Rainbow Trout,
Salmo aalrdnerl
Rainbow Trout,
Saliio galrdnerl
er*
i
"^ Guppy,
Poecllla retlculata
Guppy,
poecllli retleulata
Northern Pike,
Esox luclus
Northern Pike,
Esox luclus
Frog,
Rana catesblana
frog.
Rana catesblana
Life Stage,
Height or
Length
3.5 g
6.6 g
eggs and
larvae
eggs and
larvae
0.85 g
9-40 IM
8-12 m
eggs and
larvae
eggs and
larvae
larvae
adults
(150-250 g)
Duration
of Exposure
(hours)
96
96
96
96
96
120
24
96
96
l.p.
Injection
l.p.
Injection
Duration LC§o
of Test (tig/i)
(days)
64 0.0056
114 0.010Sb
72 NR
164 NR
72 NR
37 NR
69 NR
23 NR
23 0.001
50 NR
35 NR
(days)
(0
114
NR
NR
NR
21.7
NR
NR
23
NR
NR
Lowest Effect
Concentration
frt/*)
0.0056
0.0105
0.0001
0.001
0.010
0.1
0.0001
0.0001
0.001
NO
SOO vg/kg bw
No Effect
Concentration
(Mi/I)
0.00056
0.00105
ND
0.0001
NR
ND
0.00001
ND
0.0001
1000 pg/kg bw
250 ug/kg bw
Effect
reduced growth, food
consumption, survival
reduced growth, food
consumption, survival
temporary growth
Inhibition
teratologlc effects,
decreased survival
and growth
decreased survival
and growth, htsto-
loglcal effects
100X mortality by
3.7 days after
beginning exposure
higher Incidence
of fin necrosis
temporary Inhibition
of egg development
decreased survival
and growth
no effect on survival
metamorphosis,
histology
temporary decrease
In food consumption.
but no effects on
survival or histology
Reference
Miller et al., 1973,
1979
Miller et al., 1973,
1979
Helder. 1981
Helder, 1981
Helder, 1981
Miller et al 1973;
Norrls and Miller.
1974
Miller et al.. 1979
Helder, I960
Helder, 1980
Beatty et al., 1976
Beatty et al,, 1976
IT™ * nedlan lethal time In days after beginning exposure
b4?X mortality
SB * Sot reported; NO = Not determined
-------
Acute exposure experiments were also conducted by these researchers
(HUler et al., 1973, 1979; Morris and Hlller, 1974) with gupples, PoedHa
retlculata. Miller et al, (1973) and Morris and MUler (1974) reported the
effects of exposing gupples to nominal concentrations of 0.1, 1.0 and 10.0
vg/fi. for 120 hours followed by transfer to clean water. Some fish
(8-18%) died 1n each test concentration during the exposure period. All
treated fish died by 37 days after beginning exposure; smaller fish gener-
ally died first. F1n necrosis was observed 1n all fish surviving more than
10 days. In a later study, HUler et al. (1979) measured the Incidence of
fin necrosis 1n gupples exposed for 24 hours to much lower nominal concen-
trations of 2,3,7,8-TCDD and then maintained for 69 days. The Incidence of
fin necrosis was significantly greater 1n fish exposed to >0.8 vg/kg bw
(0.0001 vg/fi-) than 1n controls or 1n fish exposed to 0.08 pg/kg bw
(0.00001 vg/!t).
The effects of static acute exposure to 2,3,7,8-TCDD on eggs and larvae
of northern pike, Esox luc1 us. and rainbow trout, Salmo galrdnerl. were
reported by Helder (1980) and Helder (1981), respectively. In both studies,
newly fertilized eggs were exposed for 96 hours to a range of nominal
2,3,7,8-TCDD concentrations (0.0001, 0.0010, 0.010 pg/fi.) followed by
transfer to clean water. There was no significant Increase 1n egg mortality
up to the highest nominal test concentration of 0.010 pg/fi, for either
species. Significantly greater mortality occurred after hatching and during
yolk sac absorption 1n both species at concentrations as low as 0.0010
pg/a.. Total mortality of pike fry reached 9954 at 0.010 pg/fi. and 50%
at 0.0010 pg/J. by 23 days after fertilization. Total mortality of trout
fry was 26% at 0.010 vg/fi. and 12% at 0.0010 pg/st. Although cumula-
tive mortality was not significantly Increased at the lowest test concentra-
6-3
-------
tlon {0.0001 pg/fc), sublethal effects occurred 1n both species. At this
concentration, growth was significantly, but temporarily, retarded 1n both
species.
Helder (1981) also exposed Juvenile trout to nominal concentrations of
0.100 and 0.010 pg/s, for 96 hours and followed growth and survival for
72 days. Growth was significantly reduced 1n both groups. Mortality
reached 100H by 27 days at the highest concentration, but was only 754 at the
lowest concentration.
The only other study regarding the effects of acute exposure on aquatic
animals 1s that of Beatty et al. (1976), who Investigated the effects of
single 1ntraper1toneal Injections of 2,3,7,8-TCDO 1n larval and adult frogs,
Rana catesblana. Groups of 15 tadpoles and 5 adults were Injected with
2,3,7,8-TCDD 1n olive oil at maximum nominal dosages of 1000 and 500 yg/kg
bw, respectively. There were no effects on survival and metamorphosis of
larvae through 50 days after Injection, or on survival of adults for 35 days
after Injection. There was a slight, temporary decrease 1n food consumption
by adults at the highest dose. H1stopatholog1cal examination revealed no
significant lesions 1n metamorphoslzed or adult frogs. The lack of toxlclty
1n this amphibian species 1s In sharp contrast to the results previously
described with fish. Although the difference may be due, 1n part, to the
different routes of exposure, 1t 1s probable that some fish are actually
more sensitive, because toxic effects occurred 1n coho salmon at an Internal
dose of 5.4 Pg/kg bw (HUler et al., 1973, 1979).
6.1.1.2. CHRONIC TOXICITY — The effects of chronic or subchronlc
exposure to 2,3,7,8-TCDD have been reported for three species of freshwater
Invertebrates and three species of freshwater fish (Table 6-2). Miller et
al. (1973) exposed adult snails, Physa sp., adult ollgochoete worms, Paran-
ajs sp., and mosquito larvae Aedes aegyptl to a nominal Initial concentra-
6-4
-------
TABLE 6-2
Effects of Chronic or Subchronlc Exposure to 2,3.7,8-TCDD on Aquatic Animals
Species
Mosquito,
Aedes aeqyptl
Ollgochaete Worm,
Parana Is sp.
Snail,
Physa sp.
Snail,
Helosona sp.
i
01 Waterflea,
Daphnla magna
flosqultoflsh,
Sambusla afflnls
Channel Catfish,
Ictalurus punctatus
Rainbow Trout,
Sal mo galrdnerl
Life Stage,
Weight or
Length
larvae
adult
adult
adult
adult
NR
flngtrllngs
7.8 era
Duration
of Exposure
(days)
17
55
36
32
32
IS
20
105
Duration
of Test
(days)
30
55
48
46
32
15
20
IDS
Lowest Effect
Concentration
(u9/D
NO
0.2
0.2
ND
ND
0.003
0.003
2300 ug/kg
In diet
Mo Effect
Concentration
(t>9/l)
0.2
MO
ND
0.003
0.003
NO
ND
2.30 tig/kg
In diet
Effect
no effect on pupation
reduced reproduction
reduced reproduction
no apprent effects
no apparent effects
100X mortality
100X mortality
reduced survival,
food consutaptlon and
growth, Increased
fin erosion
Reference
Miller et al.,
1973
Miller et al.,
1973
Miller et al.,
1973
Yockla et al.,
1978
YocklM et al..
1978
Yockln et al..
1978
Yockln et al..
1978
Hawkes and Norrls,
1977
ND * Not determined
-------
tlon of 0.20 pg/8, for 36, 55 and 17 days, respectively. There was no
significant difference 1n total pupation or pupation rate between exposed
and control mosquito larvae during the 17-day exposure period or for the
30-day total test period. Exposure of adult snails to 0.20 pg/fc for 36
days had no significant effect on adult survival and egg production. The
number of live juvenile snails and empty juvenile shells was counted 48 days
after beginning exposure. The total snail hatch was ~30H lower (p=0.056) 1n
the treated groups, but there was no significant difference 1n the percent-
age of survival of young snails. Exposure of worms to 2,3,7,8-TCDD resulted
1n a significant decrease 1n the total number of worms at 55 days. Total
and mean dry weight were also reduced, but the variation among replicates
reduced the statistical significance of this effect to p=0.057, Indicating
that 0.20 pg/8. exerted Us principal effect on reproduction rather than
Individual worm growth.
HUler et al. (1973) also conducted chronic feeding studies with rainbow
trout. The results of this study were also reported by Hawkes and Morris
(1977). Groups of rainbow trout were fed diets containing 0.0023, 2.30 or
2300 pg/kg, 6 days/week for 105 days. The calculated doses were, respec-
tively, 0.000032, 0.036 or 21.0 pg 2,3,7,8-TCOD/kg freeze-dry bw/day.
Consumption of food containing 0.0023 and 2.3 pg/kg had no effect on
survival, food consumption, growth and fin morphology. In contrast, fish
fed the highest dose showed reduced food consumption after 10 days, reduced
growth by 7 days, fin erosion by 14 days, and mortality that began on day 33
and reached 50% by day 61 and 88% by day 71.
The only other Information concerning subchronlc toxlclty to aquatic
animals was provided by Yocklm et al. (1978), who exposed channel catfish,
Ictalurus punctatus. mosqultoflsh, Gambusla afflnls. waterfleas, Daphnla
6-6
-------
magna. snails, Helosoma sp., and algae, Oedogonlum cardlacum. to 14C-
labeled 2,3,7,8-TCDD 1n a redrculatlng aquatic model ecosystem. Soil was
treated with 100 yg/kg and flooded with water, and organisms were added 1
day after flooding. Organisms were removed periodically for measurement of
tissue residues. The mean concentration (yg/l) 1n the water, measured
by liquid scintillation counting, was 0.0034 at day 1, 0.0029 at day 3,
0.0024 at day 7, 0.0026 at day 15 and 0.0042 at day 32. The mean concentra-
tion through the 32-day period was 0.0031 vg/l. No effects over the
32-day exposure period were observed 1n algae, waterfleas or snails as
measured by reproductive activity, feeding and growth. All unharvested
mosquHoflsh died by day 14, with a mean tissue concentration of 7.2 vg/kg
bw. A second group of mosquHoflsh added at day 15 were all dead after
15-20 days. Channel catfish added at day 32 all died after 15-20 days of
exposure, with a mean tissue concentration of 4.4 yg/kg bw. These results
Indicate that 15-20 days of exposure to ~0.003 yg/8, was lethal to fish,
but had no effects on snails, waterfleas and algae.
6.1.1.3. AQUATIC PLANT EFFECTS -- As mentioned earlier, Yocklm et al.
(1978) did not observe any obvious effects of 0.003 yg/8, on the growth
of the freshwater algae, 0. cardlacum. over a 32-day period. The only other
Information concerning toxldty to aquatic plants was provided by Zullel and
Benecke (1978), who conducted contact Inhibition studies with filamentous
algae, Phorm1d1um sp. Filter paper was spotted 1n three places with 1 yg
of 2,3,7,8-TCDD. Disks (5mm diameter) of filtered algae were placed on the
spots, and the filter paper was placed 1n a petrl dish containing nutrient
media. The motmty of the algae filaments outward from the disks was
measured over a 3-hour period with a photoelectric cell. Relative to
controls, 1 vg of 2,3,7,8-TCDD caused a significant Inhibition of
6-7
-------
motmty. Although the exposure concentration 1s unknown, these results
Indicate that this algal species may be affected by contact with contami-
nated substrates (I.e., sediment).
Jackson (1972) studied the progression of mitosis 1n the African blood
Uly, Haemanthus katherlnae. endosperm cells. In this study, cells were
exposed during prophase, prometaphase, metaphase and anaphase to 2,3,7,8-
TCDD at nominal levels of either 0, 0.1 or 0.5 vg/st, and the ability of
the cells to progress to the next stage of cell division within a 2-hour
period was evaluated. Regardless of the stage of cell division during which
exposure occurred, the treatment resulted 1n an Inhibition of progression to
the next stage. The authors noted that 2,3,7,8-TCDD strongly adsorbs to
glass and speculated that the concentrations, 1n the test chamber were
actually lower than reported. It was estimated that the higher concentra-
tion may possibly be approaching 0.2 jig/2., the solubility of 2,3,7,8-
TCDD 1n water.
6.2. TISSUE RESIDUES
Levels of 2,3,7,8-TCDD 1n several species of commercial fish taken from
eastern Lake Ontario, Lake Erie and the Wei land Canal ranged from 0.002-
0.039 vg/kg In those fish with positive test results (Josephson, 1983).
Rock bass showed no detectable levels. Highest concentrations generally
occurred 1n eels (0.006-0.039 vg/kg), followed by smelt and catfish. The
high fat content In these species (37, 13 and 3.5%, respectively) may
explain, 1n part, the higher 2,3,7,8-TCDD concentrations.
Analysis by the NYS Department of Health showed levels of 2,3,7,8-TCDD
In 46 muscle (fillet) samples of Lake Ontario fish that ranged from
0.002-0.162 vg/kg 1n 45 samples and were undetectable 1n one sample (NRCC,
1981a). The fish that were sampled Included smallmouth bass, lake trout,
6-8
-------
white sucker, brown bullhead, rainbow trout, coho and chlnook salmon, and
brown trout. The Ontario Ministry of the Environment (NRCC, 1981a) reported
i
concentrations olr 2,3,7,8-TCDD ranging between 0.010 and 0.019 pg/kg 1n
fillet samples of lake trout, brown trout, white bass, white perch and smelt
1n Lake Ontario, but no detectable (<0.010 pg/kg) levels 1n fish from the
Niagara River, Lake Erie, Lake Huron or Lake Superior. Other fish residue
data summarized by NRCC (1981a) Included 2,3,7,8-TCDD concentrations 1n
positive samples ranging from 0.020-0.230 ug/kg 1n THtabawassee River,
Saglnaw Bay and other locations near Midland, MI; 0.015-0.480 pg/kg 1n the
Arkansas River; and 0.019-0.102 pg/kg 1n Lake Ontario and Niagara River.
OCDD concentrations 1n fish ranged from 0.040-0.150 pg/kg near Midland,
HI, and from 0.004-0.078 pg/kg 1n the Honesatonlc River. The levels of
2,3,7,8-TCDD In fish and shellfish as determined by various authors are
given 1n Table 6-3.
Levels ranging from 0.004-0.695 pg/kg were cited by the U.S. EPA
(1984) for the edible portion of channel catfish, carp, yellow perch, small-
mouth bass, sucker and lake trout from THtabawassee, Grand and Saglnaw
Rivers, Lake Michigan and Saglnaw Bay. The highest concentrations were
detected In bottom-feeding catfish and carp, and the lowest concentrations
were detected 1n bass, perch and suckers (Harless and Lewis, 1980b).
Young et al. (1976) measured 2,3,7,8-TCDD residue levels 1n terrestrial
and aquatic animals from contaminated areas of Eglln A1r Force Base, FL,
which had received massive amounts of herbicides, one of which (2,4,5-T) was
contaminated with 2,3,7,8-TCDD. Beach mice from contaminated areas
contained 0.540-1.30 pg/kg 1n the liver and 0.130-0.140 pg/kg 1n pelts.
Residues 1n racerunner lizards trapped from the most highly contaminated
6-9
-------
TABLE.6-3
Levels of 2t3f7f8-TCDDs In Fish and Shellfish
Type/Section
of F1sh
Sampling Site
Concentration
tppt)
Reference
I
o
Edible flesh
Catfish
Buffalo
Bottom feeder
Whole body
Rock bass
Eel, smelt and
catfish
Crayfish
Catfish, bass and
wall-eyed pike
Lake trout
Chinook salmon
Coho salmon
Rainbow trout
Brown trout
White perch
White sucker
Bayou Heto/Arkansas River
Bayou Heto/Arkansas River
Bayou Heto/Arkansas River
Bayou Heto/Arkansas River
Tone River, Japan
Lake Ontario/Lake Erie/
Weiland Canal
Lake Ontario/Lake Erie/
Helland Canal
Bergholtz Creek, Love Canal
2,4,5-T contaminated
watershed In Arkansas and
Texas; THtabawassee and
Saglnaw Rivers
Lake Ontario
Lake Ontario
Lake Ontario
Lake Ontario
Lake Ontario
Lake Ontario
Lake Ontario
480
ND (7 ppt)a-50
NO {7-13 ppt)a
77
200
ND
ppt)a
2-39
3.7
NO {5-10 ppt)a
51-107
26-39
20-26
17-32
8-162
17-26
ND {3.2)-10
Hltchum et al.. 1980
Hltchutn et al., 1980
Hltchum et al., 1980
Hltchum et al., 1980
Yamaglshl et al., 1981
Josephson, 1983
Josephson, 1983
Smith et al., 1983b
Shadoff et al., 1977;
U.S. EPA, 1980a;
Buser and Rappe, 1980
O'Keefe et al., 1983
O'Keefe et al., 1983
O'Keefe et al., 1983
O'Keefe et al., 1983
O'Keefe et al., 1983
O'Keefe et al., 1983
O'Keefe et al., 1983
-------
U-tJ \l,Ulll../
Type/Section
of F1sh
Smallmouth bass
Brown bullhead
Carp/Goldfish
Northern pike
Pumpkin seed
Rock bass
Coho salmon
Walleye pike
Smallmouth bass
Carp/Goldfish
Lake trout
Carp
Channel catfish
Sucker
Yellow perch
Coho salmon
Rainbow trout
Perch/sucker
Catfish
Carp
Sampling Site
Lake Ontario
Lake Ontario
Cayuga Creek
Cayuga Creek
Cayuga Creek
Cayuga Creek
Lake Erie
Lake Erie
Lake Erie
Lake Erie
Lake Huron
Lake Huron
Lake Huron
Lake Huron
Lake Huron
Lake Michigan
Lake Superior
Saglnaw Bay
Saglnaw Bay
Saglnaw Bay
Concentration
(ppt)
5.9
3.6
87
32
31
12
1.4-<3.5
2.6
1.6-<2.4
ND (2.6)
21
26
20
25
ND (8.7)
ND (3.8)
1.0
ND (3.8)-25
14-37
23-47
Reference
O'Keefe et a!.,
O'Keefe et al..
O'Keefe et al..
O'Keefe et al.,
O'Keefe et al..
O'Keefe et al.,
O'Keefe et al..
O'Keefe et al..
O'Keefe et al..
O'Keefe et al.,
O'Keefe et al.,
O'Keefe et al.,
O'Keefe et al.,
O'Keefe et al.,
O'Keefe et al.,
O'Keefe et al.,
O'Keefe et al.,
Nlemann et al.,
Nlemann et al.,
Nlemann et al.,
1983
1983
1983
1983
1983
1983
1983
1983
1983
1983
1983
1983
1983
1983
1983
1983
1983
1983
1983
1983
-------
TABLE 6-3 (cont.)
Type/Section
of Fish
Sampling Site
Concentration
(PPt)
Reference
Catfish
Bottom feeders
Lake trout
Rainbow trout
Ocean haddock
Carp
Carp
Carp
Carp
Lake trout
Broun trout
Yellow perch
Channel catfish
Carp
Yellow perch
Small mouth bass
Bayon Heto/Arkansas River
Bayon Heto/Arkansas River
Lake Ontario
Lake Ontario
Atlantic Ocean
Lake Huron
Saglnaw Bay
Bay Port
THtabawassee River
Lake Michigan
Lake Ontario
Woods Pond, HA
Tlttabawassee River,
Saglnaw River and
Grand River
THtabawassee River,
Saglnaw River and
Grand River
Tlttabawassee River
and Saglnaw River
Grand River
ND (3.8)
ND (6.7J-12
34-54
43
ND (4.6)
3-28
94
27
81
5
33
26
157 (13)c
55
13
8
Nlemann et al., 1983
Nlemann et al., 1983
Nlemann et al., 1983
Nlemann et al., 1983
Nlemann et al., 1983
Stalling et al., 1983
Stalling et al., 1983
Stalling et al., 1983
Stalling et al., 1983
Stalling et al., 1983
Stalling et al., 1983
Buser and Rappe, 1983
Harless and Lewis, 1982
Harless and Lewis, 1982
Harless and Lewis, 1982
Harless and Lewis, 1982
-------
TABLE 6-3 {cont.)
Type/Section
of Fish
Sampling Site
Concentration
(ppt)
Reference
I
CO
Sucker
Trout
Trout
Trout
Trout
THtabawassee River
and Saglnaw Bay
Lake Michigan
Lake Ontario at
Burlington, Canada
Lake Ontario at Toronto
Harbor, Canada
Lake Huron at
Burnt Island, Canada
10 (4)c
ND (5)c
61.2 (3.6)
32.3 (3.6)
30.4 (3.6)
Harless and Lewis, 1982
Harless and Lewis, 1982
Ryan et al., 1983
Ryan et al., 1983
Ryan et al., 1983
aNot detected and the detection limit 1s Indicated within the parentheses.
bOnly the GC/MS results of these authors are Included In tabulation
cThese are the mean concentrations In samples showing detectable levels of 2,3,7,8-TCDD.
ND « Not detected
-------
areas contained 0.36-0.37 vg/kg 1n the visceral mass and trunk, respec-
tively. Residues were also found 1n three fish species taken from a stream
and pond 1n the contaminated area. Residue levels of 0.012 vg/kg were
found 1n the viscera of sallfln shiners and 1n the bodies (heads and tails
removed) of mosqultoflsh. Samples of skin, muscle, gonad and gut of spotted
sunflsh contained 0.004, 0.004, 0.018 and 0.085 vg/kg 2,3,7,8-TCDD,
respectively. 2,3,7,8-TCDD was not detected 1n Insect larvae, snails,
diving beetles, crayfish, tadpoles and other fish species taken from water-
bodies that contained 0.010-0.035 vg/kg 1n the sediments.
Finally, the levels of 2,3,7,8-TCDD 1n wildlife have been determined by
various authors. These values are shown 1n Table 6-4. From the somewhat
higher levels of 2,3,7,8-TCDD found 1n Saglnaw Bay and 1n Lake Ontario gull
eggs (Table 6-4), Norstrom et al. (1982) Indicated the possibility of
Industrial contamination since the former 1s near a major 2,4,5-T manufac-
turing plant on the Sag1naw/T1ttabawassee River, and the latter 1s down-
stream from a 2,4,5-TCP plant at Niagara Falls, NY.
6.3. ECOSYSTEM EFFECTS
Investigations concerning the ecosystem effects of 2,3,7,8-TCDD are
restricted to the field studies of Young et al. (1975) at the Eglln A1r
Force Base. A 1-square mile area was sprayed with massive amounts of herbi-
cides over an 8-year period (1962-1970). In particular, a 92-acre test area
was sprayed from 1962-1964 with 87,186 pounds of 2,4,5-T that was contami-
nated with 2,3,7,8-TCDD. Analysis 1n 1974 of surface soils 1n this area
showed 2,3,7,8-TCDD levels of 0.010-0.710 vg/kg. Large numbers of beach
mice were trapped from contaminated and control sites and evaluated for
differences 1n organ weights and hlstopathology. The only significant
differences 1n organ weight were Increased liver weight 1n females and
6-14
-------
I HOLE O-t
TCDD Levels 1n Wildlife
2,3.7.8-TCDD Concentration {ppb)
Type of Tissue
Animal
Rabbit liver
Field mouse whole body
Hare liver
i
tn Toad whole body
Snake liver
Snake adipose tissue
Earthworm whole body
Eagle carcass
Herring gull egg
Sampling Site
Seveso, Italy
Seveso, Italy
Seveso, Italy
Seveso, Italy
Seveso, Italy
Seveso, Italy
Seveso, Italy
throughout U.S.
Saglnaw Bay,
Lake Ontario
Average3
31
4.5
7.7
0.2
2.7
16
12
<50 ppb
NR
Range
1-<1024
0.07-49
2.7-13
LS
LS
LS
LS
NR
0.043-0.093
Reference
FanelH
et al., 1980a
FanelH
et al., 1980c
FanelH
et al., 1980c
FanelH
et al., 1980c
FanelH
et al., 1980c
FanelH
et al., 1980c
Panel 11
et al,, 1980c
Helling
et al., 1973
Og1lv1e, 1981
-------
TABLE 6-4 (cont.)
2.3.7.8-TCDD Concentration (oob)
Type of
Animal
Herring gull
Herring gull
Herring gull
Herring gull
Herring gull
Herring gull
Herring gull
Turtle
Snake
Huskrat
Tissue
egg
egg
egg
egg
egg
egg
egg
egg and liver
liver and
muscle
liver
Sampling Site
Lake Superior
Lake Michigan
Lake Huron
(main body)
Lake Huron,
Saglnaw Bay, N.
Lake Huron,
Saglnaw Bay, S.
Lake Erie
Lake Ontario
Bayou Heto/
Arkansas River
Bayou Me to/
Arkansas River
Bayou Heto/
Arkansas River
Average3
0.011
0.009
0.009
0.043
0.086
0.011
0.059
0.15
0.060
ND (40 ppt)b
Range
NR
NR
NR
NR
NR
NR
NR
LS
LS
LS
Reference
Norstrom
et al., 1982
Norstrom
et al., 1982
Norstrom
et al., 1982
Norstrom
et al., 1982
Norstrom
et al., 1982
Norstrom
et al., 1982
Norstrom
et al., 1982
MHchum
et al., 1980
MHchum
et al., 1980
MHchum
et al., 1980
-------
TABLE 6-4 (cent.)
Type of Tissue
Animal
Racoon liver
Fr.og liver and
muscle
Horse fat
Horse liver
These are averages of samples
Not reported and the limit of
NR = Not reported; LS = Limited
2.3,7.8-TCDD Concentration (ppb)
Sampling Site
Average3 Range
Bayou Heto/ NO (10 ppt)b LS
Arkansas River
Bayou Heto/ >10 LS
Arkansas River
Midwest wire 0.04S LS
reclamation
Incinerator
Hldwest wire NO (<6 ppt)b LS
reclamation
Incinerator
that had above detectable levels of TCDD.
detection Indicated In parentheses
samples
Reference
MHchum
et al., 1980
Hitch urn
et al., 1980
Hryhorczuk
et al., 1981
Hryhorczuk
et al., 1981
-------
Increased spleen weight In males and females taken from the contaminated
sites; however, no hlstopathologlcal effects could be attributed to the
collection sites. Similar studies on racerunner lizards showed no signifi-
cant difference 1n relative or total body weight of animals collected from
contaminated and control sites. Sweep net surveys of the contaminated sites
for terrestrial Insects 1n 1971 and 1973 Indicated that there was a signifi-
cant Increase In the number of families and total number of Insects 1n the
contaminated test site, which was correlated with the Increase 1n vegetation
after herbicide spraying. Aquatic species diversity studies were conducted
1n 1969, 1970, 1973 and 1974 on a stream In the contaminated area and a con-
trol stream. As mentioned before, 2,3,7,8-TCDD was detected In sediments
and fish from the contaminated stream; however, there was no significant
difference 1n Ichthyofauna diversity 1n the two streams, and no significant
change 1n diversity through time In either stream. As a result, the only
effects that can be attributed to 2,3,7,8-TCDD contamination were Increased
Hver and spleen weight 1n beach mice. The ecological significance of this
effect 1s unknown, especially since no obvious detrimental effects were
observed 1n this or other species from contaminated sites.
Korfmacher et al. (1984) analyzed fat tissue and eggs from snakes for
2,3,7,8-TCDD. Water snakes were selected as a possible marker for
2,3,7,8-TCDD contamination. Three snakes were collected from Lake Dupree,
Arkansas In 1983. This lake 1s a site of 2,3,7,8-TCDD contaminated sediment
and fish (Arkansas Dept. of Pollution Control and Ecology, 1983). Two
snakes were collected from a lake evidently not contaminated with
2,3,7,8-TCDD from any Industrial source. Eggs were derived from one of the
snakes obtained from Lake Dupree. 2,3,7,8-TCDD concentration 1n the fat
material of three snakes from contaminated lake varied from 500-730 ppt, 1n
6-18
-------
the snake eggs varied from 151-294 ppt. and 1n the fat material from two
snakes from noncontamlnated lake varied from 38-378 ppt.
The only other Information pertinent to ecosystem level effects was
provided by Bollen and Norrls (1979), who Investigated the effects of
2,3,7,8-TCDD on respiration (C02 production) In forest Utter and soil
samples. LHter and soil samples were air dried, placed 1n blometer flasks,
moistened and treated with 2,3,7,8-TCDO. Concentrations as high as 0.031
vg/kg dry weight 1n Utter had no effect on respiration. Concentrations
as high as 0.052 yg/kcj dry weight 1n soil caused a slight but significant
stimulation of CO,, production. Because higher concentrations were not
tested, 1t 1s unknown whether 2,3,7,8-TCDO would have Inhibitory effects on
soil microblal populations, carbon metabolism or nutrient cycling at the
higher levels of soil contamination found 1n such contaminated areas as the
Eglln A1r Force Base test site.
6.4. SUMMARY
Almost all of the available Information concerning the toxldty of PCODs
to wildlife deals with aquatic species. Acute exposure to Initial nominal
2,3,7,8-TCDD concentrations as low as 0.0001 jig/a has been shown to
cause delayed sublethal effects 1n early life stages of northern pike and
rainbow trout (Helder 1980, 1981) and 1n adult gupples (Miller et al.,
1979). Decreased growth, food consumption and survival have been reported
In these and other fish species after acute exposure to >0.001 yg/i.
During these tests, the nominal Initial concentrations probably decreased.
rapidly because of uptake by test organisms, adsorption to the exposure
containers and perhaps volatilization. As a result, H 1s possible that
constant acute or chronic exposure to dissolved concentrations <0.0001
would produce toxic effects 1n sensitive aquatic organisms.
6-19
-------
Several studies provide evidence that 2,3,7,8-TCDD 1s less toxic to
aquatic Invertebrates and amphibians than to the tested fish species.
Subchronlc exposure to an Initial nominal concentration of 0.20 yg/a, had
no effect on mosquito population and caused a 30-50% decrease 1n reproduc-
tion of snails and ollgochoete worms (Miller et al., 1973). In contrast,
acute exposure to 0.1 yg/a. caused 100% delayed mortality 1n gupples
(Norrls and Miller, 1974) and juvenile rainbow trout {Helder 1981).
Similarly, exposure to relatively constant, measured, dissolved concentra-
tions of ~0.002-0.004yg/», 1n aquatic model ecosytems killed all exposed
mosqultoflsh and channel catfish 1n 15-20 days, but had no discernible
effects on snails and waterfleas over a total test period of 32-46 days
(Yocklm et al., 1978). The dying mosqultoflsh and catfish had mean whole-
body 2,3,7,8-TCDD concentrations of 7.2 and 4.4 yg/kg, respectively. In
contrast, single 1ntraper1toneal Injections of 2,3,7,8-TCDD at maximum doses
of 500 or 1000 yg/kg bw, respectively, had no effects on adult frogs over
a 35-day period or on frog larvae over a 50-day period (Beatty et al., 1976).
Chronic feeding studies with groups of rainbow trout showed that dally
feeding of 2300 yg/kg 1n the diet was lethal to all but two fish (88%) 1n
71 days, but no significant effects were seen 1n fish fed dally a diet
containing 2.3 yg/kg for 105 days (Hawkes and Norrls, 1977). Residue
analysis of single fish sampled at the end of the tests showed 2,3,7,8-TCDD
levels of 1380 yg/kg bw 1n one high dose fish and 1.573 yg/kg In one low
dose fish.
Although only limited Information was found concerning the effects of
2,3,7,8-TCDD on aquatic plants, 1t 1s probable that they are less sensitive
than fish. Using model ecosystems, Yocklm et al. (1978) observed no obvious
effects on algae at concentrations (0.002-0.004 g) that killed fish. Zullel
6-20
-------
and Benecke (1978) observed contact Inhibition of filamentous algae placed
1n contact with 1 $jg quantities of 2,3,7,8-TCDD spotted on filter paper.
The only available Information concerning the effects of low level
environmental exposure to 2,3,7,8-TCDD on terrestrial wildlife was reported
by Young et al. (1975), who Investigated tissue residues and several bio-
logical parameters 1n mice and lizards from contaminated and control sites
at Eglln A1r Force Base, FL. The concentrations of 2,3,7,8-TCDD 1n contami-
nated soils were 0.010-0.710 pg/kg. Mice trapped from the contaminated
site contained 0.540-1,30 vg/kg 1n the Hver and had significantly higher
spleen and Hver weights than mice from control sites. No other differences
(hlstopathology, weights of other organs, Incidence of abnormal fetuses,
etc.) were observed. Racerunner lizards from the contaminated site con-
tained 0.36-0.37 pg/kg 1n the viscera and trunk and showed no differences
1n body weight or hlstopathology compared with lizards from control sites.
Residues of 2,3,7,8-TCDD In three fish species taken from a pond and stream
adjacent to the contaminated site ranged from 0.004-0.085 j»g/kg. Sedi-
ments derived from the erosion taken from the contaminated site contained
localized concentrations of 0.010-0.035 ng/kg. PCDD residues have been
reported for numerous other fish species and other snakes from contaminated
water bodies. The PCDD concentrations (primarily 2,3,7,8-TCDD) 1n positive
fish tests ranged from 0.002-0.695 jig/kg.
6-21
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7. COMPOUND DISPOSITION AND RELEVANT PHABHACOKINETICS
7.1. ABSORPTION
Data are available regarding the absorption of 2,3,7,8-TCDD through the
gastrointestinal (61) tract and skin of experimental animals. Absorption
through the respiratory tract, however, has not been studied. Also, there
are no data on the absorption of 2,3,7,8-TGDD when mixed with other chlori-
nated compounds, which 1s presumably the case for human exposures.
7.1.1. Absorption from the Gastrointestinal Tract. Data on the 61
absorption of 2,3,7,8-TCDD are summarized 1n Table 7-1. The 61 absorption
of 2,3,7,8-TCDD has been Investigated more extensively 1n the rat than 1n
other species. When 2,3,7,8-TCDD was administered 1n the diet at 7 or 20
ppb for 42 days, 50-60% of the consumed dose was absorbed (Fries and Marrow,
1975). Administration of 2,3,7,8-TCDD by gavage 1n acetone:corn oil (1:25
or 1:9) as a single dose or as repeated doses (5 days/week x 7 weeks)
resulted 1n absorption of a larger percentage (70-86%) of the dose (Rose et
a!., 1976; Piper et al., 1973). It would appear, therefore, that the 61
absorption of 2,3,7,8-TCDD may vary, depending upon the vehicle used. The
Influence of vehicle or adsorbent on 61 absorption has been Investigated by
Polger and Schlatter (1980), using hepatic concentrations 24 hours after
dosing as an indicator of the amount absorbed. They found a linear rela-
tionship between ng 2,3,7,8-TCDD administered by gavage 1n 50% ethanol (for
doses of 12-280 ng, equivalent to 0.06-1.4 jig/kg) and the percentage of
the dose 1n hepatic tissues (36.7-51.5%). At the next higher dose of 1070
ng the percentage was 42%. Administration of 2,3,7,8-TCDD 1n an aqueous
suspension of soil resulted in a decrease 1n the hepatic levels of 2,3,7,8-
TCDD as compared with hepatic levels resulting from administration of
7-1
-------
TABLE 7-1
Gastrointestinal Absorption of 2,3,7,8-TCDD
Species
Guinea
pig
Rat
Rat
Rat
Rat
Rat
Hamster
Vehicle
NR
7 ppb,
1n diet
20 ppb,
1n diet
A:C,
1:25
A:C,
1:25
A:C,
1:9
olive
oil
Dose Schedule % Absorption
(vg/kg) Mean + SD
NR 50
single dose
0.5 yg/kg/day x 50-60
42 days
1.4 yg/kg/day x 50-60
42 days
1.0 yg/kg, 84 ± 11*
single dose
0.1 or 1.0 86 + 12*
vg/kg/day,
5 days/week x
7 weeks
50.0 vg/kg, 70
single dose
650 yg/kg, 74 ± 23*
single dose
Reference
Nolan et al., 1979
Fries and Marrow, 1975
Fries and Marrow, 1975
Rose et al . , 1976
Rose et al., 1976
Piper et al., 1973
Olson et al., 1980a
*Mean ± standard deviation
NR = Not reported; A:C = Acetone:corn oil, v:v
7-2
-------
2,3,7,8-TCDD in 50% ethanol. The extent of the decrease was directly pro-
portional to the length of time the 2,3,7,8-TCDD had been 1n contact with
the soil. McDonnell et al. (1984) observed a dose-response relation of
liver accumulation of 2,3,7,8-TCOD as a result of 1ntragastr1c exposure of
young male Hartley guinea pigs to 2,3,7,8-TCDO 1n corn oil or 1n soil (Table
7-2). In Sprague-Dawley female rats, they found as high as 40.8 ppb and
20.3 ppb liver accumulation of 2,3,7,8-TCDD by 1ntragast1c exposure to
2,3,7,8-TCDD 1n corn oil and 1n soil, respectively. Ph1l1pp1 et al. (1981)
and Huetter and Ph1l1pp1 (1982) have shown that radlolabeled 2,3,7,8-TCDD
becomes progressively more resistant with time to extraction from soil.
Polger and Schlatter (1980) also demonstrated that 2,3,7,8-TCDD mixed 1n an
aqueous suspension of activated carbon was very poorly absorbed (<0.07% of
the dose 1n hepatic tissues). In addition, Sllkworth et al. (1982) observed
an Increase 1n the LD5Q value for female guinea pigs from 2.5 to 19
yg/kg when the 2,3,7,8-TCDD was administered by gavage 1n corn oil or
aqueous methyl cellulose, respectively.
A comparative study on =;the biological uptake 1n the rabbit of 2,3,7,8-
TCDD 1n different formulations, Including accident-contaminated Seveso soil,
was conducted by Bonaccorsl et al. (1983). On the whole, the results
Indicated that soil-borne 2,3,7,8-TCDD had a b1oava1lab1Hty lower than that
of free (solvent-borne) 2,3,7,8-TCDD.
The feeding of fly ash containing PCDDs to rats 1n the diet for 19 days
resulted In considerably lower hepatic levels of PCDDs than did the feeding
of an extract of the fly ash at comparable PCDD dietary concentrations (Van
der Berg et al., 1983). The PCDDs were tentatively Identified as 2,3,7,8-
TCDD, 1,2,3,7,8-PeCDD, 1,2,3,6,7,8-HxCDD and 1,2,3,7,8,9-HxCDD. The
7-3
-------
TABLE 7-2
Liver Accumulation of 2,3,7,8-TCDD 1n Guinea P1gs
30 Days after a Single Intragastrlc Exposure to 2,3,7,8-TCDDa
Group
1
2
3
4
5
6
7
8
9
10
No. of
Animals
6
6
6
6
5d
5«
6
6
6
5e
Composition of the
Material Gavaged
Corn oil
TCDD 1n corn oil
TCDD 1n corn oil
Time Beach soil
Time Beach soil
Time Beach soil
Hlnker Stout soil
Hlnker Stout soil
Hlnker Stout soil
Time Beach soil
Total Quantity
Gavaged
0.1 ma/100 g
0.1 ma/100 g
0.1 ma/100 g
0.35 g
1.07 g
3.60 g
0.26 g
0.80 g
2.67 g
3.60 g
Dosage of TCDD
(vg/kg bw)
0
1
3
1.3
3.8
12.8
1.1
3.3
11.0
0
Average Liver
Concentration of TCDD"
ppt * SEH
ND
1.6*0.2
4.1C
13.3*2.3
<1.0
1.0*0.1
3.2C
34.3*6.0
<1.0
1.4*0.3
2.0*0.1C
25.7*5.2
ND
(unconlaminated)
-------
TABLE 7-2 (cont.)
i
Ul
Group
No. of
Animals
Composition of the
Material Gavaged
Total Quantity
Gavaged
Dosage of TCDD
(vg/kg bw)
Average Liver
Concentration of TCDDb
ppt ± SEH
11 6 Time Beach soil 2.71 g 10 45.4*8.4
{uncontamlnated
but TCDD added)
aSource: McConnell et al. (1984)
bDetect1on limit 100 ppt
cAn1mal/an1mals which died before 30 days
dOne animal died 2 days after dosing (not Included)
eOne animal died at the time of dosing
SEH = Standard error of the mean
NO = Not detected
-------
difference 1n hepatic levels noted between fly ash-treated and
extract-treated rats was greater for the more highly chlorinated Isomers
than 1t was for 2,3,7,8-TCDD.
The 61 absorption of 2,3,7,8-TCDD was also examined In the hamster, the
species most resistant to the acute toxiclty of this toxin. Olson et al.
(1980a) administered a single, sublethal, oral dose of [l,6-3H]-2,3,7,8-
TCDD In olive oil (650 yg/kg) to hamsters and reported that 7454 of the
dose was absorbed, while Nolan et al. (1979) reported that absorption 1n the
guinea pig, the most sensitive species, was ~50% following administration of
an unspecified amount of 2,3,7,8-TCDD. The vehicle and method for calculat-
ing the absorbed dose were not given 1n this report.
7.1.2. Absorption Through the Skin. Information on the absorption of
2,3,7,8-TCDD through the skin 1s extremely limited. Polger and Schlatter
(1980) administered 26 ng 2,3,7,8-TCDD 1n 50 W8. methanol to the skin of
six rats. After 24 hours, the liver contained 14.8i2.6% of the dose. By
comparing with hepatic levels obtained (1n the same study) after oral
administration 1n 50% ethanol (see Section 7.1.1.), assuming that hepatic
levels are valid estimates of the amount absorbed from both oral and dermal
routes and that absorption from methanol 1s equivalent to absorption from
50% ethanol, the amount absorbed from a dermal application can be estimated
at ~40% of the amount absorbed from an equivalent oral dose. As compared
with dermal application 1n methanol, dermal application of 2,3,7,8-TCDD to
rats 1n vaseline or polyethylene glycol resulted 1n hepatic tissue concen-
tration of 1,4 and 9.3% of the dose, respectively, but had no observable
effect on the concentration of 2,3,7,8-TCDD required to Induce skin lesions
(~1 vg) 1n the rabbit ear assay (Polger and Schlatter, 1980). Application
of 2,3,7,8-TCDD 1n a soil/water paste decreased hepatic 2,3,7,8-TCDD to ~2%
of the administered dose and Increased the amount required to produce skin
7-6
-------
lesions to 2-3 yg 1n rats and rabbits, respectively. Application 1n an
activated carbon/water paste essentially completely eliminated absorption,
as measured by percent of dose 1n the liver, and Increased the amount of
2,3,7,8-TCDD required to produce skin lesions to ~160 yg.
7.2. DISTRIBUTION
The tissue distribution of 2,3,7,8-TCDD 1n a number of species 1s
summarized 1n Table 7-3. As would be predicted from the UpophlUc nature
of this compound, accumulation tends to occur 1n tissues with a high I1p1d
content. In rats and mice, 2,3,7,8-TCDD residues are localized 1n the liver
and adipose tissue. In the rat, hepatic levels of 2,3,7,8-TCDD accounted
for -38-52% of the administered dose during the first week following oral
administration of a single dose ranging from 0.07-50 yg/kg (Piper et al.,
1973; Polger and Schlatter, 1979). The latter dose 1s within the LD5Q
range for rats. Similar results were obtained 7 days following administra-
tion of a single 1ntraper1toneal dose of 400 yg/kg of [3H]2,3,7,8-TCDD
to rats; 43% of the total dose was localized In the liver (Van Miller et
al., 1976). In two strains of mice, the liver contained ~35% of an admin-
istered dose of 2,3,7,8-TCDD 1 day after oral or 1ntraper1toneal administra-
tion (Manara et al., 1982). In both species, 1-22 days after single-dose
oral or 1ntraper1toneal administration, levels of 2,3,7,8-TCDD 1n adipose
tissue were similar to or slightly lower than levels 1n the liver, and were
considerably higher than concentrations 1n other tissues (Piper et al.,
1973; Rose et al., 1976; Van Miller et al., 1976; Manara et al., 1982),
Including the thymus (Rose et al., 1976; Van MUler et al., 1976).
In a 7-week gavage study and a 2-year dietary study of 2,3,7,8-TCDD 1n
rats, 2,3,7,8-TCDD was present 1n the liver at 3-5 times the concentration
1n adipose tissue when the dally dose or Intake of the compound was >0.01
yg/kg/day (Rose et al., 1976; Kodba et al., 1976) and was present at
7-7
-------
TABLE 7-3
Distribution of 2,3,7,8-TCDD
Species
Route of
Administration
Principal Organ Depots
Reference
OP
Rat
Rat
Rat
Rat
Rat
Rat
Mouse
Mouse
Rhesus monkey
Golden Syrian
hamster
Guinea pig
Guinea pig
oral
oral
oral
oral
oral
l.p.
oral
l.p.
l.p.
l.p. or oral
oral
l.p.
liver
liver > fat
liver > fat
liver > fat
liver > fat
liver > fat
liver > fat > kidney > lung
liver > fat > kidney > lung > spleen
fat > skin > liver > adrenals = thymus
liver > fat
fat > liver > adrenals > thymus > skin
fat > liver > skin
Fries and Marrow, 1975
Rose et al.f 1976
Piper et a!., 1973
Koclba et a!., 1978a
Allen et a!., 1975
Van MUler et a!., 1976
Manara et al., 1982
Manara et al., 1982
Van Miller et al.» 1976
Olson et al., 1980a
Nolan et al., 1979
Gaslewlcz and Neal, 1979
1.p. = intraperitoneal
-------
about the same concentration as 1n adipose tissue when the dally Intake was
0.001 ug/kg/day (Koclba et a!., 1976). As 1n the single-dose studies,
2,3,7,8-TCDD levels were considerably lower 1n other tissues, Including the
thymus, than 1n Hver or adipose tissue (Rose et a!., 1976).
There 1s some evidence of sex differences 1n tissue distribution 1n
rats. During 42 days of administration of 2,3,7,8-TCDO at 7 or 20 ppb 1n
the diet, -85% of the total body residue of male rats was located 1n the
liver, as compared with 70% 1n females (Fries and Marrow, 1975). This small
difference In distribution patterns may have resulted from sex differences
1n relative adipose tissue content.
The ability of mouse Hver to sequester 2,3,7,8-TCOD Increases with
prolonged exposure (Teltelbaum and Poland, 1978). The hepatic uptake of
[3H]2,3,7,8-TCDD 1n Swiss-Webster mice was maximal 12 hours after Intra-
perltoneal Injection. Hepatic uptake, expressed as percent of total dose,
Increased from 11.7% 1n control mice to 60.9% 1n mice that had been pre-
treated with a single dose of unlabeled 2,3,7,8-TCDD 36 hours previously.
This observation 1s consistent 'with other data that Indicate that 2,3,7,8-
TCDD 1s a potent Inducer of hepatic mlcrosomal mixed-function oxldase
(Section 8.1.1.5.) and that >90% of the hepatic 2,3,7,8-TCDD Is localized 1n
the mlcrosomes (Allen et a!., 1975). The toxldty of 2,3,7,8-TCDD 1n mice
has been demonstrated to correlate with the affinity of the receptor that
controls this Induction In m!ce (Poland and Glover, 1980).
In nonhuman primates, the liver seems to have much less of a role 1n
2,3,7,8-TCDD accumulation. Van Miller et al. (1976) have compared the
tissue distribution of [3H]2,3,7,8-TCDD 1n adult rhesus monkeys, Infant
rhesus monkeys, and Sprague-Dawley rats 7 days after a single 1ntraper1ton-
eal Injection of 400 jig 2,3,7,8-TCDD/kg bw. They found that while 43% of
the administered dose was localized 1n the livers of the rats, only 10.4%
7-9
-------
was found 1n the "livers of adult monkeys and 4.5% 1n the livers of Infant
monkeys. This difference cannot be explained by differences 1n absorption
or excretion, since these parameters were observed to be similar 1n both
species. In monkeys, larger percentages of the dose were found 1n adipose
tissue, skin and muscle than was the case for rats.
HcNulty et al. (1982) reported that 2 years after administration of a
single oral dose of 1 pg/kg of 2,3,7,8-TCDD to an adult rhesus macaque
monkey, tissue levels of the compound were 1000 ppt 1n adipose tissue and 15
ppt 1n the liver. These results Indicate that prolonged retention of
2,3,7,8-TCDD may occur 1n this species. The tissue distribution of 2,3,7,8-
TCDD 1n the guinea pig appears to be similar to the monkey, with the highest
concentration of the toxin being found 1n adipose tissue (Gas1ew1cz and
Neal, 1979; Nolan et al., 1979). The Interspedes difference 1n the tissue
distribution of 2,3,7,8-TCDD may be related to the relative adipose tissue
content of a given species and the affinity of 2,3,7,8-TCDD for the hepatic
mlcrosomal fraction; however, the significance of these differences remains
1n doubt. For example, the hepatotoxlclty of 2,3,7,8-TCDD 1n a given
species does not appear to be related to the hepatic concentration of the
toxin (Neal et al., 1982).
Very limited data are available on the tissue distribution of 2,3,7,8-
TCDD 1n humans. Facchettl et al. (1980) reported tissue concentrations of
2,3,7,8-TCDD at levels of 1-2 ng/g In adipose tissue and pancreas, 0.1-0.2
ng/g 1n Hver and <0.1 ng/g In thyroid, brain, lung, kidney and blood 1n a
woman who died 7 months after potential exposure to 2,3,7,8-TCDD from the
Seveso accident. This pattern of 2,3,7,8-TCDD distribution, however, may
not be representative for humans since the woman at the time of death had an
adenocarclnoma (which was not considered related to the accident) that
Involved the pancreas, liver and lungs.
7-10
-------
In addition, Young et at. (1983) reported preliminary results of the
analyses of adipose tissue from soldiers exposed to Agent Orange. Two
analyses were performed, one using the exact mass of 321.8936 and the other
the signal profile at masses of 321.8936 and 319.8965. Three groups were
studied consisting of 20 veterans claiming health problems related to Agent
Orange exposure; 3 A1r Force officers with known heavy exposure to Agent
Orange during disposal operations and 10 control veterans with no known
herbicide exposure. In the first group, 10 of the 20 had measurable levels
of 2,3,7,8-TCDD (5 with 5-7 ppt, 3 with 9-13 ppt, 1 with 23 and 35 ppt and
another with 63 and 99 ppt). In the second group, only two officers had
i
measurable 2,3,7,8-TCDD levels that did not exceed 3 ppt. In the 10 control
veterans, 4 had 2,3,7,8-TCDD levels between 6 and 14 ppt. Levels of
2,3,7,8-TCDD 1n adipose tissue did not appear to be associated 1n this study
with 111 health or any particular symptom; however, 1t was considered that
Information on background levels of 2,3,7,8-TCDD 1n adipose tissue was too
limited to draw any firm conclusions.
2,3,7,8-TCDD has been demonstrated to be fetotoxlc 1n the rat (Section
9.1.). The ability of 2B3,7,8-TCDD to gain access to the developing fetus
of Fischer 344 rats following a single oral dose of [14C]2,3,7,8-TCDD was
Investigated by Moore et al. (1976). They found low concentrations of
2,3,7,8-TCDD 1n the fetus at gestation days 14, 18 or 21. The radioactivity
appeared to be evenly distributed throughout the fetus on days 14 and 18j
however, Increased levels of radioactivity were detected 1n fetal Hver on
day 21.
Nau and Bass (1981) (more recently reported by Nau et al., 1982)
Investigated the fetal uptake of 2,3,7,8-TCDD 1n NMRI mice following oral,
1ntraper1toneal or subcutaneous administration of 5, 12.5 or 25 iig/kg 1n
7-11
-------
DMSO:corn oil or acetone:corn oil. The chemical was usually administered as
a single dose 2 days before sacrifice. Embryonic 2,3,7,8-TGDD concentra-
tions were maximal on gestatlonal days 9 and 10; however, low levels were
found In the embryo and fetus between gestatlonal days 11 and 18. This
sharp decrease 1n 2,3,7,8-TCDD concentration coincides with placentatlon.
2,3,7,8-TCDD concentrations 1n the placenta were an order of magnitude
greater than 1n the fetus Itself. The affinity of fetal Hver for 2,3,7,8-
TCDD was relatively low, as compared with maternal liver; however, 2,3,7,8-
TCDD levels 1n fetal Hvers were 2-4 times higher than the levels 1n other
fetal organs. An attempt was made to correlate 2,3,7,8-TCDD levels 1n the
fetuses with the observed Incidence of cleft palate, but no clear relation-
ship was observed (I.e., 5 minutes to 61 days after Injection).
Autorad1ograph1c studies of tissue localization following Intravenous
administration of [14C]2,3,7,8-TCDD 1n DMSO to three strains of mice Indi-
cated that the Hver had the highest concentration and longest retention of
-«v
*.
radioactivity 1n the body, followed by the nasal mucosa (Appelgren et al.,
1983). In pregnant mice, the concentration of radioactivity 1n the fetuses
was lower than 1n the dams, but a similar, selective labelling of the liver
and the nasal mucosa was seen 1n the fetuses at day 17 of gestation. In the
adult animals, labelling of the adrenal cortex was about equal to that of
the liver at 1 hour after dosing, but thereafter was much lower than 1n the
Hver. Labelling of the thymus, lymph nodes, bone marrow and prostate were
low at all observation times.
7.3. HETABOLISH
Vlnopal and Caslda (1973) found no evidence of water soluble metabolites
of 2,3,7,8-TCDD following Incubation with mammalian liver mlcrosomes or
7-12
-------
)ntraper)tonea1 injection Into mice. In the same experiment, only unmetab-
ollzed 2,3,7,8-TCDD was extractable from mouse liver 11-20 days after treat-
ment. Piper et al. (1973), however, detected 14C activity 1n the expired
air and urine within the first 10 days following administration to rats,
Indicating that some metabolic alteration of 2,3,7,8-TCDD occurs. Nelson et
al. (1977) found that Incubation of [i:>C]2,3,7,8-TCDD with rat hepatic
mlcrosomes resulted 1n the formation of bound radioactivity which, 1n
contrast to free 2,3,7,8-TCDD, was not ethyl acetate extractable. This
binding was found to result from ox1dative metabolism, as Indicated by a
requirement for NADPH, and could be Induced by phenobarbltal pretreatment.
Binding was not covalent, because the bound radioactivity could be extracted
with chloroform:methanol (9:1); this extracted radioactivity cochromato-
graphed with the 2,3,7,8-TCDD standard.
Ramsey et al. (1982) detected five distinct radioactive compounds 1n the
bile of rats given dally oral doses of 15 wg [14C]2,3,7,8-TCDD. Incuba-
tion of the bile with
-------
The ability of 1,6-^-2,3,7,8-1000 derived radioactivity to bind to
rat hepatic macromolecules jn. vivo was Investigated by Poland and Glover
(1979). They found maximum levels of 60 pmol 2,3,7,8-TCDD/mole of amlno
adds 1n protein, 12 pmol 2,3,7,8-TCDD/mole of nucleotlde In rRNA, and 6
pmol of 2,3,7,8-TCDD/mole of nucleotlde 1n DNA. According to the authors
this corresponds to one 2,3,7,8-TCDD-DNA adduct/35 cells (Poland and Glover,
1979). Similar results were obtained using a mouse liver mlcrosomal system
(Guenthner et al., 1979a). [3H]2,3,7,8-TCDD was found to bind to mlcro-
somal protein 120-2640 times more readily than to deprotelnlzed salmon sperm
DNA. They estimated the rate of 2,3,7,8-TCDD metabolism to be between 9000
and 36,000 times lower than the rate of P-450-med1ated benzo[a]pyrene
metabolism.
Tulp and Hutzlnger (1978) studied the metabolism of a variety of PCDDs,
Including 1,2,3,4-TCDD, 1n the rat. In d1- and higher substituted dloxlns,
only mono- and dlhydroxy derivatives were detected. Primary hydroxylatlon
occurred exclusively at the 2-, 3-, 7- or 8-pos1t1on, so the significance of
this study for the metabolism of 2,3,7,8-TCDD 1s not clear. Sawahata et al.
(1982) Investigated the metabolism of 2,3,7,8-TCDD 1n Isolated rat hepato-
cytes. The major product was deconjugated with q>-glucuron1dase, derlva-
tlzed with dlazomethane, and separated Into two compounds by HPLC. These
metabolites were subsequently Identified as l-hydroxy-2,3,7,8-TCDD and
2-hydroxy-3,7,8-tr1chlorod1benzo-p_-d1ox1n.
Polger et al. (1982a) Identified six metabolites 1n the bile of dogs
that were given [3H]2,3,7,8-TCDD. The major metabolite was 1,3,7,8-tetra-
chloro-2-hydroxyd1benzo-p,-d1ox1n. 2-Hydroxy~3,7,8-tr1chlorod1benzo-p_~d1ox1n
7-14
-------
and ],2-d1chloro-4,5-d1hydroxybenzene were also Identified as minor metabo-
lites. The structures of the three remaining metabolites were not deter-
mined; however, two appeared to be tr1chloro-d1hydroxyd1benzo-p_-d1ox1ns and
the third was apparently a chlorinated 2-hydroxyd1phenyl ether. The pres-
ence of these metabolites 1s consistent with a 1,2-arene oxide Intermediate.
Isolated rat hepatocytes 1n suspension have been used as an Ijn vitro
system for assessing 2,3,7,8-TCDO metabolism under various conditions. Data
Indicate that the rate of 2,3,7,8-TCDO metabolism 1n rat hepatocytes
correlates directly with drug Induced changes In hepatic cytochrome P-450
monooxygenase activity, suggesting that 2,3,7,8-TCDD 1s metabolized by this
enzyme (Olson et a!., 1981).
Beatty et al. (1978) found a correlation between hepatic mixed-function
oxldase (MFO) activity and the toxldty of 2,3,7,8-TCDD 1n rats. Both 1n
naturally occurring age- and sex-related differences 1n MFO activity and
following the administration of Inducers and Inhibitors of MFO enzyme
systems, hepatic HFO activity was Inversely related to toxldty that corre-
sponds to direct relationship between the 20-day LDg- and HFO activity.
The fate of 2,3,7,8-TCDD metabolites from dogs has been examined In rats
by Weber et al. (1982). 2,3,7,8-TCDD metabolites were extracted from the
bile of 2,3,7,8-TCDO-treated dogs and administered by gavage to female
Sprague-Dawley rats. The 2,3,7,8-TCDD metabolites were rapidly cleared from
the bodies of blle-duct-cannulated rats, with >85% of the dose recovered 1n
the feces, bile and urine within 24 hours. In Intact rats, only 13H of the
dose was excreted 1n the feces and urine during the first 24 hours, Indicat-
ing enterohepatlc circulation; however, the administered radioactivity was
completely eliminated within 72 hours after dosing.
7-15
-------
Polger et al. (1982a) Investigated the toxlclty of 2,3,7,8-TCDD metabo-
lites by administering bile extract from 2,3,7,8-TCDD-treated dogs to male
guinea pigs In single oral doses equivalent to 0.6, 6.0 and 60 pg of
parent compound/kg bw. Other groups of guinea pigs received bile extract
from untreated dogs or 2,3,7,8-TCDD Itself. A comparison of the mortality
data at 5 weeks after dosing Indicated that the acute toxldty of 2,3,7,8-
TCDD to guinea pigs was at least 100 , times higher than was the acute
toxldty of Its metabolites.
Olson and Blttner (1983) reported that the rate of metabolite formation
In vitro was considerably higher In hepatocytes from the hamster than 1n
hepatocytes from the rat. Qualitative evaluation of in vivo and jn. vitro
metabolites by HPLC also suggested major Interspedes variability. The
authors suggested that such differences In metabolism may partially explain
the differences 1n toxldty among species.
7.4. ELIHINATION
The following discussion assumes that elimination Is a first order
process. With the exception of the guinea pig, which may follow zero order
kinetics (Sas1ew1cz and Neal, 1979), elimination data yield a straight line
on a sem1logar1thm1c plot, Indicating a first order process. HUes and
Bruce (1976) pointed out that the studies of Allen et al. (1975) and Piper
et al. (1973) can be Interpreted equally well by either zero or first order
kinetics. The majority of the data, however, seem to support the assumption
of a first order elimination process.
2,3,7,8-TCDD 1s slowly excreted from the bodies of all species tested
(Table 7-4), with a half-life 1n the body of 10-43 days. In the SolderT
Syrian hamster, the least sensitive mammalian species to the acute toxlclty
of 2,3,7,8-TCDD, excretion occurs readily through both the urine (41%) and
7-16
-------
Elimination of 2,3,7,8-TCDD
Species
Guinea pig
Guinea pig
Rat
Rat
Rat
Rat
Monkey
(adult)
Monkey
(Infant)
Monkey
Mouse
C57BL/65
DBA/23
B6D2Fi/3*
Hamster
Hamster
Single Treatment
vg/kg (route)
2 O.p.)
1.45 (oral)
1.0 (oral)
50 (oral)
50 (oral)
400 (Up.)
400 (l.p.)
400 (1-p.)
1 (oral)
10 (l.p.)
10 (l.p.)
10 (l.p.)
650 (l.p.)
650 (oral)
Half -Life for
Elimination
(days)
30.2 t 5.8
22 - 43
31 * 6
17.4 i 5.6
21.3 ± 2.9
NT
NT
NT
365
11.0 *• 1.2
24.4 *- 1.0
12.6 * 0.8
10.8 * 2.4
15.0 * 2.5
Relative % of TCDD-Der1ved
Radioactivity
Feces
94.0
NT
>99
80.0
95.5
91.0
78.0
39.0
NR
72.0
54.0
72.0
59.0
NT
Urine
6.0
NT
<1
20.0
4.5
9.0
22.0
61.0
NR
28.0
46.0
28.0
41.0
NT
Reference
Gaslewlcz and Neal, 1979
Nolan et al., 1979
Rose et al., 1976
Piper et al., 1973
Allen et al., 1975
Van Miller et al., 1976
Van miler et al.*, 1976
Van MUler et al., 1976
McNulty et al., 1982
Gaslewlcz et al., 1983a,b
Gaslewlcz et al., 1983a,b
Gaslewlcz et al., 1983a,b
Olson et al., 1980a
Olson et al., 1980a
*0ffspr1ng of C57BL/63 and DBA/23 that are heterozygous at the Ah locus
NT « Not tested; NR = not reported
-------
feces (59%) (Olson et al., 198Qa). The high levels found 1n the urine of
Infant monkeys were probably due to the Incomplete separation of urine and
feces (Van Miller et al., 1976). In all the other species so far tested,
excretion occurs mainly through the feces (80-100%) with only minor amounts
of 2,3,7,8-TCDD metabolites found In the urine (Piper et al., 1973; Allen et
al., 1975; Rose et al., 1976; Gas1ew1cz and Neal, 1979).
Rose et al. (1976) Investigated the elimination of [1*C]2,3,7,8-TCDD
In rats given repeated oral doses of 0.01, 0.1 or 1.0 pg/kg/day Monday
through Friday for 7 weeks, or a single dose of 1.0 ng/kg. In these
studies, no 14C was excreted In the urine following a single dose; how-
ever, the urine contained 3-18% of the cumulative dose by 7 weeks. This
study Indicated that steady-state concentrations will be reached 1n the
bodies of rats In ~13 weeks. The rate constant defining the approach to
steady-state concentrations was Independent of the dosage of 2,3,7,8-TCDD
over the range studied. This 1s consistent with the observations of Fries
and Marrow (1975), who found that the total retention 1n the bodies of rats
was proportional to total Intake. When rats were maintained on a diet
containing either 7 or 20 ppb TCDD, the amount of TCDD retained 1n the body
was 5.5 times the dally Intake of TCDD at 14 days, 7.5 times the dally
Intake at 28 days, and 10.0 times the dally Intake at 42 days.
The data 1n Table 7-4 suggest some Interspedes differences 1n the half-
life for elimination (t,/2) of 2,3,7,8-TCDD. In the hamster, the least
sensitive species to the acute toxlclty of 2,3,7,8-TCDD, a mean t,/2 of
10.8 days was observed (Olson et al., 1980a,b), and 1n the guinea pig, the
most sensitive species to the acute toxlclty of 2,3,7,8-TCDD, the mean
t,/2 was 30.2 days (Gaslewlcz and Neal, 1979). The observed Interspedes
differences 1n the t, .„ of 2,3,7,8-TCDD may 1n part be related to the
7-18
-------
relative sensitivity of a given species to the acute toxiclty of
2,3,7,8-TCDD.
The Intrastraln differences In the t,,. of 2,3,7,8-TGDD 1n three mouse
strains may be due to the finding that the DBA/23 strain possesses ~2-fold
greater adipose tissue stores than the C57B1/63 and B6D2F,/J strains
(Sas1ew1ez et al., 1983b). The sequestering of the UpophlUc toxin 1n
adipose tissue stores of the DBA/20 mouse may contribute to the greater
persistence of 2,3,7,8-TCDD 1n this strain.
In all of the rat studies shown In Table 7-4, urinary and fecal elimina-
tion were monitored for a period of only 20-22 days, and from these data 1t
was assumed that elimination followed a single component, first order
kinetic model. Recently, Olson and BHtner (1983) examined the elimination
of 2,3,7,8-TCDD-der1ved radioactivity 1n rats over a 35-day period following
a single 1ntraper1toneal exposure at 1 jig 3H-2,3,7,8-TCDD/kg. They
observed first order kinetics for elimination, with a fast component having
a t,._ of 7 days (represents 13% of total elimination) and a slow compo-
nent having a t,/2 of 75 days (87% of total). The second, slow component
for elimination was evident only when urinary and fecal elimination were
monitored for >30 days. This study suggests that 2,3,7,8-TCDD may be more
persistent than earlier studies suggested. A preliminary study 1n the
rhesus monkey suggests that 2,3,7,8-TCDD may be exceptionally persistent 1n
adipose tissue. McNulty et al. (1982) estimated the apparent half-life of
2,3,7,8-TCDD 1n the fat of a monkey to be ~1 year.
Studies 1n the rat, guinea pig, hamster and mouse have found that all of
the 2,3,7,8-TCDD-der1ved radioactivity excreted 1n the urine and bile corre-
sponds to metabolites of 2,3,7,8-TCDD (Neal et al., 1982, 1984). The
apparent absence of 2,3,7,8-TCDD metabolites 1n liver and fat suggests that
7-19
-------
once formed, the metabolites of 2,3,7,8-TCDD are readily excreted. Thus,
urinary and biliary elimination of 2,3,7,8-TCDD 1s apparently dependent upon
metabolism of the toxin. Although urine and bile appear to be free of
unmetabollzed 2,3,7,8-TCOD, data from the hamster and rat Indicate that a
significant amount (10-40%) of unchanged 2,3,7,8-TCDD may be excreted Into
the feces. Unmetabollzed 2,3,7,8-TCDD thus appears to enter the Intestinal
lumen by some route other than bile for a number of days following treat-
ment. These data suggest that the jn. yl vo half-life for elimination of
2,3,7,8-TCDD may not directly reflect the rate of 2,3,7,8-TCDD metabolism In
a given animal (Neal et al., 1982, 1984). These data are consistent with
the observation of Hanara et al. (1982) that the lethal effects of 2,3,7,8-
TCDD were decreased 1n C57B1/6J mice regardless of whether the compound was
administered by gavage or 1ntraper1toneal Injection 1f the animals were
given diets containing activated carbon.
7.5. SUMMARY
Exposure to 2,3,7,8-TCDD occurs by Inhalation, dermal or 61 absorption.
Inhalation exposure to detectable levels of 2,3,7,8-TCDD 1s less likely
because of low vapor pressure of this compound; however, Inhalation exposure
could result from Inhalation of mist, dust or other contaminated partlculate
matter. Monitoring of atmospheric dust 1n the Seveso area detected 2,3,7,8-
TCDD levels ranging from 0.06-2.1 ng 2,3,7,8-TCDD/g airborne dust
(D1Domen1co et al., 1980b). This corresponds to an estimated 24-hour
Inhalation exposure of 1.4 pg assuming an average Intake of 10 m3 air
containing 0.14 mg dust/m3. No studies on the systemic absorption of
2,3,7,8-TCDD have been performed, so the significance of this route of
exposure 1n contaminated areas cannot be assessed.
7-20
-------
2,3,7,8-TCOQ 1s readily absorbed under experimental conditions (vide
ante) and following environmental contamination (Cockerham et al,, 1980;
m^
Fanelll et a!., 1980c; Walsh, 1977). Afte£^1ng absorbed, 2,3,7,8-TCDD 1s
rapidly distributed to tissues with a high I1p1d content (fat, skin,
adrenals). In most species studied, the major storage site for 2,3,7,8-TCDD
1s the liver (see Table 7-3). 2,3,7,8-TCDD exposure results In Induction of
HFO activity and a proliferation of smooth endoplasmlc retlculum, the major
subcellular storage site for 2,3,7,8-TCDD (Section 8.1.1.5.). The ability
of 2,3,7,8-TCDD to produce this effect has been correlated with the sensi-
tivity of various strains of mice to 2,3,7,8-TCDD toxicUy (Van Miller et
a!., 1976; Poland and Glover, 1980).
2,3,7,8-TCDD appears to be distributed throughout the body and stored
largely as the parent compound (Olson et a!., 1980a); however, metabolism to
more polar compounds appears to be necessary for excretion 1n the urine or
bile (Weber et al., 1982; Olson et al., 1980a; Neal et a!., 1984). Studies
have also Indicated that 2,3,7,8-TCDD was metabolized by the hepatic cyto-
chrome P-450 monooxygenase system. The structures of six metabolites In the
dog (Polger et al., 1982b) and two 1n the rat (Sawahata et al., 1982) have
been elucidated; however, the structure of the metabolites of 2,3,7,8-TCDD
have not been determined for the other species studied. Although some
[l,6-3H]-2,3,7,8-TCDD-der1ved radioactivity was capable of binding cova-
lently to cellular macromolecules (Guenthner et al., 1979b; Nelson et al.,
1977; Poland and Glover, 1979), metabolism of 2,3,7,8-TCDD seems to be
predominantly a detoxification process (Beatty et al., 1978; Polger et al.,
1982a).
7-21
-------
2,3,7,8-TCDD and Its metabolites are excreted from the body by a variety
of mechanisms. Lactatlng rats excrete 2,3,7,8-TCDD 1n the milk (Moore et
a!., 1976). 2,3,7,8-TCDD, 1,2,3,7,8-PeCDD and 1,2,3,4,6,7,8-HpCDD and OCDD
have been detected In human milk samples from Swedish and German mothers
(Rappe et al., 1985). These Investigators could detect 1,2,3,4,7,8-,
1,2,3,6,7,8- and 1,2,3,7,8,9-HxCDDs only 1n the mothers' milk from Sweden.
Piper et al. (1973) reported the excretion of [l4C]2,3,7,8-TCDD-der1ved
radioactivity 1n the feces, urine and expired air of rats given a single
oral dose of 50 pg/kg. Over a 21-day period, 53, 13 and 3% of the admin-
istered radioactivity was eliminated through the feces, urine and expired
air, respectively. This pattern of excretion seems typical of most species
studied, with the exception of the hamster, which was observed to excrete
41% of the 2,3,7,8-TCDD-der1ved radioactivity 1n the urine (Olson et al.,
1980a). In all species so far studied, metabolism and excretion are rela-
tively slow processes, with the observed Initial half-lives 1n experimental
animals on the order of a few weeks (see Table 7-4).
7-22
-------
8. TOXICOLOGY: ACUTE, SUBCHROMC AND CHRONIC
8.1. EXPERIMENTAL ANIMALS
8.1.1. Acute.
8.1.1.1. LETHAL EFFECTS — There have been studies 1n a variety of
species defining the doses necessary to cause death after acute exposure to
2,3,7,8-TCDD. A summary of the single dose LDgo data for 2,3,7,8-TCDD 1s
presented 1n Table 8-1. The dose that results 1n death varies extensively
with species, with the male guinea pig being the most sensitive species
tested (ID™ of 0.6 yg/kg) (Schwetz et al,, 1973}, and the male hamster
the least sensitive species tested (LD50 of 5051 yg/kg) (Henck et al.,
1981). The rat and monkey appear to be the second most sensitive species,
with LDgjjS between 22 and 70 yg/kg {Schwetz et al., 1973; McConnell et
al., 1978a), while other species tested (rabbit and mouse) had LO s
between 114 and 283 yg/kg (Schwetz et al., 1973; HcConnell et al., 1978b;
Vos et al., 1974). Schwetz et al. (1973) found male rats more sensitive to
2,3,7,8-TCDD, while Beatty et al. (1978) found adult female and weanling
male rats more sensitive than adult male rats (Table 8-1). In C57B1/10
mice, Smith et al. (1981) reported adult males to be far more sensitive to
the acute toxiclty of 2,3,7,8-TCDD than adult females. Thus, data on sex
differences 1n sensitivity to the acute toxiclty of 2,3,7,8-TCDD are con-
flicting and may depend on the species or strain examined.
Harris et al. (1973) studied the toxic effects of 2,3,7,8-TCDD 1n rats,
mice and guinea pigs with regard to single or multiple exposures. Similar
effects were observed after a single exposure to 2,3,7,8-TCDD as were
observed when multiple exposures totaled the same dose as received 1n the
single exposure. As Illustrated most clearly 1n rats, a single dose of 25
yg/kg, 6 weekly doses of 5 yg/kg, or 30 dally doses of 1 yg/kg were
8-1
-------
TABLE 8-1
Lethal Doses of 2,3,7,8-TCOD Following Acute Exposure
Species/Strain Sex/No. /Group
Guinea pigs/ H/NR
Hartley
Guinea pigs/ N/NR
Hartley
Guinea pigs/ M/9
Hartley
Guinea pigs/ F/6
0, Hartley
I
IVJ
Guinea pigs/ F/6
Hartley
Rats/ N/5-10
Sherman
Rats/ F/NR
Sherman
Rats/Sprague- H/6
Dawley
Rats/Sprague- F/6
Dawl ey
Route/
Vehicle
gavage/corn
oil-acetone
(9:1)
gavage/corn
oil-acetone
(9:1)
gavage/
corn oil
gavage/
corn oil
gavage/
methyl
cellulose
gavage/corn
oil-acetone
(9:1)
gavage/corn
oil -acetone
(9:1)
1. p. /olive
oil
1. p. /olive
oil
Dose Tested Duration of
(tig/kg) Observation
NR 2-8 weeks
NR 2-8 weeks
NR 30 days
0.1 42 days
0.5
2.5
12.5
20.0
0.1 12 days
0.5
2.5
12.5
20.0
8 2-8 weeks
16
32
63
NR 2-8 weeks
NR 20 days
NR 20 days
Ll>SQ
0.6
(0.4-0.9)*
2.1
(1.5-3)*
2
2.5
(1.2-5.4, 95X
confidence)
19
(15-23, 95X
confidence)
22
45
(30-66)*
60
25
Cements
Time to death was 5-34 days, the
2,3,7,8-TCDD was 91* pure
Time to death was 9-42 days, the
2,3,7,8-TCDD was 99X pure
Median time to death was 17-20 days,
marked weight loss, thymus atrophy,
Intestinal hemorrhage, no porphyrla
and only mild liver Injury
Time to first death was 32 days In
the 2.5 ug/kg group, with SOX
mortality by day 42
Time to first death was 12 days In
the 20.0 ng/kg group, with 67X
mortality by day 42
Time to death was 9-27 days, the
2,3,7,8-TCDD was 91X pure
Time to death was 13-43 days, the
2,3,7,8-TCDD was 91X pure
LD$o (tig/kg, mean + SE) adult
male. 60.2 » 7.8; weanling male,
25.2 i 1.4
Adult female had a mean » SE of
24.6 i 2.0 yg/kg
Reference
Schwetz et al.,
1973
Schwetz et al.,
1973
BcConnell et al.,
1978b
Sllkworth et al.,
1982
Sllkworth et al.,
1982
Schwetz et al.,
1973
Schwetz et al.,
1973
Beatty et al., 1978
Beatty et al., 1978
-------
TABLE 8-1 (cont.)
Species/Strain Sex/No. /Group
Monkey/rhesus F/3
H1ce/C57Bl H/14
Mce/C57Bl H/9
00
I
w
N1ce/CS7B1/10 H/5
H1ce/C57B1/H) F/5
H1ce/C57B1/6J H/NR
mce/DBA/2J M/NR
H1ce/B6D2Fi/J H/NR
Route/
Vehicle
gavage/
corn oil
gavage/corn
oil-acetone
(9:1)
gavage/
corn oil
gauge/
arachls oil
gauge/
arachls oil
1. p. /olive
oil
1. p. /olive
oil
1. p. /olive
oil
Dose Tested
(vg/kg)
0
70
350
0
100
150
200
HR
85
107
135
170
213
85
107
135
170
213
269
338
426
536
NR
NR
NR
Duration of 1059
Observation (vgAg)
>35 days <70
60 days 114
30 days 283.7
45 days 146
45 days >450
30 days 132
30 days 620
30 days 300
Comments
Weight loss, edema, severe thymus
atrophy, loss of hair, mild liver
damage
Time to death In the high dose group
was 15-20 days, bw loss, edema In
25X of treated animals, severe
thymlc and spleen atrophy, hemor-
rhage In the region of the eye and
small Intestine, liver necrosis In
the centrUobular region
Redlan time to death was 22-25
days, dose-related bw loss, thymlc
atrophy, Increased liver weight
and porphyrla, gross and historic
liver alterations, subcutaneous
edema. Intestinal hemorrhage
95X confidence limits of 111-211
tig/kg. Host deaths occurred from
22-26 days after dosing. Signs
of porphyrla, edema, hemorrhage.
1 of 4 animals died at dose of
426 pg/kg
BG02Fi/J mice are the offspring
of C57B1/6J and DBA/2J.
The BGD2-|/J mice are heterozygous
at the Ah locus.
No comment
Reference
NcConnell et
1978a
Vos et al.,
HcConnell et
1978b
Smith et al.
Smith et al.
Gaslewlcz et
1983a,b
Gaslewlcz et
19B3a.b
Gaslewlcz et
1983a,b
al.,
1974
al.,
, 1981
. 1981
al.,
al..
al..
-------
TABLE 8-1 (cont.)
Species/Strain Sex/No, /Group
Rabbits/ H&F/NR
New Zealand
Rabbits/ NW7S
New Zealand
Rabbits/ M&F/NR
New Zealand
03 Hamster/ H/6
i golden Syrian
•*»
Hamster/ M.F/S-6
golden Syrian
Hamster/ H/5
golden Syrian
Oogs/Beagle H/2
Oogs/Beagle F/2
Route/
Vehicle
gavage/corn
oil -acetone
(9:1)
l.p./
corn oil
dermal/
acetone
gavage/corn
oil -acetone
(9:1)
l.p./
olive oil
gavage/
olive oil
gavage/corn
oil-acetone
(9:1)
gavage/corn
oil -acetone
Dose Tested
NR
32
63
126
252
500
31.6
63
126
252
500
0
300
600
1000
3000
6000
0
500
1000
2000
3000
500
1000
2000
3000
3000
30
100
Duration of 10 ^Q
Observation (wg/kg)
2-8 weeks 115
(38-345)*
4 weeks NR
3 weeks 275
(142-531)*
55 days 5051
(3876-18,487,
9SX confidence)
50 days >3000
50 days 1157
2-8 weeks NA
2-8 weeks NA
Comments
Tine to death was 6-39 days, the
2,3,7,8-TCDD was 91* pure
Time to death was 6-23 days,
2-3 animals/group died In all
but the low exposure group
Time to death was 12-22 days
Tine to death was 26-43 days, the
liver and thymus appeared to be the
primary target organs, only 1 death
occurred In the 300 and 3000 ug/kg
group
Significant, dose-related decrease
In thymus weight starting at
500 wg/kg, only 2 deaths occurred
out of 11 hamsters In the 3000 wg/kg
group.
Death generally occurred between
24 and 45 days, decrease in bw above
2000 wg/kg, prollferatlve lleltls
with mild to severe Inflammation
All animals died
All animals survived
Reference
Schwetz et al.,
1973
Schwetz et al.,
1973
Schwetz et al.,
19'?
Henck et al., 1981
Olson et al., 1980b
Olson et al.. 1980b
Schwetz et al..
1973
Schwetz et al..
1973
*The number In parentheses appears to Indicate the range of lethal doses; however, the article did not specify what these numbers represented.
l.p. - IntrapeHtoneal; NR « Not reported; NA •= Not applicable
-------
all the threshold dose for observing a decrease 1n body weight, in general,
other endpolnts, Including lethality, decrease 1n thymus weight, and a no
effect level for body weight change 1n rats, mice and guinea pigs required a
specific threshold level regardless of whether this level was achieved
through a single exposure or a small number of multiple exposures.
Although 2,3,7,8-TCDO has over a 103-fold difference 1n toxldty
depending upon the species tested, some of the signs of lethal toxldty were
the same regardless of species. One of the most characteristic observations
after acute lethal exposure to 2,3,7,8-TCOD was the protracted time between
exposure and death {see Table 8-1). In determining the LDgo 1n the least
sensitive animal, the hamster, the test animals died between 24 and 45 days
after a single acute exposure (Olson et al., 1980b), and similar observa-
tions were made 1n all other species tested Including the most sensitive
species, the guinea pig, 1n which animals died up to 42 days after treatment
{Schwetz et al., 1973).
During this extended period between treatment and death the animals had
poor weight gain or loss of weight resulting 1n a "wasting syndrome" that
resembled starvation. Though weight loss 1s the primary general feature
observed 1n adult rats, In the young animals depletion of body fat results
In lean tissue formation (Peterson et al., 1984) In female Wlstar rats
Intubated with 2,3,7,8-TCDD at a dose of 100 vg/kg, the weight loss was
blphaslc (Courtney et al., 1978). The Initial weight loss occurred rapidly
during the first 7-10 days after treatment and was associated with decreased
food and water consumption. This Initial phase of weight loss was reversed
with the resumption of normal food Intake for 4 or 5 days, only to be fol-
lowed by a second, more gradual, decline 1n food and water Intake and weight
until death. Providing animals with an adequately nutritious liquid diet
8-5
-------
by Intubation did not appreciably alter the pattern of weight loss nor
affect survival. In contrast, 6as1ew1cz et al. (1980) observed that provid-
ing rats with total parenteral nutrition would prevent some of the weight
loss Induced by 2,3,7,8-TCDD; however, there was no protection from the
lethal effects of 2,3,7,8-TCDD. Seefeld and Peterson (1983) and Seefeld et
al. (1984) found that a reduction 1n food Intake caused by 2,3,7,8-TCDD 1s
primarily responsible for the loss of body weight or depressed growth rate
of rats. Pair-fed control rats lost weight at the same rate and to the same
extent as their weight-matched 2,3,7,8-TCDDtreated partners (25 or 50
jig/kg) until day 10 after treatment. At 20-35 days after treatment, the
body weight of the two groups began to diverge, with the pair-fed control
group having body weights that were 20-30 g higher than the corresponding
2,3,7,8-TCDD groups. The mortality 1n the 25 and 50 pg/kg groups was 33
and 75%, respectively, while 1n the corresponding pair-fed groups the mor-
tality was 0 and 15%. The authors proposed a hypothesis that 2,3,7,8-TCDD
lowers a regulated level or "set-point" for body weight control 1n the rat.
The ensuing change 1n food Intake was thought to occur secondarily to the
change 1n set-point {Seefeld and Peterson, 1983; Seefeld et al., 1984;
Peterson et al., 1984). Vitamin A or E did not protect or Inhibit the
decrease 1n body weight, respectively. Further, these vitamins provided
little protection against 2,3,7,8-TCDD-1nduced lethality 1n rats (Hassan et
al., 1985).
Also, severe thymlc atrophy Is universally observed 1n all species given
lethal doses of 2,3,7,8-TCDD, and since weight loss and thymlc atrophy are
both associated with malnutrition, van Logten et al. (1981) Investigated the
effects of dietary protein on the toxldty of 2,3,7,8-TCDD. Groups of
female Fischer 344 rats administered 2,3,7,8-TCDD (20 pg/kg) and main-
tained on low (3.554), normal (26%) or high (55%) protein diets maintained
8-6
-------
approximately the same amount of weight (-0.2+3, 7±6 and 7±3 g for each
dietary group, respectively) during the subsequent 10-day period. The
weight gain In treated animals was 10-18 g less than that 1n the respective
control rats. Dietary protein also had no effect on preventing or enhancing
the 2,3,7,8-TCDD Induced thymlc atrophy. Although weight loss and thymlc
atrophy were present In most species tested, there were other symptoms that
were characteristic of toxldty 1n only some species.
In the guinea pig, besides thymlc atrophy, no gross changes were
observed 1n Internal organs after a lethal oral or 1.p. dose of 2,3,7,8-TCDD
(Grelg et al.» 1973, Supta et a!., 1973). Hemorrhages were observed In a
number of organs Including the adrenal gland, urinary bladder, 61 tract and
mesenterlc lymph nodes; however, these were considered unremarkable changes
by Supta et al. (1973). H1stolog1c examination confirmed the gross observa-
tions with atrophy and lymphold cell depletion 1n the thymus, spleen and
lymph nodes, and hemorrhages observed 1n many organs. In addition, marked
hyperplasla of the urinary bladder was observed. Of particular Interest was
the absence of severe toxic effects on the Hver. Gross observation under
UV light Indicated no excess of porphyrln, while hlstologlc examinations
revealed diffuse single cell necrosis. Identical observations were made by
McConnell et al. (1978b) 1n guinea pigs administered lethal doses of
2,3,7,8-TCDD, with the additional observation that the sternal bone marrow
was hypocellular 1n all types of blood-forming cells.
Turner and Collins (1983) described some hlstologlc changes In the Hver
of guinea pigs treated with 2,3,7,8-TCDD. Groups consisting of 4-6 female
Hartley guinea pigs were treated with 2,3,7,8-TCDD at doses of 0.0, 0.1,
0.5, 2.5, 12.5 or 20 vg/kg, and 1 male guinea pig each was treated with a
dose of 0.1 or 0.5 vg/kg. The 2,3,7,8-TCDD was administered by gavage as
8-7
-------
an aqueous suspension 1n 0.7554 methyl cellulose and surviving animals were
killed 42 days after treatment. A second group of guinea pigs (6 males and
6 females/dose) were administered soot generated from a fire 1n a trans-
former cooled by polychlorlnated blphenyls and chlorinated benzenes (1, 10,
100 and 500 mg/kg). The hlstologlc observations as described were applied
1n general to both treatment groups and there was no apparent relationship
between dose and response. At the light microscope level, hepatocellular
hypertrophy, steatosls, focal necrosis, cytoplasmlc degeneration and addo-
ph1!1c hyal1n-l1ke cytoplasmlc Inclusion bodies were observed. Even though
there was no dose-response relationship for these liver lesions, the doses
spanned a range that resulted 1n the lowest dose being nonlethal (none of
the 4 female guinea pigs died during the study), while 1n the high dose
group 4 of 6 animals died before 42 days post-treatment. The L0go for
female guinea pigs was determined 1n this study to be 2.5 or 19 yg/kg bw
depending on whether the compound was administered by gavage 1n corn oil or
1n aqueous methyl cellulose (SUkworth et a!., 1982).
The greatest difference at necropsy 1n the gross and hlstologlc effects
1n rats and mice of exposure to lethal doses of 2,3,7,8-TCDO was pathologic
alterations 1n the liver, as compared with guinea pigs. An early report by
Buu-Ho1 et al. (1972) described alterations 1n the architecture of the liver
of rats within 5 days of receiving a low dose of 2,3,7,8-TCDD (10 pg/kg by
1.p. Injection). At higher oral doses of 100 or 50 »ig/kg, which killed 43
and 7% of the animals, respectively, Gupta et al. (1973) also observed
marked distortion of liver architecture 1n rats; however, only mild regener-
ative changes of the liver were observed at the sublethal dose of 5 yg/kg
administered weekly for 6 weeks. Liver toxlclty appeared to develop slowly
1n the rat with no change 1n Hver function, as Indicated by plasma protein
8-8
-------
and bMArub\n levels, or alkaline phosphatase, glutatnic-oxalaceilc trans-
amlnase (GOT) and glutamlc-pyruvlc transamlnase (GPT) activity being
detected 3 days after Intubation with 2,3,7,8-TCDD at a dose of 200 vg/kg
(Grelg et al.» 1973). B1l1rub1n levels were, however, markedly elevated
from 0.33 vg/100 ma 1n control animals to 10.97 yg/100 ml 1n treated
animals 21 days after exposure (the other parameters were not measured at
this time, although plasma protein was slightly but significantly decreased
when determined 9 days post-treatment). As In rats, the livers of mice
exposed to lethal levels of 2,3,7,8-TCDD had signs of necrotlc changes (Vos
et a!., 1974); however, Jones and Grieg (1975) reported that the centrllobu-
lar necrosis, bile duct proliferation and Hp1d accumulation were more
extreme 1n mice than 1n rats. Examination of mouse livers using long wave
UV light showed fluorescence suggestive of excess porphyrln accumulation
(HcConnell et al., 1978b). Although excess porphyrlns may be present 1n the
livers from 2,3,7,8-TCDD-exposed rats, fluorescence Is not usually observed.
Besides effects on the liver, 2,3,7,8-TCDO exposure produced other toxic
effects 1n rats and mice that were not observed or were observed to a lesser
extent 1n guinea pigs. In rats that died from 2,3,7,8-TCDD exposure, there
were extensive hemorrhages of the heart, Hver, brain, adrenal gland and 61
tract along with ulcers and necrosis of the glandular stomach, and 1n
females, atrophy of the uterus (Gupta et al., 1973), In mice, facial edema
was severe and the testicles of males appeared degenerated with necrotlc
spermatocytes and spermatozoa present (HcConnell et al., 1978b; Vos et al.,
1974). Death 1n mice was frequently attributed to terminal hermorrhages
(Vos et al., 1974).
In monkeys exposed to lethal levals of 2,3,7,8-TCDD, McConnell et al.
(1978a) [reported clinical and hlstologlc signs of toxldty, some of which
8-9
-------
were similar to those already described for other species. Severe thytnlc
atrophy and edema occurred 1n treated animals, as well as extensive weight
loss that could account for up to 38% of the body mass. As In guinea pigs,
liver Injury appeared to be mild; however, Increased serum iOT and aldolase
activity and decreased albumin levels Indicative of liver pathology occurred
near the time of death. As observed In mice, the bone marrow of monkeys was
hypocellular. In addition to the above signs of toxldty, which were
observed 1n other species as well, monkeys had progressive loss of hair,
toenalls and fingernails, with associated dermatitis consisting of the
development of a crusty texture to the skin, squamous metaplasia of sebace-
ous glands and gastric mucosal dysplasla. As with most other species, a
specific cause of death could not be determined for monkeys. Poland and
Knutson (1982) summarized the toxic response of various species to 2,3,7,8-
TCDD 1n Table 8-2.
There was very little Information on the lethal effects of PCDD con-
geners other than 2,3,7,8-TCDD. McDonnell et al. (1978b) determined the
LDgQ for nine congeners of PCDD following a single treatment by gavage 1n
mice and guinea pigs. A comparison of the LD..- expressed as ymol/kg
5U
body weight 1s presented 1n Table 8-3. The limited data suggest that con-
geners containing chlorine In the 2,3,7,8 positions were more biologically
active than congeners deficient 1n a chlorine from any one of these posi-
tions. It 'also appears that addition of one or more chlorines to 2,3,7,8-
TCDD results 1n a decrease 1n lethality. Although the congeners vary 1n
effective dose between mice and guinea pigs, the relative order of toxldty
of these congeners did not change. Also, similar effects of toxldty were
observed for all congeners as described above for 2,3,7,8-TCDD when the com-
parison was made within a single species.
8-10
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TABLE 8-2
Toxic Responses Following Exposure to 2,3,7,8-TCDD: Species Differences3
oa
i
Hyperplasla and/or metaplasia
Gastric mucus
Intestinal mucosa
Urinary tract
Bile duct and/or gall bladder
Lung: focal alveolar
Skin
Monkey
**c
4-
4-4-
4-4-
4-4-
Guinea
P1g
0
4-*
0
0
Cowb
4-
4-4-
4
*d
Rat
0
0
4-4-
0
Mouse
0
0
4-4-
0
Rabb1tb Ch1ckenb Hamster
0
4-4-
0
4-4- 0
Hypoplasla, Atrophy or Necrosis
Thymus
Bone marrow
Testicle
Other
Liver lesions
Porphyrla
Edema
4-
4-
4-
4-
0
4-
4-
4-
4-
4-
0
0
4- 4-
f
4-f
f
0
t
4-
+•
t 4-4-
f4-
f
4-
f
f
f
4-
4-f
+•
4-
0
•1-
aReferences: monkey (McConnell et al., 1978b; Norback and Allen, 1973; Allen et a!., 1977); guinea pig
(McConnell et al., 1978b; HcConnell, 1980; Moore et al., 1979; Turner and Collins, 1983);
cow (McConnell, 1980); rat (McConnell, 1980; Kodba et al., 1978a; Koclba et al., 1979);
mouse (Schwetz et al., 1973; HcConnell et al., 1978b; Vos et al., 1973); rabbit (Klmmlg and
Schultz, 1957; Schwetz et al., 1973; Vos and Beems, 1971); chicken (Schwetz et al., 1973;
Norback and Allen, 1973; Allen and Lalich, 1962; Vos and Koeman, 1970); hamster (Olson et
al., 1980b; Henck et al., 1981).
Responses followed exposure to 2,3,7,8-TCDD or structurally related chlorinated aromatic hydrocarbons.
cSymbols: 0, lesion not observed; +, lesion observed (number of "+-" denote severity); _t, lesion observed
to a very limited extent; blank, no evidence reported In literature.
Skin lesions 1n cattle are observed, but they differ from the skin lesions observed 1n other species.
Adapted from Poland and Knutson, 1982.
-------
TABLE 8-3
Estimated Single Oral LDsp - 30 Values for PCDDs3
Chlor1nat1on of PCDDs
1
2,8
2,3,7
2,3,7,8
1,2,3,7,8
1,2,4,7,8
1,2,3,4,7,8
1,2,3,6,7,8
1,2,3,7,8,9
,2,3,4,6,7,8
Guinea P1gs
(wmol/kg)b
>neo
120.41
0.006
0.009
3.15
0.185
0. 178-0. 255C
0.1 53-0. 255C
>1.400
Mice
NR
>10
0.88
0.94
>14
2.11
3.19
>3.67
NR
aSource: HcConnell et al., 19785
bSpearman-Karber method
cEst1mated range due to variability 1n replicates
NR = Not reported
8-12
-------
o. I. I .^. trrcuid un ins.
changes 1n the liver Induced by oral exposure to 2,3,7,8-TCOD have been
reported by Fowler et al. (1973), Jones and Butler (1974) and Jones (1975).
Fowler et al. (1973) treated groups of 30 male rats with a single dose of
2,3,7,8-TCDD at 0.0, 5 and 25 ng/kg by gavage. The animals were killed 1n
groups of 5 on days 1, 3, 6, 9, 16 and 28 after treatment and the livers
were prepared for hlstologlc examination. The major ultrastructural change
observed was a dose-related Increase 1n the smooth and rough endoplasmlc
retlculum (ER) In cells near the bile canallcuH. The Initial Increases
appeared at day 3, with the maximal response occurring on days 6 and 9. By
day 16 the smooth ER was nearly absent from the parenchyma! cells, although
large amounts of rough ER were stm present. By day 28 the cells had
returned to normal appearance. These changes 1n liver cells following
2,3,7,8-TCOD treatment would be consistent with the Induction of protein and
RNA synthesis.
Transmission electron microscopic observations revealed that single 1.p.
administration of 20 pg/kg of 2,3,7,8-TCDD 1n Sprague-Dawley male rats
produces necrotlzlng hepatic lesions that become progressively worse up to
the 16th week postexposure followed by gradual Improvement of the condition
and disappearance of the lesions (Weber et al., 1983).
At higher doses of 200 pg/kg, Jones and Butler (1974) observed necro-
sis and prollferatlve changes 1n the Hver of rats to be the predominant
lesions. After treatment by gavage, groups of 4 male and 4 female rats were
killed and examined on a weekly basis for 10 weeks. By the first week,
degenerating cells were observed near the central vein and these lesions
progressed to areas of focal necrosis by the sixth week. Superimposed on
8-13
-------
the necrotlc changes were hyperplasla of the viable cells with multlnucle-
t
ated cells common by the ninth week. At week 10 central vein flbrosls and
scattered necrosis remained. Fine structure observed after this large dose
of 2,3,7,8-TCDD also revealed Increases In smooth ER; however, the most
striking effect was degeneration of the plasma membrane with the resulting
fusion of parenchyma! cells. In a study of similar design, Jones (1975)
followed the distribution with time after treatment of membrane associated
ATPase activity by histochemlcal techniques. At 3 days after treatment, the
first changes In ATPase patterns were observed, with loss of activity along
the canallcular borders and some Increased activity 1n the sinusoids. The
mldzonal and perlportal zones had normal activity at this time. The loss of
ATPase activity persisted for 34-42 days and paralleled the hlstologlc
lesions described previously (Jones and Butler, 1974). In rats that sur-
vived treatment, the ATPase activity was back to normal by 9 months.
Peterson et al. (1979a) further studied the effect of 2,3,7,8-TCDD at
lower doses on hepatocyte plasma membrane ATPase activity. Liver surface
membranes (LSH) Isolated from male Holtzman rats 2, 10, 20 or 40 days after
Intubation with 2,3,7,8-TCDD at 0.0, 10 or 25 iig/kg were used for deter-
mination of Na*, K^-ATPase and Mg^-ATPase activity. The activity of
Na*, K*-ATPase was depressed to the same extent for both doses of
2,3,7,8-TCDD from day 2-40 after treatment, while a similar depression of
the Hg -ATPase activity was observed only 1n the high dose group. In the
low dose group, there was a decrease 1n Mg*+-ATPase at 20 days, but
recovery to normal levels occurred by 40 days post-treatment. It was
demonstrated that the effect of 2,3,7,8-TCDD on ATPase activity was not the
result of 2,3,7,8-TCDD Induced food deprivation and in vitro studies Indi-
cated that the loss of activity was not due to the direct Interference of
8-14
-------
2,3,7,8-TCOD wtth the enzyme. Quantitative changes (both Increases and
decreases) have been reported for the protein composition of plasma
membranes Isolated and analyzed by electrophoresls from Sprague-Dawley rats
10 days after an 1.p. Injection of 2,3,7,8-TCDD, Indicating that exposure
was actually affecting membrane components (Brewster et al., 1982).
Peterson et al. (1979a) observed a positive correlation between the
levels of LSH ATPase activity and both In v1vo cumulative biliary excretion
of ouabaln and bile flow (Wtl/m1n/g Hver). Using perfused Hver, however,
Peterson et al. (1979b) reported a segregation between LSM ATPase activity
and biliary excretion of ouabaln when 2,3,7,8-TCDD rats were exposed to the
protective agents pregnenolone-l6a-carbon1tr1le or splrenolactone. It was
concluded that LSH ATPase did not directly participate 1n ouabaln transport.
Additional studies have described the effect of 2,3,7,8-TCDD on the bil-
iary excretion of a variety of xenoblotlcs. Early studies by Hwang (1973)
Investigated 2,3,7,8-TCDD Inhibition of biliary excretion 1n male CD rats
given a single dose of 2,3,7,8-TCDD at 25 or 5 jig/kg by gavage. Animals
were examined for Indocyanlne green (ICS) excretion 1, 7 and 16 days after
treatment. Unlike Peterson et al. (1979a), Hwang (1973) observed an Inverse
relationship between 2,3,7,8-TCDD exposure and bile flow, with maximum bile
flow observed 1n the 25 jjg/kg dose group at 16 days. Even with this
Increased bile flow, however, the cumulative biliary excretion of ICG was
decreased 1n a dose-dependent manner with the greatest depression observed 7
and 16 days after the exposure to 2,3,7,8-TCDD. The levels of ICG 1n the
plasma and liver was higher 1n treated animals than 1n control animals,
while the concentration In the bile was lower, reflecting the decrease 1n
total excretion of ICG.
8-15
-------
Yang and Peterson (1977) compared the effect of 2,3,7,8-TCDD on the bil-
iary excretion of the organic neutral compound, ouabaln, with that of the
organic anlons phenol-3,6-d1faromophthale1n (DBSP) and sulfobromophthaleln
{BSPJ 1n male Holtzman rats. Animals were Intubated with 2,3,7,8-TCDD at
doses of 10 or 25 vg/kg and excretion was evaluated periodically between 2
and 4 days postexposure. The biliary excretion of ouabaln was depressed 1n
a dose-related manner starting on the second day post-treatment, with maxi-
mum depression developing between 10 and 20 days, and some recovery observed
by day 40. Decreases 1n bile flow followed a pattern similar to that
observed for ouabaln. The pattern of biliary excretion was different for
OBSP and BSP In which only a transient small decrease was observed 10 days
after exposure In the high-dose group. In the low-dose animals there was
actually an Increase at days 10 and 25 1n the excretion of the anlons. The
results obtained for DBSP and BSP differ sharply from those for the organic
neutral ouabaln or those reported by Hwang (1973) for the organic anlon ICS,
1n which a dose-related decrease In biliary excretion was observed. The
authors concluded that the effects of 2,3,7,8-TCDD on the multiple pathways
Involved 1n biliary excretion depend on the specific compound being studied.
In the guinea pig and rhesus monkey, which develop little liver pathol-
ogy after exposure to 2,3,7,8-TCDD, there was also Uttle change 1n ICG
blood clearance rates; 1n the rabbit, which develops 2,3,7,8-TCDD-1nduced
liver damage similar to the rat, there was reduced blood clearance of ICG
(Seefeld et al., 1979, 1980). In the rabbit, there were Increases 1n serum
sorbltol dehydrogenase and glutamlc pyruvlc transamlnase activity as further
Indications of 2,3,7,8-TCDD-produced Hver damage. In the monkey, which
received 2,3,7,8-TCDD by gavage at doses of 5, 25 or 75 pg/kg, there was
an Initial slight Increase In the blood clearance of ICG at 2 days post-
8-16
-------
treatment, followed 1n the two higher-dose groups by a dramatic decrease a
few days before death. Although some serum enzymes (sorbltol dehydrogenase
and glutamlc pyruvlc transamlnase) Indicative of "liver damage were elevated,
the hlstopathology of the liver was within normal limits. It appears that
major effects on biliary excretion occur only 1n species that are sensitive
to the hepatotoxlc effects of 2,3,7,8-TCDD.
Other gross signs of the hepatotoxlc effects of 2,3,7,8-TCDD observed 1n
some species Included fatty degeneration and porphyrla. Early observations
by Cunningham and Williams (1972) described a decrease 1n In vivo (1 hour
pulse) Incorporation of 3H sodium acetate Into liver I1p1ds after exposure
of male Wlstar rats to 2,3,7,8-TCDD. The rats (12-16 animals) were treated
with 2,3,7,8-TCDD at a dose of 10 vg/kg followed 1n either 3 or 7 days by
the assessment of I1p1d synthesis. At 3 days Incorporation decreased from
258 to 98 dpm/mg I1p1d 1n the control and treated animals, respectively.
There was an approximately similar decrease observed 7 days postexposure.
When Individual classes of Uplds were examined, there was a decrease 1n the
synthesis of trlglycerldes, dlglycerldes and phosphoHplds. Although
Cunningham and Williams (1972) observed that 2,3,7,8-TCDD decreased I1p1d
synthesis, Albro et al. (1978) reported an Increase 1n total Uplds 1n the
livers of rats 13 days after treatment with 2,3,7,8-TCDD at a lethal dose of
50 yg/kg. For Individual classes of Uplds there was an Increase 1n free
fatty adds and cholesterol esters; no change occurred 1n the content of
phosphollplds, free cholesterol or trlglycerldes. The fatty changes 1n the
liver were confirmed by ultrastructural examination of liver specimens. At
a sublethal dose of 10 vg/kg there was a different pattern of I1p1d accu-
mulation; trlglycerldes and fatty acids Increased and cholesterol esters
decreased. The changes 1n the I1p1d profile of the liver was attributed to
8-17
-------
2,3,7,8-TCDD Induced mobilization of body fat, a decrease 1n lysosomal add
Upase (74% decline In this enzyme 10 days after a 50 jig/kg dose of
2,3,7,8-TCDD) and an Increase 1n I1p1d peroxldatlon as Indicated by a sharp
Increase 1n the production of llpofuscln pigments,
Porphyrla was Initially characterized quantitatively 1n mice by
Goldstein et al. (1978). Groups of 12 male C57B1 mice received 4 weekly
Intubations of 2,3,7,8-TCDD at doses of 0.0, 1, 5 or 25 pg/kg, or a single
dose of 150 pg/kg followed 21-25 days after treatment by analysis of the
liver for porphyrlns. Porphyrln levels were unchanged except 1n the 25 and
150 vg/kg groups where the levels were Increased 2000- and 4000-fold,
respectively. The difference 1n responsiveness to the development of por-
phyrla was studied by Smith et al. (1981) 1n C57B1 mice that were sensitive
to, and DBA/2 mice that were Insensitive to, the toxldty of 2,3,7,8-TCDD.
Hale and female C57B1 mice had a dose-related Increase 1n hepatic porphyrlns
1n the two high dose groups 3 weeks after a single exposure to 2,3,7,8-TCDD
at 0.0, 5, 15, 50 or 75 jjg/kg; however, only minimal nondose-related
changes 1n hepatic porphyrln were observed 1n DBA/2 mice exposed to up to
1200 pg/kg. In the sensitive C57B1 mice there was only a small difference
In hepatic porphyrln between the sexes even though males were >3 times as
sensitive to the toxic effects of 2,3,7,8-TCDD than females (see Table
8-1). Results similar to those above were reported for urinary porphyrln
levels 1n male C57B1 and DBA/2 mice given 6 weekly doses of 2,3,7,8-TCDD at
25 vg/kg (Jones and Sweeney, 1980). In the sensitive strain, the Initial
elevation of porphyrln occurred 1n the second week.
In rats Increased urinary porphyrln was observed only after subchronlc
exposure to 2,3,7,8-TCDD (Cantonl et al., 1981). Female CD rats were
8-18
-------
administered weekly oral doses of 2,3,7,8-TCDD at levels of 0.01, 0.1 and
1.0 jig/kg for 45 weeks. The Initial Increase was observed 1n the high-
dose group at 3 months, and In the other two groups at 4 months, after the
start of exposure. Not only did the absolute amount of porphyrln Increase,
but the relative distribution also changed to compounds containing more
carboxyl groups. Only 1n the high dose group did the livers, at the ter-
minal necropsy, show signs of excess porphyrln under examination by UV light.
In attempts to understand the mechanism of 2,3,7,8-TCDD Induced por-
phyrla, the effects of 2,3,7,8-TCDD on the enzymes Involved In the synthesis
and catabollsm of porphyrln have been studied. Goldstein et al. (1978)
showed that 6-am1nolevul1n1c add synthetase, a rate-limiting enzyme 1n
porphyrln synthesis, was slightly Increased (2-fold) 1n male C57B1 mice
given 4 weekly doses of 2,3,7,8-TCDD at 25 vg/kg. This dose of 2,3,7,8-
TCDD Increased liver porphyrln levels 2000-fold. Catabollsm of porphyrln by
uroporphyrlnogen decarboxylase (UD) also appeared to be decreased 1n
2,3,7,8-TCDD treated mice. Smith et al. (1981) reported a decrease 1n UD
activity from ~25 to 7 n moles/hr/g liver In male and female C57B1 mice 3
weeks after a single oral exposure to 2,3,7,8-TCDD at a dose of 75 jig/kg.
No effect of 2,3,7,8-TCDD on UD activity was observed 1n DBA/2 mice that
were Insensitive to the Induction of porphyrla. A time course of changes 1n
UD activity with length of time after exposure to 2,3,7,8-TCDD Indicated a
steady decline In activity starting 3 days after exposure to 2,3,7,8-TCDD,
which continued until day 21 when the study was terminated. Sweeney and
Jones (1978) reported similar results after 5 weekly doses of 2,3,7,8-TCDD
at 25 yg/kg. In this study the UD activity declined -48% 1n C57B1 mice
8-19
-------
and only 4% 1n DBA/2 mice. Other factors besides the Increase 1n S-am1no-
levullnlc add synthetase and the decrease 1n UD activity may also partici-
pate 1n the dramatic Increase 1n Hver porphyrln In mice associated with
exposure to near lethal doses of 2,3,7,8-TCDD.
As a result of the protracted time observed between exposure to 2,3,7,8-
TCDD and the development of toxic effects, as well as the reported terato-
genlc and carcinogenic potential of 2,3,7,8-TCDO, Investigations have been
conducted to determine the Influence of 2,3,7,8-TCDD on DNA synthesis 1n the
Hver. Srelg et al. (1974) measured the in vivo Incorporation of 3H-thy-
!
m1d1ne (1 hour pulse) Into Hver DNA of male and female Porten strain rats
after a single exposure to 2,3,7,8-TCDD at doses of 10 and 200 vg/kg.
When the 2,3,7,8-TCDD was given either 0, 24 or 72 hours before a 3/4
partial hepatectomy there was only a slight, but not significant, decrease
1n thymldlne Incorporation observed when DNA synthesis was measured 24 hours
after the operation.
Although 2,3,7,8-TCDD had no effect on in vivo DNA synthesis, similar
studies by Conway and Hatsumura (1975) and Dickens et al. (1981) demon-
strated an Increase 1n thymldlne Incorporation when determined \jn vitro.
Conway and Hatsumura (1975) administered male Sprague-Dawley rats 2,3,7,8-
TCDD at a dose of 5 vg/kg followed 1n 10 days by removal of the Hver and
the in vitro determination of DNA synthesis 1n Hver slices. Incorporation
of thymldlne Into the nuclei Increased from 29 cpm/mg 1n control animals to
45 cpm/mg 1n treated animals. A similar near doubling of DNA synthesis was
observed by Dickens et al. (1981); however, when DNA synthesis was stimu-
lated by a 1/3 partial hepatectomy, thymldlne Incorporation Into liver
slices was Increased 10-fold 1n rats treated 5 days earlier with 2,3,7,8-
TCDD as compared with hepatectomlzed controls. The onset of DNA synthesis
8-20
-------
after partial hepatectomy (-20 hours) was the same In both 2,3,7,8-TCDD
treated and control animals; however, the treated animals had a more rapid
and extensive Increase 1n DNA synthesis between 20 and 32 hours after the
partial hepatectomy. The rates of DNA synthesis were again the same 1n both
groups 35 hours after the operation. It was shown by hydroxyurea Inhibition
that the DNA synthesis 1n both the treated and control animals was predomi-
nantly semlconservatlve. Further studies are needed to determine the reason
for the difference observed between in vlitro and in vivo measurements of DNA
i
synthesis 1n the liver after exposure to 2,3,7,8-TCDD.
Extensive hepatic necrosis 1n the rabbit may be responsible for death 1n
I
this species (Poland and Knutson, 1982). \
!
Besides the effects on the liver of 2,3,7,8-TCDD exposure described
above, It Is known that 2,3,7,8-TCDD 1s a potent Inducer of mlcrosomal
enzymes. These studies will be discussed 1n Section 8.1.1.5., which
describes the ability of this xenoblotlc to Induce mlcrosomal enzymes 1n a
number of tissues and organs.
8.1.1.3. EFFECTS ON OTHER ORGAN SYSTEMS — The most noticeable
feature of 2,3,7,8-TCDD toxldty 1s the loss of body weight and the apparent
"wasting away" until death. Since decreased food consumption may not total-
ly account for these findings, the effect of 2,3,7,8-TCDD on Intestinal
absorption has been studied. Madge (1977) assessed the ability of the
Intestine to absorb D-glucose, D-galactose, L-arg1n1ne and L-h1st1d1ne using
the everted Intestinal sac technique 1n CD-I mice exposed to 2,3,7,8-TCDD.
In measurements made 7 days after treatment with doses of 0.0, 10, 25, 75,
150, 200 or 300 yg/kg, D-glucose was absorbed to a lesser degree at all
doses than 1n control animals. The two low doses produced a dose-related
decrease 1n absorption; however, at doses of >75 yg/kg the decrease was
8-21
-------
uniform. At a dose of 150 pg/kg, decreased absorption of D-glucose was
slight 3 days after treatment, became maximally decreased by 7 days, and
this depressed level was maintained for 28 days, at which time the study was
terminated. Providing D-mannose to the Incubation mixture as an energy
supply Increased the absorption of D-glucose to control levels; however, the
amount of D-glucose on the serosal side was still lower than control levels.
This suggested that Intestinal utilization of D-glucose was taking place and
might account for some of the observed malabsorptlon. Treatment with
2,3,7,8-TCDD had no effect on the absorption of the other compounds Investi-
gated. In a similar experiment 1n Sprague-Dawley rats, Ball and Chhabra
(1981) also observed malabsorptlon of D-glucose. In this study, however,
absorption of leudne vas also decreased. The decrease In leudne absorp-
tion took longer to manifest Itself; a significant decrease was observed
only after 2 weeks treatment with 2,3,7,8-TCDD.
In contrast to the results observed for D-glucose, Intestinal Iron
transport was shown to be elevated by exposure to 2,3,7,8-TCDD. Manls and
K1m (1979a) examined the effect of prior treatment of male Sprague-Dawley
rats on the 30-m1nute transport of S9Fe out of a duodenal loop created by
llgatlng a section of the Intestine in situ. At single 2,3,7,8-TCDD doses
of between 22 and 84 vg/kg there was Increased serosal transfer of 59Fe
measured 48 hours after treatment. At doses >42 pg/kg the Increase was
~10054. The time after treatment at which serosol transfer was greatest was
1 day, with rapid decline 1n stimulation to near the levels of controls
observed on days 2-7. There was also an apparent effect of route of admin-
istration, with gavage treatment being more effective 1n Inducing Iron
transport than l.p. Injection. In similar experiments calcium transport was
decreased, and glulactose and prollne transport were unaffected by prior
8-22
-------
exposure to 2,3,7,8-TCDD. Hants and Kim (1979b) had Identical results when
the everted Intestinal sac was used to assess Iron transport. It was
Interesting to note that only duodenal sacs were stimulated, with no effect
of 2,3,7,8-TCDD exposure observed 1n the adjacent distal segment of the
Intestine. Increased Iron transport was also observed by Manls and K1m
(1979a) In an unidentified strain of mice. Increased Iron transport may be
one of the earliest effects of 2,3,7,8-TCDD; however, at present the toxlco-
loglc relevance of this transient disturbance In Iron transport 1s unknown.
One of the common gross observations of 2,3,7,8-TCDD toxlclty 1s severe
edema, suggestive of a breakdown 1n salt and water homeostasls. These
observations prompted Investigations to determine the effect of 2,3,7,8-TCDD
on the function of the kidney. Pegg et al. (1976) measured renal function
|n. vitro using renal cortical slices obtained from male Sprague-Dawley rats
3 and 7 days after Intubation with 2,3,7,8-TCDD at doses of 10 or 25
vg/kg. (These results were also described by Hook et al., 1977). Anlon
and cation transport were measured by the respective accumulation of
p-am1noh1ppur1c acid and N-methyln1cot1nam1de Into the cortical slices.
Anton accumulation was lower In the high dose group; cation transport was
lower at both dose levels tested. The decrease 1n anlon transport was con-
firmed 1n an In vivo study. Ammon1ogenes1s and gluconeogenesls were not
affected In 2,3,7,8-TCDD treated rats, even when the animals were made
addotlc, which suggests no effect on the kidneys' ability to maintain add
base balance. Also, sodium reabsorptlon was shown In vivo to be within
normal range. Since decreases 1n cation and anlon transport were the only
effects observed, and since these compounds are transported by a different
mechanism, the authors concluded that the effect of 2,3,7,8-TCDD was merely
8-23
-------
a general decrease 1n kidney functon reflecting the poor condition of the
treated animals (animals 1n all treated groups had decreased weight gain),
and not a cause of debilitation.
Although kidney function was only minimally affected by exposure to
2,3,7,8-TCDD, Grelg et al. (1974) demonstrated that pre-exposure to 2,3,7,8-
TCDD could reduce the ability of the rat kidney to respond to stimuli of DMA
sythesls. Folate-stlmulated DNA synthesis measured In. vivo 1n Porten strain
rats was decreased between 67 and 25% 1n animals receiving 2,3,7,8-TCDD at a
dose of 10 pg/kg on day 0-9 before administration of follc add. No sig-
nificant difference 1n folate-stlmulated DNA synthesis was observed 1f
2,3,7,8-TCDD was given 23 hours after follc add. The lack of effectiveness
of administering 2,3,7,8-TCDD shortly after treatment with follc acid sug-
gested that 2,3,7,8-TCDD did not directly Interact with cellular DNA, nor
Inhibit the protein synthesis necessary to support folate-stlmulated DNA
synthesis. Similar Inhibitory effects of 2,3,7,8-TCDD were observed when
lead acetate was used to stimulate kidney DNA synthesis. The mechanism by
which 2,3,7,8-TCDD prevents the kidney from responding to prollferatlve
stimuli 1s not known, although 1t was demonstrated that another agent
capable of Inducing mlcrosomal enzymes, 3-methylcholanthrene (3-HC), had
similar effects on the kidney.
Additionally a number a hematologlc and clinical chemistry changes have
been observed 1n the blood of laboratory animals after exposure to 2,3,7,8-
TCDD. Many of these changes, as described by Z1nkl et al. (1973), reflect
damage to previously described organ systems. In female CD rats given 30
dally doses of 2,3,7,8-TCDD at levels of 0.1, 1.0 or 10 vg/kg, the clini-
cal chemistry of the serum reflected liver damage. In the high-dose group,
serum GOT and serum GPT were elevated starting 13-17 days after Initial
8-24
-------
treatment. There was a marginal change 1n GPT 1n the mid-dose group and
lactic dehydrogenase (LDH) 1n the high-dose group, but the Increases were
only transitory. Serum cholesterol was Increased 1n the high-dose animals
starting at day 10, with a transitory Increase again observed 1n the mid-
dose group. Conversely, there was a decrease 1n serum protein from day 24
on 1n the high-dose animals. Along with these clinical chemistry changes
Indicative of liver damage, the only other major effect observed 1n the
blood was thrombocytopenla. The decrease 1n platelet count was detected
early, by day 3, 1n the 10 and 1 vg/kg groups; 1n the 0.1 vg/kg group a
significant decrease was not observed until day 17. Thrombocytopenla was
also observed 1n female guinea pigs after 8 weekly oral doses of 2,3,7,8-
TCDD at 0.2 vg/kg, and 1n mice (administered a single dose of 1.0, 10 or
50 vg/kg). In guinea pigs lymphopenla was also observed. Other hemato-
loglc changes were attributed to hemoconcentratlon.
In a more extensive Investigation of 2,3,7,8-TCDD-1nduced hyper!1p1dem1a
In male Sprague-Daw!ey rats. Poll et al. (1980) treated animals with a
single 1.p. Injection of 2,3,7,8-TCDD at 2 doses of 2.5, 5, 10 and 20
vg/kg. At day 21 after treatment there was a dose-related Increase 1n
total plasma cholesterol and high density Upoproteln cholesterol, while no
change was observed In trlglycerldes or very low and low density Upopro-
telns (VLDL and LOL, respectively). At a dose of 20 vg/kg the maximum
Increase 1n HDL cholesterol and total cholesterol occurred 30 days after
treatment, and a significant elevation was still present at 60 days after
treatment when the study was terminated. Slight changes 1n the apoproteln
of HDL from 2,3,7,8-TCDD rats and control rats were Indicative of new apo-
proteln synthesis. Although the Increases 1n HDL cholesterol may be 1n
response to eliminating excess I1p1ds, the exact function has not been
8-25
-------
clearly shown. There 1s some evidence from studies of workers exposed to
2,3,7,8-TCDD that there were reduced levels of blood HDL cholesterol and
raised total cholesterol as compared with a matched control group (Walker
and Martin, 1979).
In contrast to rats, male Hartley strain guinea pigs given a single 1.p.
Injection of 2,3,7,8-TCDD at a dose of 2 pg/kg had Increased hyperllplde-
m1a characterized by Increases 1n VLDL and LDL {Swift et a!., 1981). In
animals sacrificed 7 days after exposure to 2,3,7,8-TCDD, there was an
Increase 1n total serum Upld, cholesterol esters, trlglycerldes and phos-
phollplds, when comparison was made with pair-fed, weight-paired or ad_
libitum fed control groups. Serum-free fatty adds were not changed quanti-
tatively; however, some qualitative changes occurred, reflecting an Increase
In the types of fatty adds that were abundant 1n the adipose tissue of
guinea pigs. Anaylsls of Upoprotelns revealed a 19-fold Increase 1n VLDL,
a 4-fold Increase 1n LOL, and no change observed In the levels of HDL. The
VLDL was also qualitatively different 1n the 2,3,7,8-TCDD treated animals,
containing less cholesterol ester and an altered C apoproteln. The Import-
ance of these qualitative changes 1s unclear. The hyperI1p1dem1a may result
from the 2,3,7,8-TCDD mobilization of free fatty adds, which are then used
1n the synthesis of VLDL and are subsequently formed Into LDL. The rela-
tionship of the changes 1n serum I1p1d levels to the mechanism of 2,3,7,8-
TCDD toxldty needs further study.
Elovaara et al. (1977) observed some changes 1n blochemlcals of the
brain of male Wlstar and heterozygous Gunn rats given a single Intubation of
2,3,7,8-TCDD at a dose of 20 pg/kg. At 7 days post-treatment, there was a
small but significant decrease as compared with vehicle treated control
animals 1n both the protein and RNA content of the Wlstar rats, while levels
8-26
-------
of ac^d protelnase and DT-d^aphorase (an enzyme Induced by 2,3,7,8-TCDD In
the liver) had a small but significant Increase 1n the heterozygous Gunn
rats. There were no significant changes observed 1n homozygous rats given
2,3,7,8-TCDD at 20 yg/kg. The authors noted that add protelnase may
participate 1n chemically Induced degeneration of the brain.
8.1.1.4. IMHUNOLOGICAL EFFECTS -- During acute toxlclty studies with
2,3,7,8-TCDD, thymlc atrophy was noted as a consistent effect 1n all species
that have been Investigated. This finding suggested that 2,3,7,8-TCDD may
alter the Immune response, and Initiated 1mmunotox1c1ty studies 1n exposed
animals. In guinea pigs treated with 8 weekly oral doses of 2,3,7,8-TCDD
(0, 0.008, 0.04, 0.2 or 1.0 yg/kg bw), body weight, spleen weight and
thymus weight were depressed, adrenal weight was Increased and leukocyte and
lymphocyte counts were elevated (Vos et al., 1973). Upon histologlcal
examination, 2,3,7,8-TCDD-exposed rats had a severe depletion of lymphocytes
from the thymlc cortex (Vos and Moore, 1974). Hematologlcal changes were
noted 1n rats exposed to 10 and 14 dally doses of 10 yg/kg 2,3,7,8-TCDD
(Welssberg and Z1nkl, 1973). Increased red blood cell count, decreased
platelet count, Increased neutrophll count and Increased packed cell volumes
were reported 1n 2,3,7,8-TCDD-exposed rats. A summary of the data available
on the 1mmunotox1c effects of 2,3,7,8-TCDD 1n animals 1s presented 1n Table
8-4. A review of 1mmunotox1c1ty and 1mmunosuppress1on was reported by Vos
(1977).
Vos et al. (1973) Investigated the humoral and cell-mediated Immune
response 1n Hartley guinea pigs, CD rats and B6D2F1 mice. The humoral
Immune response was tested 1n 2,3,7,8-TCDD-treated hamsters by Injecting
tetanus toxold (subcutaneously) Into the footpad and later testing for the
concentration of tetanus antitoxin from the serum by an 1mmunod1ffus1on
8-27
-------
TABLE 8-4
liwonologlcal Effects of 2.3,7,8-TCDD In Animals
Species/ Sex Exposure Route
Strain
Nlce/B6D2F1 N gavage
N1ce/C57Bl/6 F.N Maternally
administered
(gavage)
Nice/ N gavage
C57Bl/6Jfh
CO
1
CO
Nice/Swiss N gavage
N1ce/B6C3Fl F \n vitro
(spleen cells)
Nice/Swiss- F.N maternally
Webster administered
(diet)
Nice/CD N gavage
0.
25
0.
25
0.
20
0,
50
0.
0,
20
0.
10
Dose(s) Duration of Exposure
0.2, 1.0. 5.0. 4 weeks
.0 yg/kg bw/week
1.0. 2.0, 5.0, 4 or 6 weeks (3 or 5
.0 wg/kg administrations)
0.5. 1. 5. 10. 4 weeks
tig/kg bw/week
1.5. 5, 15. 4 weeks
tig/kg bw/week
5, 5.0. 50 {ig/ml 5-60 seconds
1, 2.5. 5, 10. 10 weeks (pregestatlon
ppb (dietary) and 3 weeks post-
parturition)
0.01. 0.1. 1.0. up to 8 weeks
.0 wg/kg bw/week
NlnlMUM
Effective Dose
NA
5.0 wg/kg bw/week
5.0 wg/kg bw/week
1.0 wg/kg bw/week
25.0 yg/kg bw/week
2.0 vg/kg bw/week
1.0 wg/kg bw/week
1.5 wg/kg bw/week
50 tig/mt
2.5 ppb
2.5 ppb
5 ppb
1 ppb
NA
0.01 tig/kg bw/week
1.0 tig/kg bw/week
Parameter
bw
thyiMS weight
graft-versus-
host response
thynus weight
PHA response
skin graft
rejection
Salmonella
Infection
endotoxln (E. coll)
susceptibility
protein, DNA, and
RNA synthesis
ant) genie RBC
reaction
thywlc cortex
contact sensitivity
to DNFB
endotoxln
(Salmonella)
susceptibility
Llsterla Infection
serum ImMinoglobln
level
serum Imnunoglobln
Effect
no change
decreased
decreased
decreased
decreased
prolonged
Increased
Mortality
and
decreased
time to
death
Increased
Mortality
decreased
decreased
atrophy
decreased
Increased
aortal Hy
no change
Increased
decreased
Reference
Vos et al..
1973
Vos and
Roore. 1974
Thlgpen
et al.. 1975
Vos et al..
1978a
Luster et al..
1979a.b
Thorns and
Hlnsdlll. 1979
Sharma and
Gehrlng. 1979
Nice/CD
In vitro
10~«-10"» N
single
level
lymphocyte blasto-
genlc transforma-
tion
Increased Sharwa and
Gehrlng. 1979
-------
T/WIE 8-4 (cont.)
Species/ Sex Exposure Route Dose(s) Duration of Exposure Minimum
Strain Effective Dose
nice/Swiss- F oral (diet) 0, 10, 100 ppb 5 weeks (or wore) 10 ppb
Hebster 10 ppb
10 ppb
10 ppb
10 ppb
Nice/ H l.p. 0,1, 2, 6, single Injection 1 wg/kg
C57B1/6J 30 Mg/kg bw
1 Pi/kg
00
i
ISJ
to
H1ce/B6C3F1 N,F maternally 0,1.0,5.0, 4 days during gestation 1.0 wg/kg bw/day
administered 15.0 yg/kg bw/day and lactation
1.0 pgAg bw/day
5.0 pg/kg bw/day
Nke/C5781/& K l.p. 0, 0.4, 4.0, 4 weeks 4.0 pg/kg bw/week
40 ng/kg bw/week 0.4 tig/kg bw/week
H1ce/C578l/6 H l.p. 0, 0.004, 0.04, 4 weeks 0.004 Mg/kg bw/week
0.4 i*g/kg bw/week
Rat/CD F oral 0, 0.2, 1.0. 6 weeks 5.0 vg/kg bw/week
5.0 iig/kg bw/week 5.0 vg/kg bw/week
NA
Rat/CO F oral 0, 10 iig/kg bw/day 10, 14 days 10 tfg/kg bw/day
10 ugAg bw/day
10 wg/kg bw/day
Parameter
tetanus response
antlgenlc RBC
response
sensltlzatlon to
ONFB
resistance to
Salmonella
resistance In
Llsterla
macrophage and
natural killer
cell activity
macrophage and
natural killer
cell number
antibody
production
L. monocytogenes
"susceptibility
PYB6- tumor suscep-
tibility
bone marrow nypo-
cellularlty
thymus atrophy
cytotoxlc T-cell
response
In vitro genera-
tion of cytotoxlc
T-cells
bw
thymus weight
tuberculin hyper-
sensitivity
erythrocyte count
platelet count
neutrophll count
Effect
decreased
decreased
decreased
Increased
mortality
Increased
mortality
no change
decreased
decreased
Increased
Increased
Increased
Increased
decreased
decreased
decreased
decreased
no change
Increased
decreased
Increased
Reference
Hlnsdlll,
et al., 1980
Nantovanl
et al., 1980
Luster et al.,
1980
Clark et al..
1981
Clark et al.,
1981
Vos et al.,
1973
Helssberg and
Zlnkl, 1973
-------
TABLE 8-4 (cent.)
Species/ Sex Exposure Route Oose(s)
Strain
Rat/F-344 F.H maternally 0, 1.0, 5.0 pg/kg
administered bw/dose
Rat/Fischer F.H maternally NR
administered
(NR)
* Rat/Fischer- F.H maternally 0, 5 pg/kg bw/dose
w uistar administered
0 (NR)
Rat/Sprague- H 1.v. 0, 1 pg/kg bw
Dawley
Guinea pig/ F gavage 0, 0.008. 0.04,
Hartley 0.2, 1.0 pg/kg bw
Duration of Exposure
4 or 6 weeks (3 or 5
administrations)
4-6 weeks (during ges-
tation and neona tally)
3 or 4 applications
during gestation and
neonatally
single Injection
8 weeks
H1n1nun
Effective Dose
1.0 pg/kg bw/dose
5.0 pg/kg bw/dose
5.0 pg/kg bw/dose
5.0 pg/kg bw/dose
5.0 pg/kg bw/dose
NA
NR
NR
5 pg/kg bw/dose
5 pg/kg bw/dose
5 pg/kg bw/dose
1 pg/kg bw
0.04 pg/kg bw/week
0.04 pg/kg bw/week
0.04 pg/kg bw/week
0.2 pg/kg bw/week
Parameter
bw and thymus
weight
spleen weight
PHA response
graft-versus-host
response
skin graft
rejection
pseudorables
virus Infection
Con A and PHA
response
oxazolone skin
hypersens1t1v1ty
antibody production
to bovine gamma
globulin
PHA and Con A
response
thymus and bw
thymlc RNA
synthesis
thymlc RNA
polymerase
activity
bw
thymus weight
tuberculin hyper-
sensitivity
tetanus antitoxin
Effect Reference
decreased Vos and
Moore, 1974
decreased
decreased
decreased
prolonged
no change
decreased Moore and
Faith. 1976
decreased
no effect Faith and
Luster, 1979
decreased
decreased
until 128
days
decreased Kurl et al.,
1982
decreased
decreased Vos et al.,
decreased 1973
decreased
decreased
N = male; F = female; 1.p. •= Intraperltoneal l.v. = Intravenous; PHA
1-fluorobenzene; NA = Not applicable; NR = Not reported
Phytohemagglutlnln; Con A = Conconavalln A; R8C = red blood cell; DNF8 = 2.4-d1n1tro,
-------
technique. Cell-mediated Immunity was tested by Injecting Mycobacterlum
tuberculosis (subcutaneously) Into guinea pigs on day 35 of 2,3,7,8-TCDD
treatment (during a schedule of 8 weekly doses). Intradermal tuberculin
hypersensltlvlty was determined by measurements of skin thickening on days
47 and 54. Decreased skin hypersensltlvlty was noted 1n hamsters treated
with 0.04 g 2,3,7,8-TCDD/kg and higher doses. Decreased tetanus antitoxin
levels were evident In guinea pigs treated with 0.2 vg 2,3,7,8-TCDD/kg,
but not at lower dose levels. Vos et al. (1973) also tested the cell-
mediated Immunity 1n rats exposed to 2,3,7,8-TCDD (0, 0.2, 1.0 or 5.0
yg/kg, once weekly for 6 weeks). M. tuberculosis was Injected Into rats
by day 28 of the treatment period, followed by Intradermal hypersensltlvlty
testing on day 42. No changes 1n the thickness of skin were noted 1n
2,3,7,8-TCDD treated rats when compared with controls.
Mice were used to test the effect of 2,3,7,8-TCDD on cell-mediated
Immunity by use of the "graft-versus-host" experiment (Vos et al., 1973).
In this test, spleen cells from 2,3,7,8-TCOD-exposed mice (0, 0.2, 1.0 or
5.0 yg/kg once weekly for 4 weeks) of the C57B1/6 strain were Injected
Into the right footpad of a hybrid recipient mouse (C5781/6 x DBA-2). Donor
cells possessing sufficient activity will respond to the DBA-2 antigen on
the host cells, resulting 1n the enlargement of the popliteal lymph node.
Host cells are tolerant of the donor cells since both have C57B1/6 anti-
gens. In this test Vos et al. (1973) noted a significant (p<0.01) dose-
related decrease In the activity of 2,3,7,8-TCDD-treated spleen cells (as
measured by the degree of popliteal lymph node enlargement on the site of
the spleen cell Injection). Lymph node enlargement was significantly less
(p<0.01) 1n hybrid recipient mice receiving spleen cells from mice treated
with 5 |ig 2,3,7,8-TCDO/kg/week than from donor cells of untreated mice.
8-31
-------
Studies continued 1n an attempt to Identify the mechanism of 2,3,7,8-
TCOD-1nduced Immunodeficiency. Rats (F-344) exposed pre- and postnatally by
maternal dosing (1 or 5 yg 2,3,7,8-TCDD/kg administered to dams on days 11
and 18 of gestation and 0, 7 and 14 postnatally) had prolonged times until
graft rejection, decreased spleen cell graft-versus-host activity and
decreased binding response to phytohemagglutlnin {PHA} (Vos and Moore, 1974;
Moore and Vos, 1974). Response to conconavalln A {Con A), a humoral Immune
response, was actually Increased.
Since thymus-derlved lymphocytes (T-eells) play a central role 1n cell-
mediated Immunity and host defense mechanisms, Interest turned to these
areas of Immunology. The effect of 2,3,7,8-TCDD on host resistance to
Infection, a vital measure of Immune response, was tested by Thlgpen et al.
(1975) 1n male pathogen-free mice (C57Bl/6,Jfh). 2,3,7,8-TCDD was admin-
istered to mice at 0.5, 1, 5, 10 or 20 pg/kg once weekly for 4 weeks fol-
lowed by Inoculation with Salmonella bern 2 days after the final 2,3,7,8-
TCDD administration. Mortality rates and "time until Infection" were used
to determine the 1mmunolog1cal effect of 2,3,7,8-TCDD. A significant
(p<0.05) Increase 1n mortality and decrease 1n time of Infection were noted
1n groups treated with 1 ng/kg or higher doses of 2,3,7,8-TCDD when
compared with controls. 2,3,7,8-TCDD at 0.5 jig/kg did not alter these
parameters and was regarded as a no effect level. The Immune-resistance of
mice to J>. bern 1s therefore reduced by treatment with 1 jig 2,3,7,8-TCDD/
kg/week {for 4 weeks).
Pretreatment with 2,3,7,8-TCDD greatly enhances the susceptibility of
mice to £. coll endotoxln (Vos et al., 1978a). Injection of 250 jig of
endotoxln to mice pretreated with 0, 1.5, 5 and 15 »q 2,3,7,8-TCDD/kg
8-32
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resulted In 0/5, 1/5, 6/6 and 6/6 deaths, respectively. Mice pretreated
with 15 and 50 yg 2,3,7,8-TCDD/kg and Injected with 10 yg of endotoxln
had 1/4 and 2/4 deaths, respectively. Mice treated with lower doses of
2,3,7,8-TCDD were not susceptible to this quantity of endotoxln. Increased
mortality (2/6) 1n a control group was noted only when 500 yg of endotoxln
was administered; however, 10 jig of endotoxln was sufficient to cause
similar mortality (2/5) 1n mice treated with 50 yg 2,3,7,8-TCDD/kg.
The Immunocompetence of 5-week-old offspring of Swiss-Webster mice fed
diets containing 1, 2.5, 5, 10 or 20 ppb 2,3,7,8-TCDD was tested by several
means (Thomas and H1nsd1ll, 1979). The number of cells reactive to antl-
genlc RBC, differential white blood cell counts, organ weights, hlstopathol-
ogles, hypersens1t1v1ty to 2,4-d1n1tro-l-fluorobenzene (DNFB) and the
resistance to E.. coll Upopolysaccharlde (IPS), L1ster1a monocytogenes and
Salmonella typh1mur1uro LPS were all measured for mice exposed to different
levels of 2,3,7,8-TCDD. Adult female mice were exposed to 2,3,7,8-TCDD for
4 weeks before mating, throughout gestation and for 3 weeks postparturltlon.
Young mice being tested for 1mmunotox1c1ty were therefore exposed to
2,3,7,8-TCDD only jji utero and through lactation. The typical decrease 1n
thymus weight was noted 1n mice exposed to 2.5 and 5.0 ppb but was not evi-
dent 1n the 1.0 ppb group. A decrease 1n the number of plaque-forming cells
(RFC) reactive to sheep RBCs was significantly reduced 1n the 2.5 and
5.0 ppb 2,3,7,8-TCDD-exposed groups. (Because of the poor survival of young
1n the 10 and 20 ppb 2,3,7,8-TCDD-exposed groups, results and comparisons
were usually reported for the three lower dose groups). The humoral content
of ant1-RCD antibodies, however, was not lower In 2,3,7,8-TCDD-exposed
groups when compared with controls. A decrease 1n the skin hypersens1t1v1ty
8-33
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to DNFB following sensltlzatlon was noted 1n all 2,3,7,8-TCDD-treated groups
(only the 5-ppb group was statistically reduced from controls). 2,3,7,8-
TCDD caused an Increased susceptibility (Increased mortality level) to §_.
typh1mur1um 1n a dose-related fashion. The response to E_, coll IPS and L_.
monocytogenes was not different from controls. 2,3,7,8-TCDD exposure did
not alter the response of lymphocytes (Band T-cells) |£ vitro to Con A, nor
was m1togen-1nduced lymphocyte proliferation affected (Thomas and H1nsd1ll,
1979).
Similar findings were reported In F1scher/W1star rats exposed to
2,3,7,8-TCDD during gestation (18th day) and neonatally, or neonatally alone
(on days 0, 7 and 14) (Faith and Luster, 1979). Dams were treated with 5
g/kg 2,3,7,8-TCDD on each dose day. Typically, body weight and thymlc
weights were decreased 1n progeny, which lasted until 135 days of age. The
thymlc- and splenic-cell response to PHA and Con A was decreased 1n all
2,3,7,8-TCDD-treated animals and did not return to normal until day 270.
Delayed hypersensitive reaction was also suppressed until 270 days of age.
The production of antibodies to bovine gamma globulin, which requires
T-helper cell function, was not affected by 2,3,7,8-TCDD exposure during rat
development (Faith and Luster, 1979).
Neonatal B6C3F1 mice, exposed to prenatal (maternal dosing on day 14 of
gestation) and postnatal (days 1, 7 and 14 after birth) doses of 0, 1.0, 5.0
or 15.0 pg/kg 2,3,7,8-TCDD, were studied for 1mmunotox1c effects and host
susceptibility (Luster et a!., 1980). At the 15.0 vg 2,3,7,8-TCDD/kg dose
level, 70% of the neonates died with overt toxic effects (decreased body
weight, liver weight, spleen weight and thymus weight). Bone marrow hypo-
cellularlty and depressed macrophages-granulocyte progenitor cells and
8-34
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pleurlpotent stem cells were associated with 2,3,7,8-TCDD exposure at the
5.0 and 15.0 ug/kg dose levels. Hematologlcal changes, such as decreased
RBC count, hematocrlt and hemoglobin, and lymphocyte count showed a dose-
related response. Host susceptibility to L.. monocytogenes and PYB6-tumor
cells was tested 1n the 2,3,7,8-TCDD-exposed neonates. Death occurred-1n 73
and 40% of the JL. monocytogenes Inoculated (1.2x10® viable organisms) mice
1n the 5.0 and 1.0 vg/kg dose groups, respectively, compared with 28% of
controls. Tumor development occurred 1n 44, 60 and 22% of the neonates
Inoculated with 5xl04 tumor cells from the 5.0 jig 2,3,7,8-TCDD/kg, 1.0
pg 2,3,7,8-TCDD/kg and control groups, respectively.
H1nsd1ll et al. (1980) reported that 2,3,7,8-TCDD administered 1n the
diet of Swiss-Webster mice at 100 ppb for 5 weeks caused a marked suppres-
sion of total serum protein, gamma globulin and albumin, but an Increase 1n
B-globul1ns. At 10 ppb 1n the diet, 2,3,7,8-TCDD caused decreased Immune
response to tetanus toxold, sheep RBC, S,. typhlmurlum and L,. monocytogenes,
and lowered contact yens-ItIvlty to ONFB. This study also sugggested that
although young animals are more susceptible to 2,3,7,8-TCDD, older animals
are still Immunosuppressed and exposure jyn ute.ro. and neonatally 1s not more
crucial than 1n other periods. Vos and Moore (1974) had previously reported
that 1-month-old mice were more sensitive to 2,3,7,8-TCDD than were 4-month-
old mice (C57B1/6). Decreased body weight and thymus weight and spleen cell
response to PHA were evident at lower doses 1n 1-month-old mice than 1n
4-month-old mice.
The effect of single l.p. doses of 2,3,7,8-TCDD (1, 2, 6 and 30 vg/kg)
on peritoneal macrophage and splenic natural killer cell function 1n mice
(C57B1/6J) was studied by Hantovanl et al. (1980) and Vecchl et al. (1980).
8-35
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2,3,7,8-TCDD treatment at all dose levels did not decrease the cytostatlc
and cytocldal activity of macrophages or natural killer cells on a per cell
basis. The total number of macrophages and splenic natural killer cells
recovered from 2,3,7,8-TCDD-treated animals, however, was reduced when com-
pared with untreated controls. Harked hypocellularlty noted In the bone
marrow of 2,3,7,8-TCDD-treated mice may account for the decrease 1n peri-
pheral cell counts (McConnell et al., 1978b). The lack of macrophages and
natural killer cells was suggested as being Instrumental In the decreased
resistance to Infection common to 2,3,7,8-TCDD-exposed animals (Mantovanl et
al., 1980). Although 2,3,7,8-TCDD was a strong Immunosuppressant, animals
given a lethal dose of 2,3,7,8-TCDD did not appear to die from Infections,
nor did a germ-free environment protect them from death (Grelg et al., 1973).
The actual mechanism of 2,3,7,8-TCDD Immunotoxldty 1s unknown but
several Investigators have tested various hypotheses. Vos et al. (1973,
1978a,b) attempted to address the Indirect causes for decreased thymlc
growth and altered T-lymphocyte activity following 2,3,7,8-TCDD treatment.
Vos et al. (1973) measured serum cortlsol and cortlcosteron levels 1n guinea
pigs exposed to 2,3,7,8-TCDD to evaluate the possible Indirect Immunosup-
presslon by these hormones. There was, however, no significant difference
1n the level of these hormones between treated and control animals.
Indirect 1mmunosuppress1on of this type was unlikely. Later studies (Vos et
al., 1978a,b) Investigated the role of thymlc hormones (thymosln) on the
atrophy of the thymus during 2,3,7,8-TCDD treatment. Thymosln administered
1n conjunction with 2,3,7,8-TCDD did not protect mice from the typical
2,3,7,8-TCDD-1nduced 1mmunotox1c alterations. Thymus weight was maintained
but not Increased by thymosln, and thymus-deMved cells continued to show
8-36
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decreased responsiveness to mltogens (PHA, Con A), Thus, it 1s unlikely
that 2,3,7,8-TCDD affects the supply or synthesis of thyralc hormones which
could lead to the observed 1mmunosuppress1on.
van Logten et al. (1980) Investigated the possible Influence of the
adrenal gland, hypophysis and pituitary, and growth hormone on thymlc
atrophy and Immunosuppresslon following 2,3,7,8-TCDD exposure 1n female
F-344 rats. Adrenalectomy and exogenous growth hormone had no preventatlve
action on thymlc Involution. Hypophysectomlzed rats showed advanced thymlc
atrophy.
Sharma and Gehrlng (1979) noted that 2,3,7,8-TCDD caused stimulation of
lymphocyte transformation to blast form cells (mltotlcally active precur-
sors) when no mltogens were present 1n the culture system. This represents
a phenomenon similar to actual antlgenlc challenge. At low doses (0.01 and
0.1 yg 2,3,7,8-TCDD/kg/week for up to 8 weeks), serum 1mmunoglobul1n
levels were elevated 1n male CD-I mice. Larger doses of 2,3,7,8-TCDD (1.0
and 10 vg/kg/week) resulted 1n a decrease 1n the serum Immunoglobulln
level. It was suggested that 2,3,7,8-TCDD may elicit an antlgenlc response
either by combining with a body protein or by causing cellular or biochemi-
cal damage that releases antlgenlc proteins. Sharma and Gehrlng (1979) also
noted that thymlc atrophy was observed after 2 and 4 weeks of treatment but
not after 8 weeks. There may be a recovery of thymlc tissue, either by
Immune tolerance or Immune unresponslveness as a sort of adaptation to
\
\
2,3,7,8-TCDD-exposure and Its possible antlgenlc complex. \
Luster et al. (1979a,b) reported that 2,3,7,8-TCDD affects the Immune
system directly by altering lymphocyte function. The function of T-helper
cells was not altered, since no change 1n response to bovine gamma globulin
8-37
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(requires T-helper cell cooperation) was noted 1n W1star/F1scher and Fischer
rats exposed to 2,3,7,8-TCDD. In. vitro. 2,3,7,8-TCDD {100 ng/mil) sup-
pressed DMA, RNA and protein synthesis 1n splenic lymphold cells from B6C3F1
(Luster et al., 1979a). 2,3,7,8-TCDD, however, did not decrease the binding
of 3H-Con A to lymphocytes, Indicating that these receptors are not
blocked by 2,3,7,8-TCDD. T-lymphocytes were more susceptible to 2,3,7,8-
TCDD, measured by specific mltogen binding assays, than B-lymphocytes.
These authors (Luster et al., 1979a) suggested that 2,3,7,8-TCDD may bind
directly to the lymphocyte cell membrane and alter Its function. Faith and
Luster (1979) reported that lymphocytes from the spleen, thymus, bone marrow
and lymph nodes of Fischer rats exposed to 2,3,7,8-TCDD showed abnormal
homing patterns within the body. 2,3,7,8-TCDD exposure apparently altered
the cell surface markers so that spleen lymphocytes were taken up by the
thymus of recipient rats. These authors (Faith and Luster, 1979) suggested
that 2,3,7,8-TCDD may change cellular metabolism, which alters the cell mem-
brane constituents or may Insert directly Into the membrane. Kurl et al.
(1982) reported that 2,3,7,8-TCDD causes changes 1n thymlc transcription and
RNA synthesis that may lead to cell surface changes. Cell surface changes
could presumably result 1n altered antigen recognition and cell-to-cell
recognition, causing 1mmunosuppress1on and thymlc atrophy.
Clark et al. (1981) reported that 2,3,7,8-TCDD treatment (0.4, 4.0, 40
v9/kg weekly for 4 weeks by 1.p. Injection) caused functional Impairment
of cytotoxlc T-cells 1n C57B1/6 male mice. The authors felt that this
response was particularly sensitive to 2,3,7,8-TCDD treatment and hypothe-
sized that 2,3,7,8-TCDD directly Inhibits the function of these cells.
Contrary to the hypothesis tested by these authors and that held by Luster
8-38
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et al. (1979a,b), 2,3,7,8-TCDD treatment Impaired the generation of cyto-
toxlc T-cells by the spleen (at doses as low as 0.004 yg/kg when detected
IE vitro) but did not appear directly toxic to the cytotoxlc T-cells. At
present, the mechanism of Immunosuppresslon caused by 2,3,7,8-TCDD Is
unknown and the theories available are speculative. In a later study,
however, Clark et al. (1983) reported that a 10- to 100-fold greater dose of
2,3,7,8-TCDD was required to suppress cytotoxlc T-cells 1n DBA/2 mice as
compared with C56B1/6 mice. This Indicates that susceptibility to 2,3,7,8-
TCDD 1mmunotox1c1ty segregates with the Ah locus, which 1s consistent with a
receptor mediated mechanism. The receptor mediated mechanism was further
supported by the susceptibility of the C57B1/6 x DBA/2J hybrid mouse to
2,3,7,8-TCDD suppression of the cytotoxlc T-cells, which 1s again consistent
with the dominant Inheritance of Ah (Nagarkattl et al., 1984).
Few reports are available In which the Immunological effects of 2,3,7,8-
TCDD exposure were studied 1n humans. Regg1an1 (1980) reported that the
ImmunocapablHty of 17 people, ranging 1n age from 3-60 years, who had been
exposed to 2,3,7,8-TCDD, was normal 1n all cases. In a survey of 41 workers
exposed to 2,3,7,8-TCDD, Ward (1982) measured 1mmunoglob1n 6, A, M, D and E,
as well as lymphocytes, T-cells, B-cells, PHA response and blood cell
counts. These determinations were made 10 years after workers had developed
2,3,7,8-TCDD-1nduced chloracne. In this group of workers, there was a sig-
nificant Increase 1n the proportion of cases with reduced IgD and IgH. It
was suggested that the 2,3,7,8-TCDD-exposed group had a reduced Immune
capability and a deficiency In T-cell and B-cell cooperation. The Immuno-
toxldty of 2,3,7,8-TCDD 1n humans cannot be properly assessed because of
the paucity of data recorded soon after exposure. The most prominent
effects In animals (I.e., humoral responses) were not measured 1n humans.
8-39
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8.1.1.5. ENZYME INDUCTION BY TCDD —
8.1.1.5.1. In Cell Cultures —Although 2,3,7,8-TCDD has a very low
toxldty to cells 1n culture (Beatty et al., 1975; »Bradlaw et a!., 1976;
Knutson and Poland, 1980; Yang et al., 1983), It Is an extremely potent
enzyme Inducer 1n these systems (Kourl et al., 1974; N1wa et al., 1975;
Bradlaw et al., 1976; Malik and Owens, 1977; Malik et al., 1979; Bradlaw et
al., 1980). This enzyme Induction 1s so sensitive that 1t has been proposed
as a bloassay for detecting planar polychlorlnated organic compounds
(Bradlaw et al., 1975, Bradlaw and Casterllne, 1979; N1wa et al., 1975).
Kourl et al. (1974) found that 2,3,7,8-TCDD Induced aromatic hydrocarbon
hydroxylase (AHH) activity In cultured human lymphocytes to the same extent
as 3-MC; however, the concentration of 2,3,7,8-TCDD necessary for maximal
enzyme Induction was 40-60 times less than that of 3-MC. N1wa et al. (1975)
compared AHH Induction by 2,3,7,8-TCDD among cell cultures (H-4-II-E, VERO,
HTC, LB82, MA, Hepa-1, TRL2, JRL-2, NRKE and Chang). ED§0 values ranged
from 0.12 nM In the Hepa-1 cell line to >100 nM In the VERO and HTC cell
lines. 2,3,7,8-TCDD did not Induce AHH activity 1n LB82 cells. The respon-
siveness of AHH Induction to 2,3,7,8-TCDD was 250-900 times greater than to
3-MC. In addition, cell cultures derived from C57B1/6N mice were 16 times
as sensitive to 2,3,7,8-TCDD as cell cultures derived from DBA/2N mice. The
responsiveness of cell cultures to enzyme Induction by 2,3,7,8-TCDD 1s thus
similar to the effects seen in. vivo. The Inductive effect of 2,3,7,8-TCDD
was blocked by actlnomycln D and cycloheximlde. Implying that Induction
Involved the sythesls of new mRNA and protein. Enzyme Induction by 2,3,7,8-
TCDD, therefore, Involves an Initial RNA synthesis and continuous protein
synthesis (Malik and Owens, 1977; Malik et al., 1979).
8-40
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In all of these studies, there was no correlation between cytotoxlclty
and enzyme Induction, This Implies that, despite the correlation in. vivo
(Section 8.3.5.), there may be no direct connection between enzyme Induction
and the toxldty of 2,3,7,8-TCDD.
8.1.1.5.2. In Mice and Rats -- The effects of 2,3,7,8-TCDD on enzyme
activity 1n rats and mice have been Investigated extensively. 2,3,7,8-TCDD
has been found to alter many enzyme activities 1n a wide variety of organ
systems (vide Infra). This alteration primarily results In Increased enzyme
activity, although 2,3,7,8-TCDD has been observed to Inhibit some enzymes.
Hook et al. (1975a) reported that 2,3,7,8-TCDD supressed hepatic mlcro-
somal N-demethylat1on 1n male, but not female, rats; however, cytochrome
P-450 and benzpyrene hydroxylase activity were Increased. The suppression
of N-demethylase activity was undetectable for 73 days following a single
oral dose of 25 yg 2,3,7,8-TCDD/kg bw. The suppression of N-demethylase
activity was seen only 1n adult animals. In 10-day-old rats, 2,3,7,8-TCDD
had an Inductive effect on this activity.
The Inductive effects of 2,3,7,8-TCDD have been demonstrated to be organ
specific. Altlo and Parkkl (1978) Investigated the effects of 2,3,7,8-TCDD
on the activities of AHH, ethoxycoumarln deethylase, cytochrome C reductase,
epoxlde hydratase, UDP glucuronosyltransferase, and glutathlone S-trans-
ferase 1n the Hver, kidney, lung, small Intestine and testes of male Wlstar
rats. Honooxygenase activity was stimulated 1n the liver, lung and kidney,
but not 1n any other tissue Investigated. UDP glucuronosyltransferase
activity Increased by a factor of 7 1n the liver, by a factor of <2 1n the
kidney, and not at all 1n any other tissue. Epoxlde hydratase and gluta-
thlone S-transferase activities were not affected 1n any of the tissues
studied, although stimulation of hepatic glutathlone S-transferase has been
8-41
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reported by other Investigators (Manis and Apap, 1979). Enzyme Induction
has also been reported 1n rat mammary gland (Rlkans et al.f 1979), mouse
testes (Hattlson and Thorgetrsson, 1978), and rat prostate gland (Lee and
Suzuki, 1980), but the rat adrenal gland 1s apparently Insensitive to Induc-
tive effects of 2,3,7,8-TCDD (Guenthner et al., 1979b).
In the Hver of rats and mice, 2,3,7,8-TCDD affects a wide range of
enzymatic activities, Including DT-d1aphorase (Beatty and Neal, 1976a,b),
bH1rub1n catabollsm (KapHulnlk and Ostrow, 1978), ornlthlne decarboxylase
(Potter et al., 1982), 7-ethoxycoumar1n 0-demethylase (Greenlee and Poland,
1978), glutathlone S-transferase (Baars et al., 1978; Hanls and Apap, 1979),
aldehyde dehydrogenase (Llndahl et al., 1978; De1tr1ch et al., 1977), uro-
porphyrlnogen decarboxylase (Jones and Sweeney, 1977), 5-am1nolevul1n1c
add synthetase (Goldstein et al., 1982a; Woods, 1973), UDP-glucuronosyl
transferase (Harselos et al., 1978) and a number of mlcrosomal oxldatlve
enzyme systems (vide Infra).
2,3,7,8-TCDD 1s four orders of magnitude more potent than 3-HC as an
Inducer of hepatic AHH activity; however, the dose-response curve for the
two compounds are parallel and both produce the same maximal response
(Poland and Glover, 1974). Simultaneous administrations of maximally
Inducing doses of both compounds produced no greater response than either
alone and both produced a cytochrome with a shift In the absorption maximum
of the carbon monoxide difference spectrum from 450 to 448 nm. In a number
of studies, Increased AHH activity and cytochrome P-448 synthesis have been
separated (Chhabra et al., 1976); however, other researchers report an
apparent connection between cytochrome P-448 and AHH Induction (K1tch1n and
Woods, 1977, 1978a,b). Thus, 2,3,7,8-TCDD not only stimulates AHH activity
by Inducing cytochrome P-450 formation, but may enhance AHH activity by
other mechanisms as well.
8-42
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8.1.1.5.3. In Rabbit — The response of the rabbit 1s quite different
from that observed 1n rats and mice (Hook et al., 1975a). The only changes
1n hepatic enzyme activities observed were suppression of benzpyrene
hydroxylase and benzphetamlne N-demethylase. In the same study, blphenyl
4-hydroxylase was Induced 1n the lung and benzpyrene hydroxylase was Induced
1n the kidney. In a similar study, a hepatotoxlc dose of 2,3,7,8-TCDD (30
yg/kg) failed to alter prostaglandln synthetase activity 1n hepatic or
renal tissue (KohH and Goldstein, 1981).
In a series of studies, Johnson and Huller-Eberhard (1977a,b,c,d),
Johnson et al. (1979), Norman et al. (1978a,b), L1em et al. (1980) and Dees
et al. (1982) Isolated a series of cytochromes P-450 from rabbit liver
mlcrosomes. These cytochromes were Immunologically distinct, functioned In
different catalytic pathways, and responded differently to Induction by
polycycllc aromatic hydrocarbons. 2,3,7,8-TCDD was found to Induce two
cytochromes, designated as form 4 and form 6. Form 4 1s the major cyto-
chrome Induced 1n adult rabbit liver by 2,3,7,8-TCDD; however, form 6 1s the
major cytochrome Induced 1n newborn rabbit liver (Norman et al., 1978b),
adult rabbit lung, and adult rabbit kidney (Liem et al., 1980; Dees et al.,
1982).
8.1.1.5.4. Other Species — The guinea pig, the species most sensi-
tive to the toxic effects of 2,3,7,8-TCDD, 1s similar to the rabbit 1n Its
response to 2,3,7,8-TCDD. Blphenyl 4-hydroxylase was Induced 1n the Hver,
lung and kidney, blphenyl 2-hydroxylase was suppressed 1n the liver, and
benzpyrene hydroxylase was Induced 1n the kidney (Hook et al., 1975b).
Testlcular mlcrosomal cytochrome P-450 content was depressed following a
single oral dose of 1 yg/kg, reaching 52% of controls by 1 day and remain-
Ing at this level for 9 days (Tofllon et al., 1980). Testlcular mlcrosomal
8-43
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heme levels and 5-am1nolevul1n1c add synthetase activity were unaffected
by this treatment. In contrast to the rat, 2,3,7,8-TCDD did not Induce
DT-d1aphorase 1n brain, spleen, kidney, lung, heart or liver of male guinea
pigs (Beatty and Neal, 1978).
Aryl hydrocarbon hydroxylase and s-am1nolevu!1n1c add synthetase In
the chick embryo have been reported to be extremely sensitive to the Induc-
tive effects of 2,3,7,8-TCDD (Poland and Slover, 1973a,b), with maximal
Induction occurring with 155 pmoles/egg. This Induction 1s relatively long
lasting, with 70% of the maximum Induced activity present 5 days following a
single dose of 2,3,7,8-TCDD. Structure-activity studies demonstrated a per-
fect correspondence between the toxldty and Induction potency of a series
of d1benzo-p_~d1ox1n congeners (Poland and Glover, 1973a).
8.1.2. Subchronlc. Four laboratory studies described the systemic toxic
effects of subchronlc exposure to 2,3,7,8-TCDD 1n rodents. Also, one semi-
controlled study evaluated the toxic effects to rabbits after confinement to
an area containing soil contaminated with 2,3,7,8-TCDD. No Information was
found 1n the literature searched on the effects of subchronlc exposure to
1,2,3,7,8-PeCDD, and only one preliminary study was available describing the
effects of subchronlc exposure to a mixture of two HxCDDs 1n rats and mice.
Kodba et al. (1976) exposed Sprague-Dawley rats to 2,3,7,8-TCDD for 13
weeks. The animals 1n groups of 12 males and 12 females received the com-
pound suspended 1n acetone-corn oil (1:9) by gavage 5 days/week at doses of
0.0, 0.001, 0.01, 0.1 or 1.0 jig/kg bw. At the end of the treatment period
5 rats of each sex were killed for hlstopathologlc examination, and the
remaining animals were continued for postexposure observation. This report
on gross, hematologlc, clinical chemistry and hlstopathologlc (on animals
terminated at the Interim kill or killed when moribund) observations was
8-44
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prepared on data available 13 weeks after termination of treatment. Signs
of toxlclty were observed only at the two higher dose levels, and female
rats appeared more sensitive to the toxic effects of 2,3,7,8-TCDD. During
the study there were five treatment-related deaths 1n the high-dose group
females, with three occurring during treatment and two 1n the post-treatment
period. In male animals only two deaths occurred 1n the post-treatment
period 1n the high-dose group. Both the male and female rats of the 0.1 and
1.0 yg/kg groups had depressed body weight; however, greater relative
depression of body weight was observed 1n the high-dose females. Other
changes such as Increases 1n blUrubln concentrations, urinary copropor-
phyrln excretion, and changes In relative thymus or Hver weight to body
weight ratio occurred 1n the two high-dose female groups, but only 1n the
1.0 pg/kg male group. Although male rats had significantly decreased
hema to "logic values (packed cell volume, RBC count and hemoglobin) In the two
high-dose groups, and these values were normal 1n all female rats, the
authors pointed out that these results may have been an artifact resulting
from dehydration-Induced hemoconcentratlon 1n the female rats. No specific
data were provided, however, to support this last conclusion.
After necropsy, gross examination revealed subcutaneous edema, a
decrease 1n the size of testes and uteri, and a decrease 1n the number of
corpora lutea. H1stolog1c examination revealed Involution of the thymus,
decreased number of thymocytes, and focal necrosis and pigment accumulation
1n the Hver. These observations were made only 1n the animals of the high-
dose group, with the exception of a slight decrease 1n the number of thymo-
cytes and mild microscopic distortion of the architecture of the liver 1n
the group fed 0.1 ug/kg. Although hlstologlc evidence from animals killed
during the Interim sacrifice was consistent with the Hver and thymus being
8-45
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the primary target organs, 1n an animal that died during the study there
were signs of aortic thrombosis and adrenal hemorrhage, and 1n a second
animal there was severe anemia, suggesting possible Involvement of the hema-
topoletlc system near the time of death.
Liver toxldty was the only effect of treatment observed during hlsto-
loglc examination of rats (Osborne-Mendel) and mice (B6C3F1) administered
2,3,7,8-TCDD for 13 weeks 1n a preliminary subchronlc toxldty study
designed to define an acceptable dose for a chronic toxlclty study {NTP
1980a). The animals 1n groups of 10 males and 10 females were administered
the compound In corn oil-acetone (9:1) twice a week at doses for rats of
0.0, 0.5, 1, 2, 4 and 8 yg/kg/week, and for mice at doses of 0.0, 1, 2, 5,
10 and 20 pg/kg/week. Deaths occurred at the two high-dose levels 1n
rats, with 4 females 1n the 8 yg/kg/week and 1 1n the 4 yg/kg/week group
dying, while only 2 male rats In the 4 yg/kg/week group died. Deaths were
accompanied by severe toxic hepatitis. Hepatic lesions were observed 1n all
other rats examined 1n groups administered 1-8 yg/kg/week; however, not
all animals 1n each group were submitted to necropsy. Normal liver his-
tology was observed In the 2 male rats examined from the low-dose groups and
only threshold toxic effects occurred 1n the low-dose female rats.
Similar effects of treatment were observed In mice, with a single death
occurring 1n each sex at the high-exposure level, along with reports of
hepatic lesions on hlstologlc examination. In contrast to rats, female mice
were less sensitive to the hepatotoxlc effect of 2,3,7,8-TCDD than were the
male mice. Hepatic lesions were observed 1n all dose groups of male mice,
while the 1 and 2 pg/kg/week dose groups of female mice had normal
livers. Although the group sizes were small, making conclusions tenuous, 1t
8-46
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appeared that sex differences 1n the sensitivity to the toxic effects of
2,3,7,8-TCDO occurred, and that the more sensitive sex may vary with species
tested.
In a more extensive subchronlc study 1n rats. King and Roesler (1974)
followed the development of toxlclty by a series of Interim sacrifices
during 28 weeks of exposure to 2,3,7,8-TCDD and a 12-week post-treatment
recovery period. Groups of 35 male and 35 female Sprague-Oawley rats were
Intubated twice weekly with 2,3,7,8-TCDD 1n corn oil-acetone (9:1) at cumu-
lative doses of 0.0, 0.1 and 1 yg/kg/week. No treatment-related deaths
"-' occurred; however, 3 animals from each group of each sex were killed after
2, 4, 8 and 16 weeks, and 10 animals of each sex were killed after 28 weeks
of treatment. In addition, 3 rats of each sex were killed 4 and 12 weeks
after termination of exposure. Animals were monitored for gross changes
during the study and were examined for gross and hlstologlc changes at
necropsy.
Besides a dose-related decrease 1n body weight gain 1n male rats and a
decrease 1n body weight gain 1n the high-dose female rats, the only effect
of exposure to 2,3,7,8-TCDD was hlstologlc changes 1n the liver. Liver
pathology was normal 1n all treated groups up through the Interim kill at 16
weeks. Fatty changes 1n the liver were considered the most Important obser-
vation. The fatty changes ranged from single large I1p1d droplets In a few
centrllobular hepatocytes to I1p1d droplets 1n all centrllobular hepatocytes
with extension Into the mldzonal hepatocytes. No clear dose-response
pattern was observed 1n this study; however, 1t did appear that the severity
of fatty changes was greater 1n male rats. During the recovery period,
fatty changes progressively decreased 1n severity but were still present 1n
some treated animals 12 weeks after cessation of exposure. Other hlstologlc
8-47
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.changes observed 1n the I1ve!r predominantly 1n the animals killed at 28
weeks Included necrosis, Increased nuclear size, subtle distortion of Hver
architecture, and hyperchromatlc nuclei. All of these lesions were consid-
ered to be slight or mild, and less tox1colog1cally relevant than the fatty
changes. The data suggested that the liver was the most sensitive organ to
the toxic effect of 2,3,7,8-TCDD, and although recovery occurred after
termination of treatment, the recovery process was slow.
The recovery time was also demonstrated to be long 1n a subchronlc study
by Goldstein et al. (1982b) of 2,3,7,8-TCDD Induced porphyrla. Groups of 8
female Sprague-Dawley rats were given 2,3,7,8-TCDD 1n corn oil-acetone (7:1) *
weekly by gavage for 16 weeks at doses of 0.0, 0.01, 0.1 or 10.0 i*g/kg/
week and killed 1 week after the last treatment. Additional groups of rats
received doses of 0.0 or 1.0 jig/kg/week for 16 weeks and were allowed to
recover for 6 months. The high-dose level was lethal to all animals within
12 weeks, while the only other gross sign of toxlclty was a decrease 1n body
weight gain 1n the group receiving 1.0 yg/kg/week. After 16 weeks of
exposure to 2,3,7,8-TCDD, liver porphyrlns were elevated ~1000-fold 1n 7 of
8 animals receiving 1.0 ^g/kg/week, but only 1 of 8 animals 1n the 0.1
vg/kg/week group had elevated porphyrln levels. No effect was observed 1n
the low-dose animals. After a 6-month recovery period the porphyrln level
1n animals exposed to 1 ^g/kg/week was still 100-fold higher than values
1n the control group. A similar pattern was observed for urinary excretion
of uroporphyrln. The rate-limiting enzyme 1n heme synthesis, 6-am1nole-
vullnlc add synthetase, was also elevated at both the time of termination
of treatment and at the end of the recovery period; however, other enzymes
that were Increased after 10 weeks of treatment, cytochrome P-4SO, AHH and
glucuronyl transferase, returned to near normal levels by 6 months. It was
8-48
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clear that a 6-month recovery period from subchronlc exposure to 2,3,7,8-
TCDD at a dose of 1.0 ^g/kg/week was not sufficient for complete reversal
of 2,3,7,8-TCDD Induced porphyrla.
In addition to the above laboratory studies, Strlk and de W1t (1980)
attempted to Investigate the toxlcologlc effect on rabbits of exposure to a
natural environment that was contaminated with 2,3,7,8-TCDD. Groups of 20
female rabbits and 1.male rabbit were housed for 5 months 1n pens, located
1n five separate areas, on soil that had been contaminated with 2,3,7,8-
TCDD. The soil had been cleaned by replacement or cultivation before
Initiation of the study. The levels of 2,3,7,8-TCDD before cleaning were
from 0.8-23.2 vg/m3; however, the levels of contamination after cleaning
were not determined. At the end of 5 months liver histology, Including the
localization of porphyrln, was examined, and the levels of cytochrome P-450
and P-420 were determined along with urinary levels of total porphyrln,
creatlnlne and D-glucar1c-ac1d. All of the parameters examined were con-
sidered to be within the normal range. Since exposure data were not avail-
able, the negative results of this study cannot be compared with the con-
trolled subchronlc laboratory studies already described.
Information on the subchronlc toxldty of HxCDD was provided 1n a pre-
liminary range-finding study for a chronic bloassay conducted by NTP (1980b)
on a 1-2 mixture of 1,2,3,6,7,8- and 1,2,3,7,8,9-HxCDD. Osborne-Mendel rats
and B6C3F1 mice 1n groups of 10 males and 10 females were administered the
HxCDD mixture 1n corn oil-acetone (9:1) by gavage twice a week for 13 weeks.
The total weekly doses given rats were 0.0, 2.5, 5, 10, 50 and 100 yg/kg;
mice received weekly doses of 0.0, 1.25, 2.5, 5, 10 and 50 yg/kg. At week
10 of the study, the body weight 1n rats was decreased In a dose-related
manner to a maximum of ~20% 1n the high-dose group. In mice, body weight
8-49
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was also decreased 10-20% 1n the treated animals; however, there appeared to
be no correlation with dose. At the end of the study the animals were
killed and necropsies were performed on selected animals. In both species
liver pathology was observed, with threshold to moderate hepatotoxldty
occurring at doses of 5 and 10 pg/kg/week for male and female rats,
respectively, and at 10 jig/kg/week for both sexes of mice. At higher
exposures, splenic hyperplasla and cortical atrophy of the thymus were also
detected 1n rats. In rats 1t was unclear whether the low-dose animals were
free of any pathologic findings or none were subjected to necropsy. In mice
1t was stated that no changes were observed In males exposed to 2,3,7,8-TCDD
at 1.25 yg/kg/week or 1n females exposed to 1,25 or 2.5 yg/kg/week.
Although the data are limited, 1t appears that the same target organs are
sensitive to the toxic effects of both 2,3,7,8-TCDD and this mixture of
HxCDD.
In addition, a second subchronlc range finding study conducted by NTP
(1980c) evaluated the dermal toxlclty of the above mixture of HxCDD. Groups
of 10 male and 10 female Swiss-Webster mice were treated by dermal applica-
tion 3 times/week for 13 weeks. The doses used were from 0.01-50 yg/
application with the test compound dissolved 1n acetone. There was 100%
mortality 1n the 25 and 50 yg/app 11 cation groups and 80% mortality 1n the
10 ng/appHcatlon group. On hlstologlc examination, there were signs of
liver damage at the lowest dose tested 1n both sexes; however, the Incidence
and degree of damage were not well correlated to the dose applied.
8.1.3. Chronic. The toxic effects, other than neoplasla, of long-term
exposure to 2,3,7,8-TCDD have been studied 1n rats and mice. The primary
purpose of many of the studies 1n rodents was to assess the carclnogenldty
of 2,3,7,8-TCDD. The observation of non-neoplast1c systemic toxic effects
8-50
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in these studies was often limited, and observations were made near the end
of the natural Hfespan when conditions associated with aging may have
obscured some effects produced by 2,3,7,8-TCDD. Long-duration toxlclty
assays were also conducted 1n monkeys. Many of the same organs 1n monkeys
as 1n rodents were adversely affected by long-term exposure to 2,3,7,8-TCDD;
however, the monkeys also developed severe skin and stomach lesions. Table
8-5 summarizes the toxic effects of chronic exposure to 2,3,7,8-TCDD and
provides Information on the exposure levels that result In the observed
effects. There also are data on the chronic toxldty of a mixture of
1,2,3,6,7,8- and 1,2,3,7,8,9-HxCDD. No Information was found 1n the litera-
ture search on the effects of chronic exposure to 1,2,3,7,8-PeCDD.
8.1.3.1. STUDIES ON LABORATORY RODENTS — In an early study, Van
MUler et al. (1977a,b) defined the dietary level of 2,3,7,8-TCDD that
adversely affected the longevity of rats following chronic exposure. Groups
of 10 male Sprague-Dawley rats were maintained for 78 weeks on diets con-
taining 1, 5, 50, 500, 1000, 5000, 50,000, 500,000 or 1,000,000 ppt of
2,3,7,8-TCDD. Survival was monitored during the study or at termination 95
weeks after Initiation of treatment. No animals survived until the end of
the study at the five highest exposure levels. The respective week after
the start of treatment 1n which the first death occurred 1n these high-dose
groups was 31, 31, 3, 2 and 2 weeks, with all animals 1n groups >50 ppb dead
by week 4. The mortality rate 1n the 0.0, 1, 5, 50 and 500 ppt groups at 95
weeks was 60, 20, 40, 40 and 50%. Although the small number of animals 1n
each group makes It Impossible to precisely define a dose-response relation-
ship. It was apparent that exposure to >1 ppb curtailed survival.
8-51
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TABLE 8-5
Effects of Chronic Exposure to 2,3,7,8-TCDD In Laboratory Rodents
GO
i
VI
ro
Species/Strain Sex/Ho.
Rat/ H/10
Sprague-Oawley
H/10
H/10
H/10
H/10
H/10
H/10
Rats/ H&F/50&50
Sprague-Oawley
Dose Treatment
Schedule
0.0 ppt NA
1 ppt continuous In
diet for 78 weeks
5 ppt continuous In
diet for 78 weeks
50 ppt continuous In
diet for 78 weeks
500 ppt continuous In
diet for 78 weeks
1000 and 5000 ppt continuous 1n
diet for 78 weeks
50,000. 500,000 and continuous In
1,000,000 ppt diet for 78 weeks
-2193 ppt continuous In
(0.1 vg/kg/day) diet for 2 years
Duration
of Study
95 weeks
95 weeks
95 weeks
95 weeks
95 weeks
95 weeks
95 weeks
2 years
Parameters
Monitored
survival
survival
survival
survival
survival
survival
survival
extensive hlsto-
pathology, hema-
tology, urine
analyses, and
clinical chemistry
Effects of Treatment
40X survived until 95
weeks, the first death
occurred at week 68
BOX survived until 95
weeks, the first death
occurred at week B6
60X survived until 95
weeks, the first death
occurred at week 33
60X survived until 95
weeks, the first death
occurred at week 69
SOX survived until 95
weeks, the first death
occurred at week 17
No animals survived until
95 weeks, the first death
occurred at week 31
No animals survived until
95 weeks, the first deaths
occurred at weeks 2 and 3
Cumulative mortality,
Increased (F);
bw gain,
decreased (H,F);
Red blood cell count,
decreased (H.F);
Packed cell volume,
decreased (H,F);
Hemoglobin,
decreased (H,F);
Retlculocytes,
Increased (H.F);
White blood cell count,
decreased (F);
Serum glutamlc pyruvlc
transamlnase. Increased (F)
G-Glutamyl transferase,
Increased (F);
Alkaline phosphatase,
Increased (F);
Reference
Van HI Her
et al., 1977a
Koclba et al
1978a, 1979
-------
inOLC O-3
Species/Strain Sex/No. Dose
Rats/
Sprague-Dawley
(cont.)
Rat/ MF/50&50 -208 ppt
Sprague-Oawley (0.01 yg/kg/day)
00
^ MF/50&SO -22 ppt
w (0.001 pg/kg/day)
Rat/ MF/75&75 0.0 wg/kg/week
Osborne -Mendel
N&F/SO&50 O.S ,,g/kg/week
MF/50&50 O.OS Vf/kg/week
Mtf/SOftSO 0.01 Vi/kg/week
N\ce/B6C3Fl MF/75&75 0.0 »g/kg/week
MF/5Qi,5Q O.S yg/kg/week (H)
2.0 ng/kg/week (F)
Treatment
Schedule
continuous In
diet for 2 years
continuous In
diet for 2 years
NA
administered by
gavage biweekly
for 104 weeks
administered by
gavage biweekly
for 104 weeks
adntnlstered by
gavage biweekly
for 104 weeks
NA
administered by
gavage biweekly
for 104 weeks
Duration Parameters
of Study Monitored
2 years extensive Msto-
pathology. heM-
tology, urine
analyses and
clinical chemistry
2 years extensive Msto-
pathology, urine
analyses and
clinical chealstry
106 weeks extensive hlsto-
pathology
10? weeks extensive hlsto-
pathology
107 weeks extensive hlsto-
pathology
107 weeks extensive hlsto-
pathology
105-106 weeks extensive hlsto-
pathology
107 weeks extensive hlsto-
pathology
Effects of Treatment Reference
Urinary coproporphyrln, Koclba et al.,
Increased (F); 1978a. 1979
Urinary uroporphyrtn,
Increased (F);
Urinary delta-aalno-
levullnlc acid,
Increased hepatic
degeneration,
Increased (N,F)
Urinary coproporphyrln, Koclba et al.,
Increased (F); 1978a, 1979
Urinary uroporphyrtn,
Increased (F);
Hepatic degeneration.
Increased (H,F)
No differences In values
obtained fro* control
antMls
Toxic hepatitis; NIP, 1980a
0/74 (H|, 0/75 (FJ
Toxic hepatitis;
14/50 (H), 32/50 (F)
Toxic hepatitis;
0/50 (N). 1/50 (F)
Toxic hepatitis;
1/50 (H), 0/50 (F)
Toxic hepatitis; NTP, 1980a
1/73 (N), 0/73 (F)
Toxic hepatitis;
44/50 (N), 34/47 (F)
-------
TABLE 8-5 (cont.J
at
Species/Strain
N1ce/B6C3F1
(cont.)
Mice/Swiss
Sex/No. Dose
M&F/5Q&50 0.05 vg/kg/week (H)
0.2 pg/kg/week (F)
mf/SO&SO 0.01 vg/kg/week (M)
0.04 Mg/kg/week (F)
H/38 0.0 wg/kg/week
M/44 0.007 vg/kg/week
H/44 0.7 ,»g/kg/»«ek
M/43 7.0 vg/kg/week
Treatment Duration
Schedule of Study
administered by 10? weeks
gavage biweekly
for 104 weeks
administered by 10? weeks
gavage biweekly
for 104 weeks
NA 588 days
administered by 649 days
gavage weekly
for 1 year
administered by 633 days
gavage weekly
for 1 year
administered by 424 days
gavage weekly
for 1 year
Parameters
Monitored
extensive nlsto-
pathology
extensive hlsto-
pathology
histology on all
organs
histology on all
organs
histology on all
organs
histology on all
organs
Effects of Treatment Reference
Toxic hepatitis; HTP. 19BOa
3/49 (M), 2/48 (F)
Toxic hepatitis;
5/44 (H). I/SO (F)
Dermatitis and Toth et al.,
amyloldosls; 0/38 1978, 1979
Dermatitis and
amyloldosls; 5/44
Dermatitis and
amyloldosls; 10/44
Early mortality.
dermatitis and
amyloldosls; 17/43
NA » Not applicable
-------
Increased mortality was also observed 1n female Sprague-Dawley rats
maintained for 2 years on a diet that provided a 2,3,7,8-TCDD dose of 0.1
pg/kg/day, while no Increased mortality was observed 1n male rats at this
dose or In animals receiving doses of 0.01 or 0.001 pg/kg/day (Kodba et
al., 1978a, 1979). The average dietary levels of 2,3,7,8-TCDD associated
with these doses were 2193, 208 and 22 ppt. Interim hematologlc, clinical
chemistry and urine analyses revealed treatment-related changes 1n a number
of parameters In the high-dose group, along with some of the same changes
occurring 1n the mid-dose group, albeit to a lesser degree (see Table 8-6).
At termination of the study, gross and hlstologlc examination Indicated that
the liver was the most severely affected organ, with degenerative, necrotlc
and Inflammatory changes observed. Increases In urinary excretion rates of
coproporphyrln and uroporphyMn 1n the high and middle dose females were
consistent with the observed liver damage. Again, primary liver Injury was
dose-related with the lowest dose representing a NOEL. Although the group
sizes (50 males and 50 females 1n the treated groups, and 85 males and 86
females In the- control groups) were reported, the description of the experi-
mental results did not enumerate the number of animals affected.
When 2,3,7,8-TCDD was administered by gavage 1n corn oil-acetone (9:1)
at dose levels of 0.0, 0.5, 0.05 or 0.01 yg/kg/week, "toxic hepatitis" was
observed respectively 1n male Osborne-Mendel rats at Incidences of 0/74,
14/50, 0/50 and 1/50, and 1n female rats at Incidences of 0/75, 32/49, 1/50
and 0/50 (NTP, 1980a). Toxic hepatitis was defined as "llpldosls (llpoldo-
sls) and hydropic degeneration of the cytoplasm of the hepatocytes" 1n the
central, mldzonal and, at times, peripheral portions of the liver. No other
8-55
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non-neoplast1c lesions were observed even though extensive hlstologlc exami-
nations were performed. The two preceding studies support a NOEL for rats
of -0.001 vg/kg/day, with a LOAEL of 0.05 pg/kg/day, and a PEL for liver
Injury and possibly decreased survival of 0.5 ng/kg/day.
Non-neoplast1c effects of chronic exposure to 2,3,7»8-TCDD In mice have
been briefly decrlbed 1n studies Investigating the carcinogenic potential of
2,3,7,8-TCDD. In an NTP (1980a) bloassay, extensive hlstologlc examinations
were performed on B6C3F1 mice treated biweekly with 2,3,7,8-TCDD by gavage
1n corn oil-acetone (9:1) for 104 weeks followed by an additional 3-week
observation period. The doses for male animals were 0.0, 0.01, 0.05 and 0.5
pg/kg/week, and for female animals, the doses were 0.0, 0.04, 0.2 and 2.0
ng/kg/week. The only non-neoplast1c lesion was toxic hepatitis, which
occurred 1n males at Incidence of 1/73, 5/49, 3/49 and 44/50, and 1n females
at Incidences of 0/73, 1/50, 2/48 and 34/47, respectively, 1n the control,
low-, medium- and high-dose groups. In a second study, weekly Intubation of
2,3,7,8-TCDD at doses of 0.0, 0.007, 0.7 or 7.0 vg/kg/week for 1 year
resulted 1n amyloldosls of the kidney, spleen and liver, and dermatitis at
the time of death 1n male Swiss mice (Toth et a!., 1978, 1979). The Inci-
dence of these lesions 1n the control, low-, medium- and high-dose groups,
respectively, was 0/38, 5/44, 10/44 and 17/43. In the high-dose group, the
amyloldosls was extensive and considered to be the cause of early mortal-
ity. The amyloldosls may have resulted from the chronic dermal Inflammation
produced by the treatment. From the limited data presented 1n these
studies, 1t appears that mice and rats were approximately equally sensitive
to the toxic effects of 2,3,7,8-TCDD following chronic exposure. Severe
toxic effects were observed at doses of 1 ng/kg/day (early mortality) and
8-56
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0.28-0.07 ufl/kg/day (toxic hepatitis), while a LOAEL for dermatitis and
amyloldosls of 0.001 jig/kg/day was reported. A NOAEL for mice was not
clearly defined by these studies.
The only Information available on the effects of chronic exposure to
HxCDD was provided by an NTP (1980c) bloassay of a 1:2 mixture of
1,2,3,6,7,8- and 1,2,3,7,8,9-HxCOD. Male and female Sprague-Dawley rats and
B6C3F1 mice were exposed biweekly to this mixture for 104 weeks and followed
for an additional 3-4 weeks before the terminal kill. Both male and female
rats and male mice received doses of 0.0, 1.25, 2.S and 5.0 yg/kg/week;
female mice received doses of 2.5, 5.0 and 10 jig/kg/week. The treated
male and female rats had a dose-related decrease 1n body weight gain during
the latter portion of the study, and the high dose females had reduced sur-
vival. No gross signs of toxldty were observed 1n mice of either sex. Al-
though extensive hlstologlc examinations were performed, the only treatment-
related effect was toxic hepatitis, which was defined as "degenerative
hepatocytlc changes and/or necrosis associated with mild flbrosls and Infil-
tration." The Incidence of this lesion 1n control-, low-, medium- and high-
dose groups, respectively, was as follows: male rats - 0/75, 28/48, 35/50
and 34/48; female rats - 0/73, 33/50, 37/50 and 44/50; male mice - 0/75,
28/50, 35/50 and 34/49; and female mice - 0/75, 33/50, 37/50 and 44/50. The
severity of the toxic hepatitis was dose-related; however, 1t 1s unclear how
severely the liver was damaged at any of the doses. In rats and mice, all
doses of this mixture of 1,2,3,6,7,8- and 1,2,3,7,8,9-HxCDD represented FELs
for liver toxldty.
8.1.3.2. STUDIES IN NONHUHAN PRIMATES — Initial studies Indicating
the effect of chronic exposure to PCDDs Including 2,3,7,8-TCDD 1n nonhuman
8-57
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primates was conducted using "toxic fat," a contaminated poultry feed addi-
tive, which resulted 1n the death of a large number of chickens (Allen and
Carstens, 1967). Groups of 4-5 monkeys, Hacaca mulatta. were fed diets con-
taining 0.0, 0.125, 0.25, 0.5, 1.0, 5.0 and 10% toxic fat until death.
There was a dose-associated shortening of survival time: monkeys 1n the
high-dose group survived only for an average of 91 days and animals 1n the
low-dose group survived an average of 445 days (data for control animals
were not provided). During the course of treatment the animals were moni-
tored for hematologlc and gross clinical changes as well as hlstologlc
changes 1n the Hver evaluated through needle biopsy samples. At death,
major organs were preserved for hlstologlc evaluation. Since both clinical
and hlstologlc changes, especially near the time of death, appeared similar
regardless of dose, the data and observations were combined for all dose
groups.
During the course of the study, the monkeys consumed less food as com-
pared with controls, and progressively lost weight. Gross clinical signs of
Intoxication during the last 60 to 30 days of life Included generalized
edema and alopecia. At necropsy, the heart was observed to be hypertrophlc
and 8 of the 27 animals treated with the "toxic fat" had small gastric
ulcers. At the light microscopic level, the liver had developed moderately
distorted architecture with vacuolated cells containing neutral fat. The
sternal bone marrow was nearly devoid of blood-forming elements, which was
consistent with the observed decrease 1n packed blood cell volume and RBC
counts. Also, electron micrographs revealed derangement of the rough .endo-
plasmlc retlculum and a loss of Mbosomes, which the authors suggested may
have resulted 1n the observed decrease 1n serum proteins. Skeletal muscle,
lungs, GI tract, skin and heart had signs of edema as observed under the
8-58
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light microscope-, the electron micrographs of the heart revealed vascular
degeneration which, If present 1n the other tissues, would have accounted
for the generalized edema. It was apparent that the active component of
"toxic fat" affected many essential biologic processes 1n the monkey.
Chemical analysis of the "toxic fat" has since shown that the fat contained
PCDDs of which TCDDs represented 64% by mass (Norback and Allen, 1973).
Allen et al. (1977) also assessed the toxldty of 2,3,7,8-TCDO Itself
Incorporated Into the diets of female rhesus monkeys. The animals were
maintained for 9 months on diets containing 500 ppt of 2,3,7,8-TCDD, and the
animals that survived treatment were observed for an additional 4 months.
During the course of the study, the monkeys were observed for clinical signs
of toxldty, monitored for hematologlc changes and, following death or the
termination of the study, were subjected to complete autopsies. Since no
control animals were Included 1n this study, the data were compared with
pre-exposure values where possible.
As observed 1n monkeys fed "toxic fat," the monkeys fed 2,3,7,8-TCDD
lost hair and developed swollen eyelids and perlorbltal edema after 3 months
of treatment. Blood parameters Including hemoglobin levels and hematocrlt
decreased; however, blood proteins (total serum protein and albumin/globulin
ratio) were not altered except 1n terminal animals. In the three animals
that survived the 9-month exposure period, the toxic symptoms continued to
develop during the 4 months of observation. The hematologlc changes
observed during the treatment period were consistent with the microscopic
findings at autopsy of bone marrow degeneration. It was suggested that
decreased platelet levels resulted 1n poor clotting and the widespread
hemorrhage observed 1n many organs, which was particularly severe 1n the
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stomach. Also, the decreased RBC count and resultant loss of oxygen-carry-
ing capacity resulted 1n an Increase 1n cardiac workload and hypertrophy of
the heart. Cellular hypertrophy, hyperplasla and metaplasia of the epithe-
lium of the salivary gland, bile duct, lung and stomach were also observed
microscopically. Although many effects of treatment were observed, 1t was
concluded that the ultimate cause of death was related to the severe pan-
cytopenla.
The total dose of 2,3,7,8-TCDD used over 9 months 1n this study by Allen
et al. (1977) was estimated to be between 2 and 3 yg/kg/day, which 1s
approximately the same dose that resulted 1n severe toxic effects following
chronic exposure 1n rats and mice. Schantz et al. (1979) reported 1n an
abstract that similar, though less severe, effects were observed 1n female
monkeys following chronic 1ngest1on of diets containing 50 ppt of 2,3,7,8-
TCDD. It was also noted that this exposure resulted 1n a decreased ability
to successfully bear young (see Allen et al., 1977, 1n Section 9). It 1s
apparent that the data available for nonhuman primates do not permit the
determination of a NOAEL.
8.2. HUMAN
8.2.1. Acute Exposure. Symptoms of acute exposure to materials that con-
tained 2,3,7,8-TCDD are nausea and vomiting, headache and signs of Irrita-
tion to the eyes, skin and respiratory tract. Acute exposure to chemicals
contaminated with 2,3,7,8-TCDD may also result 1n drenching and sweating
with extensive dehydradtlon and weight loss, Increase 1n body temperature,
severe respiratory distress, fatty degeneration of liver, cyanosis, elevated
blood urea nitrogen level, followed by fast deterioration of general condi-
tion and death from acute congestive heart failure (Regg1an1, 1982; Hay
1982). Initially a chemical burn-type cutaneous reaction will occur (pos-
sibly because of other chemicals), usually followed by chloracne after
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several days to weeks (Taylor, 1979). Chloracne Is the most characteristic
and frequently observed dermal lesion produced by 2,3,7,8-TCDD and other
chlorinated aromatic hydrocarbons 1n humans (Crow, 1981; Taylor, 1979).
This lesion consists of hyperplasla and hyperkeratosls of the InterfolUcu-
lar epidermis, hyperkeratosls of the hair follicle, especially at the
1nfund1bulum, and squamous metaplasia of the sebaceous glands that form
keratlnaceous comedones and cysts (Klmbrough, 1974). These cutaneous erup-
tions of comedones, cysts and possibly pustules 1n severe cases, usually
occur on the face and shoulders (Crow, 1978a; Passl et al., 1981). The
persistence of chloracne varies greatly, with severe cases lasting for up to
15 years, while mild cases may resolve 1n a matter of months. Similar epi-
dermal changes have been produced by 2,3,7,8- TCDD 1n rhesus monkeys
(McConnell et al., 1978a; Allen et al., 1977), the ear of the rabbit (Polger
and Schlatter, 1980), and hairless mice (Knutson and Poland, 1982). These
changes have not generally been observed 1n other laboratory animals, such
as guinea pigs, hamsters, rats and mice.
Chronic exposure to 2,3,7,8-TCDD has probably occurred most 1n chemical
Industry workers exposed to low levels of this contaminant during the manu-
facture of 2,4,5-T on a dally basis. Chloracne 1s generally the first
symptom noted 1n chronic exposure. Systemic symptoms, Including altered
function of the neuromuscular system, liver, kidneys, and pancreas, altered
blood chemistry (serum b1!1rub1n, GOT, GPT, llpld and cholesterol levels),
porphyrla cutanea tarda, hyperplgmentatlon and hyperkeratosls, have also
been reported 1n Individuals that have had chronic 2,3,7,8-TCDD exposure
(Crow, 1978b, 1981). A combination of acute, high-level exposure to
2,3,7,8-TCDD followed by chronic exposure for many years (or a lifetime) has
been noted for residents of areas where PCDDs have been accidentally
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released Into the environment (Taylor, 1979). Residents of Seveso, Italy,
for example, where an explosion of a reactor vessel used to manufacture
2,4,5-T released PCDDs and other chemicals Into the atmosphere, were exposed
acutely for a few days and are now exposed dally to diminishing levels of
PCDDs 1n the soil.
The first cases of chloracne associated with exposure to PCDDs occurred
after a 1949 explosion In a chemical factory producing 2,4,5-T 1n NHro, WV
(Holmstedt, 1980). A total of 228 workers were exposed. Symptoms Included
nausea, headaches, fatigue, muscular aches and pains, and chloracne (Zack
and Susklnd, 1980). Chemical tests revealed elevated llpld levels and
prolonged prothrombln time. Chronic symptoms, lasting up to 2 years, were
severe aches and pains, fatigue, peripheral neuropathy and some residual
cmoracne. Four additional Industrial explosions were reviewed by Holmstedt
(1980). In 1953, 15 workers were exposed during an accident at a factory
(BASF) 1n Ludwlgshafen, Germany. Most of the workers developed chloracne,
while 21 workers developed nervous system and Internal organ damage 1n
addition to severe chloracne. In 1963, an explosion at a 2,4,5-T producing
factory 1n Amsterdam resulted 1n the exposure of 106 men to chlorinated
dloxln by-products. Chloracne was the most common symptom, occurring 4-6
weeks after exposure. As a result of a similar exothermic explosion at the
Coalite and Chemicals plant (England) In 1968, which manufactured 2,4,5-tr1-
chlorophenol, at least 90 workers were exposed to dloxlns. Clinical exami-
nations, Including Hver function tests, full blood counts and uMnalysIs,
were conducted on 14 employees who were 1n the building at the time of the
explosion (May, 1973; Hay, 1982). Eleven of these 14 men showed abnormal
liver function (zinc turbidity, thymol turbidity and serum transamlnase) and
altered hematologlcal parameters or glucosurla. Later, after normal plant
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operations were resumed, additional workers apparently were exposed to
2,3,7,8-TCDD by contact and developed chloracne. Seventy-nine cases of
chloracne developed by the end of 1968. The condition appeared on the face
1n all cases; however, other parts of the body were affected 1n more severe
cases (May, 1973).
The most recent and extensively studied chemical plant explosion
occurred on July 10, 1976, at the ICMESA (Industrie Ch1m1che-Meda-Soc1eta
Az1onar1a) plant at Seveso, Italy. This accident, caused by the release of
the reactor contents Into the atmosphere, exposed workers and residents
(>8655 people) of the area to 2,3,7,8-TCDD 2,4,5-tMchlorophenol (6aratt1n1,
1982; PocchlaM et a!., 1983). A total of 447 patients developed chloracne
and some complained of nausea, vomiting, headache, diarrhea, hyperhldrosls
and Irritation of the eyes (Taylor, 1979). Serious cases of chloracne and
dermal blistering occurred 1n children and appeared within several weeks of
their exposure (G1anott1, 1977; Crow, 1981; Taylor, 1979). Pocch1ar1 et al.
(1979) cited unpublished data reported to the Lombardy Regional Authority
(Boerl, 1978; Ch1app1no et al., 1978; S1rch1a, 1978) on the health effects
of 2,3,7,8-TCDO to children and adults at Seveso. Reduced peripheral nerve
conduction velocities were noted 1n adults and children, with abnormalities
being more frequent In people residing nearer the chemical plant. The
Immunology of a group (n=45) of exposed children was compared with a similar
unexposed group. No significant differences were noted; however, total
serum complement activity, lymphocyte blastogenlc response and peripheral
blood lymphocytes were elevated to some degree 1n the exposed children
(Tognonl and Bonaccorsl, 1982). Exposure to 2,3,7,8-TCDD has been associ-
ated with Irtcreased serum glutamate-oxalacetate transamlnase (GOT), serum
GPT and gamma-glutamyl transferase (g-GT) levels 1n exposed children
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(Pocch1ar1 et al., 1979). Compared with normal values for "healthy"
Individuals, lympho- cyte aberrations appeared more frequently; however, the
findings were not statistically significant.
A comparison between children (under age 15) who developed chloracne and
children of the same area who did not develop skin lesions was reported by
/
Caramaschl et al. (1981). A significant Increase 1n the frequency of head-
aches and eye Irritation (p=0.01), GI tract symptoms (nausea, vomiting, loss
of appetite, abdominal pain or gastritis) (p=l.6x10"*), and abnormal g-GT,
serum GPT and am1nolevu!1n1c add levels (p=2.3x!0~«, 0.035 and
1.2xlO~5, respectively) was noted 1n those children who had chloracne
(Caramaschl et al., 1981). Ideo et al. (1982) measured urinary D-glucar1c
add levels to assess liver mlcrosomal enzyme activity 1n 67 children
exposed to 2,3,7,8-TCDD at Seveso. A significant (p<0.05) Increase 1n the
glucarlc add levels, used to Indicate Increased mlcrosomal enzyme activity,
was noted 1n exposed children 3 years after the accident when compared with
unexposed children (n=86).
The decontamination and cleanup of the ICHESA plant at Seveso began 1n
Hay, 1980, and the possible contamination of clean-up workers was closely
monitored and safety measures were Implemented (Ghezzl et al., 1982).
_/
Laboratory tests on the blood (GOT, GPT, g-GT, alkaline phosphatase, bHI-
rubln, hemoglobin, cell counts, thromboplastlc partial time, albumin, gamma
globulin, cholesterol and trlglycerldes) and urine (porphyrln) of the
workers were performed and compared with pre-employment values {of the same
group of workers) and with a nonexposed group. No significant changes were
noted, but exposure to 2,3,7,8-TCDD was believed to be minimal. A recent
review of the Seveso Incident, Including Us history and human health
effects, Is reported by Tognonl and Bonaccorsl (1982).
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Three cases of accidental exposure to PCDDs (Isomer not specified) while
scientists were attempting to prepare a pure standard 1n the laboratory were
reported by Oliver (1975). All three laboratory scientists reported the
same general symptoms: chloracne (within several weeks after exposure), GI
pains, headaches, fat1gabH1ty and hypercholesterolemla (occurring 2-3 years
after exposure). One case reported loss of mental and muscular coordination
and blurred vision. Most symptoms of the patients subsided with time.
Since PCBs and PBBs can cause neurotoxlc and behavioral effects (Safe,
1984; Agarwal et al., 1981; Anderson et al., 1978) and their toxic effects
may be mediated by the same cytosollc receptor protein as 2,3,7,8-TCDD, 1t
may be Important to determine whether 2,3,7,8-TCDD has any neurotoxlc
activities (Sllbergeld, 1984; Safe, 1985).
Additional reports of toxic effects as a result of acute 2,3,7,8-TCDD
exposure 1n humans were noted by Klmbrough et al. (1977). Children were
exposed to soil 1n horse arenas (1n Eastern Missouri) sprayed with oil con-
taminated with 2,4,5-tMchlorophenol (5000 ppm 1n the soil) and 2,3,7,8-TCDD
(30 ppm 1n the soil). A 6-year-old girl developed headaches, diarrhea,
eplstaxls and hemorrhaglc cystitis, and became lethargic. Two 3-year-old
boys developed chloracne -1.5 months after playing 1n a contaminated horse
arena. Three additional Individuals who had exposure to the arenas devel-
oped less severe symptoms of headache, skin lesions and polyarthralgla. The
girl was re-examined 5.3 years following exposure to the soil of the horse
arena and showed no residual signs of toxldty (Beale et al., 1977). Addi-
tional data on these or other cases from Eastern Missouri were not available.
8.2.2. Chronic Studies. Poland et al. (1971) reported a health survey
study of 73 men employed 1n the manufacture of 2,4,5-T. These workers, how-
ever, were also exposed to d1- and trlchlorophenols, PCDD contaminants and
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2,4-D. Thirteen employees developed moderate to severe chloracne; another
35 had minimal "active acne" (cysts, comedones or pustules). Other com-
plaints noted by the workers were eye Irritation, hyperplgmentatlon and
hlrsutlsm. Gastrointestinal symptoms (nausea, vomiting, diarrhea, abdominal
pain or blood In the feces) were reported by 22 of the 73 workers. Findings
as to cardiovascular, hepatic, pulmonary and neurological function were
regarded as unremarkable and unrelated to occupational exposures. The
authors noted that exposure was to several compounds and to assign a causa-
tive agent(s) would be conjecture (Poland et a!., 1971).
In a brief report, Walker and Martin (1979) reported on some of the
clinical findings of eight men who had contracted chloracne as a result of
occupational exposure to 2,3,7,8-TCDD. Five men had elevated g-ST and tr1-
glycerlde levels. All eight men had decreased levels of high-density Upo-
proteln (HDL) cholesterol and elevated total/HDL cholesterol ratios con-
sistent with higher than average risk of 1schaem1c vascular disease. Abnor-
mal I1p1d levels, reported 1n 6 men, were attributed to enzyme Induction.
May (1982), however, observed no differences 1n trlglycerlde, cholesterol,
alkaline phosphatase D glucarlc add or g-GT levels 1n 41 workers exposed to
2,3,7,8-TCDD. These determinations were made 10 years after the workers had
developed 2,3,7,8-TCDD chloracne.
Blelberg et al. (1964) found 29 workers In a chemical plant manufactur-
ing 2,4-chlorophenol and 2,4,5-trlchlorophenol exhibiting features of chlor-
acne. These patients were tested for the presence of porphyrla cutanea
tarda (PCT). This Investigation revealed evidence of varying degrees* of
severity of PCT In 11/29 workers, but the authors could not determine any
quantitative relationship between the chloracne and PCT. Urinary uropor-
phyrlns were elevated 1n all 11 cases. A number of workers were noted to
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have fiyperplgmentatlon, h1rsut1sm» fragility of the skin and veslculobulbous
eruptions on exposed areas of the skin. In this paper the authors suggested
PCT 1s perhaps an acquired disease occurring after various Insults to the
Hver (Blelberg et al., 1964).
In a survey of 204 employees engaged 1n the manufacture of 2,4,5-T for 1
month to 10 years, Ott et al. (1980) reported no cases of chloracne, por-
phyrla cutanea tarda or other effects Indicative of dloxln exposure. Maxi-
mum allowable 2,3,7,8-TCDD levels 1n the final product were <1 mg/kg 1n 1966
and <0.1 mg/kg 1n 1972. Estimates of TWA exposure to 2,4,5-T ranged from
0.2-0.8 mg/m3, so that 2,3,7,8-TCDD levels would be exceedingly low. Cook
et al. (1980) reported chloracne, from slight to severe cases, 1n 49 of 61
employees exposed to 2,3,7,8-TCDD during the manufacture of trlchlorophenol.
Changes 1n Industrial and personal hygiene techniques decreased potential
exposure to 2,3,7,8-TCDD and subsequent chloracne. Additional toxic effects
were not reported. The National Institute for Occupational Safety and
Health (NIOSH) In a survey of workers at a St. Louis, MO, trucking terminal
contaminated with 2,3,7,8-TCDD (subsoil concentration of 2,3,7,8-TCDD was as
high as 17 ppb) found one of the long-term former workers had developed
porphyrla cutanea tarda and anglosarcoma of the right 1l1um (Hope et al.,
1984). Pazderova-Vejlupkova et al. (1981) reported that 80 workers
developed chloracne, nausea, fatigue and weakness In the lower extremities
while engaged 1n the production of 2,4,5-sodlum trlchlorophenoxyacetate and
trlchlorophenoxyacetate butylester. Prominent clinical symptoms among 55 of
the 80 workers Included hypercholesterolemla, hyperHpemla and hyperphospho-
Upemla, Increased plasma alpha and gamma globulins, and decreased plasma
albumin. Porphyrla cutanea tarda was observed 1n 11 of the 55 workers
tested. In some cases Illness subsided, while other cases became more
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severe during a 3-4 year follow-up period. Long-term pathological symptoms
(remaining evident 5 years after exposure) Include deviations 1n I1p1d
metabolism, abnormal glucose tolerance and high urinary excretion of uropor-
phyrlns (Pazderova-Vejlupkova et a!., 1981). Polyneuropathy, usually of the
lower extremities, occurred during the period of Illness and remained evi-
dent after 4 years. Singer et al. (1982) also Indicated a decrease 1n nerve
conduction velocities of sural nerves 1n workers exposed to phenoxy add
herbicides (average exposure, 7 years) when compared with a similar group of
nonexposed workers (40.3 m/sec 1n exposed vs. 42.8 m/sec 1n nonexposed,
p=0.02). Although the causative agent 1s not known, PCDD contaminants are
suggested.
The toxic effects attributed to 2,3,7,8-TCDO exposure were studied over
a 10-month period 1n a group of 78 Vietnam veterans who claimed to have been
exposed to Agent Orange (Bogen, 1979). Symptoms reported by the veterans
Included gastrointestinal complaints (anorexia, nausea, diarrhea, constipa-
tion, abdominal pain), Joint pain and stiffness, and neurological complaints
(numbness, dizziness, headaches, depression and bouts of violent rage).
These patients had previously been chronically 111 and had frequent Infec-
tions and allergies (Bogen, 1979). This study was apparently based on per-
sonal evaluations of health 1n a survey-type format. No control group was
used for comparison and no clinical or medical evaluations of health were
made. Host of these complaints are nonspecific, judgmental and occur com-
monly In the general public.
In an effort to evaluate the toxic effects attributed to 2,3,7,8-TCDD as
a contaminant of Agent Orange, Stevens (1981) estimated a minimum toxic dose
of 2,3,7,8-TCDD and determined the amount of this contaminant to which
veterans may have been exposed during Agent Orange spraying. Based on
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studies 1r> which rhesus monkeys were fed small amounts of dietary 2,3,7,8-
TCDD and analogy with human data on the minimum toxic dose of 2,3,7,8-tetra-
chlorod1benzo-p_-furan (TCDF), the cumulative minimum toxic dose of 2,3,7,8-
TCDD 1n man was estimated to be 0.1 yg/kg (Stevens, 1981). Based on
application rates (4,1 g Agent Qrange/m2} and 2,3,7,8-TCDO concentration
In the herbicide (2 ppm), the average concentration of 2,3,7,8-TCDO on
sprayed surfaces of Vietnam was estimated to be ~8 vg/m2. Based on
accidental exposures to 2,3,7,8-TCDO 1n humans (Industrial accidents,
Eastern Missouri cases), Stevens (1981) estimated an average Intake transfer
factor (ratio of absorbed compound to environmentally available compound) of
1-.20SO for 2,3,7,8-TCOD. Assuming this absorption-to-exposure ratio and
even assuming that a soldier was directly sprayed (exposed to 8 pg/m2)
for each day of his 1-year service 1n Vietnam, his cumulative Intake would
be only 1.4 wg or 0.02 vg/kg of 2,3,7,8-TCDD (Stevens, 1981). Based on
these calculations and assumptions, Stevens (1981) reported that 5 years of
direct dally contact with Agent Orange would be necessary to reach a toxic
level of 2,3,7,8-TCDD and felt that claims of Illness caused by 2,3,7,8-TCDD
1n Agent Orange were without merit. Exception 1s made, however, for certain
workers (forest Industries) who may have been exposed to 2,4,5-T and
2,3,7,8-TCDD for many years.
8.3. MECHANISM OF TOXICITY
A number of studies have attempted to determine the mechanism of toxlc-
1ty of 2,3,7,8-TCDD. The ultimate purpose 1s to provide a better estimate
of man's relative sensitivity to 2,3,7,8-TCDD and other compounds having a
similar mode of action. Specifically, these studies may be able to explain
the reason for the marked Interspecles differences 1n 2,3,7,8-TCDD toxldty
and, thus, help determine 1f humans possess factors that are associated with
sensitivity to 2,3,7,8-TCOD toxldty. i
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8.3.1. Receptor-Mediated Toxldty. Pharraacogenetlc studies have played
an Important role 1n understanding the biologic and toxic effects of drugs
and xenoblotlcs. Nebert and coworkers have shown that carcinogenic poly-
cycllc aromatic hydrocarbons (PAHs) Induce the cytochrome P-450-dependent
monooxygenase AHH In certain responsive strains of mice (e.g., C57B1/6J,
BALBc, CSHF/He), whereas this PAH Induction activity 1s minimal or nonexis-
tent 1n nonresponslve strains (DBA/2J) (Nebert, 1979, 1982; Nebert and
61elen, 1972; Nebert and Jensen, 1979; Nebert et a!., 1972, 1981, 1983).
The gene complex responsible for the Induction of AHH and several other
enzymes has been designated the Ah locus that comprises regulatory, struc-
tural and possible temporal genes. Extensive studies on genetically Inbred
responsive and nonresponslve mice (and their backcrosses) Indicate that
these differences are related to the Aromatic Hydrocarbons (Ah) regulatory
gene (termed "Ah complex" or "AH cluster") and Its gene product, the Ah
cytosollc receptor protein. This receptor protein Interacts with PAH
Ugands and the resultant PAH:Ah receptor complex translocates Into the
nucleus and presumably Initiates the Induction of AHH by a process compar-
able to that proposed for the steroid hormones.
Since the carcinogenic and toxic effects of PAHs are dependent on their
oxldatlve metabolism to reactive electrophlllc forms, It 1s not surprising
that the Ah receptor plays an Important role 1n mediating their toxlclty and
carclnogenlclty (Kourl, 1976; Kourl et a!., 1974; Benedict et a!., 1973;
Shum et a!., 1979; Thomas et a!., 1973; Legraverend et a!,, 1980; Duran-
Reynolds et a!., 1978; Robinson et al., 1975; Mattlson and Thorgelrsson,
1979). Responsive mice are more susceptible to the toxic (Inflammation,
fetotoxlclty, primordial oocyte depletion) and carcinogenic effects of PAH
at organs/tissues 1n direct contact with the applied chemical; In contrast,
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nonresponslve mice are more susceptible to the tumorlgenlc effects of PAHs
at tissue/organ sites remote from the Initial site of exposure to the PAHs.
These differences 1n susceptibility are due to several factors Including
AHH-med1ated toxlcatlon and detoxlcatlon.
2,3,7,8-TCDD can produce dermal lesions Including epidermal hyperplasla,
hyperkeratosls and squamous metaplasia of the sebaceous glands 1n hairless
mice (HRS/J), homozygous for hr/hr locus, but not 1n heterozygous (hrA) or
normal haired wild type {+/+) mice. These effects on the skin seem to be
mediated through the Ah receptor (Poland, 1984).
8.3.1.1. 2,3.7,8-TCDD: SEGRESATION OF ACTIVITY WITH THE Ah LOCUS —
Genetic studies also support the role of the Ah receptor 1n mediating the
toxic and biologic effects of 2,3,7,8-TCDD. Initial studies by Poland and
coworkers (Poland et al., 1974, 1983; Poland and Glover, 1975; Nebert et
a!., 1975} demonstrated that the mlcrosomal AHH-lndudng activity of
2,3,7,8-TCDD and 3-MC 1n several genetically Inbred mice strains were
similar. Like HC and related PAHs, 2,3,7,8-TCDD Induced AHH 1n several
responsive mouse strains (I.e., C5781/6J). In contrast to 3-MC, 2,3,7,8-
TCDD Induced mlcrosomal AHH 1n the DBA/2J nonresponslve mice; however, the
ED50 for this biologic response was significantly higher than values
reported for the responsive mice. In genetic crosses between responsive
C57B1/6 and nonresponslve DBA/2 mice 1t was also shown for both 3-MC and
2,3,7,8-TCDD that the trait of responsiveness 1s Inherited 1n a simple
autosomal dominant mode (Poland and Knutson, 1982). It has been suggested
that the observed differences 1n the activities of 3-MC and 2,3,7,8-TCDD are
related to their relative Ah receptor affinities (Poland and Knutson, 1982}
and the pharmacoklnetlc and metabolic factors that would more rapidly
diminish the "available" concentrations of 3-MC caused by metabolism and
excretion.
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Several studies with 2,3,7,8-TCDD 1n genetically Inbred mice support the
receptor mediated hypothesis. The Induction of UDP-glucuranosyl transfer-
ase, DT dlaphorase, 5-am1nolevul1n1c add, glutath1one-S-transferase B,
T-aldehyde dehydrogenase and chollne klnase by 2,3,7,8-TCDD or 3-MC In
genetically Inbred mice have also been shown to segregate with the Ah locus
{Beatty and Neal, 1976b; Owens, 1977; Klrsch et al., 1975; Dietrich et al.,
1977; Ishldate et al., 1980; Poland and Glover, 1973a). Toxicology studies
with genetically-Inbred mice confirm the role of the Ah locus 1n mediating
several toxic effects Including porphyrla, 1mmunotox1c1ty a wasting syn-
drome, thymlc atrophy and cleft palate formation (Jones and Sweeney, 1980;
Poland and Glover, 1980; Courtney and Moore, 1971; Vecchl et al., 1980,
1983). Poland et al. (1982) also linked the tumor-promoting activity of
2,3,7,8-TCDD 1n hairless mice to the cytosollc receptor. In vitro studies
with XB cells 1n culture also support the role of receptor 1n mediating a
dose-related cell kerat1n1zat1on by 2,3,7,8-TCDD that resembles some of the
characteristics of chloracne (Knutson and Poland, 1980). This cell line Is
also responsive to AHH Induction and contains a cytosollc receptor binding
protein. Although the murlne Ah receptor has not been characterized,
several studies confirm that a protein with high affinity for 3-MC and
2,3,7,8-TCDD 1s present 1n low concentrations 1n the hepatic (-30-50 fmolar)
and extrahepatlc tissues of responsive C57B1/60 mice (Greenlee and Poland,
1979; Okey et al., 1979, 1980; Poland et al., 1976; Mason and Okey, 1982;
6as1ew1cz and Neal, 1982; Okey and Vella, 1982; Okey, 1983; Nebert et al.,
1983). In responsive C57B1/6J mice and Sprague-Dawley rats, but not 1n
nonresponslve DBA/2J mice, the Ah receptor can be Induced by pretreatment
with phenobarbHal, which 1s the only known agent at present that has been
demonstrated to affect tissue concentrations of the receptor (Okey and
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Vella, 1984). Although the Ah receptor has not been detected 1n the cytosol
of DBA/2J mice, after the administration of radlolabeled 2,3,7,8-TCDD to
these mice, some of the radlolabel 1s detected In the nuclei of the non-
responsive mice. Moreover, the sedimentation characteristics of the
[3H]-2,3,7,8-TCDD:nuclear protein complex In DBA/2J mice are similar to
those observed with the bound Ah cytosollc receptor protein 1n C57B1/6J mice
using a sucrose density gradient centrlfugatlon separation technique (Okey,
1983). The cytosollc Ah receptor protein migrates Into the nucleus of the
cell only after binding with 2,3,7,8-TCDD (Nebert and Jensen, 1979; Nebert,
1980; Greenlee and Poland, 1979; Okey et al., 1979, 1980; Tukey et a!.,
1982; Gonzalez et al., 1984), and this parallels the observations noted for
the Interactions between steroids and their receptor proteins. The 2,3,7,8-
TCDD 1nducer-Ah receptor complex undergoes a temperature-dependent step
before gaining high affinity for DNA (Okey et al., 1980; Klmura et al,,
1984). The 2,3,7,8-TCDD Ah-receptor complex thus binds to the nucleus and
regulates the transcription of cytochrome P,-450, which represents the
gene product of Ah-structural loci, 1n mouse hepatoma cells 1n culture
(WhHlock et al., 1984; Elsen, 1984) and 1n mice with various Ah genotypes
(E1sen, 1984). This results 1n Induction of AHH activity which may remain
elevated for a prolonged period. Such prolongation of activity may be
because cytochrome P,-45Q mRNA remains elevated even after 1 week follow-
ing single exposure to 2,3,7,8-TCDD (Elsen, 1984).
In elucidating the mechanisms of 2,3,7,8-TCDD Induced teratogenlc effect
1n the formation of cleft palate 1n C57 mouse fetus, the presence of
Ah-receptor In the palatal shelves of the embryo seems to be necessary for
alteration/Inhibition of terminal differentiation of the medial epithelial
cells In the palate (Denker and Pratt, 1981; Pratt, 1983; Pratt et al..
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1984a,b). Pratt and Willis (1985) have even suggested utilizing growth
Inhibition of an established line of human embryonic palatal mesenchymal
cells for Ijn vitro short-term screening for assessment of the teratogenlc
potential of environmental agents.
The presence of Ah-receptor have been detected 1n normal lung, liver,
kidney, spleen and Intestine from human fetus. In addition, normal lung
tissue from 10 of the 50 Individuals examined were found to have Ah-receptor
(Roberts et a!., 1985). Ah-receptor has also been observed 1n cell lines of
human squamous cell carcinoma at a concentration of 5-10 fmol/mg (Hudson et
a!., 1983; Roberts et al.f 1985). Whether variation 1n Ah-receptor content
1n human 1s genetically determined and Is a critical determinant of Individ-
ual susceptibility to PCDDs 1s not known and warrants further Investigation.
8.3,1.2. 2,3,7,8-TCDD AND RELATED TOXIC HALOGENATID ARYL HYDROCARBONS:
STRUCTURE-ACTIVITY CORRELATIONS — The evidence for a receptor mediated
mechanism of action for 2,3,7,8-TCDD 1s supported by data reported for the
effects of other halogenated aryl hydrocarbons 1n genetically Inbred mice
i
and other diverse animal species. A number of reviews and comparative
studies (Allen et a!., 1979; Klmbrough, 1974; Klmbrough et a!., 1978;
HcConnell and Moore, 1979; Taylor, 1979) clearly Indicate that the toxic
halogenated mixtures and Individual compounds (Including the PCDDs, PCDFs,
PCBs and PBBs) elicit similar toxic and biologic responses that Include 1) a
wasting syndrome which 1s manifested by a progressive weight loss and
decreased food consumption by the treated animals; 2) skin disorders Includ-
ing acneform eruptions or chloracne, alopecia, edema, hyperkeratosls, and
hypertrophy of the Nelbomlan glands; 3) lymphold Involution and atrophy;
4) porphyMa (resembling porphyrla cutanea tarda); 5) endocrine and repro-
ductive disorders; 6) modulation of chemical cardnogenesls; and 7) the
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induction of numerous enzymes Including the cytochrome P-448 (or P-450c)
dependent monooxygenases. It 1s apparent that the effects of these com-
pounds are not manifested 1n all the animal species tested. McDonnell and
Moore (1979) summarized the pathologic findings observed 1n several animal
species after pretreatment with PCDDs, PCDFs, PCBs and PBBs; these data
Illustrate the different species and organ/tissue susceptibilities to these
compounds. It 1s also evident that for most of these effects, all the toxic
halogenated aromatlcs elicit similar effects 1n these species that also
contain the cytosollc receptor protein (Carlstedt-Duke, 1979; Carlstedt-Duke
et al., 1979, 1981; Okey, 1983; Okey and Vella, 1982; Mason and Okey,
1982). These observations support a common mechanism of action for all the
toxic halogenated aryl hydrocarbons (Poland and Knutson, 1982; Safe et al.,
1982; McConnell and Moore, 1979).
Several reports have demonstrated the effects of structure on the activ-
ity of PCDDs. The most active member of this group is substituted 1n the
lateral 2, 3, 7 and 8 positions; activity 1s decreased with 1) decreasing
lateral substHuents, and 2) increasing Cl substitution. Moreover, for
several PCDDs, there 1s an excellent correlation between the toxlcity of
Individual PCDD congeners 1n guinea pigs and mice (McConnell et al., 1978b)
and their AHH Induction potencies 1n chick embryos and rat hepatoma H-4-II-E
cells 1n culture and their binding affinities for the C57B1/6J mouse hepatic
cytosollc receptor protein (Poland et al., 1976, 1979; Bradlaw et al., 1980;
Bradlaw and Casterllne, 1979). Comparable structure-activity correlations
have been reported for the PCDFs 1n which the most active compound, 2,3,7,8-
TCDF, 1s an approximate Isostereomer of 2,3,7,8-TCDD (Poland et al., 1979;
Poland and Knutson, 1982). Moreover, like the PCDDs, there was an excellent
correlation among the toxldty of several Individual PCDFs (Yoshlhara et
8-75
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al., 1981), their AHH Induction potencies 1n rat H-4-II-E hepatoma cells and
binding affinities to male Wlstar rat hepatic cytosollc receptor protein
(Bandlera et al., 1983).
Correlations between structure-activities of PCDDs and Ah-receptor site
binding, AHH Induction potencies and systemic toxldty have also been sug-
gested (Safe et al., 1984). 2,3,7,8-TCOD, the Homer substituted with Cl 1n
all four lateral positions 1s most active for all of the above three param-
eters. Increased or decreased substitution of 2,3,7,8-substltuted PCDDs
tend to decrease receptor binding affinity and toxic action.
The most active PCB congeners, 3,4,4' ,5-tetra-, 3,3' ,4,4'-tetra-,
3,3' ,4,4' ,5-penta- and 3,3' ,4,4' ,5,5'-hexachlorob1phenyl, are substituted at
both para and at two or more meta positions. The four coplanar PCBs Induce
rat hepatic mlcrosomal AHH and cytochromes P-450a, P-450c and P-450d and
resemble 3-HC and 2,3,7,8-TCDD 1n their mode of Induction of the cytochrome
P-450 Isozymes (34) (Parkinson et al., 1980a,b, 1983; Safe et al., 1982;
Sawyer and Safe, 1982; Poland and Glover, 1980; Goldstein et al., 1977).
Like Aroclor 1254, all the monoortho and at least eight dlorthg-chloro
analogs of the coplanar PCBs exhibited a "mixed-type" Induction pattern and
Induced mlcrosomal AHH, DHAP N-demethylase and cytochromes P-450a to P-450e
(Parkinson et al., 1983, 1980a,c). Quantitative structure-activity rela-
tionships (QSARs) within this series of PCBs were determined by comparing
their AHH Induction potencies (EC) In rat hepatoma H-4-II-E cells and
their binding affinities (£0™) for the 2,3,7,8-TCDD rat cytosollc recep-
tor protein (Sawyer and Safe, 1982; Bandlera et al., 1983). The results
showed that there was an excellent correlation between AHH Induction
potencies and receptor binding avidities of these compounds and the order or
activity was coplanar PCBs (3,3' ,4,4'-tetra-, 3,3' ,4,4' ,5-penta- and
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S,S' ,4,4' ,5,5'-hexachloroblphenyls) > 3,4,4',5-tetrachloroblphenyl > mono-
ortho coplanar PCBs > dlortho coplanar PCBs. It was also apparent that the
relative toxlcltles of this group of PCBs paralleled their biological
potencies {Blocca et al., 1981; Yoshlhara et a!., 1979; Marks et al.» 1981;
McKlnney et al., 1976; Yamamoto et al., 1976; Ax and Hansen, 1975; Kurokl
and Hasuda, 1977).
The coplanar and monoortho coplanar PCBs also exhibit differential
effects 1n the Inbred C57B1/6J and DBA/23 mice. These compounds Induce AHH
and cause thymlc atrophy 1n the former "responsive" mice whereas at compar-
able or higher dose* none of these effects are observed 1n the nonresponslve
DBA/2J mice (Parkinson et al., 1982). The results obtained for structurally
diverse PCDDs, PCBs and PCDFs clearly support the role of the receptor pro-
tein 1n Initiating the broad spectrum of biologic and toxic effects elicited
by these chemicals. Bandlera et al. (1983) demonstrated that the 2,3,7,8-
TCDD receptor protein 1s not only susceptible to halogen substitution
patterns but also the structure of the substituent. The cytosol receptor
binding avidities and AHH Induction potencies 1n rat hepatoma H-4-II-E cells
for several 4'-X-2,3,4,5-tetrachlorob1phenyls were remarkably dependent on
the structure of the X substltuent. The binding data for 13 different sub-
stltuents was subjected to multlparameter regression analysis to correlate
binding avidities with the physical and chemical characteristics of the
critical lateral X substltuents. The equation
log (1) m 1.530 + 1.47 1 + 1.09 HB + 4.08
?C50
showed that Ugand binding was dependent on substltuent electronegatWHy
(cr), I1poph1l1c1ty (1) and hydrogen binding (HB) with a correlation
coefficient (r) equal to 0.978 for 13 different substltuents.
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Dependency of llgand-receptor complex and the biological activity of
PCDDs on their electronic and geometric structure Investigated by an ^
vitro molecular fragment analysis has also been suggested (Cheney, 1982).
The receptor mediated hypothesis for the mechanism of action of 2,3,7,8-
TCDD stm requires further confirmation and numerous problems must be
clarified. For example:
1. Several cell culture lines that appear to have the Ah receptor
are highly resistant to the toxldty of TCDD; the nonrespon-
slve HTC and responsive H-4-II-E cell lines (I.e., for AHH
IndudblHty by TCDD) do not possess cytosollc receptor; how-
ever, the nonresponslve HTC cells possess more nuclear recep-
tor binding protein than the responsive H-4-II-E cells (Okey,
1983; Okey et al., 1980).
2. Hepatic cytosollc receptor levels 1n rats (Wlstar and Sprague-
Dawley), C57B1/6J mice, hamsters and guinea pigs are compar-
able (Gas1ew1cz et al., 1983b); however, their susceptibility
to the biologic and toxic effects of TCDD are highly variable:
guinea pigs are highly susceptible to the lethal effects of
TCDD (LDso = 1-2 tig/kg) whereas the susceptibility of the
other species follows the order rat > G57B1/63 mice > DBA/2J
mice > hamster (Neal et al., 1982).
3. "Responsiveness" of the mouse to 2,3,7,8-TCDD Induced toxldty
seems to be highly dependent on the genetic conditions, as
regards the Ahb allele gene, of the animal. However, cell
lines "nonresponslve" to P]-450 Induction by 2,3,7,8-TCDD
have also been found to possess Ah-receptor protein (Guenthner
and Nebert, 1977).
Ah receptor protein 1s also present 1n human tissue (Roberts
et al., 1985). Whether variation 1n Ah-locus Is critical for
Individual susceptibility to toxldty by PCDDs remains to be
demonstrated 1n human population.
8.3.2. Metabolism. The metabolism of 2,3,7,8-TCDD has been examined 1n
the guinea pig, rat, mouse and hamster. Urine and bile from 14C-TCDD-
treated animals were found to be free of unmetabollzed 2,3,7,8-TCDD, demon-
strating that metabolism was required for elimination through these routes
(Olson et al., 1983). The direct Intestinal elimination of unchanged
2,3,7,8-TCDD 1n feces suggests, however, that some routes of excretion may
not be dependent on prior metabolism of the toxin (Olson et al., 1983).
8-78
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Thus, 1t 1s not possible to directly correlate the half-life for elimination
of 2,3,7,8-TCDD with Its in vivo rate of metabolism 1n a given species, The
relative persistence of 2,3,7,8-TCDD 1n a given species may be related to
the la vivo rate of 2,3,7,8-TCDD metabolism, excretion of the toxin not
dependent upon metabolism (direct Intestinal elimination, lactation, sebum),
and the relative tissue distribution of 2,3,7,8-TCOD, particularly to
adipose stores. Qualitative and quantitative differences 1n the metabolism
and disposition of 2,3,7,8-TCDD have been observed between various species,
and these may 1n part be related to the remarkable Interspedes differences
1n sensitivity to 2,3,7,8-TCDD toxlclty (Olson et a!., 1983).
Polger et al. (1982a) suggested that 2,3,7,8-TCDD metabolism represents
detoxification, since they observed relatively Uttle toxlclty In guinea
pigs given extracts of dog bile containing 2,3,7,8-TCDD metabolites. How-
ever, a recent study proposes that metabolites of 2,3,7,8-TCDD may Inhibit
uroporphyrlnogen decarboxylase activity and lead to 2,3,7,8-TCDD-1nduced
porphyrla {De Verneull et al., 1983). Current data on the structural
Identification of 2,3,7,8-TCDD metabolites suggest that reactive epoxlde
Intermediates may be formed during metabolism (Polger et al., 1982b;
Sawahata et al., 1982). Poland and ilover (1979) reported that the maximum
possible In. vivo covalent binding of l,6-9H-2,3,7,8-TCDD derived radio-
activity to hepatic DNA was 4 orders of magnitude less than the levels of
binding observed with other chemical carcinogens. The study found much
higher levels of 2,3,7,8-TCDD derived radioactivity bound to hepatic protein
of the rat. No data 1s available, however, on the degree 2,3,7,8-TCDD
derived radioactivity Is bound to tissues of various species of laboratory
animals, which have demonstrated remarkable variability 1n sensitivity to
2,3,7,8-TCDD. While biliary excretion products may represent detoxified,
8-79
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polar metabolites of 2,3,7,8-TCDD, 1t remains to be shown whether unexcreted
reactive metabolites Initiate some of the toxic responses associated with
exposure to this toxin.
8,3.3. - Vitamin A Depletion. Many of the toxic effects of 2,3,7,8-TCDD
resemble the effects of vitamin A deficiency, such as epithelial lesions,
keratosls and Immunosuppresslon. The administration of a single oral dose
of 0.1, 1.0 or 10 ng 2,3,7,8-TCDD/kg bw produces a dose-related decrease
1n the hepatic storage of retlnol 1n Sprague-Dawley rats (Thunberg, 1984;
Thunburg et al., 1979, 1980). The authors suggested, but did not demon-
strate, that the low storage of retlnol 1n the 2,3,7,8-TCDD-treated animals
1s the result of an Increased turnover of retlnol.
Hakansson and Ahlborg {1985} pretreated male Sprague-Dawley rats with
2,3,7,8-TCDD at 10 pg/kg bw 4 days before the oral administration of 1200
Ill/kg of1 retlnyl acetate. One hundred ninety-two hours postadmlnlstratlon
of retlnyl acetate the 2,3,7,8-TCDD-pretreated rats excreted 41% of the
retlnyl acetate compared to the control excreting only 3054. After 2,3,7,7-
TCDD treatment the decrease 1n vitamin A content was 39-53, 19-67 and 18-44%
1n the liver, Intestine and ep1d1dym1s, respectively. 2,3,7,8-TCDD treat-
ment also Influenced vitamin A content 1n the thymus, Initially Increasing
by 42% 1n 6 hours and then decreasing by 40% In 192 hours as compared to the
controls. 2,3,7,8-TCDD pretreatment Increased the vitamin A content 1n the
kidney 3-30 times that of the control. It 1s Important to note that the
kidney becomes the primary vitamin A storage organ In vitamin A deficient
animals (Johnson and Baumann, 1947; Moore and Sharman, 1950). In a similar
study Thunberg and Hakansson (1983) has also found an Increase of vitamin A
storage 1n the kidney after a single oral dose of 2,3,7,8-TCDD 1n male
Sprague-Dawley ratsv Results from these observations suggest strongly that
8-80
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pretreatment with a single oral dose of 2,3,7,8-TCDD can affect both storage
and excretion of retlnyl acetate as well as the vitamin A storage 1n several
tissues.
These results suggest that an Induced vitamin A deficiency may be
responsible for some, but not all, of the toxic effects produced by 2,3,7,8-
TCDD. At the highest dose of 2,3,7,8-TCDD, dietary retlnol supplements
could not fully compensate for the 2,3,7,8-TCDD-produced decrease 1n hepatic
retlnol content.
8.3.4. Llpld Peroxldatlon. Increased Upld peroxldatlon has been sug-
gested as a possible mechanism of 2,3,7,8-TCDD-1nduced toxldty (Sweeney and
Jones, 1983). This hypothesis 1s based on the following limited pieces of
evidence. First, Iron deficiency Inhibits in vitro I1p1d peroxldatlon {Bus
and Gibson, 1979; Sweeney et a!., 1979) and reduces the hepatotoxlc effects
of 2,3,7,8-TCDD (Sweeney et a!., 1979). Secondly, Upofusdn pigments,
by-products of I1p1d peroxldatlon, are Increased 1n the heart muscle of rats
treated with 2,3,7,8-TCDD (Albro et al., 1978). Thirdly, Sweeney and Jones
(1983) reported that administration of the antloxldant butylated hydroxyanl-
sole (BHA) at a level of 0.75% 1n the diet provided some protection from
2,3,7,8-TCDD-1nduced prophyrla and neutral I1p1d accumulation. At this dose
level of BHA, 4 of the 6 mice (sex not specified) tested were protected;
however, at a lower dose (0.25%), all animals were protected from these
toxic effects. No beneficial effects were observed when the antloxldant
vitamin E (0.01%) was Included 1n the diet.
Recently, Stohs et al. (1983) obtained direct evidence that 2,3,7,8-TCDD
accelerates I1p1d peroxldatlon 1n Sprague-Dawley rats. Groups of 4-8 female
rats were treated for 3 days with 2,3,7,8-TCDD at doses of 0, 10, 20 or 40
vg/kg by gavage (In a corn oil vehicle). At days 1, 6 and 11 after the
8-81
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last treatment the animals were sacrificed and I1p1d peroxldatlon was deter-
mined 1n Isolated liver mlcrosomes by the reaction of formed malondlaldehyde
with th1obarb1tur1c add. At all sacrifice periods, Increased I1p1d peroxl-
datlon was observed and the Increase was dose-related. The maximal Increase
detected on day 6 after the last treatment was 5- to 6-fold greater than 1n
the controls. In addition, these workers measured I1p1d peroxldatlon jjj.
vivo by the determination of conjugated dlenes 1n rats receiving 2,3,7,8-
TCDD at 40 ^g/kg. Using this latter method, similar Increases 1n I1p1d
peroxldatlon were detected, although the maximal Increase of 2.35-fold was
observed at day 1 postexposure rather than day 6. The authors suggested
that the in vivo formation of reactive free radicals during llpld peroxlda-
tlon could account for the nonspecific nature of 2,3,7,8-TCDD toxlclty.
Since 0-carotene can quench singlet oxygen pO^) and vitamin E 1s an
antloxldent, Hassan et al. (1985) studied the effects of vitamins A and E on
2,3,7,8-TCDD Induced I1p1d peroxldatlon. Vitamin A was found to Inhibit
I1p1d peroxldatlon, elevated the activity of glutathlone peroxldate and
prevented a 2,3,7,8-TCDD-1nduced decrease 1n 6SH content 1n the liver.
Vitamin E markedly Inhibited rolcrosomal I1p1d per1ox1dat1on, but did not
have any effect on glutathlone peroxldase activity or glutathlone content.
8.3.5. Endocrine Imbalance. Some of the toxic response to 2,3,7,8-TCDD,
Including hlrsutlsm and diminishing libido, Indicate that 2,3,7,8-TCDD may
produce some of Its toxlclty through endocrine disturbances (Oliver, 1975).
Nlenstedt et al. (1979) reported that a single oral dose of 20 vg 2,3,7,8-
TCDD/kg bw significantly reduced testosterone eatabollsm. Catabollsm of
exogenous estrogen 1n ovarlectomlzed rats 1s also decreased by 2,3,7,8-TCDD
pretreatment (Sh1ver1ck and Muther, 1982). In this study, there was a 57K
Increase 1n serum estrone concentrations following administration of 10 mg
8-82
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estrone/100 g bw/day for 4 days to either control or 2,3,7,8-TCDD pretreated
ovarlectomlzed rats. No differences were observed 1n the Increase 1n
uterine wet weight following estrone administration 1n control and 2,3,7,8-
TCDD pretreated rats. Thus, the uterotrophlc response was not altered by
any 2,3,7,8-TCDD-med1ated change 1n estrone disposition,
Sh1ver1ck and Muther (1983) also measured estradlol metabolism 1n female
Holtzman rats given 2,3,7,8-TCDD at a dose of 1 jig/kg bw on days 4-19 of
gestation. At this fetal toxic dose, the catechol estrogen formation abil-
ity of Isolated liver mlcrosomes from the dams was decreased 50% when mea-
sured on day 20 of gestation. These mlcrosome preparations had a 4-fold
Increase 1n the 7a-hydroxylat1on of testosterone, while there was no
change 1n the 16a- or 60-hydroxylase activity. Although steroid metabo-
lism was altered 1n mlcrosomes Isolated from 2,3,7,8-TCOD-treated pregnant
rats, similar exposure of pregnant rats on days 4-15 of gestation resultd In
no change 1n circulating levels of serum !73-estrad1ol. The authors sug-
gested that other mechanisms besides liver metabolism of steroids may be
Involved 1n the fetotoxlc effect of 2,3,7,8-TCDD.
Gustafsson and Ingelman-Sundberg (1979) observed that 2,3,7,8-TCDD pro-
duced greater change 1n steroid metabolism In female Sprague-Dawley rats
than 1n male rats of the same strain, resulting 1n a liver enzyme pattern
displaying less sex differentiation than 1n unlnduced rats. Based on this
result, they propose that some of the effects of 2,3,7,8-TCDD resulted from
an Interaction with the hypothalamo-pltultary axis, rather than from a
direct effect on steroid metabolism.
Since glucocortlcold hormones are known to have a catabollc effect on
lymphold tissues, such as the thymus and spleen, and these tissues degener-
ate after exposure of rats to 2,3,7,8-TCDD, Neal et al. (1979) Investigated
8-83
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the ability of 2,3,7,8-TCDD to either stimulate the production or mimic the
effects of these hormones. In male Sprague-Dawley rats treated by gavage
with 2,3,7,8-TCDD at a dose of 50 vg/kg (the ~LD5Q), there was a slight
depression 1n blood glucocortlcolds during post-treatment days 1-4, followed
by an ~2.5-fold Increase on post-treatment days 7 and 14. While 1n competi-
tive binding assays between 2,3,7,8-TCDD and a synthetic hormone, dexametha-
sone, 2,3,7,8-TCDD had no affinity for the hormone receptor. Thus, 2,3,7,8-
TCDD may have stimulated glucocortlcold production but was not able to mimic
the action of these hormones by binding to the glucocortlcold receptor. It
was determined, however, that the Increase 1n glucocortlcolds was likely not
to participate 1n the toxldty of 2,3,7,8-TCDD through adrenal hyperfunc-
tlon, since prior adrenalectomy did not provide any protection from the
lethal effects of !2,3,7,8-TCDD 1n rats.
8.4. SUMMARY
8.4.1. Experimental Animal Data. A wide range of lethal doses has been
reported for 2,3,7,8-TCDD depending on the species tested. The male guinea
pig was the most sensitive, with an LD5Q value of 0.6 pg/kg; the male
hamster was the least sensitive, with an LDKn value of 5051 yg/kg
DU
(Schwetz et a!., 1973; Henck et al., 1981). At least for acute exposure,
the toxldty of 2,3,7,8-TCDD appears to depend on the total dose admin-
istered over a given time and not on whether exposure occurs through a
single treatment or a limited number of multiple treatments. Unlike most
lethal exposures to toxicants, death resulting from a lethal exposure to a
single dose of 2,3,7,8-TCDD occurs long after treatment (5-45 days, see
Table 8-1). The most common symptoms after lethal exposure were weight
loss, often characterized as "wasting away," and thymlc atrophy. Although
liver damage was not observed 1n the guinea pig, the most sensitive species
8-84
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to 2,3,7,8-TCDQ, extensive liver damage was reported 1n rats and mice (Qupta
et al., 1973). In general, no specific cause of death could be Identified.
In a limited comparison of the LDcn for 9 congeners of PCDDs, It appeared
DU
that biologic activity required chlorine 1n the 2,3,7,8-posltlons (McConnell
et al., 1978b), with 2,3,7,8-TCDD being the most potent congener.
The liver has been studied extensively with regard to 2,3,7,8-TCDD acute
tox1c1ty 1n rats and mice. Single high doses, 200 pg/kg» of 2,3,7,8-TCDD
produced liver necrosis 1n rats (Jones and Butler, 1974); however, lower
doses of 5 and 25 yg/kg produced fatty changes and proliferation of the ER
(Fowler et al., 1973). Along with Increases 1n ER, there was an associated
marked Increase In MFO activity (see Section 8.I.I.S.). Additional membrane
changes Included degeneration of the plasma membrane with loss of ATPase
activity. In species sensitive to the hepatotoxlc effects of 2,3,7,8-TCDD,
there was also a decreased ability to excrete some xenoblotlcs Into the bile
(Yang and Peterson, 1977; Hwang, 1973). Porphyrla was also observed, with
the mouse being more sensitive than the rat. In addition to effects on the
liver, 2,3,7,8-TCDD also affects Intestinal absorption by Increasing and
decreasing the absorption of specific nutrients. In some species, the cel-
lularlty of the blood was decreased,
Effects of 2,3,7,8-TCDD exposure on the Immune system have been studied
extensively. 2,3,7,8-TCDD Is undlsputably an acute 1mmunotox1c substance 1n
animal models, causing decreases 1n thymlc and splenic weight and hindering,
predominantly, cell-mediated Immunity. T-lymphocyte function 1s primarily
affected, although a reduction 1n the Immune response to a thymus-lndepen-
dent antigen (type III pneumococcal polysaccharlde) has been reported fol-
lowing 2,3,7,8-TCDD exposure (Vecchl et al., 1980). 2,3,7,8-TCDD presumably
affects lymphocytes or thymlc cells directly, since several studies have
8-85
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negated Indirect routes of 1mmunosuppress1on {hormonal controls), 2,3,7,8-
TCDD at 1mmunotox1c levels that alter all function, however, 1s not directly
cytotoxle to lymphocytes (Kodba and Schwetz, 1982). Its effects may be
reversible after long recovery periods {Faith and Luster, 1979).
2,3,7,8-TCDD has been shown to alter serum Immunoglobln levels 1n mice
at oral doses as low as 0.01 and 0.1 jig/kg/week when administered for up
to 8 weeks (Sharma and Gehrlng, 1979). Thomas and H1nsd1ll (1979) reported
reduced hypersens1t1v1ty to DNFB, decreased Immune response to £. coll IPS
and decreased thymlc weight 1n young mice exposed to 2.5 and 5 ppb 2,3,7,8-
TCDD {0.33 and 0.65 ng/kg) through maternal dosing. Thlgpen et al. (1975)
postulated a NOEL of O.S vg 2,3,7,8-TCDD/kg/week for 4 weeks, but more
precise tests of 1mmunotox1c1ty suggest a lower NOEL would be appropriate,
especially for neonatal and young animals.
The mechanism of 2,3,7,8-TCDD-1nduced 1mmunotox1c1ty 1s not yet known.
2,3,7,8-TCDD 1s not likely to decrease Immune responsiveness through an
endocrine control. 2,3,7,8-TCDD may act as an antlgenlc agent causing
1mmunosuppress1on and thymlc atrophy (Sharma and Sehrlng, 1979). It has
also been suggested that 2,3,7,8-TCDD attaches to the cell membrane of
T-lymphocytes, altering the cell surface, which could Interfere with antigen
and cell-to-cell recognition (Luster et al., 1979a,b; Faith and Luster,
1979).
In subchronlc toxlclty studies 1n rats and mice, the liver appeared to
be a target organ. The Induction of liver damage after repeated exposure to
small doses of 2,3,7,8-TCDD was shown 1n rats. H1stolog1c changes 1n the
liver of rats killed 2, 4, 8, 16 and 28 weeks after exposure to weekly doses
of 1 jig/kg bw revealed no fatty changes until week 28; however, 12 weeks
after termination of the 28-week exposure, there was stm evidence of fatty
8-86
-------
changes In the liver (King and Roesler, 1974). A similar long Induction
period was observed by Goldstein et al. (1982b) for porphyMn accumulation
1n the Hver of rats. Following 16 weeks of exposure to 2,3,7,8-TCDD and a
6-month postexposure period, porphyrln levels were stm elevated. The only
study 1n mice (NTP, 1980a) described toxic hepatitis as the only effect of
subchronlc exposure to low levels of 2,3,7,8-TCDD. In these and other sub-
chronic studies, NOELs of 0.01 vg/kg/day (Kodba et al., 1976), 0.5 vg/
kg/week (NTP, 1980a) and 0.01 vg/kg/week (Goldstein et al., 1982b) have
been reported for rats. In mice, a NOEL of 2 pg/kg/week was obtained 1n
females, while males exposed to 1 yg/kg/week (the lowest dose tested)
developed toxic hepatitis. Similar hepatic lesions were observed after
exposure to a mixture of HxCDDs with NOELs of 2.5 and 1.25 yg/kg/week
reported for rats and mice, respectively (NTP, 1980b).
In chronic toxlclty studies 1n rats and mice, 1t was again the Hver
that appeared to be the most sensitive organ. Changes 1n the liver of rats
Included Initially fatty Infiltration, and at higher doses, necrosis. The
studies 1n rats Indicated that 0.001 vg/kg/day was a NOEL, while 0.05 and
0.1 yg/kg/day were the NOAEL and PEL for liver damage (Kodba et al.,
1978b, 1979; NTP, 1980a). In mice, a NOEL was not determined, with the
lowest doses tested, 0.0015 and 0.006 yg/kg/day, producing liver damage 1n
male and female B6C3F1 mice (NTP, 1980a), while the lowest dose tested In
Swiss mice, 0.001 yg/kg/day, produced amyloldosls of the kidney, spleen
and liver (Toth et al., 1978, 1979). In nonhuman primates, chronic exposure
to 2,3,7,8-TCDD 1n the diet at 50 or 500 ppt resulted 1n hair loss, edema
and pancytopenla (Allen et al., 1977; Schantz et al., 1979). Data were not
available to determine a NOEL for monkeys. Also, 1n the only study avall-
8-87
-------
able for 1,2,3,6,7,8- or 1,2,3,7,8,9-HxCDD, the lowest doses tested, 1.25
and 2.5 vg/kg/week for males and females, respectively, produced toxic
hepatitis and represented a PEL (NTP, 1980b).
8.4.2, Human Data, There seems to be general agreement that exposure to
2,3,7,8-TCDD, whether acutely or chronically, leads to chloracne, altered
liver function, hematologlcal abnormalities, porphyrla cutanea tarda, hyper-
pigmentation and hlrsutlsm. Recently, Susklnd and Hertzberg (1984) have
demonstrated an association between exposure to 2,4,5-T contaminated with
2,3,7,8-TCDD and the history of 61 ulcer. No evidence of Increased risk for
cardiovascular disease, hepatic disease, renal damage or central or peri-
pheral nervous system problems could be found 1n a group of workers exposed
to 2,4,5-T following a run way reaction (Susklnd and Hertzberg, 1984). How-
ever, occupational or accidental exposure to 2»3,7,8-TCDD has been shown to
produce neurological ailments In addition to the above ailments. .The neuro-
logical problems Include peripheral polyneuropathles. Impairment of sensory
functions Including sight disorders, loss of hearing, taste and sense of
smell, central lassitude, weakness, Impotence and loss of libido (Regglanl,
1982; Klmbrough et a!., 1984). Only one estimate was available, which
speculates a cumulative minimum toxic dose of 0.1 jig/kg for man (Stevens,
1981). The available follow-up reports and ep1dem1olog1cal studies, primar-
ily on populations exposed occupatlonally, accidentally or 1n Vietnam, Indi-
cate that toxic effects noted soon after exposure to 2,3,7,8- TCDD may sub-
side or may persist for many years.
8.4.3. Mechanisms of Toxldty. In the preceding sections, five possible
mechanisms by which 2,3,7,8-TCDD may produce Its toxic effects were
reviewed. The data suggest that metabolism of 2,3,7,8-TCDD 1s a detoxifica-
tion process, resulting 1n the production of metabolites that are less toxic
8-88
-------
than the parent compound, although Intermediate or minor metabolites of
2,3.7,8-TCDD may be Involved 1n toxldty. VHamln A depletion. Increased
I1p1d peroxldatlon and effects on the hypothalamo-pHuHary axis have all
been Implicated as possible mechanisms for 2,3,7,8-TCDD-1nduced toxic
response. It seems probable that these mechanisms are responsible for some,
but not all, of the toxic effects of 2,3,7,8-TCDD.
The major mechanism of 2,3,7,8-TCDD toxldty that has received Intense
Investigation Involves effects mediated by specific cytosollc receptors pro-
duced by the Ah locus. The toxldty of various dloxlns has been correlated
with binding to the cytosollc receptor and enzyme Induction 1n a wide range
of animal species and under a variety of experimental conditions (vide
ante). While these studies have been done 1n several species, species
differences 1n the toxic response to 2,3,7,8-TCDD do not correlate with
species differences 1n receptor concentration or affinity, or with the
degree of enzyme Induction. It thus appears that the toxldty of 2,3,7,8-
TCDD may be mediated by binding to the cytosollc receptor responsible for
enzyme Induction; however, this theory does not apply 1n various species,
and cell culture studies Indicate that enzyme Induction 1s not necessarily a
cytotoxlc process.
8-89
-------
9. TERATOGENICITY AND OTHER REPRODUCTIVE EFFECTS
9.1. STUDIES ON EXPERIMENTAL MAMMALS
9.1.1. 2,3,7,8-TCDD Administered as a Contaminant of Other Chemicals.
Courtney et al. (1970a,b) were the first to report that 2,4,5-T was capable
of causing teratogenlc effects 1n rats and mice. In these studies, rats and
two strains of mice were exposed subcutaneously or orally to 2,4,5-T con-
taining 30 ppm 2,3,7,8-TCDD. The mixture was teratogenlc and fetotoxlc to
mice at >46.4 mg/kg. Rats were more sensitive, exhibiting fetotoxlc
responses at 10 mg/kg for this 2,4,5-T/2,3,7,8-TCDD mixture. Since this
Initial report, research has focused on determining the role of 2,3,7,8-TCDD
contamination 1n eliciting the teratogenlc response. These studies are
summarized In Table 9-1.
Neubert and Dlllmann (1972) conducted a detailed study to determine the
significance of 2,3,7,8-TCDD contamination. These Investigators assayed
three 2,4,5-T samples: a highly purified sample containing <0.02 ppm
2,3,7,8-TCDD (referred to as Sample A), a purified sample Identical to that
used by Roll (1971) that contained 0.05+0.02 ppm 2,3,7,8-TCDD (Sample B),
and a commercial sample containing an undetermined quantity of 2,3,7,8-TCDD
(Sample C). All three samples Induced cleft palates at sufficiently high
doses (30-90 mg/kg). In terms of the number of fetuses with cleft palate/
the total number of fetuses, the dose/response pattern observed by Neubert
and Dlllmann (1972) was similar to that observed by Roll (1971) using a
similar grade of 2,4,5-T. In addition to the three 2,4,5-T samples, Neubert
and DUlmann (1972) also assayed a sample of 2,3,7,8-TCDD alone and 1n
various combinations with the highly purified sample of 2,4,5-T. This
approach allows at least partial quantification of the significance of
2,3,7,8-TCDD contamination 1n 2,4,5-T-1nduced cleft palates. When the
9-1
-------
TABLE 9-1
Studies on Iht Potential Teratogenic Effects of 2,3,7,8-TCOD Contaminated 2,4,5-T
Species/Strain Vehicle
NIce/NNRI Rape-seed oil
Form of
2,4,5-T TCOD Level Oally
acid <0.02 ppm 8, IS,
{Sample A) 45, 60,
and 120
Dose
30,
90
rag/kg
Treat-
ment
Days
6-15
Obser-
vation
Day
18
Haternal Response
No toxic effects;
decreased maternal
weight at doses of
90 ng/kg and
greater
Fetal Response
Significant Increases In
the Incidence of cleft
palates at doses above
30 mg/kg (see text for
additional details).
Significantly decreased
(p<0.005) fetal weight
at all dose levels.
Reference
Neubert and
Dlllmann, 1972
NIce/NNRI Rape-seed oil
acid 0.05*0.02 ppn 30, 60 and 6-15
(Sample B) 90 mg/kg
to
NJ
NIce/NNRI
NIce/NMRI
Rape-seed oil
Rape-seed oil
add MR {Sample C) 90 mg/kg
butyl NR
ester
12 and 17
mg/kg
6-15
6-15
18 No toxic effects;
decreased maternal
weight at 90 mg/kg
18 No toxic effects
but decreased
maternal weight
16 No toxic effects
NIce/NNRI NR
acid 0.05*0.02 ppm 20, 35, 60. 6-15
90 and 130
ng/kg
NR
Nice/CD-I Corn o11:acetone acid <0.05 ppm 115 mg/kg
(9:1)
10-15
IB
Toxic effects
observed at 90
and 130 mg/kg
No significant
effect on weight
gain or llver-to-
bw ratios
Increases In the Incidence
of cleft palate at 60 and
90 mg/kg; significant
(p<0.005) at all dose
levels
Increase In the Incidence
of cleft palate; signifi-
cant (p<0.005) decrease
In fetal weight
Significant decrease In
fetal weight but no effect
on mortality; Increase In
the frequency of cleft
palate similar to that seen
with add (see text)
Increases In the percent-
age of resorptlons and/or
dead fetuses at 90 and 130
mg/kg; Increases In the
Incidence of cleft palate
and retardation of skeletal
development at 35 rag/kg
and above
No effect on fetal mortal-
ity or fetal weight but
an Increase In the Inci-
dence of cleft palate
Neubert and
Olllminn, 1972
Neubert and
Dlllmann. 1972
Neubert and
Dlllmann, 1972
Roll, 1971
Courtney, 1977
-------
1ABIE 9-1 (cont.J
Species/Strain Vehicle
form of
2,4,5-T TCOD Level
Treat- Obser-
Dally Dose ment vatlon Maternal Response
Days Day
Fetal Response
Reference
H1ce/C57BL/6 Honey: water
0:1)
acid 30 pom
46.4 and 113 6-14 18 NR
wg/kg
10
i
HIce/AKR
Rats/Sprague-
Oawley
(groups of
25 ratsj
Rats/Mtstar
Rats/Histar
Honey:water
(1:1)
Gavage/hydroxy-
propyl-methyl-
cellulose
add 30 ppn
add 0.5 ppm
113 mg/kg
6-15
19
1, 3, 6. 12 or 6-15
24 mg/kg/day
Savage/aqueous
gelatin or
corn oil
add <0.5 mg/kg
25. 50, 100 or
150 mg/kg/day
6-15
Savage/aqueous
gelatin or
corn oil
butyl
-------
TABLE 9-1 |cont.)
Species/Strain
Rats/Holtzman
Vehicle
6avage/l:l
solution of
Form of
2,4,5-T TCDD Levtl
acid 30 ppm
Dally
4.6,
46,4
Dose
10.0 and
mg/kg/day
Treat-
ment
Days
10-15
Obser-
vation Maternal Response
Day
20 HR
fetal
Significant
Increases In
Response
(p<0.01)
fttal nwr-
Reference
Courtney
et al., 1970a
,b
honey and water
Rats/CD
Savage/15% acid 0.5 ppm
sucrose solution
us
i
Rats/strain Gavage/methocel
not specified
acid O.S ppm
Rats/strain Gavage/methocel acid 0.5 ppm
not specified
Syrian Gavage/acetone, acid <0.1-4.5ppnt
hamsters/ corn oil, and
Hesocrlcetus carboxymethyl
euratus cellulose In
ratio of 1:5.8:10
10.0, 21.5,
46.4 and 80.0
mg/kg/day
50 rag/kg
100 Dig/kg
20, 40, 80
and 100 mg/kg
6-15 20 Reduced maternal
weight gain at the
2 higher dose
levels (p<0.05)
and Increased
llver-to-bw ratio
at the highest dose
level (p<0.05)
6-15 NS Ho effect on mor-
tality or bw gain
6-10 NS Increased mor-
tality and
decreased bw gain
6-10 14 NS
talHy at the 2 higher
dose levels; dose-related
Increases In tht percent
of abnormal fetuses per
litter; a high Incidence
of cystic kidneys In
treated groups
Increase In the Incidence
of kidney anomalies, but
no Increase in cleft
palate
No significant effect on
fetal mortality or fetal
weight; a significant
(p<0.05) Increase In the
Incidence of delayed
ossification
Increase in the Incidence
of delayed ossification
and poorly ossified or
malallgned sternebrae
(p<0.05>
Dose-related Increases In
fetal mortality, gastro-
intestinal hemorrhages,
and fetal abnormalities;
see text for discussion
of effect TCDD level on
development
Courtney and
Hoore, 1971
Sparschu
et al.. 1971a
Sparschu
et al., Wla
Collins et al.
NS = Not specified; NR = Not reported
-------
"Htter H used as the basic experimental unit, the Incidences of cleft
palate (number of Utters with cleft palate/total numbers of litters) versus
the dose can be plotted on log dose/problt response paper, correcting for
background response using Abbott's equation. According to this method, the
ED5Q (by eye-fH) for cleft palate Induction are as follows:
2,3,7,8-TCDD: 4.6 wg/kg bw
2,4,5-T (Sample A): 115 mg/kg bw
2,4,5-T (Sample B): 46 mg/kg bw
If the assumption were made that all teratogenlc activity 1n the 2,4,5-T
samples were attributable to 2,3,7,8-TCDD contamination, the expected EDgo
for samples A and B would be 230,000 mg/kg (0.0046 mg/kg x 0.02 ppm"1) and
92,000 mg/kg (0.0046 mg/kg x 0.05 ppm"1), respectively. Since the
observed ED,-0 was lower by a factor of over 1000, this suggests that
2,3,7,8-TCDD 1s not the sole factor 1n 2,4,5-T-1nduced cleft palate.
The nature of possible Interaction between 2,4,5-T and 2,3,7,8-TCDD 1s
more difficult to define. Based on assays of five mixtures of 2,3,7,8-TCDD
and the highly purified 2,4,5-T, Neubert and Dlllmann (1972) noted a greater
than additive effect on the Induction of cleft palates. A similar conclu-
sion can be reached If one assumes that Sample A was a "totally pure" sample
of 2,4,5-T. Using the assumptions of simple similar action (Flnney, 1971)
and treating Sample B as a mixture of 2,3,7,8-TCDD and 2,4,5-T, the expected
EDgg for Sample B would be 119.8 mg/kg. The observed value of 46 mg/kg
again suggests a greater than additive effect. A more detailed statistical
analysis of these data, however, would be required to support the assump-
tions of simple similar action or Independent joint action that are Implicit
1n these analyses. Furthermore, the Inability to define precisely the
9-5
-------
levels of 2,3,7,8-TCDD 1n the 2,4,5-T samples and the possible significance
of other contaminants would preclude an' unequivocal Interpretation of the
results of the analysis.
Nevertheless, three of the studies summarized 1n Table 9-1 (Neubert and
DUlmann, 1972; Roll, 1971; Courtney, 1977) have demonstrated the Induction
of cleft palate 1n mice by using 2,4,5-T samples containing 2,3,7,8-TCDD
levels of 0.05 + 0.02 ppm or less. Although 2,3,7,8-TCDD contamination 1s
undoubtedly a factor 1n the teratogenlc activity of 2,3,7,8-TCDD contami-
nated 2,4,5-T, the above analysis suggests that 2,3,7,8-TCDD contamination
1s not the sole factor, and that some teratogenlc activity must be attrib-
uted to 2,4,5-T Itself or other contaminants 1n 2,4,5-T.
9.1.2. 2,3,7,8-TCDD Studies In Mice. Courtney and Moore (1971) tested a
purified sample of 2,3,7,8-TCDD for teratogenlc potential. A summary of
this study and others assessing the teratogenlc potential of purified
2,3,7,8-TCDD are presented 1n Table 9-2. CD-I, DBA/2J and C57B1/6J mice
were given subcutaneous Injections of 2,3,7,8-TCDD at 1 or 3 jig/kg/day on
days 6-15 of gestation 1n the study by Courtney and Moore (1971). This dose
regime did not result 1n maternal toxlclty, although an Increase 1n the
maternal Hver/bw ratio was observed 1n DBA/23 and C57B1/6J mice. 2,3,7,8-
TCDD had no measurable effect on fetal mortality; however, anatomical
abnormalities were observed 1n all strains and at all dose levels, with
C57B1/6J being the most sensitive strain. The abnormalities observed were
cleft palate and unspecified kidney anomalies.
Moore et al. (1973) treated pregnant C57B1/6 mice with an oral dose of
2,3,7,8-TCDD at 1 or 3 pg/kg/day on days 10-13 of gestation, or 1 vg/kg
on day 10 of gestation. At the high dose level, the average Incidence of
cleft palate was 55.4%. Kidney anomalies (hydronephrosls) were observed on
9-6
-------
TABLE 9-2
Studies on the Potential leratogenlc Effect of 2,3,7,8-tCDB
Species /Strain
Mouse/C57B1/6
Rouse/AKR
Mouse/CD-I
«ouse/DBA/2J
House/C57Bl/6J
Nouse/C57B1/6
Mouse/CD-I
us
i
House/CF-1
House/NHRl
Rat/CD
Rat/Sprague-
Dawley
Rat/His tar
Vehicle
DHSO or
honey: water
(1:1)
DHSO
acetone:
corn oil
DHSO or
corn oil
corn oil/
acetone
<98:2J
rape-seed
oil
DHSO
corn oil/
acetone
corn oil/
anlsole
Dally Dose
21.5. 46,4.
113.0 mg/kg
0.5, 1, 3 ug/kg
1. 3 pg/kg
25. 50, 100,
200, 400 yg/kg
0.001, 0.01,
0.1, 1.0.
3.0 jig/kg
0.3, 3.0, 4.5.
9.0 tig/kg
0. 0.5,
2.0 pg/kg
0, 0.03, 0.125,
0.5. 2.0 and
8.0 tig/kg
0.0, 0.125.
0.25. 1, 2, 4,
8, 16 vg/kg
Treatment
Days
6-14 or 9-17
6-15
10-13 or 10
7-16
6-15
6-15
6-15, 9 and
10, or 13
and 14
6-15
6-15
Observation
Day
iga
17a or 18
18*
18°
183
18
20a
20*
22
Haternal Response
Increased liver/
bw ratio
Increased liver/
bw ratio
none reported
Increased liver/
bw ratio
none reported
no effect observed
none reported
vaginal hemorrhage
at 2.0 and
8.0 )ig/kg
maternal toxklty
observed at or
above 1 i>g/kg
Fetal Response
fetocldal, cleft palate.
cystic kidney
cleft palate,
kidney anomalies
cleft palate, kidney
anomalies
cleft palate, hydronephrotlc
kidneys, hydrocepttalus, open
eyes, edema, petechlae
cleft palate, dilated renal
pelvis
fetocldal at the high dose,
cleft palate at doses at or
above 5 yg/kg
kidney malformations at
both dose levels
Intestinal hemorrhage at
0.125 and 0.5 ug/kg.
fetal death at higher doses,
subcutaneous edema
Increased fetal death
observed at or above
1 vg/kg. subcutaneous
Reference
Courtney
et al., 1970b
Courtney and
Hoore, 1971
Roore et al..
1973
Courtney, 1976
Smith et al.,
1976
Neubert and
Dlllmann, 1972
Courtney and
Hoore, 1971
Sparschu
et al., 1971b
Khera and
Ruddlck, 1973
edema and hemorrhages 1n
the 0.25-2 vg/kg groups
-------
TABLE 9-2 (conl.)
Species/Strain
Rat/Sprague-
Dawley
Rat/Sprague-
Dawley
lO
i
00
Rabbit/
New Zealand
Vehicle
corn oil/
acetone
(9:1)
diet
corn oil/
acetone
(9:1)
Dally Dose
0.1, 0.5.
2.0 pg/kg
0.001, 0.01
and 0.1 ug/kgc
0.0, 0.1,
0.25, 0.5
and 1 iig/kg
Treatment
Days
1-3
throughout
gestation
6-15
Observation
Day
21
post-
parturition
28
Maternal Response
decrease In bw
gain In the high
dose group
low fertility at
0.01 and 0.1 wg/kg
decreased bw at
0.01 and 0.1 v9/kg
dilated renal pelvis
maternal toxlclty
at doses of 0.2S
pg/kg and above
Fetal Response
decreased fetal weight In
the 0.5 and 2 gg/kg group
low survival at 0.01 and
0.1 ug/kg, decreased bw at
0.01 yg/kg. slight dilated
renal pelvis at 0,001 ^g/kg
In the FI but not succeeding
generations''
Increases In extra ribs and
total soft tissue anomalies
Reference
Glavlnl
et al..
Murray
et al,.
Glavlnl
et al.,
19B2a
1979
1982b
'First day of gestation designated day zero
°F1rst day of gestation designated day one
cThe high dose level (0.1 Mg/kg/day) was discontinued due to very low fertility In adults
dfilsbet and Paxton (1982) re-evaluated the study by Hurray et al. (T979) using different statistical methods and considered the effects In the 0,001 »g/kg
group to be statistically significant.
-------
an average of 95.1% of the fetuses/lUter, with 83.1% having bilateral
kidney anomalies. When the dose was decreased to 1 yg/kg/day, the average
Incidence of cleft palate dropped to 1.9%; however, the Incidence of kidney
anomalies remained relatively high, with an average Incidence of 58.954. On
the average, bilateral kidney anomalies occurred 1n 36.3% of the fetuses/
Utter. A single dose of 1 ug/kg on day 10 of gestation produced kidney
anomalies 1n 34.3% of the fetuses; however, no cleft palates were observed.
When G57B1/6 mice were treated with 1 pg/kg on day 10 of gestation and
were then allowed to Utter, the detection of kidney lesions on postnatal
day 14 was found to depend largely on whether the pups nursed on a 2,3,7,8-
TCDD-treated mother. When pups from a 2,3,7,8-TCDD-treated mother nursed on
control mice, kidney anomalies were found 1n only 1/14 Utters. In
contrast, when pups from control mothers nursed on 2,3,7,8-TCDD-treated
mice, kidney anomalies were observed 1n 4/14 Utters. In the pups exposed
to 2,3,7,8-TCDD both In. utero and during the postnatal period, kidney
anomalies were observed In 5/7 litters. Kidney anomalies observed following
In utero exposure or exposure through the milk were similar, and these
kidney anomalies may not be considered a purely teratogenlc response.
Neubert et al. (1973) reviewed what was known of the embryotoxlc effects
of 2,3,7,8-TCDD 1n mammalian species. Also reported were their own studies
and previous work (Neubert and DUlmann, 1972) using NMRI mice, In which
cleft palate was observed to be a common abnormality; however, no kidney
anomalies were reported. Neubert and Dlllmann (1972) administered 2,3,7,8-
TCDD by gavage to 20 female mice on days 6 through 15 of gestation at doses
of 0.3, 3.0, 4.5 and 9.0 ug/kg. At day 18 of gestation, extensive
reabsorptlon was observed 1n the high-dose group with 6/9 Utters totally
resorbed. In the few surviving fetuses, there was an 81% Incidence of cleft
9-9
-------
palate. At lower doses, there were 9 and 354 Incidences at doses of 4.5 and
3.0 yg/kg, respectively, and no cleft palates were observed 1n 138 fetuses
examined 1n the 0.3 yg/kg group. Fetal mortality was Increased at the 9.0
yg/kg dose 1f animals were treated only on days 9 through 13; however, the
Incidence of cleft palate remained high at a frequency of 60%. In a series
of experiments to determine the time of gestation at which 2,3,7,8-TCDD was
effective 1n Inducing cleft palate, mice were treated for a single day
between days 7 and 13 of gestation with 2,3,7,8-TCDD at a dose of 45
yg/kg. A maximum number of Induced cleft palates occurred when animals
were treated on either day 8 or 11 of gestation; exposure to 2,3,7,8-TCDD
after day 13 of gestation produced no cleft palates In the fetuses,
Courtney (1976) compared the teratogenlc potential of 2,3,7,8-TCDD
administered orally with 2,3,7,8-TCDD administered subcutaneously. CD-I
mice were dosed with 2,3,7,8-TCDD on days 7 through 16 of gestation at
levels of 25, 50, 100, 200 or 400 yg/kg/day; the 400 yg/kg dose was not
used 1n animals treated by subcutaneous Injection. Doses of 200 or 400
yg/kg/day produced vaginal bleeding and high rates of abortion. A dose of
100 yg/kg/day was fetotoxlc, resulting In decreased fetal weight and
survival. Anatomic abnormalities were observed at all dose levels, with
cleft palate and hydronephrotlc kidneys being most common. Other abnormal-
ities observed Included hydrocephalus, open eye, edema and petechlae.
Subcutaneous administration of 2,3,7,8-TCDD produced a greater teratogenlc
response at a lower dose than oral administration, with abnormalities
observed 1n 87% of the fetuses following subcutaneous administration and 42%
after oral administration of a dose of 25 yg/kg/day.
The effects of 2,3,7,8-TCDO on the Incidence of fetal anomalies were
also studied by Smith et al. (1976) 1n CF-1 mice. The mice were given
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0.001-3.0 >ig 2,3,7,8-TCDD/kg/day by gavage from day 6 through 15 of gesta-
tion. The Incidence of cleft palate was found to be significantly Increased
1n 1.0 and 3.0 pg/kg/day dose groups, and the Incidence of kidney
anomalies was significantly Increased at 3.0 yg/kg/day. There were no
observable teratogenlc effects 1n the study at 0.1 jig/kg/day; however,
some were noted at lower dose levels, although not statistically signif-
icantly elevated.
Poland and Glover (1980) compared cleft palate formation by 2,3,7,8-TCDD
1n the responsive C57B1/63, the nonresponslve DBA/23 and the hybrid B6D2F1/J
strains of mice. Female mice were mated with male mice of the same genetic
strain, and on day 10 of pregnancy the pregnant mice were given a single
subcutaneous dose of 3.0, 10.0 or 30.0 pg/kg of 2,3,7,8-TCDD dissolved 1n
p-d1oxane or the solvent (control) alone (0.4 ma/kg). On day 18, the
animals were killed and the number of cleft palates and resorbed fetuses was
determined. At doses of 3.0 and 10.0 pg/kg of 2,3,7,8-TCDD, cleft palates
(3% Incidence among live fetuses) were observed only 1n the C57B1/63 mice at
the higher dose level. At a dose of 30 pg/kg, the Incidence of cleft
palates among live fetuses for the C57B1/63, B6D2F1/3 and DBA/2J mice was
54, 13 and 2%, respectively. This study also reported that cleft palate
formation was significantly higher 1n several other responsive mouse strains
compared with nonresponslve mice. At a dose level of 30 pg/kg of
2,3,7,8-TCDD, the Incidence of cleft palates among live fetuses for the
responsive C57B1/63, A/3, BALB/cByO and SEC/1REJ mice was 54, 73, 65 and
95%, respectively. The only responsive mouse (CBA/3) strain that was
resistant to 2,3,7,8-TCDD-med1ated cleft palate was also resistant to the
teratogenlc effects of cortisone. In contrast, the Incidence of cleft
palates 1n the nonresponslve DBA/2J, RF/3, AKR/J, SWR/3 and 129/3 mice was
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between 0 and 3% at the 30 vg/kg dose level. In a reciprocal blastocyst
transfer study between 2,3,7,8-TCDD "responsive" (NMRI) and "nonresponslve"
(DBA) strains of mice 1t has been demonstrated that 2,3,7,8-TCDD exposure
(30 jig/kg bw) on day 12 of gestation developed cleft palate 1n 75-100% of
NMRI fetuses, Irrespective whether these embryo were kept 1n their own
(NHRI) dams or transferred to DBA dams. However, none of the 2,3,7,8-TCDD
exposed DBA fetuses transferred to NMRI dams or kept 1n their own (DBA) dams
had cleft palate (D"Argy et al., 1984). These results suggest that the
responsive mice, containing high levels of the Ah receptor, are highly sus-
ceptible to the effects of 2,3,7,8-TCDD 1n producing cleft palate, whereas
the nonresponslve mice, which contain low (or 0) levels of the Ah receptor
protein, are resistant to this teratogenlc effect of 2,3,7,8-TCDD. These
data and other results (Hassoun and Dencker, 1982) suggest that cleft palate
formation elicited by 2,3,7,8-TCDD segregates with the Ah locus.
Dencker et al. (1981), Pratt (1983) and Pratt et al. (1984a,b) found an
association between 2,3,7,8-TCDD-1nduced cleft palate and Ah activity In
mice. Significant concentrations of TCDD have been detected 1n the placenta
of pregnant TCDD-dosed mice, resulting 1n cleft palate Induction 1n the
fetus without any apparent effect 1n the dams. Sensitivities to TCDD vary
among strains. The AKR strain lack Ah receptors and remain Insensitive to
cleft palate whereas the C57 strain possess Ah responsiveness and are sensi-
tive to TCDD-1nduced cleft palate. These observations further prove that
the Ah locus 1s the cause of strain differences 1n cleft palate production.
It 1s thought that TCDD forms a complex with the Ah receptor and becomes
Incorporated Into the chromatln. This alters the terminal differentiation
of the medial epithelial cells 1n the palate.
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9.1.3. 2,3,7,8-TCOD Studies In Rats. In an early study, Courtney and
Moore (1971) tested the teratogenlc potential of 2,3,7,8-TCDD 1n pregnant
rats (CD) Injected subcutaneously on a dally basis with 2,3,7,8-TCDD (0.5 or
2 vg/kg) 1n DMSO on days 6 through li, days 9 and 10, or days 13 and 14 of
gestation and examined on day 20 of gestation. Kidney malformations were
observed 1n fetuses exposed to 2,3,7,8-TCDD. In the group exposed transpla-
centaily at a dose of 0.5 pg/kg, 4/6 Utters had fetuses with kidney mal-
formations (average number of kidney defects/Utter was 1.8). An 11 and 34%
Incidence of kidney anomalies occurred 1n groups exposed to 2,3,7,8-TCDD on
days 9 and 10, and 13 and 14, respectively. In addition, six hemorrhaglc 61
tracts were observed 1rj the treated group (these data .were not enumerated
with respect to dose); however, this was considered a primary fetotoxlc
effect of 2,3,7,8-TCDD and not a malformation.
2,3,7,8-TCDD was administered by gavage to groups (10-14 animals/group)
of pregnant Sprague-Dawley rats at dose levels of 0, 0.03, 0.125, 0.5, 2.0
or 8.0 jig/kg/day on days 6 through 15 of gestation (Sparschu et al.,
1971b). No adverse teratogenlc effects were reported 1n fetuses exposed
transplacentally at the 0.03 vg/kg level. At the 0.125 yg/kg level,
three dead fetuses were reported,, fetal weights were slightly depressed, and
Intestinal hemorrhage was noted 1n 18 of 127 examined fetuses. In the group
given doses of 0.5 pg/kg, the number of viable fetuses was reduced,
resorptlons were Increased, 6 dead fetuses were reported, and 36 of 99
fetuses suffered an Intestinal hemorrhage. In the 2.0 jig/kg group, only 7
live fetuses were reported (occurring 1n only 4/11 Utters), 4 having
Intestinal hemorrhage. Early and late resorptlons were prevalent. No live
fetuses, but many early resorptlons, were reported In the group exposed to
8.0 yg 2,3,7,8-TCDD/kg/day. Subcutaneous edema appeared dose-related.
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occurring 1n a considerable number of fetuses from the higher dose groups.
Hale fetuses appeared to be more susceptible to 2,3,7,8-TCDD exposure;
however, there was no significant difference 1n the sex ratio of live
fetuses.
Khera and Ruddlck (1973) tested a wide range of 2,3,7,8-TCDD doses for
teratogenlc and fetotoxlc potential. Groups of 7-15 Wlstar rats were
Intubated with 2,3,7,8-TCDD at doses of 0.125, 0.25, 1, 2, 4, 8 or 16
vg/kg on days 6 through 15 of gestation. At day 22 of gestation, there
were no live fetuses 1n groups exposed to >4 pg/kg, and reduced Utter
size was observed 1n the 1 and 2 pg/kg group. Unspecified maternal toxlc-
1ty was reported In all groups where there was fetal mortality. In groups
exposed to 0.25-2 pg/kg, there were fetal anomalies observed as either
gross or microscopic lesions consisting of subcutaneous edema of the head
and neck, and hemorrhages 1n the Intestine, brain and subcutaneous tissue.
The Incidences of grossly observed lesions were 0/18, 2/11, 7/12 and 11/14
1n the control, 1, 1 and 2 pg/kg dose groups, respectively (the study was
conducted 1n two parts, and the 1 vg/kg dose was repeated). With regard
to the other dose levels tested, the table enumerating the results had an
entry of "not done." The Incidence of microscopically observed lesions for
the control, 0.25, 0.5, 1, 1 and 2 pg/kg groups was 0/10, 1/33, 3/31,
3/10, 3/6 and 3/7, respectively. There were no effects of treatment
observed 1n the 0.125 pg/kg group.
Khera and Ruddlck (1973) also exposed dams to 2,3,7,8-TCDD at doses of
0.125, 0.25, 0.5 and 1 pg/kg on days 6 through 15 of gestation and allowed
the dams to litter and wean the pups. In this experiment, maternal toxldty
was reported 1n the 0.5 and 1 vg/kg group. At birth, there were fewer
viable pups, and the pups had lower body weight 1n all but the 0.125
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group. At weaning on day 21 after birth, there were no surviving pups in
the 1 jig/kg group, and 40% of the pups 1n the 0.5 wg/kg group did not
survive. Fostering pups from dams exposed to 2,3,7,8-TCDO at 1 yg/kg onto
control dams did not appreciably Increase survival, while fostering control
pups onto dams exposed to 2,3,7,8-TCDD did not Increase pup mortality.
These data suggest that poor pup survival was a result of delayed toxlclty
from in. uterq exposure to 2,3,7,8-TCDD.
61av1n1 et al. (1982a) assessed the effect of small doses of 2,3,7,8-
TCDD administered during the prelmplantatlon period 1n Sprague-Daw!ey rats.
The animals, In groups of 20, were treated by gavage with 2,3,7,8-TCDD at
doses of 0.0, 0.1, 0.5 and 2 pg/kg on days 1-3 of gestation. (The legends
to the tables 1n this paper Indicated that the low dose was 0.125 vg/kg.)
At day 21 of gestation, no toxic effects were observed 1n the dams except
for a decrease from 19.3-12.9 g 1n average maternal weight gain 1n the high
dose animals as compared with controls. In the fetuses, weight was signifi-
cantly reduced (p<0.05) 1n the 0.5 and 2 vg/kg groups. Malformed Utters
and malformation/fetuses examined were 2, 5, 5 and 6, and 2/270, 8/260,
5/255 and 8/253, respectively, 1n the control 0, 0.1, 0.5 and 2 yg/kg
groups; however, these Increases 1n the treated animals were not statisti-
cally significant. The anomalies observed were restricted to cystic kidney.
This exposure to 2,3,7,8-TCDD early 1n pregnancy did not affect Implantation
frequency, and the decrease 1n fetal weight was considered a result of
2,3,7,8-TCDD delayed Implantation.
In a second study, S1av1n1 et al. (1983) administered the same doses of
2,3,7,8-TCDD (0.0, 0.125, 0.5 or 2 yg/kg) dally to 15 female CRCD rats per
group by gavage 1n corn o1l:acetone (9:1) for 2 consecutive weeks before
mating. Females that did not become pregnant during three estrous cycles
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were necropsled to determine signs of toxldty, while pregnant animals were
allowed to proceed to day 21 of gestation, at which time necropsies were
performed with particular emphasis on reproductive organs and reproductive
success. At the lowest dose tested (0,125 vg/kg), there were no overt
clinical signs of toxldty 1n the dams or adverse effects 1n any of the
fetal parameters examined. At the 0.5 and 2 yg/kg levels, average
maternal weight was decreased. Also, one animal 1n each of these groups did
not become pregnant, although necropsy did not reveal any obvious dysfunc-
tions. The only other overt sign of toxldty was Ustlessness during the
treatment period 1n the animals of the high-dose group. The only signif-
icant (p<0.01) fetal effect observed 1n the 0.5 vg/kg group was an
Increase 1n postlmplantatlon losses from 2.9% 1n the control group to 10.2%.
In the high-dose group, there were decreases 1n corpora lutea and Implanta-
tions (averages of 17.6% In control and 14.9% 1n treated animals, and 15.5%
1n control and 12.0% In treated animals, respectively), and Increases 1n
both pre- and postlmplantatlon losses of 11.7% for controls and 19.5%
(p<0.05) 1n treated animals, and 2,9% 1n control and 30.3% (p<0.001) 1n
treated animals, respectively. In addition to these signs of fetal toxlc-
1ty, 9/10 Utters In the high-dose group contained at least one malformed
fetus as compared with 1/13, 2/13 and 2/13 1n the control, 0.125 and 0.5
wg/kg groups. The predominant fetal malformations were cystic kidney and
dilated renal pelvis, which have been observed 1n other studies 1n which
2,3,7,8-TCDD was administered during gestation.
The reproductive effects of 2,3,7,8-TCDD were also studied 1n a 3-gener-
atlon study using Sprague-Daw!ey rats (Hurray et a!., 1979). Throughout the
study, animals were continuously maintained on diets providing doses of 0,
0.001, 0.01 or 0.1 pg 2,3,7,8-TCDD/kg/day. The parental group (fQ) was
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maintained for 90 days on the test diets before mating. The ffl rats were
mated twice, producing the filial generations (f,. and f-io)- Selected
f,D and f0 rats were mated at ~130 days of age to produce the f« and
IB c c
f- Utters, respectively. In later generations, the high-dose group (0.1
pg 2,3,7,8-TCDD/kg/day) was discontinued because few offspring were pro-
duced In this group. At the Intermediate dose (0,01 pg/kg/day), 2,3,7,8-
TCDD caused lower body weight 1n exposed rats of both sexes (f, and f2).
At the low dose, no toxic effects were discerned.
Fertility was greatly reduced 1n the fQ generation exposed to 0.1 pg
2,3,7,8-TCDD/kg/day. At 0.01 pg 2,3,7,8-TCDD/kg/day, fertility was
significantly (p<0.05) reduced 1n the f1 and f2 rats. Fertility 1n rats
(of any generation) exposed to 0.001 pg 2,3,7,8-TCDD/kg/day was not
different from that of control rats. Decreases In Utter size were noted In
the f,. group exposed to 0.1 pg/kg/day and the fp and f,. Utters
exposed at 0.01 pg/kg/day. Statistically significant decreases 1n fetal
survival throughout gestation were noted 1n f_ and f_ Utters of the
0.01 tig 2,3,7,8-TCDD/kg/day exposed dams. At 0.001 pg 2,3,7,8-TCDD/kg/
day, a decreased gestatlonal survival was reported for the f? Utters, but
not for other generations. Decreased neonatal survival was noted among
f.|A and f2 pups exposed to 0.01 pg 2,3,7,8-TCDD/kg/day, but not among
fio or f0 pups. Postnatal body weights of the f~ and f- Utters at
ID J C. J
0.01 pg 2,3,7,8-TCDD/kg/day were significantly depressed. At the low dose
(0.001 pg 2,3,7,8-TCDD/kg/day), necropsy of 21-day-old pups revealed a
statistically significant (p<0,05) Increase 1n dilated renal pelvis 1n the
f1 generation. Subsequent generations at this dose level or any at the
Intermediate dose (0.01 pg 2,3,7,8-TCDD/kg/day) did not have a significant
Increase 1n this abnormality. Significantly decreased thymus weight and
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Increased liver weight were reported 1n the fg generation, but not 1n the
f, generation (f_ generation data not obtained) of the Intermediate dose
group. Hurray et al. (1979) concluded that 2,3,7,8-TCDD Ingested at 0.01 or
0.1 pg/kg/day Impaired reproduction among rats, and NOAELs were associated
with 0.001 vg 2,3,7,8-TCDD/kg/day.
Nlsbet and Paxton (1982) reevaluated the primary data of Hurray et al.
(1979) using different statistical methods. From this reevaluatlon 1t was
concluded that 2,3,7,8-TCDD significantly reduced the gestatlonal Index,
decreased fetal weight, and Increased I1ver-to-body weight ratios and the
Incidence of dilated renal pelvis 1n both lower-dose groups. Nlsbet and
Paxton (1982) concluded that the dose of 0.001 pg/kg/day was not a NOAEL
In this study. The FIFRA Scientific Advisory Panel has also reviewed the
data from this 3-generat1on study and concluded that the effects observed at
the 0.001 vg/kg dose were not consistent enough between the different
generations to consider them treatment-related (U.S. EPA, 1979b). Although
the panel considered the data suggestive of an embryotoxlc effect, they
concluded that 0.001 tig/kg represented a NOEL.
Crampton and Rogers (1983) 2,4,5-tr1chlorophenoxyacet1c add (2,4,5-T)
contaminated with 30 ppb of TCDD appears to have behavlorally teratogenlc
effect 1n Long-Evans rats at doses as low as 6 mg 2,4,5-T/kg bw administered
to mother rats on day 8 of gestation.
9.1.4. 2,3,7,8-TCDD Studies 1n Rabbits and Ferrets. A report by G1av1n1
et al. (1982b) describes the effects of exposure to 2,3,7,8-TCDD on fetal
development 1n rabbits. Groups of 10-15 New Zealand rabbits were adminis-
tered 2,3,7,8-TCDD by gavage at doses of 0.0, 0.1, 0.25, 0.5 and 1 vg/kg
on days 6 through 15 of gestation. The dams were examined for Implantation
sites, resorptlons and live fetuses, and the fetuses were examined for
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malformations on day 28 of gestation. Decreased maternal weight gain and
unspecified signs of maternal toxldty occurred In dams exposed to 2,3,7,8-
TCDD at doses of >0.25 yg/kg. At doses of 0.5 and 1 yg/kg, there were 2
and 4 deaths, respectively, among the dams. There were Increases 1n abor-
tions and resorptlons at a dose of >0.25 yg/kg, and no live fetuses were
detected 1n the high dose group. In the fetuses, the most common observa-
tion was a -significant Increase 1n extra ribs from 33.3% 1n the controls to
82, 66.6 and 82% 1n the 0.1, 0.25 and 0.5 yg/kg dose groups. Although
there was no significant Increase 1n specific soft-tissue anomalies, there
was an Increase from 0/87 to 3/78, 2/33 (p<0.05) and 2/28 (p<0.05) 1n total
soft-tissue anomalies 1n the control, 0.1, 0.25 and 0.5 yg/kg groups. The
most prevalent soft-tissue anomaly was hydronephrosls, which the authors
pointed out was a common finding In rat fetuses exposed to 2,3,7,8-TCDD Vn
utero. These effects were considered to be signs of embryotoxldty rather
than a teratogenlc effect.
In addition to the fetotoxlc effects of prenatal exposure to 2,3,7,8-
TCDD, Norman et al. (1978b) demonstrated that 2,3,7,8-TCOD could Induce
liver mlcrosomal enzymes following in utero exposure. Pregnant New Zealand
rabbits were given subcutaneous Injections of 2,3,7,8-TCDD at a dose of 30
nmol/kg (9.6 yg/kg) on day 24 of gestation, and the livers of newborns
were examined for enzyme activity within 12 hours after birth. While this
treatment Increased the liver cytochrome P-450 levels In the adults ~2-fold,
from 1.8-3.7 nmol/mg protein, the Increase In the newborns was ~5-fold, from
0.3-1.6 nmol/mg protein. SDS-polyacrylam1de gel electrophoresls revealed
that 2,3,7,8-TCDD Induced a single form (form 6} of cytochrome P-450, and
that this form was one of the two that were also Induced by 2,3,7,8-TCDD 1n
the adult Hver. The Identity of form 6 was confirmed by 1mmunolog1c
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reaction and Its peptlde fingerprint. It was shown that Induction of cyto-
chrome P-450 1n newborns resulted 1n levels of benzo(a)pyrene hydroxylase
and 7-ethoxy-resoruf1n-0-deethylase activity similar to adult levels. The
consequence to the newborn of these changes 1n the development of liver
mlcrosomal enzymes has not been established.
Huscarella et al. (1982) reported 1n an abstract the fetotoxlc and
teratogenlc' effects of subcutaneously administered 2,3,7,8-TCDD on ferrets.
An unspecified number of animals received 1, 6, 13.5, 20, 30 or 60 yg of
2,3,7,8-TCDD/kg on day 18 of gestation or two doses given on days 18 and 20
of gestation at one-half the level of the single dose. The animals were
examined on day 28, 29 or 30 of gestation and the results were reported
without reference to specific experimental groups. In all test groups there
were Increases 1n fetal deaths and resorbed fetuses, along with growth
retardation. Terata observed Included unilateral and bilateral patalos-
ch1s1, open eyelids, anasarca and brachygnathla. The author concluded that
2,3,7,8-TCDD was a teratogen 1n ferrets.
9.1.5. 2,3,7,8-TCDD Studies 1n Nonhuman Primates. Dougherty et al.
(1975) found no evidence of teratogenldty or embryotoxldty 1n rhesus
monkeys that were given on days 22-38 of gestation dally oral doses (1n
gelatin capsules) of up to 10 mg/kg/day of 2,4,5-T containing 0.05 ppm
2,3,7,8-TCDD. The 2,3,7,8-TCDD dose at the highest dose level of 2,4,5-T
administered (10 mg/kg/day) would correspond to 0.5 vg 2,3,7,8-TCDD/kg/
day. Palate closure 1n the monkey, however, occurs on gestatlonal days
42-44 and the kidney 1s also a late developing organ.
Adverse effects of exposure to 2,3,7,8-TCDD on reproductive success 1n
monkeys have also been described. Schantz et al. (1979) fed a diet contain-
ing 50 ppt 2,3,7,8-TCDD to rhesus monkeys for 20 months. Seven months Into
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the study the female monkeys were bred to control males. There were four
abortions and one stillbirth; two monkeys did not conceive even though they
were mated repeatedly; and two monkeys carried their young to term. The
total 2,3,7,8-TCDD Intake over the 7 months was estimated by the authors to
be 0.35 vg/kg, corresponding to a calculated dally dose of 0.0015 jig
2,3,7,8-TCDD/kg/day.
Allen et al. (1979) and Barsottl et al. (1979) fed adult female rhesus
monkeys for 6-7 months on diets containing 50 or 500 ppt of 2,3,7,8-TCDD.
These exposure levels correspond to total doses per animal at the end of 7
months of 1.8 and 11.7 v9 2,3,7,8-TCDD. Although menstrual cycles were
not affected 1n either treatment group, 5/8 animals In the high-dose group
had either decreased serum estradlol or decreased progesterone levels.
Hormone levels were normal 1n the low dose animals. At 7 months, the
females were bred with nonexposed males, and 6/8 and 3/8 females 1n the low-
and high-dose groups, respectively, were Impregnated. The animals were
continued on treatment during pregnancy. Of the Impregnated animals, 4/6
and 2/3 had spontaneous abortions, while the remaining Impregnated animals
had normal births. All of the control females (one group of 8 and another
group of unspecified size) conceived and gave birth to "normal" offspring.
The high dose resulted 1n the death of five animals between the 7th and 12th
month of treatment.
HcNulty (1978) treated pregnant rhesus monkeys by gastric gavage to
2,3,7,8-TCDD In a vehicle of corn o1l:acetone solution. Group I animals
were administered total dosage of 5 jig/kg bw (two animals), 1 ^g/kg bw
(four animals) and 0.2 tig/kg bw (four animals) 1n nine divided doses, 3
times/week during weeks 4, 5 and 6 (days 20 through 40) after conception.
Group II, consisting of 12 animals, received single doses of 1 yg/kg bw of
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2,3,7,8-TCDD on days 25, 39, 35 and 40 after conception. Three animals were
exposed In each of these 4 days. The vehicle control group, consisting of
11 animals, was treated with corn o1l:acetone only, on the same schedule as
Group I animals. Both of the females that received the highest dose (5
vg/kg) had fetal losses. In the next lower-dosed animals (1 vg/kg 1n
both groups), 12 of 16 females had fetal losses; and In the lowest-dosed
animals (0.2 vg/kg 1n Group I), one abortion occurred 1n four pregnancies.
Maternal toxIcHy was observed 1n many of these treated females. The
difference 1n frequency of fetal loss between all pregnant animals given 1
vg/kg and the rate of historical abortion 1n the author's breeding colony
was found to be significant. The author concluded that short exposure to 1
vg/kg bw of 2,3,7,8-TCDD during early pregnancy results 1n fetal loss 1n
rhesus monkeys. In a recent report, McNulty (1984) reveals that he failed
to detect any malformations 1n the fetus but observed widespread maternal
toxldty and fetocldal effects 1n monkeys as a result of 1ntragastr1c
exposure to 2,3,7,8-TCDD.
9.1.6. Studies 1n Chickens. The effects of 2,3,7,8-TCDD on the develop-
ment of the heart 1n chicken embryos was studied by Cheung et al. (1981) as
a consequence of the known Induction of hydroperlcardlum by 2,3,7,8-TCDD 1n
adult chickens and the relation between changes 1n hemodynamlcs and cardio-
vascular malformation. Groups of at least 20 White-Leghorn eggs were
Injected with 2,3,7,8-TCDD 1n acetoneicorn oil (0.5:9.5 v/v) on day zero of
embryo development. Administered doses ranged from 0.009-77.5 pmol/egg
(0.00029-2.5xlO~2 vg/egg) 1n 5 v&. The embryos were examined on day
14 of development. A dose-related Increase 1n cardiovascular malformations
was observed with 1 pmol/egg resulting 1n malformations 1n 50% of the
embryos. Increases In all types of malformations (ventricular septa!
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defect, aortic arch anomaly, aorllc arch anomaly and ventricular septa!
defect, and conotruncal malformations) occurred. Hydroperlcardlum was
observed 1n some embryos {not enumerated), but It could not be concluded
that this was the cause of the cardiovascular malformations. Malformed legs
and crossed beaks associated with m1cropthalm1a was observed In treated
embryos, however, the Incidence, 7/284 and 2/284, respectively, was low.
9.1.7. Studies of the Teratogenlc and Reproductive Effects of HxCDD. In
addition to 2,3,7,8-TCDD, the teratogenlc potential of a related chlorinated
d1benzo-p_-d1ox1n compound, HxCDD (congeners not specified), has been Invest-
igated 1n rats. Pregnant Sprague-Dawley rats were treated by gavage with
0.1, 1.0, 10 or 100 pg HxCDD/kg/day on days 6-15 of gestation {Schwetz et
a!., 1973). Treatment with high levels of HxCDD (10 and 100 wg/kg) was
highly lethal to fetuses during late gestation. There was a significant
dose-related Increase In late resorptlons from 0% (at 0.1 yg/kg/day) to
79% (at 100 jig/kg/day). Decreases 1n the weight and length of surviving
fetuses were due to HxCDD. The Incidences of cleft palate, subcutaneous
edema, malformed vertebrae and split sternebrae were significantly Increased
In fetuses of rats treated with 100 yg HxCDD/kg/day. No Increase 1n fetal
anomalies was noted In fetuses exposed to 0.1 jig HxCDD/kg, and only
subcutaneous edema was more prevalent 1n groups exposed at 1 or 10 yg
HxCDD/kg/day when compared with controls.
Pertinent Information regarding the teratogenldty or reproductive
effects of PeCDDs was not located 1n the available literature.
9.2. STUDIES ON HUMAN POPULATIONS
A positive association between 2,4,5-T exposures and Increases 1n birth
defects or abortions has been reported 1n human populations 1n Oregon (U.S.
EPA, 1979c), New Zealand (Hanlfy et a!., 1981), and Australia (Field and
9-23
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Kerr, 1979). A lack of any such association has been reported 1n human
populations 1n Arkansas (Nelson et al., 1979), Hungary (Thomas, 1980b), New
Zealand (Dept. of Health, New Zealand, 1980; McQueen et al., 1977), and
Australia (Aldred, 1978). Almost all of the reports are geographic correla-
tion studies, and because of the uncertainties Inherent 1n this type of
ep1dem1olog1c Investigation, as well as the difficulties 1n distinguishing
the effects of 2,4,5-T from those of 2,3,7,8-TCDD contamination, none of the
reportedly positive associations unequivocally Identify either 2,4,5-T or
2,3,7,8-TCDD as the causative agent. Similarly, the reportedly negative
associations do not rule out 2,4,5-T or 2,3,7,8-TCDD as potential teratogens
or abortlfadents 1n humans.
Based on a report of a high Incidence of abortions 1n a small group of
women living around Alsea, Oregon, who may have been exposed to the herbi-
cide 2,4,5-T from aerial spraying (Smith, 1979), the U.S. EPA (1979c)
Initiated a study, often referred to as the "Alsea II study," to determine
1f spontaneous abortion rates differed between the exposed and unexposed
populations, 1f spontaneous abortion rates evidenced seasonal variation 1n
these two groups, and 1f such seasonal variations were associated with
2,4,5-T spray application.
The Spontaneous Abortion Rate Index, as defined by the U.S. EPA, 1s
"basically the ratio of the number of hospitalized spontaneous abortions to
the number of births corresponding to the spontaneous abortions, based on
the residence zip code of the women contributing to each event." Upon
completion of the study, the U.S. EPA concluded that (1) the 1972-1977
Spontaneous Abortion Rate Index for the study area was significantly higher
than 1n the Rural Control Area or the Urban area; (2) there was a statisti-
cally significant seasonal cycle 1n the abortion Index 1n each of the areas
with a period of ~4 months. In particular there was an outstanding peak 1n
9-24
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the study area In June; and (3) there was a statistically significant corre-
lation between the Spontaneous Abortion Rate Index and spray patterns 1n the
study area when a lag-time of 2 or 3 months was Included. The U.S. EPA
concluded, however, that "This analysis 1s a correlational analysis, and
correlation does not necessarily mean causation,"
H1lby/ et al. (1980), citing three critiques of the Alsea II study (not
published 1n the open literature), state that the statistical method and
basic design of the Alsea II study were sufficiently flawed to make this
study of no use 1n human risk assessment. The Alsea II study has also been
reviewed by a panel of scientists who, 1n a published report of their
meeting, also concluded that the basic design of the study was Inadequate to
demonstrate either an effect or absence of an effect of exposure to 2,4,5-T
(Coulston and Olajos, 1980). The major Inadequacies of the study were that
the data collection methods were likely to result 1n the underestimation of
abortions, particularly 1n the urban area (the Incidence of abortions 1n all
three groups was within the expected background rate of 8-15%); only a small
part of the area from which the exposed subjects were selected was actually
sprayed with 2,4,5-T, and the study was not controlled for other factors
such as age, smoking habits and alcohol consumption, which may affect the
spontaneous abortion rate. Based on a new report by Smith (1979), the U.S.
EPA Is attempting or has attempted to correlate 2,3,7,8-TCDO levels 1n the
affected areas with the observed rate of abortion. No published reports
have been located on the outcome of this effort.
Nelson et al. (1979) noted a general Increase 1n the reported Incidence
of facial cleft 1n both high and low exposure groups 1n Arkansas from
1948-1974. In this study, exposure estimates were based on average
9-25
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rice production In different areas of Arkansas, and the Incidence of cleft
palate was determined by screening birth certificates and checking records
of the Crippled Children's Services. No consistent exposure/effect correla-
tions were noted, and the general Increase with time In the Incidence of
facial clefts was attributed to better reporting procedures; however, there
does not have to be a direct correspondence of malformations 1n human beings
and experimental animals.
Of the four reports available from New Zealand (Dept. of Health, New
Zealand, 1980; McQueen et al., 1977; Hanlfy et al., 1981; Smith et a!.,
1982a), the report by the Department of Health 1s essentially anecdotal,
Involving two women who gave birth to malformed children (one with an atria!
septal defect and a malformation of the trlcuspld valve of the heart, and
the other with biliary atresla). In both cases, exposure to 2,4,5-T could
not be ruled out. Based on an analysis of spraying records, the time course
of the pregnancies and plant damage near the women's homes, however, the
Department of Health, New Zealand (1980) concluded that there was Insuffic-
ient evidence to Implicate 2,4,5-T spraying as a causative factor. Even 1f
the spraying had been Implicated, a lack of Information on 2,3,7,8-TCDD
levels 1n the spray and the absence of any monitoring data on 2,4,5-T or
2,3,7,8-TCDD would limit the usefulness of this report.
The study by McQueen et al. (1977) 1s not published 1n the open litera-
ture but 1s summarized by MUby et al. (1980). According to the summary,
McQueen et al. (1977) "...examined the epidemiology of neural-tube defects
In three areas 1n New Zealand and concluded 'there 1s no evidence to
Implicate 2,4,5-T as a causal factor 1n human birth defects."' No addi-
tional details are provided.
9-26
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et al. (1981) performed an ep1dem1olog1c study 1n Northland, New
Zealand, 1n areas where spraying of 2,4,5-T was done by various companies
for a number of years. The rate of birth defects was obtained from an
examination of hospital records In seven nonoverlapplng areas on a monthly
basis over a period extending from 1959-1977. The rate of birth defects
from 1959-1965 represented the rate for a nonexposed population since this
was prior to the use of 2,4,5-T, while the Incidence of birth defects from
1972-1976 represented the rate for the exposed population. During the time
of the survey there were 37,751 births, 436 stillbirths, 264 deaths shortly
after birth, and 510 congenital anomalies. Three categories of birth
defects, heart abnormalities, hypospadlas and eplspadlas, and talipes, had
elevated rate ratios of >1 (p=0.05) In comparisons between the exposed
(1972-1976) and control (1959-1965) populations. Exposure estimates were
made for the seven areas and for different years using company records of
aerial spraying and a model that factored 1n assumed fractional removal
rates/month (this factor was assumed to be either 1.0 or 0.25). Comparisons
of the rate of specific malformations with exposure demonstrated a statisti-
cally significant association between the occurrence of talipes and exposure
when the fractional removal rate was assumed to be 0.25. There was, how-
ever, no statistically significant association where 1.0 was used as the
fractional removal rate.
Smith et al. (1982a) Investigated the outcome of pregnancy 1n families
of professional 2,4,5-T applicators and agricultural contractors 1n New
Zealand. Agricultural contractors were chosen as the control population
since both sprayers and contractors were of the same economic group with
similar outdoor occupations. The. survey was conducted by mall with 8954 of
the chemical applicators responding and 83% of the agricultural contractors
9-27
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responding to questions asking whether they used 2,4,5-T and Its temporal
relationship to reproductive histories regarding birth, miscarriages, still-
births and congenital defects. The relative risks of congenital defects and
miscarriages were 1.19 (0.58-2.45% confidence limits) and 0.89 (0.61-1.30%
confidence limits) for the wives of chemical sprayers as compared with the
wives of agricultural contractors. These data Indicate that exposure of
fathers and mothers (I.e., while cleaning clothes) had no effect on the
outcome of pregnancy. Biases that may have affected the results, such as
the age of the mother at childbirth, smoking habits and birth to Maori
parents were Investigated and eliminated as possible confounders.
The two reports from Australia (Aldred, 1978; Field and Kerr, 1979) also
present apparently conflicting results. The report by Aldred (1978) 1s not
published 1n the open literature, but the following summary Is taken from
Hllby et al. (1980): "The report concluded that birth defects In a group of
babies born 1n the [Yarram] district 1n 1974 and 1976 could not be attrib-
uted to exposure to 2,4,5-T or 2,4-D." Additional details that might be
useful 1n assessing the rationale for this statement are not provided 1n the
summary. The report by Field and Kerr (1979) plotted the Incidence of
neural-tube defects (anencephaly and menlngomyelocele) 1n New South Wales,
Australia, over the years 1965-1975, and the usage of 2,4,5-T 1n all of
Australia during the previous years. The authors noted a decrease In the
Incidence of neural-tube defects expected on the basis of the plotted line
In 1975 and 1976, when Australia Instituted monitoring of 2,4,5-T to ensure
a 2,3,7,8-TCDD level <0.1 ppm. The data were not tested for significance;
although Field and Kerr (1979) Indicate that they consider the ep1dem1olog1-
cal data on neural-tube defects to be "relatively complete," they do not
comment on the Increasing Incidence of neural-tube defects during the time
9-28
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period of this study and whether or not an Increase 1n the thoroughness of
reporting neural-tube defects could have contributed to the apparent corre-
lation of 2,4,5-T exposure with these defects. A replottlng of the data
suggests that the Incidence of cleft palate correlates better with 2,4,5-T
usage than with time. Nonetheless, the appropriateness of correlating
2,4,5-T usage 1n all of Australia with the Incidence of defects 1n one area
of Australia 1s questionable.
Thomas (1980b) used an approach similar to that of Field and Kerr (1979)
on data from Hungary. One major difference, however, 1s that Thomas (1980b)
compared the Incidence of stillbirths, cleft Up, cleft palate, splna
blflda, anencephalus and cystic kidney disease 1n all of Hungary between
1976 and 1980 with 2,4,5-T use 1n 1975 1n all of Hungary. Because Hungary
requires compulsory notification of malformations diagnosed from birth to
age 1 year, because a relatively large percentage (55%) of the Hungarian
population lives 1n rural areas where 2,4,5-T exposure may be expected to be
greatest, and because annual use of 2,4,5-T 1n Hungary had risen from 46,000
kg In 1969 to 1,200,000 kg 1n 1975, Thomas (1980b) considered Hungary to be
"...probably the best country In which to examine possible health effects of
this herbicide." All Indices of birth defect rates decreased or remained
stable over the period of study.
In addition to contamination of 2,4,5-T being a potential source of
2,3,7,8-TCDD exposure, 2,3,7,8-TCDD 1s also an Inadvertent contaminant of
2,4,5-trlchlorophenol (TCP). Chronic exposure to 2,3,7,8-TCDD may occur
during the manufacture of TCP and high level acute exposure to 2,3,7,8-TCDD
has occurred after an accident 1n July, 1976 at the ICHESA TCP chemical
factory 1n Seveso, Italy (Bonaccorsl et a!., 1978). In this accident, the
reaction used to produce TCP became uncontrolled, producing conditions
9-29
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favorable for 2,3,7,8-TCDD formation before venting the contents of the
chemical reactor Into the atmosphere. The resulting cloud of chemicals
settled over a heavily populated area. Although the amount of 2,3,7,8-TCDD
released was not known, the reported cases of chloracne, a symptom of acute
exposure to 2,3,7,8-TCOD, Indicated that exposure to 2,3,7,8-TCDD had
occurred. Some preliminary results are available from ep1dem1olog1c studies
of reproductive events 1n the Inhabitants of Seveso, and recently a study
has become available on the reproductive history of men employed 1n the
chemical manufacturing Industry with possible chronic exposure to 2,3,7,8-
TCOD {Townsend et al., 1982).
Ep1dem1olog1c studies to determine the reproductive effects In Individ-
uals exposed to 2,3,7,8-TCDD and TCP following the accidental contamination
of a populated area around Seveso, Italy, are not completed. The Incidence
of spontaneous abortions occurring between March 1976 and January 1978 have
been reported for Inhabitants 1n the area around Seveso by Bonaccorsl et al.
(1978), Regg1an1 (1980) and B1sant1 et al. (1980). The spontaneous abortion
rate 1n the contaminated area for the three trimesters following the acci-
dent was 13.1, 11.0 and 13.05%, which was similar to the worldwide li-20%
frequency of spontaneous abortion. Subdividing the contaminated area Into
highly, moderately, and least contaminated, and examining the rates for each
area Individually, also failed to demonstrate any change 1n the spontaneous
abortion rate. The Incidence rates of malformations also were examined;
however, the numbers were too few for meaningful assessment. There are
several Inadequacies 1n these studies that might make them Insensitive 1n
detecting reproductive effects. The authors noted that there are many
difficulties 1n Interpreting these data. Adequate data on the Incidence
9-30
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rates of spontaneous abortions and birth defects were not adequately avail-
able for the region before the accident as a result of suspected under-
reporting. There was Inadequate reporting even after the accident because
of political turmoil with regard to the management of health services.
Also, an unknown number of pregnancies were surgically aborted for fear of
2,3,7,8-TCDD-lnduced birth defects. In a recent review of the progress of
epidemlologic Investigations of the Seveso accident, Tognonl and Bonaccors!
(1982) Indicated that the data on spontaneous abortions and malformation
rates still needed verification, and that these data were too preliminary to
allow for conclusions.
Townsend et al. (1982) Investigated the reproductive history of wives of
employees potentially exposed to 2,3,7,8-TCDD during chlorophenol production
1n Midland, HI. A total of 930 potentially exposed males were Identified
who had worked for >l month between January 1939, and December 1975, 1n a
job with potential 2,3,7,8-TCDD exposure. Exposure estimates of low,
moderate and high were made by an Industrial hyglenlst primarily from Job
description and surface contamination data; however, the high potential
exposure group was reserved for process workers during 1963-1964 when
changes 1n operations resulted 1n a number of cases of chloracne. The
control population was an equal number of male employees not Involved 1n any
process that might Involve exposure to 2,3,7,8-TCDD and matched for date of
hire. In these groups, 586 wives were Identified and 370 agreed to partici-
pate as the exposed group, while 345 wives of a potential control group of
559 agreed to participate. After Identification of the participants, a
personal Interview was conducted with the wives to determine pregnancy
outcome. Of the total of 737 conceptions 1n the exposed category and 1785
conceptions In the control category (conceptions that occurred 1n the
9-31
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exposed group before work records Indicating potential exposure to 2,3,7,8-
TCDO were placed 1n the control group), there was no statistically signifi-
cant Increase 1n spontaneous abortions, stillbirths. Infant deaths or
selected congenital malformations. Sample sizes were too small to provide
meaningful data 1f the populations were subdivided by extent of exposure.
The authors suggested that many confounding factors could account for these
negative results, such as the Inappropriate selection of the populations,
the use of "exposed" persons 1n both exposed and control groups, unidenti-
fied covarlables and low power; however, It was asserted that these results
were consistent with animal data, which report that paternal exposure to
2,3,7,8-TCDD does not affect the conceptus.
Poole (1983), 1n testimony before the House Committee on Science and
Technology, described a reanalysls of the primary data used by Townsend et
al. (1982). In this reanalysls, the relative risk of cleft palate and cleft
Up were reported to be 1.9 {90% confidence Intervals of 1.0-3.6) 1n the
years 1971-1974 for both the control and exposed groups (the comparison
population was not described). At the same House Committee hearing, Houk
(1983) presented data from the Birth Defect Monitoring Program of the
Centers for Disease Control on the yearly rate of cleft palate alone or
cleft Up with or without cleft palate for births 1n Midland County,
Michigan (the site of Dow's chlorophenol production facility) during the
years 1970-1981. The data Indicated an Increased rate for these defects of
between 50 and 100% 1n the years 1971-1975, with the rate returning to
normal from 1976-1981. The observed Increase was statistically signif-
icant 1f the rates for cleft palate alone and cleft Up with or without
cleft palate were combined; however, 1t was the opinion of Houk (1983) that
these defects should not be combined since the causal mechanism may be
9-32
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different. The Michigan Department of Public Health (1983a) also reported
these results and, in addition, demonstrated that the same results occurred
1f the comparison was made with other counties 1n Michigan as well as with
the general population of the United States. It was noted 1n this report
that "runs" of Increases 1n oral cleft for successive years have occurred In
six other counties with no obvious chemical exposure. The Michigan Depart-
ment of Public Health (1983a) Interpreted the data to Indicate that a more
detailed case control study was necessary to determine 1f any common factors
may exist, such as exposure to chemicals contaminated with 2,3,7,8-TCDD.
A similar but limited study of the reproductive history of the wives of
employees of the Long Island Railroad was performed by Honchar for NIOSH
(1982). The employees were concerned about the use of 2,4,5-T for mainte-
nance along the right-of-way. There were 170 live births as Indicated by
union files during the study period from 1975-1979. For each birth. Insur-
ance claims were reviewed to determine any health problems during the first
year of life. The Incidence of major birth defects was underrepresented In
the study population when compared with data from the Metropolitan Atlanta
Congenital Defects Program (3 observed and 3.81 expected). Some minor
health problems (I.e., tear duct obstruction) were elevated; however, the
authors considered this to have resulted from diagnostic bias. It was
concluded that no association between birth defects and exposure to 2,4,5-T
was demonstrated 1n this study.
To test any possible association between birth defects and exposure to
Agent Orange In Vietnam veterans, Erlckson et al. (1984) conducted a case-
control study on newborns with various types of congenital defects 1n the
metropolitan Atlanta area during the years 1968 through 1980. Though most
of the Vietnam veterans received from the Army Agent Orange Task Force an
9-33
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estimated opportunity Index score regarding their exposure to Agent Orange,
25% of the Vietnam veterans Interviewed 1n this study felt that they were
exposed and approximately an equal proportion did not know 1f they were
exposed to Agent Orange. Increased estimated risks for fathering babies
with 1) splna blflda, 2) cleft Up with or without cleft palate and 3) cer-
tain tumors were found 1n this study. However, the authors concluded that
"Vietnam veterans who had greater estimated opportunity for Agent Orange
exposure did not seem to be at a greater risk for fathering babies with all
types of defects combined" (Erlckson et a!., 1984).
9.3. OTHER REPRODUCTIVE EFFECTS
The effects of a mixture of 2,4,5-T, 2,4-D and 2,3,7,8-TCDD (simulated
Agent Orange; however, the free adds were used rather than butyl esters to
eliminate problems of volatility) on the fertility and reproductive capaci-
ties of male C57B1/6 mice were studied by Lamb et al. (1980, 1981a). Groups
of 25 mice were treated with dietary levels of the three compounds so that
the dally doses/kg bw were 40 mg each of 2,4,5-T and 2,4-D, and 2.4 jig of
2,3,7,8-TCDD (Group II); 40 mg each of 2,4,5-T and 2,4-D and 0.16 pg of
2,3,7,8-TCDO (Group III); or 20 mg each of 2,4,5-T and 2,4-D and 1.2 vg of
2,3,7,8-TCDD (Group IV). A vehicle control group (Group I) was given a diet
containing 2% corn oil. An 8-week exposure period was followed by an 8-week
observation period during which fertility and reproductive assessments were
conducted. Sperm concentrations, sperm motmty and sperm abnormalities
were evaluated. In addition, the males were mated with virgin females (3
females/week for 8 post-treatment weeks) to assess mating frequency, average
fertility, percent Implantations and resorptlons, and percent fetal malfor-
mations. There was no significant decrease 1n any of the parameters used as
a measure of fertility and reproductive capacity 1n any groups of treated
9-34
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mice when compared with controls. Lamb et al. (1981b), In a further report
of this work. Indicated that germ cell toxldty was not apparent and
survival of offspring of exposed mice was unaffected. No external, visceral
or skeletal terata were noted 1n offspring whose sires were exposed to the
phenoxy adds/2,3,7,8-TCDD mixture 1n this study. The only effects noted
were dose-related decreases 1n body weight 1n the treated males, and these
effects were reversed when treatment was terminated,
9.4. SUMMARY
2,3,7,8-TCDD has been demonstrated to be teratogenlc 1n all strains of
mice tested. The most common malformations observed are cleft palate and
kidney anomalies; however, other malformations have been observed occasion-
ally. With an MED of 1 Pg/kg/day for mice, 2,3,7,8-TCDD 1s the most
potent teratogen known. At higher doses, 2,3,7,8-TCDD has a marked feto-
toxlc effect, as measured by decreased fetal weight and Increased fetal
toxldty. Hemorrhaglc GI tract has been associated with 2,3,7,8-TCDD fetal
toxldty.
In rats, 1t has also been observed that 2,3,7,8-TCDD produced terato-
genlc and fetotoxlc responses 1n all strains tested. In this species, the
most common fetal anomalies observed were edema, hemorrhage and malformation
of the kidney with effects observed at doses of >0.1 ^g/kg/day. In
addition, there 1s some evidence that 2,3,7,8-TCDD can Induce mlcrosomal
enzymes 1n the fetus exposed jn, utero. and this Induction Is accompanied by
damage to the fine structure of the liver cell; however, other reports
Indicate that enzyme Induction occurs only after birth following exposure to
2,3,7,8-TCDD through the mother's milk. As 1n mice, hemorrhaglc SI tracts
have been observed 1n rat fetuses exposed in utero to 2,3,7,8-TCDD.
9-35
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Rabbits and monkeys are also susceptible to the fetotoxlc effects of
2,3,7,8-TCDD; however, the studies of these species have been too limited to
clearly evaluate a teratogenlc response or define a threshold dose for
fetotoxldty.
A number of studies, mostly correlation studies, have been conducted on
groups of persons exposed to 2,3,7,8-TCOD as a contaminant of the herbicides
2»4»5-T or the chemical of TCP. Although some studies have shown a positive
association between exposure to 2,4,5-T and birth defects or abortions,
other studies have not. In Investigations concerning potential exposure to
2,3,7,8-TCDD through the manufacture of TCP, there has been no positive
substantiated association between exposure and reproductive difficulties.
In these studies, exposure was always mixed, with 2,3,7,8-TCDD being only a
minor component. Hence, It Is not possible to attribute with certainty any
positive finding to 2,3,7,8-TCDD. It 1s also possible, since levels of
2,3,7,8-TCDD contamination of 2,4,5-T and TCP were only estimated, that the
negative results reflect the exposure was too low or the study designs too
Insensitive to elicit a detectable response. From an extensive review of
d1ox1n-1nduced animal and human reproductive toxlclty data by Hattlson et
al. (1984) and another review of 15 reports dealing with human exposure to
dloxlns and reproductive effects by Hatch (1984), 1t can be concluded that
ep1dem1olog1c observations from well designed studies are warranted before
deriving any conclusion on dloxln-lnduced reproductive toxlclty In humans.
Although the evidence from human studies 1s Insufficient to prove 2,3,7,8-
TCDD Is teratogenlc, the animal data clearly Indicate teratogenlc or feto-
toxlc effects 1n all animal species tested.
9-36
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10. MUTAGENICITY AND OTHER INDICATIONS OF GENOTOXICITY
10.1. RELEVANT STUDIES
10.1.1. Assays 1n Microorganisms. Short-term 1_n vitro test systems have
been developed to assess the biologic, toxic and genotoxlc effects of
chemicals. These assays have proven to be useful Indicators of potential
activity of diverse Industrial chemicals, a broad range of drugs and xeno-
blotlcs, carcinogens and crude environmental extracts. The most widely used
short-term test system, the Ames test for bacterial mutagenesls, employs
several strains of Salmonella typhlmurlum that are highly susceptible to the
effects of mutagenlc chemicals. Despite the obvious utility of the Ames
test and related short-term assays, their predictive capabilities (I.e., the
correlation between bacterial mutagenldty and carc1nogen1c1ty) have not
been fully assessed (Bartsch et al., 1982).
Mutagenldty assays 1n microorganisms have been used to assess the
genotoxlc effects of 2,3,7,8-TCDD; however, the results of most of these
assays have Indicated little, potential for mutagenlc effects (Table 10-1).
Hussaln et al. (1972) exposed S. typhlmurlum h1st1d1ne-dependent strains
TA1530 and TA1532 1n liquid suspension to 2,3,7,8-TCDD followed by plating
Into selective medium to observe reversion to prototypes. No Increase 1n
the reversion rate was observed with strain TA1530 at exposure levels of 1
and 10 yg/ml. These exposures resulted 1n cell survivals of 90 and <1%,
respectively. In strain TA1532, Increased reversion frequency was not
observed at 2,3,7,8-TCDD concentrations of <2-3 Vg/m9., which resulted 1n
a 0-5054 decrease 1n survival; however, at 2,3,7,8-TCDD levels that resulted
1n a 99% decrease 1n survival, there was an Increased number of revertant
colonies/surviving cells. This positive response 1s questionable because of
the extremely high toxldty observed. The dose levels were not specified.
10-1
-------
ISJ
TABLE 10-1
The Results of HutagenlcHy Assays for 2,3,7.8-TCDD In Salmonella typhlaurlp
Type of Assay
Spot test
Plate
Incorporation
Plate
Incorporation*
Fluctuation
test
Spot test
Plate
Incorporation
Plate
Incorporation
Suspension
assay
Suspension
assay
S-9 TA98
f/- NT
+/- NT
*/- 0
+/- 0
NT
*• 0
NT
NT
*/- 0
TA1530
NT
NT
0
0
0
NT
NT
0
NT
1A1535
0
0
0
0
NT
0
NT
NT
0
1A1537
0
0
0
0
NT
0
0
NT
0
Strains
TA1538
0
0
0
0
NT
0
NT
NT
NT
of Salnonella typtilMirlm
TA1532
0
0
0
0
t
NT
NT
QR
NT
TA19SO
NT
NT
0
0
NT
NT
NT
NT
NT
TA1975
NT
NT
0
0
NT
NT
NT
NT
NT
TA1978
NT
NT
0
0
NT
NT
NT
NT
NT
646
NT
NT
0
0
0
NT
NT
NT
NT
TA100
NT
NT
0
0
NT
0
NT
NT
0
TA1531
NT
NT
NT
NT
QR
NT
NT
NT
NT
TA1534
NT
NT
NT
NT
QR
NT
NT
NT
NT
Reference
HcCann, 1978
KcCann, 1978
Gilbert et al..
Gilbert et al..
Seller. 1973
Gelger and Neal,
Gelger and Neal,
Hussaln et al..
Hortelmns et al
1984
1980
1980
1981
1981
1972
* i
•The assay was performed under both aerobic and anaerobic conditions.
NT = Not tested; QR = Questionable response; 0 « Negative response; » •= Positive response
-------
The source of the 2,3,7,8-TCDD sample studied 1n this paper was the Food and
Drug Administration, and Us reported purity was 99%. Also, Seller (1973)
observed a positive mutagenlc response 1n a spot test of 2,3,7,8-TCDD per-
formed In the absence of a metabolic activation system. However, the purity
of the sample studied was not provided. In tester strains 646 and TA1530,
the ratio of revertants/108 cells 1n the treated plates divided by spon-
taneous revertants/103 cells was <1. In strains TA1531 and TA1534, the
ratio was between 1 and 2, which was considered a "doubtful" mutagenlc
response, while 1n strain TA1532, the ratio was >10. There was no mention
of the 2,3,7,8-TCDD levels tested 1n this assay. The positive controls,
dlethylsulfate, 2-am1nopur1ne and 2-am1nofluorene, produced ratios of 2 to
5, <1 and 5 to 10, respectively, 1n strain TA1532. In both the study by
Hussaln et al. (1972) and the study by Seller (1973), 2,3,7,8-TCDD produced
a positive mutagenlc response only 1n the S. typhlmurlum strain TA1532,
which 1s sensitive to frameshlft mutagens.
Hussaln et al. (1972) also performed a mutagen1c1ty test of 2,3,7,8-TCDD
1n two other microblal test systems. A positive response was observed 1n
Escherlchla coll Sd-4 as Indicated by a reversion to streptomycin Indepen-
dence. In this assay, cells were treated 1n suspension for 1 hour with
2,3,7,8-TCDD at 0.5-4 pg/ma,. The greatest mutation frequency (256
mutants x 10~8, as compared with the control frequency of 2.2 mutants x
10~8) occurred at a dose level of 2 pg/ma,. The absolute number of
colonies/plate was 7 for the control and 46 for the treated plate. The dose
of 2 pg/mft. caused an 89% decrease In cell survival. A duplicate sample
resulted 1n an 82% decrease 1n survival and a mutation frequency of
34xlO~8. These results Indicate that the reproduc1bH1ty of the assay may
not have been perfect, but both results are well above the control value of
10-3
-------
2.2x10 8. A dose-response relationship was not observed, Indicating that
the results at 2 jig/mi are only suggestive of a positive response. In
addition, the positive results were obtained at a concentration of 2,3,7,8-
TCDD (2 jig/ma.) that was well above solubility 1n water (0.2 yg/t),
which also casts doubt on the significance of the positive result. In the
second test system, the ability of 2,3,7,8-TCDD to Increase prophage Induc-
tion 1n E.. coll K-39 cells was examined. The vehicle control, DMSO, Inhib-
ited prophage Induction as compared with the untreated controls, while the
most effective dose level of 2,3,7,8-TCDD (0.5 vg/mt) resulted 1n an
Increased prophage Induction as compared with the vehicle control but not as
compared with the untreated controls. Hussaln et al. (1972) concluded that
2,3,7,8-TCDD was capable of causing Increases 1n the reverse mutation rate
1n £. coll Sd-4 and that 2,3,7,8-TCDD had a weak ability to Induce prophage
1n £. coll K-39 cells.
The studies that followed these two early reports of Hussaln et al.
(1972) and Seller (1973) failed to detect mutagenlc activity of 2,3,7,8-TCDD
1n S. typhlmurlum. Wassom et al. (1978) cited a personal communication from
HcCann (1978), *h1ch reported that 2,3,7,8-TCDD was Inactive 1n both the
spot test and plate Incorporation assay with S>. typhlmurlum strains TA1532,
TA1535, TA1537 and TA1538. Doses and other experimental protocols were not
mentioned except that the tests were performed both with and without
metabolic activation. Gilbert et al. (1980) reported that 2,3,7,8-TCDD gave
"substantially negative results" with S. typhlmurlum strains TA98, TA100,
TA1530, TA1535, TA1537, TA1538, G46, TA1532, TA1950, TA1975 and TA1978.
Both the standard plate Incorporation assay and the bacterial fluctuation
test were used, and both were performed with and without S-9 prepared from
the livers of Aroclor 1254 pretreated rats. In the plate Incorporation
assay, the test compound was tested at 1-2000 yg/plate under both aerobic
10-4
-------
and anaerobic conditions. Details were not provided for the fluctuation
assay. It Is difficult to assess possible reasons for the conflicting
results between the earlier studies and these later mutagenldty assays,
since Information on experimental conditions was limited 1n the negative
studies.
In an attempt to resolve the conflicting results and observe a mutagenlc
response, Gelger and Neal (1981) tested 2,3,7,8-TCDD 1n the standard plate
Incorporation assay using S-9 prepared from different sources. In order to
maximize the amount of compound tested, dloxane, a better solvent for
2,3,7,8-TCDD than the commonly employed DMSO, was used. Even with the use
of dloxane, the limited solubility of 2,3,7,8-TCDD allowed only 20 »q/
plate to be tested, a dose that was shown to be nontoxlc to the cells. The
S-9 used 1n these assays was prepared from the Hvers of Aroclor 1254
pretreated male Sprague-Dawley rats and male Golden Syrian hamsters, and
from 2,3,7,8-TCDD Induced male hamsters. In all assays at 2,3,7,8-TCDD
concentrations of 0.2, 2, 5 or 20 »ig/plate, and regardless of the source
of the S-9, there was no observed mutagenlc response. In further attempts
to duplicate the previous positive results, Gelger and Neal (1981) tested
the same concentrations of 2,3,7,8-TCDD 1n strain TA1537, a more sensitive
direct descendent of strain TA1532, for mutagenlc activity 1n the absence of
S-9. Again, no Increase 1n the number of revertants was observed. In
assays either with or without S-9, positive controls had predictable
Increases 1n the number of revertant colonies. The authors concluded that
2,3,7,8-TCDD was not active under the conditions of this assay; however,
testing at higher concentrations may elicit a positive response. It was
also noted that many other polychlorlnated aromatic compounds are not
mutagenlc 1n the Ames test, even though there 1s positive evidence of
carc1nogen1c1ty.
10-5
-------
The National Toxicology Program (NTP) provided data on 2,3,7,8-TCDD from
four assay systems: the S. typhlmurlum (strains TA98, TA100, TA1535 and
TA1537) h1st1d1ne reversion assay, the sex-linked recessive lethal test In
Drosophlla. and cytogenetlc studies (sister chromatld exchange and chromo-
some aberrations) 1n Chinese hamster ovary cells. Negative results were
obtained In all of these assays (Hortelmans et al., 1984; Z1mmer1ng et al.,
1985; NTP, 1985).
Hutagenlc effects of 2,3,7,8-TCDD 1n yeast were observed by Bronzettl et
al. (1983). Positive results for reversion and gene conversion were
obtained jm vitro and 1n the host-mediated assay. The jji vitro experiments
yielded small dose-related Increases 1n trp convertants and llv rever-
tants. An S10 metabolic activation system was required. Exposure of the
yeast to 2,3,7,8-TCDD at the highest level tested (10 iig/mi) resulted 1n
16% survival and yielded 4-fold Increases 1n reversion and gene conversion.
In the host-mediated assay, male mice were exposed to 25 vg of
2,3,7,8-TCDD/kg (Bronzettl et al., 1983). After 5, 10, 20 or 30 days, 0.2
mil of a yeast culture (4 x 108 cells) was Instilled retroorbltally.
Four hours later, the liver and kidneys were removed and the yeast cells 1n
these organs were assayed for mutagenlc responses. Increases (4- to 6-fold)
1n reversion and gene conversion were observed 1n yeast cells obtained from
the livers and kidneys. The toxic response of the animals to an exposure of
25 pg/kg was not described 1n this report. The positive results described
1n this paper suggest that 2,3,7,8-TCDD 1s mutagenlc In yeast, but more
definitive studies are needed before a firm conclusion can be drawn.
Hay (1982) has found that 2,3,7,8-TCDD dissolved 1n DMSO transformed
baby hamster kidney cells (BHK) in vitro. The dloxln Isomers
2,8-d1chloroand 1,3,7-tr1chlorod1benzo-p_-d1ox1n also transformed BHK cells,
10-6
-------
tot the response was weak. The unchlorlnated d1benzo-p_-d1oxln and the tuny
chlorinated octachlorod1benzo-|)-d1ox1n were both negative 1n the BHK assay
(I.e., there was no cell transformation).
Abernethy et al. (1985) failed to transform CSH/IOT^ cells 1n cul-
ture by single treatments with 0.06 mM to 5 p dosage of 2.3.7.8-TCDD or
Initiate transformation In these treated cells by subsequent exposure with
tumor promoter 12-0-tetradecanoylpharbol-13-acetetate (TPA). However, these
authors could transform C3H/10 T, ,~ cells 1n vitro by N-methyl-N'-n1tro~N-
I/c. —
nltrosoguanldine (MNN6) and this transformation could be enhanced by subse-
quent treatment with low concentration (> 4 pM) of 2,3,7,8-TCDD. Maximum
enhancement was observed at a concentration of 40 pH of 2.3.7.8-TCDD. This
study Indicates that 2,3,7,8-TCDD Induced promotional activities can be
observed 1n C3H/10 T, .„ cells 1n cultured.
Rogers et al. (1982) reported that 2,3,7,8-TCDD Induced mutations 1n the
excess thymldlne, thloguanlne and methotrexate selective systems In L5178Y
mouse lymphoma cells 1n culture. However, no significant mutation was noted
1n ouabln or cytoslne arablnoslde selective systems.
10.1.2. Interactions with Nucleic Acids, in vitro reactions of 2,3,7,8-
TCDD with bacterlophage Qfi RNA were evaluated by Kondorosl et al. (1973).
Active RNA was purified from QB phage followed by Incubation for 1 hour at
37°C with 0.0, 0.2, 2.0 or 4.0 Vg/m8, of 2,3,7,8-TCDD. At all concentra-
tions tested, 2,3,7,8-TCDD had no effect on the transfect1v1ty of QB RNA.
Other compounds tested Included the alkylatlng agents methyl, ethyl and
Isopropyl methane-sulfonate, and dlethyl pyrocarbonate, all of whlfcti Inacti-
vated QB RNA under the same experimental conditions. The authors suggested
that 2,3,7,8-TCOD Inactivity 1n this assay Indicated that 2,3,7,8-TCDD was
10-7
-------
an Intercalating agent, and hence would require double stranded DNA 1n order
to Interact. The data presented 1n this study, however, were Insufficient
to support this conjecture.
In vivo binding of radlolabeled 2,3,7,8-TCDD to liver macromolecules was
studied 1n Sprague-Dawley rats by Poland and Glover (1979), Both male and
female animals were administered [1»6-3H]2,3,7,8-TCDD 1.p. at a dose of
7.5 pg/kg. This dose corresponded to a tritium level of 0.87 mC1/kg. The
animals were killed 12, 48 and 168 hours after treatment, or 24 hours after
treatment when the animals were pretreated with the enzyme Inducers pheno-
barbltal or unlabeled 2,3,7,8-TCDD. Following sacrifice, Isolation of
macromolecules, and removal of free labeled 2,3,7,8- TCDD, the amount of
label bound to protein, RNA and DNA was determined. The greatest nonex-
tractable binding of labeled 2,3,7,8-TCDD occurred to protein; however, the
amount of label bound was small and only amounted to 0.03-0.1% of the total
radioactivity administered. The total amount of label associated with RNA
and DNA was, respectively, only 50 and 4 cpm above background. Time after
exposure, sex or prior enzyme Induction had no significant effect on
2,3,7,8-TCDD binding. As a result of the extremely low levels of radioacti-
vity associated with RNA and DNA, 1t 1s uncertain whether 2,3,7,8-TCDD truly
binds covalently to these macromolecules and, 1f so, whether there 1s any
biological significance to this low level of apparent binding.
10.1.3. Cytogenetlc Effects of 2,3,7,8-TCDD. The effects of 2,3,7,8-TCDD
exposure on the extent of chromosomal aberrations 1n the bone marrow of male
rats were reported 1n an abstract by Green and Moreland (1975). In the
Initial experiment, no Increase In chromosomal aberration was observed after
five dally gavage treatments at a 2,3,7,8-TCDD dose of 10 pg/kg. In the
second portion of this study, rats were exposed by a single Intraperltoneal
10-8
-------
Injection of 2,3,7,8-TCOO at 5, 10 or 15 ^g/kg j)r a single gavage treat-
ment at 20 vg/kg. The animals at the two highest exposure levels were
killed 24 hours post-treatment, while the remaining animals were killed 29
days post-treatment. Again, no Increase 1n chromosomal aberrations was
observed, except 1n the positive control group exposed to trlethylenemela-
mlne.
In a later report, a small but significant Increase 1n chromosomal aber-
rations was observed 1n the bone marrow cells of male and female Osborne-
Hendel rats (Green et a!., 1977). Bone marrow cells for cytogenetlc
analysis were obtained from Osborne-Mendel rats used 1n a range-finding
study preliminary to a chronic bloassay (Green et a!., 1977). The animals
1n groups of 8 males and 8 females received twice weekly Intubations of
2,3,7,8-TCOD at respective doses of 0.25, 1.0, 2.0 and 4.0, or 0.25, 0.5,
2.0 and 4.0 ng/kg for 13 weeks. Because 1t was not required for the
range-finding study, a control group was not Included. Bone marrow cells
were analyzed for abnormalities and cells 1n mitosis 1n the animals that
survived to the end of the study (4-8 animals/group). The only significant
Increases 1n chromosomal aberrations 1n comparison with the low dose group
were 1n males at 2 and 4 pg/kg and females at 4 jig/kg. The greatest
Incidence observed was 4.65% of the cells with chromosomal breaks 1n the
high-dose males; this was considered only weakly positive. The weak
response, as well as the lack of data from control animals and the reported
difficulty of obtaining cells from the high-dose animals as a result of
2,3,7,8-TCDD toxlclty, makes the conclusion from this study that 2,3,7,8-
TCDD produced chromosomal breaks tenuous.
10-9
-------
A similar weak response was observed by Loprleno et al. (1982) 1n male
and female CD-I mice that received an 1ntraper1toneal Injection of 2,3,7,8-
TCDD at a dose of 10 pg/kg. At 96 hours post-treatment, there was a
significant (p<0.01) Increase 1n bone marrow cells with gaps and chromatld
aberrations. When chromosomal aberrations were analyzed at 24 hours post-
treatment, there was no significant change 1n the Incidence of cells with
aberrant chromosomes. The study was continued with a more extensive experi-
ment using CD-COBS female rats. The rats were treated weekly by gavage
(vehicle acetone-corn oil 1:6) at doses of 0, 0.01, 0.10 or 1.00 yg/kg for
45 weeks. Analysis of bone marrow cells for chromosomal aberrations 24
hours after the last treatment failed to detect significant Increases.
Czelzel and Klraly (1976) reported an Increased Incidence (p<0.001) of
chromatld-type and unstable chromosome aberrations 1n the peripheral lympho-
cytes of workers exposed to the herbicides 2,4,5-trlchlorophenoxyethanol
(2,4,5-TCPE) and Bumlnol. The 2,3,7,8-TCDD levels In the final product were
<0,1 mg/kg; however, the exposure levels for Individual workers were not
available.
Hulcahy (1980) reported no Increased Incidences of chromosomal aberra-
tions 1n the lymphocytes of 15 soldiers exposed to Agent Orange. The expo-
sure was for 6-15 months and all subjects complained of symptoms, Including
skin eruptions, which they associated with Agent Orange. The analyses were
performed with lymphocytes obtained ~10 years after the last exposure, and
comparisons were made with eight subjects who had no history of exposure to
2,3,7,8-TCDD. Neither sister chromatld exchange nor structural aberrations
Including both gaps and breaks were Increased. The authors noted that the
long time between exposure and analysis may have accounted for the negative
results.
10-10
-------
In addition, Regglanl (1980) and Mottura et al. (1981) studied the
2,3,7,8-TCDD exposed Inhabitants 1n Seveso. Regg1an1 (1980) examined 4
adults and 13 children (3-13 years) for chromosomal aberrations within 2
weeks of the accident. These 17 Individuals were examined to support claims
of and determine extent of Injury. Although burn-like skin lesions 1n these
17 Individuals Indicated chemical exposure, no Increase 1n chromosomal aber-
rations was detected. The methods of performing the analyses and the actual
number of aberrations detected were not described. Similar negative results
were reported In an abstract by Mottura et al. (1981). In this study, sub-
jects were chosen from the area of heavy contamination following the acci-
dent (acute high level exposure), from the working population of the plant
(chronic low level exposure) and a nonexposed control population. The num-
ber of subjects 1n each group was not provided. The specimens were examined
by three Independent laboratories and no laboratory reported an Increase 1n
chromosomal aberrations, although there was a significant difference 1n the
reported scores between laboratories. There was no Information 1n this
abstract on the extent of Individual exposure or the length of time that
elapsed between the accident and obtaining samples for analyses of chromo-
somal aberrations.
Tenchlnl et al. (1979) also conducted a cytogenetlc study of the exposed
Individuals at Seveso, Italy and of the aborted fetal tissue from exposed
mothers. No significant chromosomal aberrations could be observed 1n the
peripheral lymphocytes of the exposed 1d1v1duals. But aborted fetuses
showed a nonsignificant Increase 1n .chromosomal abnormalities compared to
the spontaneously aborted fetuses as observed In the general population. In
a subsequent study, Tenchlnl et al. (1983) observed a significant Increase
10-11
-------
1n the frequencies of aberrant cells and 1n the average number of aberra-
tions per damaged cell 1n fetal tissues from exposed pregnancies. This 1s a
potentially Interesting observation, but the study has the following pit-
falls. First, the controls were nonconcurrent. This 1s a major problem 1n
the Interpretation of the results from pregnancies before and after expo-
sure. Second, cells carrying the chromosomal aberrations described are not
expected to survive more than one cell cycle, but 1n this study cells were
examined that had undergone several cell divisions. This casts doubt on the
validity of a positive result.
D1Lern1a et al. (1982) conducted additional studies on lymphocytes pre-
pared 1n 1976 and 1979 from eight persons considered acutely exposed to
2,3,7,8-TCDD 1n the Seveso accident, eight ICMESA factory workers (con-
sidered chronically exposed), and 14 control subjects (eight had chromosome
preparations made In 1976 and six In 1979). Cells were examined for average
number of SAs (Satellite Associations; evidence for functional Mbosomal
genes), both on a cell basis and for the large acrocentMc chromosomes
(D group chromosomes). There was no change 1n the frequency of SAs on a per
cell basis 1n any of the groups as compared to control values, nor 1n D
group chromosomes from acutely exposed subjects examined Immediately after
the accident. There was, however, a decrease 1n the average frequency of
SAs 1n group D chromosomes of acutely exposed subjects examined In 1977 and
1n ICHESA workers at both the 1976 and 1979 examinations. Although the bio-
logic relevance of these observations has not yet been confirmed, D1Lern1a
et al. (1982) observed a similar decrease 1n SAs after exposure of lympho-
cytes to x-1rrad1at1on. It was concluded that the decrease 1n SAs may have
resulted from mutagenlc damage to functional nucleolar organizing regions.
10-12
-------
10.2. SUMMARY
A limited number of Initial studies on the mutagenldty of 2,3,7,8-TCDD
In bacteria reported positive results 1n SL typhlmurlum strain TA1532 1n the
absence of a mammalian metabolic activation system (Hussaln et a!., 1972;
Seller, 1973). More recent attempts to repeat these results with strain
TA1532 or related strains have failed (Gelger and Neal, 1981; Nebert et a!.,
1976; Gilbert et al., 1980; McCann, 1978). These authors have also reported
no Increase In mutation rate when 2,3,7,8-TCDD was tested In the presence of
a mammalian metabolic activation system. In other jjn vitro assays, 2,3,7,8-
TCDD has produced a positive response 1n reversion to streptomycin Indepen-
dence 1n £. co.1.1 Sd-4 cells and questionable positive response with prophage
Induction 1n £. coll K-39 cells {Hussaln et al., 1972). Also, 2,3,7,8-TCDD
has been reported to be mutagenlc 1n the yeast S. cerevlslae in both the in
vitro assay with S-10 and the host-mediated assay (Bronzetti et al., 1983).
Rogers et al. (1982) also reported positive mutagenldty results in the
mouse lymphoma assay system. In the £. coll studies, the poor survival of
the cells or the Interference of the vehicle solvent, DMSO, with the assay
makes the evaluation of the studies difficult. With the data available, H
is not possible to resolve the conflicting reports on the mutagenlc poten-
tial of 2,3,7,8-TCOD,
Overall, the data Indicate little potential for the Interaction of
2,3,7,8-TCDD with nucleic adds or the ability of 2,3,7,8-TCOD to produce
chromosomal aberrations. Kondorosi et al. (1973) demonstrated that 2,3,7,8-
TCDD did not react with RNA In. vitro 1n the absence of a metabolic activa-
tion system. In. vivo studies using radiolabeled 2,3,7,8-TCDO indicated some
association of nonextractable label with RNA and DNA (Poland and Glover,
1979); however, the level of bound label was very low. Similar marginal
10-13
-------
data were available on the clastogenlc effect of 2,3,7,8-TCDD. Although two
lH v^vo studies In rats (Green and Moreland, 1975; Loprleno et al., 1982)
failed to demonstrate treatment-related chromosomal aberration, a second
study by the same authors (Green et al», 1977) using a longer exposure
period reported a small Increase 1n the number of aberrations. A similar
small Increase was observed by Loprleno et al. (1982) following a single
1ntraper1toneal Injection of 2,3,7,8-TCDD 1n mice. In humans exposed to
2,3,7,8-TCDD during the manufacture of 2,4,5-TCPE and Bumlnol, Czelzel and
Klraly (1976) reported an Increase 1n the number of chromosomal aberrations;
however, no Increase was detected 1n Individuals exposed to 2,3,7,8-TCDD
following an Industrial accident 1n Seveso, Italy (Regg1an1, 1980; Mottura
et al., 1981; Tench1n1 et al., 1979). In contrast, Tench1n1 et al. (1983)
reported positive results 1n a Seveso study, but this study has problems.
The studies of the clastogenlc effect of 2,3,7,8-TCDD were presented with
little or no experimental detail to assist 1n evaluating the merits of the
reports. The data available are too limited to Indicate whether 2,3,7,8-
TCDD can Interact with nucleic adds or produce chromosomal aberrations.
The differences among the results reported could be due to several
factors, such as treatment protocols, solubility problems, purity of the
samples tested and the high toxlclty of 2,3,7,8-TCDD. This chemical may be
a weak mutagen, but because 1t 1s very toxic, the dose range for detecting a
positive genetic effect may be very narrow. Therefore, additional experi-
mentation 1s necessary before any conclusive determination can be made.
Suggested further testing Includes the ability of 2,3,7,8-TCDD to Induce
forward mutations In mammalian cells In culture, additional yeast and bac-
terial studies and the sex-Hnked recessive lethal test In Drosophlla.
Pertinent Information regarding the mutagenldty of PeCDDs and HxCDDs
were not located In the available literature.
10-14
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11. CARCINOSENICITY
The purpose of this section 1s to provide an evaluation of the likeli-
hood that 2,3,7,8-tetrachlorod1benzo-p_-d1ox1n (TCDD), and a mixture of
1,2,3,7,8,9- and !,2,3,6,7,8-hexachlorod1benzo-p_-d1ox1n (HxCDD), are human
carcinogens and, on the assumption that they are human carcinogens, to
provide a basis for estimating their public health Impact, Including a
potency evaluation, 1n relation to other carcinogens. The evaluation of
carclnogenlclty depends heavily on animal bloassays and epldemlologlc
evidence. However, Information on mutagenldty and metabolism, particularly
1n relation to Interaction with DNA, as well as to pharmacoklnetlc behavior,
has an Important bearing on both the qualitative and quantitative assessment
of carclnogenlclty. The available Information on these subjects Is reviewed
1n other sections of this document. This chapter presents an evaluation of
the animal bloassays, the human epldemlologlc evidence, the quantitative
aspects of assessment, and finally, a summary and conclusions section
dealing with all of the relevant aspects of carclnogenlclty.
11.1. ANIHAL STUDIES
11.1.1. Studies Using 2,3,7,8-TCDD. The polychlorlnated d1benzo-p_-d1ox1ns
(PCDDS), 2,3,7,8-TCDD and a mixture of 1,2,3,7,8,9- and 1,2,3,6,7,8-HxCDD,
have been tested for carclnogenlclty 1n rats and mice by administering the
compound 1n the diet and by gavage. Also, the tumor Incidence In native
mice Inhabiting an area with heavy exposure to the herbicide Agent Orange
has been assessed and compared with mice from an uncontamlnated habitat.
The results of these bloassays are discussed 1n this section. Along with
studies using the oral route, both 2,3,7,8-TCDD and a mixture of
1,2,3,7,8,9- and 1,2,3,6,7,8-HxCDD have been tested for tumor1gen1dty by
n-i
-------
dermal application. Using the skin two-stage tumor1gen1c1ty model, 2,3,7,8-
TCOD has been tested for promoting and Initiating activity as well as anti-
carcinogenic activity. Other model systems have been used to a more limited
extent 1n studies of the effect of 2,3,7,8-TCDD on the carcinogenic poten-
tial of chemical carcinogens.
11.1.1.1. VAN MILLER ET AL. (ORAL) RAT STUDY (1977a,b) — In a limited
study, Van Miller et al. (1977a,b) maintained small groups of male Sprague-
Dawley rats on diets containing 2,3,7,8-TCDD. The animals, In groups of 10,
were fed diets containing 0.0, 0.001, 0.005, 0.05, 0.5, 1.0, 5.0, 50, 500 or
1000 ppb of 2,3,7,8-TCDD for 78 weeks. As determined from the food consump-
tion of two animals from each group, these exposure levels corresponded to
doses of 0.0, 0.0003, 0.001, 0.01, 0.1, 0.4, 2.0, 2.4, 240 and 500 jig/kg/
week, respectively. At week 65 of treatment, all surviving animals were
examined by laparotomy, and biopsy samples were obtained from any gross
tumors. Following termination of treatment, the animals were observed for
an additional 17 weeks before sacrificing all surviving animals. Necropsy
was performed on animals killed when moribund, found dead or killed at
termination of the study, and the animals were examined for both gross and
microscopic lesions. Intake and mortality are shown 1n Table 11-1.
All animals 1n groups maintained on diets containing 1-1000 ppb of
2,3,7,8-TCDD were dead by week 90 of treatment; the first deaths 1n groups
at the 1000 and 1 ppb levels were observed at 2 weeks and 31 weeks of
treatment, respectively. Animals exposed to 0.001-0.5 ppb of 2,3,7,8-TCDD
had similar food consumption and survival as control animals; however, all
treated animals had hlstopathologlc degenerative changes 1n the kidneys.
11-2
-------
TABLE 11-1
2,3,7,8-TCDD Intake and Mortality 1n Hale Sprague-Dawley Ratsa
Doseb
{ppb)
0.0
0.001
0.005
0.05
0.5
1
5
Weekly Dose/Rat
(vg/kg bw)
0.0003
0.001
0.01
0.1
0.4
2.0
Meek of
First Death
68
86
33
69
17
31
31
Number of Rats
Dead at 95th Week
6/10 (60%)
2/10 (20%)
4/10 (40%)
4/10 (40%)
5/10 (50%)
10/10 (100%)
10/10 (100%)
aSource: Van Miller et al.t 1977a,b
bRats at 50, 500 and 1000 ppb dose levels were all dead within 4 weeks.
11-3
-------
Complete necropsies were done and samples of tissues were taken for
microscopic examination from the control groups and each treatment group
(Laboratory audit* and personal communication with author).
Special staining methods were used as an aid 1n the diagnosis of neo-
plasms. Various benign and malignant tumors were found 1n each treatment
group. No tumors were observed 1n the controls (Table 11-2).
Statistically significant Increases of squamous cell tumors of the lungs
and neoplastlc nodules of the liver were observed 1n rats Ingesting 5 ppb
TCDD (Table 11-3). In addition, two animals 1n the 5 ppb dose group and one
animal 1n the 1 ppb dose group had Hver cholanglocardnomas, which are rare
In Sprague-Dawley rats. These results provide evidence of a carcinogenic
effect.
The observation of no tumors of any kind 1n the controls 1s unusual for
Sprague-Dawley rats. In addition, the reporting of the study was not exten-
sive. These factors may tend to lessen the reliance that can be placed on
the positive results of this study. However, this study 1s suggestive of a
carcinogenic response upon exposure to TCDD 1n rats.
11.1.1.2. KOCIBA ET AL. (ORAL) RAT STUDY (1978a) — Although this
study was published as Koclba et al., 19?8a, a fuller version was submitted
1n an unpublished report (Kodba et al.t 1977).
In this study, groups of 50 Sprague-Dawley rats (Spartan substraln) of
each sex were maintained for up to 2 years on diets providing 0.1, 0.01 or
0.001 ng/kg/day 2,3,7,8-TCDD. Vehicle control groups consisted of 86
animals of each sex. The test was appropriately conducted with the
*The audit of this study brought out the fact that 1t was Intended to be
only a range-finding study. Therefore, only small numbers of animals were
used. This may have made the study relatively Insensitive for detecting
carcinogenic effects at doses <1 ppb.
11-4
-------
TABLE 11-2
Benign and Malignant Tumors In Rats Ingesting 2,3,7,8-TCDDa
Doseb
0
1 ppt
5 ppt
50 ppt
500 ppt
1 PPb
5 ppb
Benign
0
0
1
2
2
0
8
Malignant
0
0
5
1
2
4
2
Number of
Tumors
0
0
6d
3^
49
§1
103
Number
With
0/10
0/10
5/10
3/10
4/10
4/10
7/10
of Rats
Tumors
«K)C
(0%)
(50%)e
(30%)
(40%)h
(40%)
(70%)
Source: Van Miller et a!., 1977a,b
Rats at dose levels of 50, 500 and 1000 ppb were all dead within 4 weeks.
C40 male rats used as controls for another study, received at the same
time and kept under Identical conditions, did not have neoplasms when
killed at 18 months.
^1 rat had ear duct carcinoma and lymphocytlc leukemia
1 adenocarclnoma (kidney)
1 malignant hlstlocytoma (retroperltoneal)
1 anglosarcoma (skin)
1 Leydlg cell adenoma (testls)
e3 rats died with aplastlc anemia
^1 flbrosarcoma (muscle)
1 squamous cell tumor (skin)
1 astrocytoma (brain)
Si fibroma (striated muscle)
1 carcinoma (skin)
1 adenocarclnoma (kidney)
1 scleroslng semlnoma (testls)
1 rat had a severe Hver Infarction
11 rat cholanglocardnoma and malignant hlstlocytomas (retroperltoneal)
1 anglosarcoma (skin)
1 glloblastoma (brain)
1 malignant hlstlocytoma (retroperltoneal)
3l rat had squamous cell tumor (lung) and neoplastlc nodule (liver)
2 cholanglocardnomas and neoplastlc nodules (liver)
3 squamous cell tumors (lung)
1 neoplastlc nodule (liver)
11-5
-------
TABLE 11-3
Liver Tumors 1n Rats Ingesting 2,3,7,8-TCDDa
Dose (ppb)
0
1
5
Neoplastlc
Nodules
0/10 (0%)
0/10 (0%)
4/10 (40%)
p=0.043c
Cholanglocardnomas
0/10 (0%)
1/10 (10%)
2/10 (20%)b
Squamous Cell
Tumors of the Lungs
0/10
0/10
4/10 (40%)
p=0.043c
aSource: Van HUler et al., 1977a,b
two animals had both neoplastlc nodules of the liver and cholanglo-
cardnomas.
cp-values calculated using the Fisher Exact Test.
11-6
-------
high-dose group given a dose which Induced signs of tissue toxldty, reduced
weight increments in both sexes, and shortened lifespans 1n female rats.
Clinical tests performed at intervals during the study monitored organ
specific toxldty, particularly of the Hver. Pathologic examinations In-
cluded hlstopathologlc evaluation of all major tissues 1n both the high-dose
and control animals, but only of selected tissues identified as possible
target organs and suspect tumors in lower-dose group;;. This approach is
suitable for the Identification of a carcinogenic effect, but does not
determine actual tumor Incidences 1n all groups except in those organs Iden-
tified as target organs. It, therefore, 1s adequate to define dose-response
relationships only 1n these target organs. Tissues examined from most
animals in all dose groups Included liver, lungs, kidneys, urinary bladder,
tongue, brain, testes/ovaries and prostate/uterus. For these tissues, a
quantitative analysis can be performed using the actual number of tissues
examined hlstopathologlcally for animals at risk. For other tissues
(excluding skin, mammary glands and nasal turblnates/hard palate), actual
tumor Incidence cannot be evaluated for the two lower doses. For skin,
mammary glands and nasal turblnates/hard palate, the number of animals
necropsled Is the appropriate denominator to determine Incidence, because
detection of these tumors 1s based on observation of the tumor at necropsy.
A laboratory audit of this study by H. Spencer and W.S. Woodrow, Hazard
Evaluation Division, Office of Pesticide Programs, U.S. EPA, did not reveal
significant new information. Re.vJewers concluded that the study was
properly conducted, adhering to the accepted procedures (Spencer and
Woodrow, 1979).
Based on data reported for food consumption, body weight and dietary
level of TCDD, the dally doses were reasonably constant for most of the
11-7
-------
study, although somewhat below the value expected 1n most groups during the
third month.
High early mortality was observed 1n all groups 1n this study but was
only statistically significant 1n the high-dose group. The survival curves
show progressive mortality beginning as early as the 12th month and leading
to 5054 mortality by 21 months.* The effects of this early mortality are a
reduction 1n expected tumor Incidence because of a truncated latency period,
and a reduction 1n sensitivity of the study because of a reduction 1n number
of animals at risk during the time of expected tumor manifestation. Cumula-
tive mortality and Interval mortality rates are given 1n Tables A-l to A-4
of Appendix A (Clement Associates, 1979).
The results of this study provide substantial evidence that 2,3,7,8-TCOD
Is carcinogenic In rats. 2,3,7,8-TCDD Induced a highly statistically
significant Increase of both hepatocellular carcinomas and hepatocellular
neoplastlc nodules 1n female rats at doses of 0.1 and 0.01 pg/kg/day (2200
and 210 ppt 1n the diet, respectively). The Increase of hepatocellular
carcinomas alone, 1n the high-dose females, was also highly significant. In
addition, at the highest dose level, 2,3,7,8-TCDD Induced a statistically
significant Increase 1n stratified squamous cell carcinomas of the hard
palate and/or nasal turblnates 1n both males and females, squamous cell
carcinomas of the tongue 1n males, and highly significant keratlnlzlng squa-
raous cell carcinomas of the lungs In females (Tables 11-4, 11-5 and 11-6).
*In the 0.001 group of males, 44% of the animals had died by 18 months. The
mortality patterns were analyzed by the Whltney-Wllcoxon test and the
Kolmogorov-Slmonov test. These tests showed that mortality was signifi-
cantly higher 1n the high-dose females than 1n controls, and while Indica-
tions of Increased mortality were found 1n other groups, they were not part
of a consistent pattern.
11-8
-------
TABLE 11-4
Hepatocellular Carcinomas and Hepatocellular Hyperplastlc Nodules
1n Female Sprague-Dawley Rats Maintained on Diets Containing 2,3,7,8-TCODa
Dose Level
(vg/kg/day)
0
0.001
(22 ppt)
0.01
(210 ppt)
0.1
(2200 ppt)
Hepatocellular
Hyperplastlc
Nodules
8/86 (9K)
3/50 (6%)
18/50 (36%)
23/49 (48%)
Hepatocellular
Carcinomas**
1/86
0/50
2/50
11/49
(p»5.6
(1%)
(0%)
(4%)
(22%)
X 10~5)
Total Number
With Both
Types of Tumors^
9/86 (10%)
3/50 (6%)
18/50 (36%)c
(p=4.36 x 10~«)
34/50 (71%)
(p=4.56 x 10~13)
aSource: Kodba et al., 1977
bp-values calculated using the Fisher Exact Test (one-tailed).
cTwo rats had both hepatocellular carcinomas and hyperplastlc nodules.
11-9
-------
TABLE 11-5
Tumor Incidence 1n Female Rats Fed Diets Containing 2,3,7,8-TCDDa
Dose Level Stratified Squamous Cell Keratlnlzlng Squamous
(jig/kg/day) Carcinomas of Hard Palate Cell Carcinomas of
or Nasal Turblnates Lungs
0 1/54 (254) 0/86 (0%)
0,001 0/30 (0%) 0/50 (0%)
(22 ppt)
0,01 1/27 (4K) 0/50 (0%)
(210 ppt)
0.1 5/24 (21%) 7/49 (14%)
(2200 ppt) (p=0.01)b (p=0.0006)6
aSource: Kodba et al.» 1977
bp-values calculated using the Fisher Exact Test (one-tailed).
11-10
-------
TABLE 11-6
Tumor Incidence 1n Male Rats Fed Diets Containing 2,3,7,8-TCDDa
Dose Level Stratified Squamous Cell Hard Palate/Nasal Turblnates
(vg/kg/day) Carcinomas of the Tongue Stratified Squamous Cell Carcinoma0
0 0/76 (OH) 0/51 (Q%)
0.001 1/49 (2%) NS 1/34 (3%) NS
(22 pot)
0.01 1/50 (2%) NS 0/27 (Q%) NS
(210 ppt)
0.1 3/42 (7%) (p=4.3 x 10~2)c 4/30 (13%) (p=0.016)c
(2200 ppt)
aSource: Kodba et al., 1977
blncludes examinations from both original and updated report (5/20/79).
cp-values calculated using the Fisher Exact Test,
NS = Not significant at p=0.05.
11-11
-------
Dr. Robert S-d1ox1n and a pentachlorod1benzo-jj-
dloxln. The presence of 0.1-0.2% hexachlorod1benzo-£-d1ox1n was also
detected by gas chromatography and mass spectrometry.
11-12
-------
TABLE 11-7
Dow 2,3,7.8-KDD Oral Rat Study by Dr. Koclba, WHh Dr. Squire's Review (8/15/80)
Sprague-Dawley Fenale Rats - Spartan Subs train (2 years)atb
Dose Levels (uq/kq/day)
0 (control) 0.001
Tissues and Diagnoses
S K S K
Lung
Squanous cell carcinomas 0/86 0/86 0/50 0/50
Nasal turblnate/hard palate
squamous cell carcinomas 0/54 1/54 0/30 0/30
Liver
Neoplastlc nodules/
hepatocellular carcinomas 16/86 9/86 8/50 3/50
(p=4.37 x 10"«)
Total combined 16/86 9/86 8/50 3/50
(each animal had at least 19X 10X 16X 6X
one tumor above) (p=4.37 x 10"*)
0.01
S K
0/49 0/49
1/27 1/27
27/50 18/50
(p=2.42 x 10"*) (p*4.37 « 10"*)
27/50 18/50
54X 34X
(p=2.42 x 10"») {p=4.37 x 10~«)
0.
S
8/4? (1?X)
(p=1.61 x 10"«)
5/22 (23X)
|p«1.43 x 10"»)
33/47 (70X)
(p=4.92 x 10~»)
34/47
72X
(p=1.20 X 10"»)
1
K
7/49 (14X)
5/24 (21X)
(p-9.46 x 10"'}
34/48 (71X)
(p=9.53 x 10~a»
34/49
69X
(p=2.13 x 10"1'
^Source: Koclba et al., 1977; Squire, 1980
bp-values calculated using the Fisher Exact Test.
S ° Or. Squire's hlstopathologlc analysis; K - Or. Koclba's hlstopathologlc analysis
-------
TABLE 11-8
Dow 2,3,7,8-TCDD Oral Rat Study by Dr. Kodba, H1th Dr. Squire's Review (8/15/80)
Sprague-Dawley Hale Rats - Spartan Substraln (2 years)*
Tissues and Diagnoses
0 (control)
S K
Dose Levels («q/kq/day)
0.001
0.01
0.1
Nasal turblnate/hard palate
squamous cell carcinomas
Tongue
squamous cell carcinomas
0/55 0/51 1/34 1/34 0/26 0/27 6/30 (20%) 4/30 (13%)
(p=1.36 x 10"3) (p=1.6 x 10~2)
0/77 0/76 2/44 1/49 1/49 1/49 3/44 (7%) 3/42 (7X)
(p=4.60 x 10~2) (p=4.34 x 10~2)
Total - 1 or 2 above 0/77
(each rat had at least
one tumor above)
2/44
554
1/49
2%
9/44
20X
(p=6.28
x 1Q~5)
*p-values calculated using the Fisher Exact Test.
S = Dr. Squire's hlstopathologlc analysis
K = Dr. Koclba's hlstopathologlc analysis
-------
In rats, a dose-related depression In mean body weight gain became
evident 1n the males after week 55 of the bloassay and 1n the females after
week 45,
The results of hlstopathologlc diagnosis of primary tumors caused by the
oral administration of 2,3,7,8-TCDO are presented 1n Table 11-9. In male
rats an Increased Incidence of folllcular-cell adenomas or carcinomas of the
thyroid was dose-related and was statistically significantly higher In the
low-, mid- and high-dose groups than In the vehicle controls. In addition,
a statistically significant Increase 1n subcutaneous tissue flbromas was
found 1n males of the high-dose group.
In female rats, a statistically significant Increase of each of the
following tumors was found 1n the high-dose group: hepatocellular carci-
nomas and neoplastlc nodules (p=0.001), subcutaneous tissue flbrosarcomas
(p=0.023) and adrenal cortical adenomas (p=0.039), as shown 1n Table 11-10.
These results confirm the carcinogenic effect observed 1n the Kodba et
al, (1978a) study using Sprague-Oawley (Spartan substraln) rats.
11.1.1.4. TOTH ET AL. (ORAL) HOUSE STUDY (1979) — This study Investi-
gated the cardnogenlcHy of 2,3,7,8-TCDO 1n Swiss mice. Ten-week-old
outbred Sw1ss/H/R1op mice were used. 2,3,7,8-TCDO was administered 1n a
sunflower oil vehicle by gavage to groups of 45 male mice once a week at
doses of 7.0, 0.7 and 0.007 yg/kg bw for a year (groups 9, 10, 11, respec-
tively, In Table 11-11). Matched male vehicle controls were administered
sunflower oil once a week. Matched controls to a companion study Investi-
gating the cardnogenlcHy of (2,3,5-tr1chlorophenoxy)ethanol (TCPE) contam-
inated with low levels of 2,3,7,8-TCDD, were administered carboxymethyl
cellulose (the vehicle used 1n that study) once a week. Two untreated
controls were also maintained.
11-15
-------
TABLE 11-9
Incidence of Primary Tumors In Hale Rats Administered 2,3,7,8-TCDD by Gavage3
Type of Tumor Vehicle Control
Subcutaneous tissue 3/75 (4%)
Fibroma
Liver
Neoplastlc nodule
or hepatocellular
carcinoma 0/74 (OX)
Adrenal
Cortical adenoma 6/72 (8%)
Thyroid
Folllcular cell
adenoma 1/69 (IX)
Thyroid
FolUcular cell
adenoma or
carcinoma 1/69 (IX)
Lou Doseb
0.01
1/50 (2X)
0/50 (OX)
9/50 (18X)
5/48 (10X)
p= 0,042
5/48 (10X)
p=0.042
wQ/kg/week
Hid Ooseb
0.05
3/50 (6X)
0/50 (OX)
12/49 (24X)
6/50 (16X)
p=0.021
8/50 (16X)
p=0.004
High Ooseb
0.5
7/50 (14X)
p=0.048
3/50 (6X)
9/49 (18X)
10/50 (20X)
p=0.001
11/50 (22X)
p<0.001
aSource: NTP, 1980a
bp-values calculated using the Fisher Exact Test.
-------
TABLE 11-10
Incidence of Primary Tumors 1n Female Rats Administered
2,3,7,8-TCDD by Savage3
uQ/kq/week
Type of Tumor Vehicle Control
Low Dose*5
0.01
H1d Dose
0.05
High Doseb
0.5
Subcutaneous tissue
Mbrosarcoma
Liver
Neoplastlc nodule
Liver
Neoplastlc nodule
or hepatocellular
carcinoma
Pituitary
Adenoma
Adrenal
Cortical adenoma
0/75 (0%)
5/74 (7%)
5/75 (7%)
1/66 (254)
11/73 (15%)
2/50 (4%) 3/50 (6%) 4/49 (8%)
p=0.023
1/49 (2%) 3/50 (6%) 12/49 (24%)
p-0.006
1/49 (2%) 3/50 (6%) 14/49 (29%)
p=0.001
5/47 (11%) 2/44 (5%) 3/43 (7%)
p=0.044
8/49 (16%) 4/49 (8%) 14/46 (30%)
p=0.039
aSource: NTP, 1980a
kp-values calculated using the Fisher Exact Test.
11-17
-------
TABLE 11-11
Cumulative Data on Tumor Incidence3
oo
TCPF.&
Group (mg/kg)
1 67.0
2 70.0
3
4 7.0
5 7.0
6 0.7
7
8
9
10
11
12
Treatment
TCDD
(vg/kg)
0.112
(1.6 ppal
0.007
{0,1 ppm)
control
0.07
(10 ppm)
0.0007
(0.1 PfKl)
0.00007
(0.1 ppm)
»-
control
7.0
0.7
0.007
"-
Vehicle*
(«gAg)
50
50
50
50
50
50
10
10
10
10
Sex
H
F
H
F
N
F
N
F
N
F
H
F
N
F
H
F
N
H
N
N
Effective
Number of
Nice
88
83
98
96
93
84
93
96
94
93
97
94
96
84
96
91
43
44
44
38
Number
of Tumor
Bearing
Nice
69
61
78
59
63
57
79
60
77
71
78
64
74
55
78
57
27
36
39
27
Liver
(X)
42«J
7
57e
9
24
4
25
10
23
8
24
5
32
4
32
4
13
21
13
7
(18)
(8)
(58}
(9)
(26)
(5)
(27)
(10)
(24)
(9)
(25)
(5)
(33)
(5)
(33)
(*)
(30)
(48)
(29)
(18)
Number of
Lung
50
52
18
39
44
41
38
38
50
42
51
38
44
38
38
31
11
18
27
15
Animals with Tumors of:
Lvmphomas
7
15
11
15
8
23
18
19
23
36
20
22
14
18
22
24
6
12
10
6
Other
Organs
16
25
16
23
17
13
22
19
17
21
17
21
22
17
15
19
7
4
6
7
Average
Llfespan
595
652
571
582
577
639
641
589
660
590
643
566
615
565
651
549
424
633
649
588
"Source: Totti et al., 1979
TCPE = TrIchlorophenoxy ethanol
cCarboxymethyl cellulose In groups 1-8, sunflower oil In groups 9-12.
dp<1X
ep<0-1X
-------
This study appears to have been generally well conducted. However, the
administration of 2,3,7,8-TCDD over a period of only 1 year, which 1s far
short of the life expectancy of the mice used, made the study relatively
Insensitive. Animals were followed for their entire lifetimes. Autopsies
were performed after spontaneous death or when the mice were moribund, and
all organs were examined h1stolog1cally. Sections were stained with
hematoxylln and eosln for light microscopy. Pathological findings were
evaluated and analyzed statistically. The findings of the 2,3,7,8-TCDD
study and the comparison study on TCPE are given 1n Table 11-11.
Analysis of the results of this study focused on the Incidence of liver
tumors 1n the groups treated with 2,3,7,8-TCDD and the Incidence of these
tumors 1n the matched controls (group 12) and 1n the males 1n the three
other control groups. Males 1n groups 3 and 8, the two untreated control
groups, had 26% and 33% liver tumors, respectively (p<0.20). The carboxy-
methyl cellulose male controls (group 7) had 33% (32/96) liver tumors. No
significant differences 1n liver tumors were observed when males 1n all four
control groups were compared with each other (p<0.05). Nevertheless, there
was evidence that the Incidence of liver tumors 1n the control groups was
associated with the average Hfespan 1n the respective groups. The two
groups that had <600 days average survival (groups 3 and 12) had the fewest
liver tumors (26 and 18%, respectively). On the other hand, the two groups
that had an average survival of >600 days (groups 7 and 8), had 33% liver
tumors each. The test for linear trend (tumors vs. days of average
survival) was not quite significant (p=0.065).
Among the three treatment groups (groups 9, 10 and 11), the middle dose
(0.7 yg/kg) showed the highest Incidence of liver tumors (21/44 = 48%).
11-19
-------
This Incidence was significantly higher than the Incidence of liver tumors
1n either the sunflower oil controls (p<0.01) or the pooled controls (all
four control groups combined) (p<0»025).
The highest-dose group (7.0 yg/kg) had an Increased Incidence of liver
tumors compared with the matched sunflower oil controls (13/43 = 3054), but
this Increase was not statistically significant (p=0.11). The Incidence of
Hver tumors 1n the high-dose group was comparable with that of the pooled
controls. The highest-dose group, however, had a much reduced average sur-
vival 1n comparison with any of the control groups (only 424 days compared
with 577, 588, 615 and 651 days 1n the four control groups). This poor
survival may have accounted for the lack of a statistically significant
Increase 1n liver tumors 1n the high-dose group. Furthermore, If time-to-
tumor data had been available, 1t 1s likely that the high-dose group would
have shown a significant decrease 1n t1me-to-tumor compared with the con-
trols. Therefore, the Increase 1n Hver tumors that was observed 1n the
high-dose group 1n comparison with the matched control group, although not
statistically significant, 1s considered to be consistent with an oncogenlc
effect.
In conclusion, the results of this study provide suggestive evidence of
an oncogenlc effect.
11.1.1.5. NATIONAL TOXICOLOGY BIOASSAY PROGRAM (ORAL) HOUSE STUDY
(1980a,b) — A cancer bloassay for the possible carc1nogen1dty of 2,3,7,8-
TCOO was tested by the Illinois Institute of Technology 1n mice under a
contract sponsored by the NCI.
In the mouse study* groups of 50 B6C3F1 mice of each sex were admlnls-
stered 2,3,7,8-TCDD suspended 1n a vehicle of 9:1 corn oil-acetone 2 days/
week for 104 weeks at doses of 0.01, 0.05 and 0.5 pg/kg/week for male mice
11-20
-------
and 0.04, 0.2 and 2.0 ^g/kg/week for female mice. Seventy-five mice of
each sex were used as vehicle controls. One untreated control group of 25
mice of each sex was present In the 2,3,7,8-TCDO treatment room. One
untreated control group of 25 mice of each sex was present In the vehicle
control room. In mice, the mean body weight gain In the treated groups was
comparable with that of the vehicle control groups. However, the mean body
weight of the treated mice was lower when 1t was compared with untreated
controls.
The results of the hlstopathologlc diagnosis of primary tumors are pre-
sented 1n Table 11-12. The results Indicate that, 1n male mice, 2,3,7,8-
TCOO Induced a statistically significant Incidence of hepatocellular carci-
nomas (p=0.002) and both hepatocellular carcinomas and neoplastlc nodules
combined (p=<0.001) 1n male mice of the high-dose group.
In female mice, 2,3,7,8-TCDD Induced statistically significant Increases
of hepatocellular carcinomas (p=0.014) and both hepatocellular adenomas and
carcinomas (p=0.002) In the high-dose group. In addition, a statistically
significant Increase 1n tumor Incidences of flbrosarcoma, hlstlocytlc
lymphoma, thyroid folUcular-cell adenoma and cortical adenoma or carcinoma
were also observed 1n the high-dose group (Table 11-13).
The Incidence of liver tumors observed 1n this study confirms the
earlier observations of an Increase In Hver tumors 1n the male mouse study
performed by Toth et al. (1979).
11.1.1.6. OTHER RELATED STUDIES —
11.1.1.6.1. P1tot et al. Promotion Study 1n Rats (1980) — P1tot et
al. (1980) Investigated a two-stage model of hepatocardnogenesls. Twenty-
four hours after a partial hepatectomy (to enhance cell proliferation),
female Sprague-Dawley rats were divided Into seven groups (Table 11-14).
11-21
-------
TABLE 11-12
Incidence of Primary Tumors 1n Male Mice Administered
2,3,7,8-TCDD by Gavage3
Type of Tumor Vehicle Control
ug/kg/week
Low Dose
0.01
M1d Dose
0.05
High Doseb
0.5
Liver
Hepatocellular
adenoma
Liver
Hepatocellular
carcinoma
Liver
Hepatocellular
adenoma and
carcinoma
7/73 (10%) 3/49 (6%) 5/49 (10%) 10/50 (20%)
8/73 (11%) 9/49 (18%) 8/49 (16%) 17/50 (34%)
p=0.002
15/73 (21%) 12/49 (24%) 13/49 (27%) 27/50 (54%)
p=<0.001
aSource: NTP, 1980a
bp-values calculated using the Fisher Exact Test.
11-22
-------
TABLE 11-13
Incidence of Primary Tumors 1n Female Mice Administered
2,3,7,8-TCDD by Gavage3
ug/kg/week
Type of Tumor Vehicle Control
Low Dose
0.04
Mid Dose
0.2
High Doseb
2.0
Subcutaneous tissue
Flbrosarcoma
Hematopo1et1c
system
H1st1ocyt1c
lymphoma
Hematopo1et1c
system
All lymphoma
Hematopo1et1c
system
Lymphoma or
leukemia
Liver
Hepatocellular
carcinoma
Liver
Hepatocellular
adenoma or
carcinoma
Thyroid
Folllcular-cell
adenoma
1/74 (1%) 1/50 (2%) 1/48 (2%) 5/47 (11%)
p=0.032
9/74 (12%) 4/50 (8%) 4/48 (17%) 14/47 (30%)
p=0.016
18/74 (24%) 11/50 (22%) 13/48 (27%) 20/47 (43%)
p=0.029
18/74 (24%) 12/50 (24%) 13/48 (27%) 20/47 (43%)
p»0.029
1/73 (1%) 2/50 (4%) 2/48 (4%) 6/47 (13%)
3/73 (4%) 6/50 (12%)
0/69 (0%) 3/50 (6%)
6/48 (13%) 11/47 (23%)
p=0.002
1/47 (2%) 5/46 (11%)
p=0.009
^Source: NTP, 1980a
bp-values calculated yslng the Fisher Exact Test.
11-23
-------
TABLE 11-14
Promoting Effect of 2,3,7,8-TCDD on Hepatocardnogenesls by a Single Dose of
D1ethyln1trosam1ne (DEN) and Partial Hepatectoray (PH)a*B
Group
No.
1
2
3
4
5
6
7
Treatment
PH + DEN
PH * TCDD (low dose)
PH f TCDD (high dose)
PH * Phenobarbltal
PH + DEN + TCDD
(low dose)
PH + DEN + TCDD
(high dose)
PH f DEN + Phenobarbltal
NC
4
5
5
6
5
7
4
No. of Enzyme-Altered
foci per cm3 of Liver
346 i 65
46 * 15
76 t 20
138 + 40
1582 * 300
1280 t 40
1510 ± 185
Percent Liver Volume Which
Is Enzume-Altered foci
5.0
0.1
0.1
0.1
7.8
35.0
5.0
Number of Rats
with Carcinoma
0
0
0
0
°d
5/7e (p=0.0075)f
2
aSource: P1tot et al., 1980
^Female rats (200 g) were Intubated where shown with DEN. Seven days later TCDD (Injected subcutaneously)
or phenobarbltal (0.05% In the diet) administration was begun and continued for 28 weeks at which time the
animals were sacrificed and the livers examined. The low and high doses of TCDD were 0.14 and 1.4 yg/kg/
2 weeks, respectively, administered subcutaneously. DEN was given at a dose of 10 mg/kg. See text for
further details.
°Denotes the number of animals used In each group.
Three rats showed "neoplastlc nodules."
eOne rat showed a "neoplastlc nodule."
p-value calculated using the Fisher Exact Test.
-------
The animals 1n groups 1, 5, 6 and 7 received d1ethyln1trosam1ne (DEN), The
rats 1n group 1 were then maintained on a standard laboratory diet for 32
weeks. The rats 1n groups 2 and 3 received no DEN, but starting 1 week
after hepatectomy received biweekly subcutaneous Injections of 0,14 or 1.4
vg/kg of 2,3,7,8-TCDD 1n corn oil for a period of 28 weeks (2,3,7,8-TCDD
was 98.6% pure and provided by Dow Chemical Co.). Groups 5 and 6 received
DEN, and 1 week later were Initiated on a regimen of 14 biweekly Injections
of 0.14 and 1.4 jig/kg of 2,3,7,8-TCDD. The animals 1n group 4 received
0.05% sodium phenobarbltal 1n the diet starting 1 week after partial
hepatectomy for 28 weeks, and the animals 1n group 5 received DEN and 1 week
later were also administered 0.05% sodium phenobarbltal 1n the diet for the
duration of the experiment. At the end of the experiment, rats were killed
and sections of the Hver were removed and frozen on solid COp. Serial
sections of the frozen blocks of liver were cut and stained consecutively
for glucose-6-phosphatase (G&Pase), canallcular ATPase, glutamyl transpeptl-
dase (GGTase) with hematoxylln and eosln. The number of enzyme-altered foci
were determined from photographs of h1stochem1cally stained sections.
Hepatocardnomas were diagnosed by standard hlstopathologlcal criteria.
*
The results presented 1n Table 11-14 showed that the number of foci with
single enzyme changes, the number of foci with multiple enzyme changes, and
the total liver volume, substantially Increased with the administration of
2,3,7,8-TCDD. No carcinomas were detected 1n four rats treated with DEN
only, but five of seven rats treated biweekly with 2,3,7,8-TCDD at 1.4
pg/kg 1n addition to DEN had hepatocellular carcinomas, and six of seven
rats had hepatocellular carcinomas or hepatocellular neoplastlc nodules with
a statistical significance (p=0.0075). Three of five rats treated biweekly
with 2,3,7,8-TCDD at 0.14 vg/kg 1n addition to DEN had hepatocellular
11-25
-------
neoplastlc nodules (p=0.083). Rats receiving only 2,3,7,8-TCDD after
partial hepatectomy showed no significant Increase 1n enzyme-altered foci
and no neoplasla.
The results of this study provide evidence that 2,3,7,8-TCDD acts as a
potent promoter 1n this two-stage model of hepatoeardnogenesls, causing
Increased neoplasla and Increases 1n enzyme-altered foci at exceedingly low
levels.
11.1.1.6.2. National Toxicology Bloassay Program Skin Painting Study In
Mice (19805) — This cancer bloassay of 2,3,7,8-TCDD for possible cardno-
genlclty 1n Swiss-Webster mice was tested by the Illinois Institute of
Technology under a contract sponsored by NCI. In this study, groups of 30
male and female Swiss-Webster mice were used. 2,3,7,8-TCDD 1n acetone
suspension was applied to the skin of mice 3 days/week for 104 weeks. Male
mice received 0.001 vg 2,3,7,8-TCDD per application, and the female mice
received 0.005 vg 2,3,7,8-TCDD per application.
In another experiment, the same number of animals were pretreated with
one application of 50 vg 7,l2-d1methylbenz(lJanthracene (DMBA*) 1n
0.1 ml acetone 1 week before 2,3,7,8-TCDD application was Initiated.
Forty-five mice of each sex received 0.1 ml acetone 3 times/week and 30
animals of each sex were used as untreated controls; no DMBA control was
used.
In the male and female groups of mice treated with 2,3,7,8-TCDD or
2,3,7,8-TCDO following a single application of DMBA, mean body weights were
not affected as compared with the vehicle controls. Mean body weights of
*DMBA obtained from K and K Laboratories (Cleveland, Ohio). Its purity was
not evaluated by NCI, but was stated by the manufacturer to be at least 95%,
11-26
-------
treated and vehicle control groups of females were lower than those of
untreated controls. Mean body weights of males were less than that of
untreated controls.
The results of hlstopathologlc diagnosis are shown 1n Table 11-15. The
results show that 2,3,7,8-TCDD Induced statistically significant (p<0.05)
Increases of Hbrosarcoma 1n the Integumentary systems of female mice
treated with 2,3,7,8-TCDD alone and 2,3,7,8-TCOD following a single Initial
application of DMBA.
11.1.1.6.3, Berry et al. Skin Painting Study In Mice (1978, 1979) —
Berry et al. (1978) applied 2,3,7,8-TCDD 1n acetone solution at 0.1
pg/mouse twice weekly for 30 weeks to the skin of 30 female Charles River
CD-I mice after Initiation with a single dermal application of the known
skin carcinogen DMBA In acetone. After 30 weeks of promotion with 2,3,7,8-
TCDD, no paplllomas were observed on the DMBA-1n1t1ated mice. In the posi-
tive controls, DHBA-1n1t1ated mice were treated with 12-Q-tetradecanoyl-
phorbol-13-acetate (TPA) for 30 weeks; 92% of these mice developed tumors.
Berry et al. (1979) also studied the effects of treatment with 2,3,7,8-
TCDD and 7,12-d1methylbenz(a)anthracene (DHBA) 1n a two-stage tumorlgenesls
bloassay 1n mouse skin. In this study, tumors on the shaved skin of female
CD-I mice were Initiated by topical application of DMBA and were promoted
with TPA. Pretreatment with 2,3,7,8-TCDD markedly Inhibited the Initiation
of tumors by DMBA. The effects were greatest when 2,3,7,8-TCDD was applied
3-5 days before Initiation and were negligible when 1t was applied only 5
minutes before Initiation. The Inhibition was almost complete (94-96%) when
a single dose of 1 pg of 2,3,7,8-TCOD/mouse was applied, but was only
slightly less effective (89%) when the dose was Increased to 10 jig/mouse.
11-27
-------
TABLE 11-15
Incidence of Primary Tumors 1n Mice Administered 2,3,7,8-TCDO
or 2,3,7,8-TCDD Following DMBA by Dermal Application3
Type of Tumors Vehicle Control
Dose Levels0
TCDD
DMBA (50 jig)
plus TCDD
Integumentary
system
Flbrosarcoma
3/42 (7%)
HALE
0.001
x 3/weeks 0.001 yg x 3/weeks
6/28 (21%)
p=0.08
6/30 (20%)
p=0.10
Flbrosarcoma
2/41 (5%)
FEMALE
0.005 v»
8/27 (30%)
p=0.007
3/weeks
0.005
x 3/weeks
8/29 (28%)
p-0.010
aSource^~NTP, 19805
kp-value calculated using the Ftsher Exact Test.
11-28
-------
The time course of the Inhibitory effects was closely parallel to the time
course of Induction of arylhydrocarbon hydroxylase 1n the skin of the mice.
It was also associated with substantial reduction 1n the covalent binding of
the DMBA metabolite to DNA and RNA, but with no change 1n their binding to
protein.
The same authors also reported Inhibitory effects of 2,3,7,8-TCDD on the
Initiation of mouse skin tumors by benzo(a)pyrene (BaP), although the effect
was not as great (maximum 65%) with BaP as with DMBA.
11.T.I.6.4. Cohen et al. Skin Painting Study 1n Mice (1979) — Cohen
et al. (1979) showed that pretreatment of mice with dermally applied
2,3,7,8-TCDD resulted 1n the Inhibition of skin tumor Induction by subse-
quent treatment with DMBA and BaP. The Inhibition of skin carclnogenesls by
BaP 1n mice after pretreatment with 2,3,7,8-TCDD was associated with an
Increase 1n covalent binding of BaP metabolites to DNA, RNA and protein (In
contrast to the results with DMBA, which showed a reduction In binding to
DNA and RNA). However, the BaP metabolites that were bound to DNA and RNA
1n mice pretreated with 2,3,7,8-TCDD differed from those In untreated mice.
In particular, pretreatment with 2,3,7,8-TCDD markedly reduced the formation
of the presumptive ultimate carcinogenic metabolite of BaP, 7,8-d1ol-9,lO-
epoxy-BaP and Its covalent binding with guanoslne 1n DNA.
11.1.1.6.5. Kourl et al. Mouse Study (1978) ~ This study was designed
as an Investigation of the cocardnogenlc activity of 2,3,7,8-TCDD adminis-
tered to mice 1n conjunction with subcutaneous administration of 3-methyl-
cholanthrene (3-MC). Two Inbred strains 1n mice, C57BL/6Cum (abbreviated
B6) and DBA/2Cum (abbreviated D2), were used. These strains are responsive
and nonresponslve, respectively, to the Induction of aryl hydrocarbon
hydroxylase (AHH) by 3-MC.
11-29
-------
Groups of mice of both sexes were Injected subcutaneously at 4-6 weeks
of age with either 150 pg of 3-MC dissolved 1n trloctanoln or with trloc-
tanoln alone. Some groups were also Injected with 2,3,7,8-TCDD dissolved 1n
£-d1oxane, either simultaneously with the administration of 3-MC or 2 days
earlier. Two doses of 2,3,7,8-TCDD (1 vg/kg and 100 vg/kg) were used,
and the effects of both Intraperltoneal and subcutaneous Injections were
Investigated.- Two sets of experiments Involving 29 groups of mice were
conducted ~1 year apart (Tables 11-16 and 11-17).
After treatment, the mice were observed for 36 weeks, during which time
they were palpated weekly for the presence of tumors; latency was calculated
when the subcutaneous tumors became 1 cm 1n diameter. Only tumors charac-
terized hlstologlcally as flbrosarcomas at the site of Inoculation were
considered. It 1s unclear whether or not these were the only tumor types
observed. The term "carcinogenic Index" used by the authors was defined as
the percentage of tumor Incidence 8 months after treatment divided by the
average latency 1n days multiplied by 100. No details were given of the
number of animals 1n each group at the start of each experiment, but the
numbers dying 1n the first 28 days and the numbers at risk (surviving 36
weeks) were tabulated. The results of this study are shown 1n Tables 11-16
and Tr-1-7.
No subcutaneous tumors were observed 1n controls or 1n mice treated with
2,3,7,8-TCDD alone. In B6 (responsive) mice, the administration of
2,3,7,8-TCDD did not significantly enhance the Induction of tumors by 3-HC.
However, 1n both experiments Involving D2 (nonresponslve) mice, the adminis-
tration of 2,3,7,8-TCDD simultaneously with 3-HC appeared to enhance the
carcinogenic response. The "carcinogenic Index" Increased from 1-6 1n
groups treated with 3-MC alone to 14 In the group treated subcutaneously
11-30
-------
TABU It -16
Effects of Intraperltoneal Administration of 2,3,7.8-TCDO on 3-HC-In1t1ated Subcutaneous Tumors3
i
W
Treatment
Inbred
Strain -2 Days
B6 l.p. p-dloxln
1.p. TCDO (100 pg/kg)
None
None
None
None
None
l.p. TCDD (100 pg/kg)
l.p. TCDD (1 pg/kg)
02 l.p. p-dloxane
l.p. TCDD (100 pg/kg)
None
None
None
None
None
l.p. TCDD (100 pg/kg)
l.p. TCDD (1 pg/kg)
s
s
s
1
1
s
1
1
s
s
s
s
s
s
1
1
s
\
1
s
s
s
.c.
.c.
.c.
• p.
• p.
.c.
.p.
.p.
.c.
.c.
.c.
.c.
.c.
.c.
• p.
• p.
.c.
.p.
• p.
.c.
.c.
.c.
0
Days
trloctanoln
trloctanoln
3-HC
TCDD
TCDD
3-HC
TCDD
TCDD
3-HC
3-HC
3-HC
(100 pg/kg)
(100 pg/kg) <-
(1 pg/kg)
(1 pg/kg) f
trloctanoln
trloctanoln
3-HC
TCDD
TCDD
3-HC
TCDD
TCDD
3-HC
3-HC
3-HC
(100 pg/kg)
(100 pg/kg) f
(1 pg/kg)
(1 pg/kg) *
No. of Mice
Dying Because
of Treatment0
1
20
1
20
30
4
6
20
6
6
24
3
30
43
5
5
20
6
No. of Hlce
at Risk for
Tumorsc
39
27
36
30
43
46
27
25
23
22
25
34
38
43
48
34
28
31
No. of
Hlce with
Tumors*'
0
0
29
0
33
0
27
21
16
0
0
1
0
10
0
5
0
0
X of H1ce
with Tumors
0
0
81
0
71
0
100
84
70
0
0
3
0
23
0
15
0
0
Average
Latency
(days)
125
123
132
129
140
217
178
199
Carcinogenic
Index6
65
63
76
65
50
1
13*
7
aSource: Kourl et al., 1978
During the first 28 days following treatment.
C0ef1ned as the number of mice surviving the 36-week observation period.
At the end of the 36-week experiment.
Percentage of Incidence of tumors, divided by the average latency 1n days, multiplied by 100 (8).
flh1s carcinogenic Index value lies outside (greater than) the 99X confidence Interval (I.e., p<0.01) constructed from seven different
studies over the past 5 years during which 150 pg of 3-HC was given s.c. to 0? mice. These studies Included ?95 I)? Ice, the mean -- 5 0 for
all seven studies was a carcinogenic Index of 5.43 - 2.70.
-------
TABIE 11-17
Effect of Intraperltoneal or Subcutaneous Administration of ?,3,7,8-TCOD Given 2 Days Before or Simultaneous
With Subcutaneous Administration of 3-HC on Tumor{genesis In D? M1cea
Treatment
-2 Bays
None
l.p. p-dloxane
l.p. TCOB (100 wg/ko,)
None
__>
— > None
W
fSJ
None
None
None
None
None
None
s
s
s
1
1
s
1
s
s
s
s
s
s
s
s
.c.
,c.
,c.
•p.
.p.
.c.
•p.
.c.
.c.
.c.
.c.
.c.
.c.
.c.
.c.
0 Days
3-HC
3-HC
3-HC
p-dloxane * s.c. 3-8C
TCDD (100 jig/kg) *
3-HC
TCOD (1 jig/kg) *
3-HC
p-dloxane » s.c. 3-HC
TCDD (100 wq/kg)
TCOO (100 iig/kg) »
3-HC
TCDO (1 vg/kg)
TCDD (1 ng/kg) »
3-HC
No. of Hlce
Dying Because
of Treatment
0
10
35
5
38
2?
?
8
18
?
?
No. of nice
at Risk for
Tumors
30
40
65
45
6?
78
68
4?
82
48
96
Ho. of
H1ce with
Tumors
3
3
9
5
U
8
8
0
46
0
?1
% of Bice
ill th Tumors
10
10
14
11
?7
10
12
0
55
0
21
Average
latency
(days)
177
194
145
176
183
162
180
145
154
Carcinogenic
Index
6
5
10
6
15l>
6
6
3Bb
14"
aSource; Kourl et al., 1978
''These carcinogenic Index values lie outside the 99X confidence Interval.
-------
wHh 2,3,7.8-TCDD at 1 wg/kg, and 13-15 1n the groups treated IntrapeM-
toneally with 2,3,7,8-TCDD at 100 yg/kg. The authors concluded that
2,3,7,8-TCDD acts as a cocardnogen, possibly as an Inducer of AHH at the
site of Inoculation.
A more appropriate statistical analysis would be a comparison of tumor
Incidence 1n 2,3,7,8-TCDD-treated groups with tumor Incidence 1n correspond-
ing 3-HC-treated groups within the same experiment. The results of this
analysis are given 1n Table 11-18.
From these results, the CAG concluded that the experiment adequately
demonstrated the enhancement by 2,3,7,8-TCDD of tumor Induction when
2,3,7,8-TCDD was administered simultaneously with 3-HC at the higher dose
(100 vg/kg). The reported results at the lower dose (1 vg/kg) are not
statistically significant unless the reduction 1n latency 1s taken Into
account, which 1s difficult to do rigorously. Despite defects 1n reporting
(failure to specify the Initial number of animals 1n each group and to
report tumor Incidence by sex), the results provide evidence that 2,3,7,8-
TCDD acts as a cocardnogen. The failure of 2,3,7,8-TCDD to Induce tumors
when administered alone was not unexpected since only a single dose was
administered and the duration of the study was very short (36 weeks).
11.1.1.6.6. Poland et al. Study (1982) — Poland et al. (1982)
described studies which Indicate that genetic differences 1n mice affect the
tumor-promoting capacity of 2,3,7,8-TCDD 1n the mouse skin two-stage tumorl-
genesls model. Both 2,3,7,8-TCDD and TPA were compared for tumor-promoting
activity 1n DHBA-1n1t1ated HRS/J mice that were either heterozygous (hour/*)
or homozygous (hour/hour) for the recessive "hairless" trait. Promotion
with biweekly applications of 2 yg of TPA for 25 weeks resulted 1n
papHloma Incidences of 100 and 70% 1n (hourA) and (hour/hour) mice,
11-33
-------
TABLE 11-18
Incidence of Tumors 1n Mice Treated With 3-MG
and WHh 3-HC and 2,3,7,8-TCDDa
Experiment
1
2
2
2
Dose of
TCDD
(vg/kg)
100
100
100
1
Tumor Incidence
Route of
Administration
Intraperltoneal
Intraperltoneal
subcutaneous
subcutaneous
TCDO and 3-MC
10/43
17/62
46/82
21/98
p-Value°
3-MC
1/34 p=0.01
5/45 p=0.03
5/42 p=3.0 x 10~7
5/45 p=0.1
aSource*. Kourl et al., 1978
Dp-value calculated using the Fisher Exact Test (one-tailed).
11-34
-------
respectively. Promotion of OMBA-initiated (hour/*) mice with 2,3,~7,8-TCDD
(50 rig/application for 8 weeks followed by 20 ng/application) did not result
in the formation of tumors, while promotion of (hour/hour) mice resulted in
both the same incidence and multiplicity of tumors as observed in TPA-pro-
moted mice. With either DMBA or methyl-N-nitrosoguanidine (MNNG)-inltiated
(hour/hour) mice, the effective dose of 2,3,7,8-TCDD was -100-fold less than
TPA on a molar basis. Histologic examination of the skin showed that TPA
produced both acute inflammation and hyperplasia in (hour/*) and (hour/hour)
mice, while 2,3,7,8-TCDD produced hyperplasia and hyperkeratosis only in
(hour/hour) mice with no inflammatory response. The lack of a 2,3,7,8-TCDD-
Induced inflammatory response suggested to the authors that 2,3,7,8-TCDD
promoted skin papillomas in (hour/hour) mice by a mechanism different from
TPA.
11.1.1.6.7. DiGiovanni et al. Study (1977, 1980) — Investigations
have also been conducted on the effects of prior or simultaneous treatment
with 2,3,7,8-TCDD on the subsequent development of skin tumors by chemical
carcinogens. When 2,3,7,8-TCDD (0.1 yg) was administered simultaneously
with DMBA (200 nmol) to the backs of CD-I mice in a single Initiation dose,
the skin papllloma incidence following promotion with TPA was nearly the
same as when DMBA alone was used as the Initiator (DiGiovanni et al., 1977).
Although simultaneous exposure to 2,3,7,8-TCDD and DMBA did not appreciably
affect tumor yield, Berry et al. (1979) demonstrated a marked 93% decrease
1n the incidence of DMBA-initiated tumors when CD-I mice were pretreated 3
days before DMBA initiation with 1 pg/mouse of 2,3,7,8-TCDD. The time of
treatment with 2,3,7,8-TCDD in relation to Initiation was shown to be
critical 1n the antitumorigenic effects of 2,3,7,8-TCDD (Berry et al., 1979;
11-35
-------
D1G1ovann1 et al., 1979a, 1980), as shown 1n Figure 11-1. Maximum tumor
Inhibition of between 86 and 94% occurred when pretreatment was between 1
and 5 days before Initiation. If pretreatment was 10 days before DHBA
Initiation, the tumor yield was decreased by 78%, while 2,3,7,8-TCDD treat-
ment 5 minutes before or 1 day after DHBA Initiation had no effect on tumor
yield. There was some Indication of an Inverse relationship between the
pretreatment dose of 2,3,7,8-TCDD (3 days before DHBA Initiation) and the
Incidence of tumors. 2,3,7,8-TCDD doses of 0.0, 0.01, 0.1 and 2 yg/mouse
resulted 1n decreased tumor yields, respectively, of 0, 83, 92 and 96%
(D161ovann1 et al., 1979a). Also under similar experimental conditions
Cohen et al. (1979) observed a 75% decrease 1n the Incidence of skin tumors
In Sencar mice pretreated with 1 ng of 2,3,7,8-TCDD 3 days before Initia-
tion by DHBA.
D1S1ovann1 et al. (1980) Investigated the antltumorlgenlc effect of
2,3,7,8-TCDD 1n CD-I mice with chemical carcinogens other than DHBA (see
Figure 11-1). As observed with DHBA, exposure to 2,3,7,8-TCDD 3 days! before
Initiation with either benzo(a)pyrene (BaP) or 3-MC resulted 1n a decrease
1n tumor yield as compared with acetone-pretreated animals; however! pre-
treatment with 2,3,7,8-TCDD 5 minutes before or' 1 day after Initiations was
Ineffective 1n changing the tumor yield. The maximum decrease 1n tumor
production was 86 and 57%, respectively, for BaP and 3-MC Initiated mice. A
different temporal relationship was observed 1n the ability of 2,3,7,8-TCDD
to Inhibit tumor formation by BaP-d1ol-epox1de as compared with
the
previously studied polyaromatlc hydrocarbons (PAH). When 2,3,7,8-TCDD was
applied 3 days or 5 minutes before, or 1 day after Initiation with BaP-diol
epoxlde, there was an 81.5 and 49% decrease 1n tumor yield. Examination of
PAH metabolism 1n the skin of mice treated with 2,3,7,8-TCDD showed
11-36
-------
140 —
-10
TIME OF APPLICATION IN DAYS
(RELATIVE TIME TO THE APPLICATION OF THE INITIATOR
FIGURE 11-1
Time-Dependent Inhibition by 2,3,7,8-TCDD of Tumor Initiation
Summary of the time-dependent Inhibitory effect of 2,3,7,8-TCDD on tumor
Initiation by DMBA U), BaP (o), 3-NC (A) and BaP-d1ol-epox1de (o).
Animals were Initiated with 10 nmol DNBA, 100 nmol BaP, 100 nmol 3-MC and
200 nmol BaP-d1ol-epox1de and promoted 1 week later with twice weekly appli-
cation of TPA.
11-37
-------
21-fold Increase 1n aryl hydrocarbon hydroxylase (AHH) activity 72 hours
after treatment (D1G1ovann1 et al., 1980). The In vitro metabolism of DHBA
by dermal homogenates from 2,3,7,8-TCDD-treated mice Indicated both qualita-
tive and quantitative changes 1n metabolism (Cohen et al., 1979; D1G1ovann1
et al., 1979a; Berry et al., 1979). The similarity 1n the time frame of AHH
Induction and the ant1tumor1gen1c effect of pretreatment with 2,3,7,8-TCDD
suggested that the ant1tumor1gen1c properties of 2,3,7,8-TCDD resulted from
2,3,7,8-TCDD Induced alteration 1n the metabolism of the Initiating chemi-
cal. Although metabolic change was a possible mechanism for the Inhibition
of DHBA, 3-HC and BaP Initiation, the ability of 2,3,7,8-TCDD to Inhibit
tumor yield when administered 1 day after Initiation with BaP-d1ol-epox1de
Indicated by D1G1ovann1 et al. (1980) that more than one mechanism may
participate 1n the ant1carc1nogen1c effect of 2,3,7,8-TCDD.
11.1.1.6.8. Cockerham et al. 1980 Field Study on Beach Mice -- Cocker-
ham et al. (1980) performed a field study on beach mice, Peramyscus polle-
notus. that Inhabited an area which was heavily treated with the herbicide
2,4,5-T, of which 2,3,7,8-TCDD was a contaminant. Analysis of the soil 1n
the contaminated area revealed average 2,3,7,8-TCDD levels of 150 ppt at the
surface. Measured levels of 2,3,7,8-TCDD 1n the liver of beach mice from
the contaminated area were determined to be 1300 ppt 1n males and 960 ppt 1n
females. Detection of 2,3,7,8-TCDD 1n the liver Indicates that the compound
was absorbed; however, since seeds 1n the area did not contain 2,3,7,8-TCDD,
1t was believed that the animals Ingested the compound from contaminated
dust while grooming. In the 10 male and 5 female animals captured 1n the
contaminated area, there were no hlstopathologlc differences, Including neo-
plastlc lesions, observed 1n the liver as compared with 9 male and 6 female
mice captured 1n a noncontamlnated area. The only observed difference 1n
11-38
-------
the two groups of mice was a statistically significant {95% confidence)
Increase 1n Hver-to-body weight ratios. The authors back-calculated from
the 2,3,7,8-TCDD levels of the liver and estimated a dally 2,3,7,8-TCDD dose
of 0.0012 vg/kg bw. It was noted that this exposure was much lower than
the exposures used 1n laboratory studies to produce tumors.
11.1.2. Studies Using HxCDD.
11.1.2.1. NATIONAL TOXICOLOGY BIOASSAY PROGRAM (ORAL) STUDY IN RATS AND
MICE (NTP, 1980d) -- Although 1,2,3,6,7,8-HxCDD and 1,2,3,7,8,9-HxCDD have
not been tested Individually for carc1nogen1c1ty, the NTP has performed a
chronic bloassay In both Osborne-Hendel rats and B6C3F1 mice to determine
the carc1nogen1c1ty of a mixture of 1,2,3,6,7,8- and 1,2,3,7,8,9-HxCDD {NTP,
1980d). The mixture consisted of 31% of the 1,2,3,6,7,8-HxCOD congener and
67% of the 1,2,3,7,8,9-HxCDD congener, with a total HxCDD purity of 98%.
The following Impurities were detected 1n HxCDD used for this bloassay:
PeCDD, 0.04%; TCDD, 0.09%±0.03%; TMCDD, 0.004%; DCDD, 0.004% and Bromo
PeCDD, <0.004%. The specific Isomers of these Impurities were not Identi-
fied. The compound was protected from light during storage, and every 3
months a stock acetone suspension was prepared. The working solution was
administered to the test animals 1n corn oil-acetone (9:1) by gavage 2
times/week. All treated groups consisted of 50 animals of each sex, while
the control groups, both vehicle and untreated controls, consisted of 75
animals of each sex. The male and female rats, and the male mice received
HxCDD doses of 0.0, 1.25, 2.5 and 5 pg/kg/week, and the female mice
received doses of 0.0, 2.5, 5.0 and 10 yg/kg/week. Treatment was con-
tinued for 104 weeks followed by a 3- to 4-week observation period.
Complete necropsies, Including extensive hlstologlc examinations, were
performed on animals at the time of natural death, when moribund or at the
termination of the study.
11-39
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A decrease 1n body weight gain was seen at the two higher exposure
levels. A dose-related "toxic hepatitis" that was noninflammatory and
consisted of degenerative changes 1n the liver, eos1noph1!1c foci of cellu-
lar alteration, mild flbrosls and bile duct hyperplasla was also observed.
Cytomegaly and I1p1dos1s were Included 1n these degenerative changes. The
only neoplastlc lesions that appeared to be treatment-related were neoplas-
t1c nodules of the liver and hepatocellular carcinomas (Table 11-19). The
combined Incidences of these tumors 1n male rats were 0/74, 0/49, 1/50 and
4/48, while 1n female rats the Incidences were 5/75, 10/50, 12/50 and 30/50
for the control, low-, medium- and high-dose groups, respectively. The
Incidence of liver tumors 1n male rats showed a positive dose-related trend
by the Cochran-Armltage test; the Incidence 1n the high-dose male rat group
was statistically different from the control group by the Fisher exact test
(p=0.022) but the requirements by NTP for overall significance were not met
based on the Bonferronl Inequality. The NTP thus concluded that the
evidence for the carc1nogen1c1ty of HxCDD 1n male rats was Inconclusive. In
female rats, the Cochran-Armltage test was significant at p<0.001, and the
liver tumor Incidence of the high-dose animals was significantly (pO.OOl)
different from that of the control group, as well as with the mid-dose group
(p=0.006).
Subsequent to the release of the NTP gavage study of HxCDD 1n rats and
mice (NTP, 1980d), several pathologlsts reevaluated the microscopic slide
material of the female rats. These reviews resulted from a report by Squire
(1983) which stated that many of the entitles diagnosed as tumors by NTP
were actually nonneoplastlc regenerative nodules; but his report concluded
that the HxCDD bloassay still provided evidence of a weak hepatocardnogenlc
11-40
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TABLE 11-19
Liver Tumor Incidences 1n Male and Female Osborne-Mendel Rats
Administered HxCDD for 104 Weeks3
Diagnoses
Neoplastlc
nodule (NN)
Hepatocellular
carcinoma (HC)
Combined NN + HC
Neoplastlc
nodule (NN)
Hepatocellular
carcinoma (HC)
Combined NN + HC
aSource: Adapted
b
Treatment Group
Untreated Vehicle Low Dose M1d Dose
Control Control
MALE
2/75b 0/74 0/49 1/50
0/75 0/74 0/49 0/50
2/75 0/74 0/49 1/50
FEMALE
1/73 5/75 10/50 12/50
p=0.026 p=0.006
0/74 0/75 0/50 0/50
1/73 5/75 10/50 12/50
p=0.026 p=0.006
from NTP, 1980c
No. of rats with lesion
High Dose
3/48
1/48
4/48
p=0.002c
30/50
p=6.94xlO~"
4/50
p=0.024
30/50
p=6.94xlQ~il
No. of rats examined microscopically
cp-values calculated using the Fisher Exact Test.
11-41
-------
effect 1n rats and mice. Drs, R. Schueler and B. Haberman also reported
discrepancies 1n the diagnoses of liver tumors from the NTP gavage study.
Their findings were reported 1n an Internal U.S. EPA memorandum from CAG to
J. Benin (U.S. EPA, 1983b) with an attached report prepared by Dr. R.
Schueler, Research Pathology Associates, Inc. (Schueler, 1983). Finally,
Dr. E. HcConnel of NTP requested that Dr. P. Hlldebrandt of Tracor-JHco,
Inc., review the microscopic slides of the HxCOD bloassay (gavage) 1n the
female rat; his findings (HUdebrandt, 1983) agreed closely with those of
Drs. Schueler and Haberman. Dr. Hlldebrandt's findings (Table 11-20),
although not as statistically significant as the original NTP findings,
still confirmed that the HxCDD mixture administered by gavage produced an
Increased Incidence of Hver tumors 1n treated female rats as compared with
control animals, as well as an Increase In "toxic hepatitis."
In mice there were no gross signs of HxCDD toxldty; however, as
observed 1n rats, there was a dose-related Incidence of "toxic hepatitis"
consisting of degenerative liver changes and/or necrosis associated with
cellular Infiltration and mild flbrosls. The only neoplastlc changes that
were treatment-related were Increases 1n hepatocellular adenomas and carci-
nomas (Table 11-21). The adenomas were characterized as groups of cells
with a uniform cell type that did not conform to the lobular architecture
and which caused compression of the surrounding normal liver, while the
carcinomas contained cells with greater hlstologlc deviations, disorganized
growth and more cells 1n mitosis. A few liver tumors 1n control and dosed
groups metastaslzed to the lungs. The Incidence of hepatocellular adenomas
or carcinomas were 15/73, H/50, 14/49 and 24/48 1n male mice, and 3/73,
4/48, 6/47 and 10/47 1n female mice of the control, low-, medium- and high-
dose groups, respectively. In both male and female mice, the liver tumor
11-42
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TABLE 11-20
Liver Tumor Incidences 1n Female Qsborne-Mendel Rats Administered
HxCDD by Gavage for 104 Weeks3
Diagnoses
Neoplastlc
nodule (NN)
Hepatocellular
carcinoma (HC)
Combined NN * HC
aSource: Adapted
b
Untreated
Control
1/7 3b
0/73
1/73
Vehicle Low Dose
Control 1.25
2/75 5/50
0/75 0/50
2/75 5/50
yg/kg/week
M1d Dose
2.5
7/50
p=0.02c
0/50
7/50
p=Q.Q2
High Dose
5
16/50
p=6.0xlO~«
2/50
18/50
p=7.3x!0~7
from HUdebrandt, 1983
No. of rats
with lesion
No. of rats examined microscopically
cp-values calculated using the Fisher Exact Test.
11-43
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TABLE 11-21
Liver Tumor Incidences 1n Male and Female B6C3F1 Mice Administered
HxCDD by Gavage for 104 Weeks3
Diagnoses
Hepatocellular
adenoma (HA)
Hepatocellular
carcinoma (HC)
Combined HA + HC
Hepatocellular
adenoma (HA)
Hepatocellular
carcinoma (HC)
Combined HA +• HC
aSource: Adapted
b
Tnr* ^ r4£»n/"Q — — -..
Untreated
Control
15/75b
12/75
27/75
2/74
0/74
2/74
from NTP, 1980c
Treatment Group
Vehicle Low Dose M1d Dose
Control
!
HALE !
7/73 5/50 9/49
8/73 9/50 5/49
15/73 14/50 14/49
FEMALE
2/73 4/48 4/47
1/73 0/48 2/47
3/73 4/48 6/47
High Dose
15/48
p=0.003c
9/48
24/48
p=7.33x!0~4
9/47
p=0.003
2/47
10/47
p=0.004
No. of rats with lesion
No. of rats examined microscopically
cp-values calculated using the Fisher Exact Test.
11-44
-------
showed a significant dose-related trend by the Cochran-Armltage
test, and the Incidence of tumors 1n the high-dose group was significantly
higher than the Incidence 1n the control group by the Fisher exact test.
The obvious question was raised concerning the presence of tetrachloro-
d1benzo-p-d1ox1n as an Impurity (0.09%) 1n the test material, which may have
contributed to the observed liver tumor Incidence. The analysis presented
1n Table 11-22 shows that the calculated 95% upper-limit liver cancer
response due to 0.09% TCDD Impurity Is so low as compared with the observed
liver cancer response due to HxCDD 1n this cancer bloassay study, 1t 1s
reasonable to conclude that the Impurity 1n the test material did not
contribute significantly to the observed carcinogenic response for HxCDD.
HcGaughy and R1sp1n (1985) made the following comments regarding the
three documents listed below, which evaluated Issues that have been raised
with respect to the NCI/NTP HxCDD carc1nogen1c1ty bloassay on rats and mice:
1. The responses outlined by the Office of Health and Environ-
mental Assessment {OHEA} were prepared for presentation to
EPA's Science Advisory Board on November 28, 1984.
2. The document entitled "Response to Comments" was prepared by
Agency staff and a consultant pathologist.
3. A memorandum from Dr. John Doull» member of EPA's Science
Advisory Board, concerning the HxCDD audit by Dr. G. Schoenlg.
The above documents respond to questions that were raised
concerning many aspects of the bloassay study. These questions
relate, for example, to allegations of problems In:
• test procedures, such as problems 1n preparation of the
test material; flaws 1n methods of administration; flaws
1n recorclkeeplng procedures and practices.
pathology practices, such as non-uniform and substandard
tissue harvesting practices; non-uniform hlstologlc pro-
cedures; bias 1n histology review; and deficiencies 1n
correlation between gross and microscopic observations.
11-45
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TABLE 11-22
Liver Tumor Response for HxCDD (Observed)
and TCDD Contaminant (Calculated)
Animal
Rat (OH)
Male
HxCDD Dose Liver Cancer
( ^g/kg/week ) Response
Observed
5 4/48b
0.09% TCOO
Contaminant
Dose3
(vg/kg/week)
0.0045
Liver Cancer
Response
Calculated 95%
Upper Limit
TCDD has shown
Female
House (B6C3F1}
18/50C
0.0045
no effect 1n
NCI study
0.02/50d
Hale
Female
5
10
24/48e
10/47f
0.0045
0.009
0 . 20/48d
0.22/476
alt 1s assumed that all of the contaminant Is 2,3,7,8-TCDD.
bNTP reviewed
cRe-evaluat1on by Hlldebrandt (see Table 11-35)
dBased on response 1n NCI 2,3,7,8-TCDD study; see Table B-10.
eBased on response 1n NCI 2,3,7,8-TCDD study; see Table B-ll.
fBased on response 1n NCI 2,3,7,8-TCDD study; see Table B-12.
11-46
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• alleged bias 1n the above practices with respect to
treated and control animals.
pathology Interpretation: disagreement 1n conclusions
reached by different pathologlsts.
From our review of these documents we conclude that there were
Indeed some procedural flaws during the 1n-l1fe portion of the
study, and there were minor recordkeeplng problems. The management
of a two-year rodent study 1s a very complex undertaking. It "is
therefore not surprising that the procedural and recordkeeplng
deficiencies highlighted by the two audits occurred. They do not
Invalidate the study.
The detailed review by Agency staff and Dynamac Corporation of
Dr. Schoenlg's findings concerning room bias did not substantiate
his allegations. Similarly, review of Dr. Schoenlg's criticism of
the hlstologlc practices did not reveal meaningful deficiencies 1n
tissue harvesting, preparation of microscopic slides, and hlsto-
loglc diagnoses.
Differences 1n Interpretation among pathologlsts have pre-
viously been addressed by the Agency (see the OHEA document
attached hereto). The slight differences In Interpretation among
the different pathologlsts do not alter the conclusion as to the
carcinogenic potential of HxCDD.
We conclude that the HxCDD bloassay 1s valid, and that 1t can
appropriately be used for the assessment of the carcinogenic poten-
tial of HxCDD.
Under the test conditions of this bloassay, the 1:2 mixture of
1,2,3,7,8- and 1,2,3,7,8,9-HxCDD was carcinogenic, as Indicated by a statis-
tically significant Increased incidence In tumors of the liver In female
rats and 1n both male and female mice, and by a borderline liver tumor
response 1n male rats.
11.1.2.2. NATIONAL TOXICOLOGY BIOASSAY PROGRAM SKIN-PAINTING STUDY IN
MICE (NTP, 1980b,c) — Both 2,3,7,8-TCDD (NTP, 1980b) and a 2:1 mixture of
1,2,3,6,7,8- and 1,2,3,7,8,9-HxCDD (NTP, 1980c) have been tested 1n mice for
tumorlgenlc potential by dermal application. These studies were conducted
under the NTP and the description of the chemicals used was the same as
previously presented 1n the discussion of NTP (1980a,d). There was no
11-47
-------
Information found 1n the literature searched on the tumorlgenlc effect of
1,2,3,7,8-PeCDD following dermal exposure. The tumorlgenlc response after
chronic dermal exposure to HxCDD was presented 1n Table 11-23,
In both NTP bloassays {1980b,c>, groups of 30 male and 30 female Swiss-
Webster mice were treated with 100 yfi. of a solution of the test compound
1n acetone 3 times/week for 104 weeks. Groups of 45 animals were employed
as vehicle controls, and 2 groups of 15 animals were used as untreated
controls. The concentration of 2,3,7,8-TCDD used resulted 1n a dose of 0.01
vg/appl1cat1on 1n male mice and 0.005 jig/appl1cat1on 1n female mice; the
concentration of HxCDD used resulted 1n a dos« of 0.005 jig/application for
the Initial 16 weeks of the study, followed by a subsequent Increase to 0.01
vg/appl!cation for the remainder of the study. Subchronlc toxlclty
studies used to define the dose levels for the chronic bloassay Indicated
that all the doses used resulted 1n some liver damage but no Increase In
mortality. In the chronic study, animals were killed when moribund at the
termination of the study and examined for gross tumors. Microscopic exami-
nations were also made of all major organs.
In mice exposed to 2,3,7,8-TCDD (NTP, 1980b), there was no treatment-
related difference 1n body weight of either sex between exposed animals and
control groups; however, male mice treated with 2,3,7,8-TCDD had a signifi-
cant shortening of Hfespan. Nontumor1gen1c hepatic lesions were observed
1n treated female mice; no mention was made of these lesions occurring 1n
male mice. The only tumors that were treatment-related were Integumentary
system flbrosarcomas, with tumors developing on or near the site of applica-
tion. The Incidence of these tumors 1n male mice was 3/42 and 6/28, and 1n
female mice the Incidences were 2/41 and 8/27, respectively, for the vehicle
control groups and the treated animals. Only the tumor Incidence In female
11-48
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lABLt II-23
Carclnogenldty Bloassays of 2,3,7,8-TCDD and HxCDD by Dermal Application to M1cea
Compound
2,3,7,8-TCDD
2,3,7,8-TCDD
HxCDD
Sex
M
M
M
F
F
F
M
M
Doseb
0.01 tig/application
0.0 pg/appllcatlon
(vehicle control)
0.0 vg/appl1 cation
(untreated control)
0.005 jig/appll cation
0.0 vg/app 11 cation
(vehicle control)
0.0 pg/appli cation
(untreated control)
0.01 jig/app!1cat1onc
0.0 |ig/appl1cat1on
(vehicle control)
Duration
of
Exposure
104 weeks
104 weeks
NA
104 weeks
104 weeks
NA
104 weeks
104 weeks
Target Organ
Integumentary
system
Integumentary
system
Integumentary
system
Integumentary
system
Integumentary
system
Integumentary
system
lung
lung
Tumor Type
flbrosarcoma
flbrosarcoma
flbrosarcoma
flbrosarcoma
flbrosarcoma
flbrosarcoma
alveolar/
bronchlolar
carcinoma
alveolar/
bronchlolar
carcinoma
Tumor
Incidence
&/28
3/42
0/28
7/28
2/41
1/27
5/30
1/41
-------
TABLE 11-23 (cent.)
i
in
o
Compound Sex
HxCDD (cont.) H
HxCDD F
F
F
Doseb
0.0 yg/appH cation
(untreated control)
0.01 }ig/appl1cat1onc
0.0 jig/application
(vehicle control)
0.0 yg/appHcaUon
(untreated control)
Duration
of
Exposure
NA
104 weeks
104 weeks
NA
Target Organ
lung
skin
skin
skin
Tumor Type
alveolar/
bronchlolar
carcinoma
flbrosarcoma
flbrosarcoma
flbrosarcoma
Tumor
Incidence
4/28
4/27
2/41
0/30
^Source: NTP, 1980b,c
&The compound was applied 3 times/week 1n 100 ji£ of acetone.
cFor the Initial 16 weeks of the study, the dose was 0.005 tig/appHcation.
NA = Not applicable
-------
mice was statistically (p=Q.Q07) greater than control values; however, life
table analyses Indicated that the time to tumor was shorter 1n both male and
female treated mice. The Incidence of tumors 1n untreated and vehicle
control groups was similar.
In the bloassay of HxCDD (NTP, 1980c), no gross or nonneoplastlc hlsto-
loglc effects associated with treatment were observed. Although there was a
slight Increase 1n the Incidence of skin flbrosarcomas In female mice, this
Increase was significant 1n comparison with the vehicle control group, but
not significantly different from the untreated control group. The opposite
occurred with the Incidence of alveolar/bronchlolar carcinomas of the lung
1n male mice, which was significantly elevated 1n comparison with untreated
but not vehicle-treated controls. It was concluded that although dermal
exposure to 2,3,7,8-TCDD resulted 1n a carcinogenic response 1n both male
and female Swiss-Webster mice, dermal exposure to a mixture of 1,2,3,7,8-
TCDD and 1,2,3,7,8,9-HxCDD did not result In a carcinogenic response under
the conditions of this bloassay. A summary of the cardnogenldty bloassays
1s given 1n Table 11-24.
11.1.3. Summary of Animal Cardnogenldty. In a preliminary study by Van
Miller {1977a,b}, 2,3,7,8-TCDD was tested for cardnogenldty following oral
administration to rats. At the five highest dietary levels, 0.005, 0.05,
0.5, 1.0 and 5.0 ppb, which allowed long-term survival of the animals, an
Increased Incidence of total tumors was observed. In animals at an exposure
level of 0.001 ppb and 1n the control animals there were no tumors. This
study, however, provides only suggestive evidence of a carcinogenic response
since no Increase 1n slte-spedflc tumors was detected and the group sizes,
-10 animals/group, were too small for an assessment of a treatment-related
response. In a second, more extensive study by Kodba et al. (1978a) a
positive carcinogenic response was detected. In this study the estimated
n-5i
-------
TABLE 11-24
Carctnogenklty Btoassays of PCDD Administration by the Oral and Dermal Route
Exposure
Route/ Species/Strain Sex Dose or Exposure
Compound
Gavage/ rats/ H 0.0 ng/kg/week
2,3,7,8-TCOD Osborne-Hendel
0.1 yg/kg/week
0.05 ug/kg/week
-j 0.5 vg/kg/week
i
IVJ
Gavage/ rats/ F 0.0 wg/kg/week
2,3,7,8-TCOD Osborne-Hendel
0.1 tig/kg/week
0.05 ug/kg/week
0.5 vg/kg/week
Gavage/ m1ce/B6C3F1 N 0.0 ug/kg/week
2,3,7,8-TCDD
0.1 ug/kg/week
Duration
of
Treatment
104 weeks
104 weeks
104 weeks
104 weeks
T04 weeks
104 weeks
104 weeks
104 weeks
104 weeks
104 weeks
Duration
of Study
105 weeks
107 weeks
107 weeks
105 weeks
105 weeks
107 weeks
10? weeks
107 weeks
105 weeks
107 weeks
Vehicle
corn oil-
acetone
corn oil-
acetone
(9:1)
corn oll-
acetone
(9:1)
corn oil-
acetone
(9:1)
corn oll-
acetone
(9:1)
corn oil-
acetone
corn oll-
acetone
(9:1)
corn oll-
acetone
corn oil-
acetone
(9:1)
corn oll-
acetone
(9:1)
Tumor Type
folltcular-een adenomas
or carcinoma of the
thyroid
folllcular-cell adenomas
or carcinoma of the
thyroid
folltcular-cell adenomas
or carcinoma of the
thyroid
folllcular-cell adenomas
or carcinoma of the
thyroid
neoplastlc nodule or
hepatocellular carcinoma
of the liver
neoplastlc nodule or
hepatocellular carcinoma
of the liver
neoplastlc nodule or
hepatocellular carcinoma
of the liver
neoplastlc nodule or
hepatocellular carcinoma
of the liver
hepatocellular carcinoma
hepatocellular carcinoma
Tumor Reference
Incidence
1/69 UTP, 1980a
5/48
8/50
11/50
5/75
1/49
3/50
14/49
8/73 NTP, 1980a
9/49
-------
TABLE 11-24 (cont.)
Exposure
Route/ Species/Strain
Compound
Gavage/ m1ce/B6C3M
2,3,1,8-TCDO
(cont.)
Gavage/ m1ce/l6C3F1
2,3,7,8-TCDD
i
01
w
Oral/ rat/
2.3.7,8-TCDD Sprague-Dawley
Sex Dose or Exposure
0.05 pf/kg/week
0.5 ng/kg/week
F 0.0 wo,/kg/week
0.04 vg/kg/week
0.2 yg/kg/week
2.0 jig/kg/week
H 0.0 ppb
0.001 ppb
0.005 ppb
0.05 ppb
0.5 ppb
1.0 ppb
5.0 ppb
Duration
of
Treatment
104 weeks
104 weeks
104 weeks
104 weeks
104 weeks
104 weeks
78 weeks
78 weeks
78 weeks
78 weeks
78 weeks
78 weeks
78 weeks
Duration
of Study
107 weeks
107 weeks
10S weeks
107 weeks
107 weeks
107 weeks
95 weeks
95 weeks
95 weeks
95 weeks
95 weeks
95 weeks
95 weeks
Vehicle
corn oll-
acetone
(9:1)
corn oil-
acetone
(9:1)
corn oil-
acetone
(9:1)
corn oil-
acetone
(9:1)
corn oll-
acetone
(9:1)
corn oil-
acetone
(9:1)
In diet
In diet
In diet
In diet
In diet
In diet
In diet
Timor Type
hepatocellular carcinoma
hepatocellular carcinoma
hepatocellular carcinoma,
foTUcular-cell adenomas
of the thyroid
hepatocellular carcinoma,
folltcular-cell adenomas
of the thyroid
hepatocellular carcinoma,
folllcular-cell adenomas
of the thyroid
hepatocellular carcinoma,
folllcular-cell adenomas
of the thyroid
all tumors
all tumors
all tumors
all tumors
all tumors
all tumors
all tumors
Tumor
Incidence
8/49
17/50
1/73
0/69
2/50
3/50
2/48
1/47
6/47
5/46
0/10
0/10
5/10
3/10
4/10
4/10
7/10
Reference
HTP, 19803
DTP, 1980a
Van Miller
et al.. 1977a
-------
TABLE 11-24 (cont.)
I
Ul
Exposure Duration
Route/ Species/Strain Sex Dose or Exposure of Duration Vehicle
Compound Treatment of Study
Oral/ rat/ H 0.0 ng/kg/day 105 weeks 105 weeks In diet
2,3,7,8-TCDO Spragae-Bauley
0.001 vg/kg/day 105 weeks 105 weeks In diet
Oral/ rat/ H 0.01 vg/kg/day 105 weeks 105 weeks In diet
2,3,7,8-TCBB Sprague-Oawley
0.1 vg/kg/day 105 weeks 105 weeks In diet
Oral/ rat/ F 0.0 tig/kg/day 105 weeks 105 weeks In diet
2,3,7,8-TCOO Sprague-
Dawley
* 0.001 ng/kg/day 105 weeks 105 weeks In diet
0.01 vg/kg/day 105 weeks 105 weeks 1n diet
Tumor Type
squamous cell carcinoma
of the hard palate,
squamous cell carcinoma
of the tongue,
adenoma of the adrenal
cortex
squamous cell carcinoma
of the hard palate,
squamous cell carcinoma
of the tongue.
adenoma of the adrenal
cortex
squamous cell carcinoma
of the hard palate.
squamous cell carcinoma
of the tongue,
adenoma of the adrenal
cortex
squamous cell carcinoma
of the hard palate,
squamous cell carcinoma
of the tongue.
adenoma of the adrenal
cortex
hepatocellular carcinoma.
squamous cell carcinoma
of the tongue.
squamous cell carcinoma
of the lung
hepatocellular carcinoma.
squamous cell carcinoma
of the tongue,
squamous cell carcinoma
of the lung
hepatocellular carcinoma.
squamous cell carcinoma
of the tongue,
squamous cell carcinoma
of the lung
Tumor
Incidence
0/85
0/85
0/85
0/50
1/50
0/50
0/50
1/50
2/50
4/50
3/50
5/50
0/86
0/86
0/86
0/50
0/50
0/50
2/50
1/50
0/50
Reference
Koclba
et al., 1978a
Koctba
et al., 1978a
Koclba
et al., 1978a
-------
TABLE 11-24 (cont.)
Exposure
Route/
Compound
Oral/
2,3.7,8-TCDD
Gavage/
2.3.7,8-TCDD
Oral/
2,3,7,8-TCDD
Gavage/HxCDD
Gavage/HxCDD
Species/Strain
rat/
Sprague-Dawley
mice/Swiss/
H/Rlop
mice/
Peramyscus
pollenotus
rats/
Osborne-Hendel
rats/
Osborne-Hendel
Sex Dose or Exposure
F 0.1 pg/kg/day
H 0.0 pg/kg/week
0.007 pg/kg/week
0.7 pg/kg/week
7.0 pg/kg/week
H&F 0.0012 pg/kg/day
0.0 pg/kg/day
H 0.0 pg/kg/week
(vehicle control)
H 1.25 pg/kg/week
2.5 pg/kg/week
5.0 pg/kg/week
Duration
of
Treatment
105 weeks
365 days
365 days
365 days
365 days
NA
NA
104 weeks
104 weeks
104 weeks
104 weeks
Duration
oF Study
105 weeks
588 days
649 days
633 days
424 days
NA
NA
105 weeks
106 weeks
107 weeks
107 weeks
Vehicle
In diet
sunflower
oil
sunflower
oil
sunflower
oil
sunflower
oil
contami-
nated soil
contami-
nated soil
corn oll-
acetone
(9:1)
corn oil-
acetone
(9:1)
corn oil-
acetone
corn oil-
acetone
Tumor Type
hepatocellular carcinoma,
squamous cell carcinoma
of the tongue,
squamous cell carcinoma
of the lung
liver tumors
liver tumors
liver tumors
liver tumors
liver
liver
liver neoplastlc nodules
or hepatocellular
carcinoma
liver neoplastlc nodules
or hepatocellular
carcinoma
liver neoplastlc nodules
or hepatocellular
carcinoma
liver neoplastlc nodules
or hepatocellular
Tumor Reference
Incidence
11/49 Koclba
et al., 1978i
4/49
7/49
7/38 Toth et al . ,
1979
13/44
21/44
13/43
0/15 Cockerhan
et al.. 1980
0/15
0/74 NTP. 1980d
0/49 NTP. 1980d
1/50
4/48
carcinoma
-------
TABU 11-24 (cont.)
Exposure
Route/ Species/Strain Sex Dose or Exposure
Compound
Gavage/HxCBD rats/ F 0.0 j»g/kg/week
Osborne-Hendel
1.25 yg/kg/week
2.S tig/kg/week
5.0 yg/kg/week
__, Gavage/HxCOD n!ce/B6C3F1 N 0.0 fig/kg/week
_j
en
9*
1.25 ng/kg/week
2.5 wg /kg/week
5.0 nf/kg/week
Gavage/HxCDD m1ce/B6C3Fl F 0.0 tig/kg/week
2.5 ug/kg/week
5.0 yg/kg/week
10.0 ug/kg/week
Duration
of Duration
Treatment of Study
104 weeks 105 weeks
—
104 weeks 107 weeks
104 weeks 107 weeks
104 weeks 107 weeks
104 weeks 105 weeks
104 weeks 108 weeks
104 weeks 107 weeks
104 weeks 108 weeks
104 weeks 106 weeks
104 weeks 108 weeks
104 weeks 108 weeks
•
104 weeks 107 weeks
Vehicle
corn oll-
acetone
(9:1)
corn oil-
acetone
(9:1)
corn oil-
acetone
(9:1)
corn oil-
acetone
(9:1)
corn oil-
acetone
(9:1)
corn oll-
acetone
(9:1)
corn oll-
acetone
(9:1)
corn oil-
acetone
(9:1)
corn oil-
acetone
(9:1)
corn oil-
acetone
(9:1)
corn oil-
acetone
(9:1)
corn o11-
acetone
(9:1)
TiMor Type
liver neoplastlc nodules
or hepatocellular
carcinoma
liver neoplastlc nodules
or hepatocellular
carcinoma
liver neoplastlc nodules
or hepatocellular
carcinoma
liver neoplastlc nodules
or hepatocellular
carcinoma
hepatocellular adenenss
or carcinomas
hepatocellular adenomas
or carcinomas
hepatocellular adenomas
or carcinomas
hepatocellular adenomas
or carcinomas
hepatocellular adenomas
or carcinomas
hepatocellular adenomas
or carcinomas
hepatocellular adenomas
or carcinomas
hcpatocellular adenomas
or carcinomas
Tmor Reference
Incidence
5/75 KIP, 1980d
10/50
12/50
30/50
15/73 NTP, 1980d
14/50
14/49
24/48
3/73 NTP, 19BOd
4/48
6/47
10/47
NA . Not available
-------
Intake of 2,3,7,8-TCDD from the diet was 0,0, 0.001, 0.01 and 0.1
day. In the high-dose group, both male and female animals had significant
Increases 1n site-specific tumors. The target organs and tumor types 1n
male animals were squamous cell carcinomas of the tongue, squamous cell
carcinomas of the hard palate and nasal turblnates, and adenomas of the
adrenal cortex; 1n female animals the target organs and tumor types were
hepatocellular carcinomas, squamous cell carcinomas of the tongue and nasal
turblnates, and squamous cell carcinomas of the lung. The data demonstrate
that dietary exposure to 2,3,7,8-TCOD at levels that produce a dally dose of
0.1 wS/kg results 1n Increased tumor Incidences 1n both male and female
rats.
Under the National Toxicology Program, 2,3,7,8-TCDD was tested for
carclnogenldty 1n rats following administration by gavage (NTP, 1980a).
Both male and female animals were exposed to weekly doses of 0.0, 0.01, 0.05
and 5 yg/kg bw. The only tumors that appeared to be treatment-related
were folUcular cell adenomas or carcinomas of the thyroid In male animals,
and neoplastlc nodules or hepatocellular carcinomas of the liver 1n female
animals. The Incidence of these tumors was significantly greater than
control 1n the high-dose groups, and the Incidence of both tumors showed a
positive dose-related trend. Under the conditions of this assay, 2,3,7,8-
TCDD was concluded to be carcinogenic 1n both male and female rats.
Further studies In mice exposed by gavage have provided support for the
carclnogenldty of 2,3,7,8-TCDD. Toth et al. (1979) exposed male mice to
2,3,7,8-TCDD at doses of 0.0, 0.007, 0.7 and 7.0 yg/kg/week 1n a study to
determine whether 2,4,5-TCPE, Its contaminant 2,3,7,8-TCDO or both were
carcinogens. At the 0.7 yg/kg/week level there was a significantly
Increased Incidence of liver tumors. Liver tumors were not significantly
Increased 1n the high-dose group; however, early mortality In this group may
11-57
-------
have precluded observing late-developing tumors. Similar Increased Inci-
dences of liver tumors were observed 1n the NTP (1980a) study 1n the high-
dose male mice exposed to 0.5 yg/kg/week and 1n the high-dose female mice
exposed to 2 jig/kg/week of 2,3,7,8-TCDD by gavage. Female mice also had
an Increased Incidence of folUcular-cell adenomas of the thyroid. In both
studies, 2,3,7,8-TCDD was carcinogenic to mice, with effective doses ranging
between 0.5 and 2 vg/kg/day, depending on sex and the Individual study.
The mouse skin two-stage tumor1gen1c1ty model has also been used to test
the carcinogenic potential of 2,3,7,8-TCDD. Following long-term dermal
application 3 times/week of 2,3,7,8-TCDD at levels of 0.01 and 0.005
jig/appHcatlon to male and female mice, respectively, there was an
Increased Incidence of skin tumors only 1n female mice (NTP, 1980b). Along
with the Indication that 2,3,7,8-TCDD was a complete carcinogen 1n this
system, D1G1ovann1 et al. (1977) reported that 2,3,7,8-TCDD was also a tumor
Initiator 1n mouse skin. The ability of 2,3,7,8-TCDD to Initiate tumors,
however, has yet to be confirmed since appropriate vehicle and promotion-
only control groups were not Included. Attempts to demonstrate tumor-pro-
moting activity with 2,3,7,8-TCDD on mouse skin have produced negative
results 1n some assays (NTP, 1980b; Berry et al., 1978, 1979); however,
Poland et al. (1982) reported that 2,3,7,8-TCDD was a tumor promoter when
tested on the skin of mice homozygous for the "hairless" trait, but not 1n
mice heterozygous for this recessive trait. P1tot et al. (1980) also
reported that 2,3,7,8-TCDD was a promoter for DEN-1n1t1ated hepatocardno-
genesls 1n rats following parenteral administration of the compounds. On
mouse skin, 2,3,7,8-TCDD was a complete carcinogen and possibly a tumor
Initiator, while no tumor-promoting activity could be attributed to 2,3,7,8-
TCDD 1n the assays. In rat Hver Initiated with DEN, 2,3,7,8-TCDD was a
tumor promoter.
11-58
-------
In studies of the Interaction of 2,3,7,8-TGQD with other chemical
carcinogens, Kourl et al. (1978) reported that 2,3,7,8-TCDD was a cocar-
dnogen with 3-MC when administered by subcutaneous Injection. In the mouse
skin bloassay, Initiation with simultaneous administration of 2,3,7,8-TCDD
and DHBA, however, did not affect tumor yield (D1G1ovann1 et al., 1977).
Similarly, no effect was observed when 2,3,7,8-TCOD was administered either
Immediately before (5 minutes) or 1 day after DHBA Initiation (Berry et al.,
1979; D1G1ovann1 et al., 1977, 1979b; Cohen et al., 1979). When treatment
with 2,3,7,8-TCDD occurred 1-10 days before DHBA Initiation, 2,3,7,8-TCDD
demonstrated a potent antlcardnogenlc action. Although 1-5 days prior
exposure to 2,3,7,8-TCDD Inhibited tumor Initiation by BaP, 3-HC and BaP-
d1ol-epox1de, the tumor Initiating ability of the latter compound was also
Inhibited when 2,3,7,8-TCDD exposure occurred either 5 minutes before or 1
day after Initiation (D1G1ovann1 et al., 1980). The Increased AHH activity
resulting from 2,3,7,8-TCDD exposure may account for the ant1carc1nogen1c
activity by altering the metabolism of the Initiating compound; however,
D1G1ovann1 et al. (1980) suggest that the Inhibition of the Initiating
activity of BaP-d1ol-epox1de 1 day after Initiation Indicates that more than
one mechanism participates 1n the antlcardnogenlc activity of 2,3,7,8-TCDD.
HxCDD has also been tested for cardnogenldty In rats and mice treated
by gavage and by dermal application to mice (NTP, 1980c,d). In these
studies, a 1:2 mixture of 1,2,3,6,7,8- and 1,2,3,7,8,9-HxCDD was tested. In
the oral study, animals received HxCDD at doses of 0.0, 1.25, 2.5 or 5.0
jjg/kg/week, except for female mice, which received 0.0, 2.5, 5.0 and
10.0 yg/kg/week. In both species and either sex only tumors of the liver
occurred at a significantly greater Incidence than controls. In male rats
and male and female mice, the Hver tumor Incidence was significantly
11-59
-------
Increased over control values only In the high-dose groups, while 1n female
rats the Incidence was significantly greater at both the medium- and high-
dose levels. In the study of HxCDD cardnogenldty 1n mouse skin conducted
by NTP (1980c), there were no treatment-related tumors 1n either the car-
clnogenlclty bloassay or the tumor promotion assay using DMBA as an Initi-
ator. It was concluded that this mixture of HxCDD was carcinogenic to rats
and mice following administration by gavage; however, there was no tumorl-
genlc activity when HxCDD was applied to mouse skin.
No chronic animal bloassays were found 1n the literature searched on the
cardnogenldty of 1,2,3,7,8-PeCDD.
11.2. CASE REPORTS AND EPIDEMIOLOGICAL STUDIES*
11.2.1. Case Reports, Observations of an unusual occurrence of relatively
rare soft-tissue sarcomas were first made by Harden (1977), Of some 87
patients seen from 1970-1976 at the Department of Oncology, University
Hospital, Umea, Sweden, seven Individuals with soft-tissue sarcomas were
Identified. All seven had had occupational exposure to phenoxy acids 10-20
years earlier. The tumors were 2 lelomyosarcomas, 1 llposarcoma, 1 rhabdo-
myosarcoma, 1 myxoflbrosarcoma and 2 additional sarcomas of which the hlsto-
pathology was uncertain, but one was probably a neuroflbrosarcoma and the
other a rhabdomyosarcoma. The clustering of this rare tumor type among
these patients prompted the author to suggest that ep1dem1olog1cal studies
be done to determine 1f exposure to phenoxy acids and the Impurities they
contain are related to the occurrence of soft-tissue sarcomas.
*Port1ons of this section were taken from U.S. EPA (1980c).
11-60
-------
lack and Susklnd (1980) reported a soft-tissue sarcoma death 1n a cohort
study of workers exposed to 2,3,7,8-TCDD 1n a trlchlorophenol process
accident 1n NHro, Nest Virginia. This tumor, a fibrous hlstlocytoma, was
noted by the author as a rare event. This study, referred to as the Nltro
study, 1s discussed later.
Cook et al. (1980) 1n a cohort mortality study of 61 male employees of a
tMchlorophenol manufacturing area, who exhibited chloracne following a 1964
exposure Incident, noted four deaths by the end of his study period, one of
which was due to a flbrosarcoma. The authors did not seem to attribute any
special significance to this finding at the time.
Ott et al. (1980) 1n a cohort mortality study of 204 employees exposed
to 2,4,5-T during Its manufacture from 1950 to 1971, found no soft-tissue
sarcomas among 11 deaths that had occurred by 1976. One of these 11 deaths
was due to a malignant neoplasm.
In a review of the studies of Zack and Susklnd (1980), Cook (1980), an
unpublished study by Zack (1n which a Uposarcoma was found), a study by Ott
et al. (1980) and Honchar and Halperln (1981) noted 3 (2.9X) soft-tissue
sarcomas 1n a total of 105 deaths. Among U.S. males aged 20-84, 0.07% of
the deaths were reported as soft tissue sarcomas (ICO 171, 8th Revision,
1975)* Indicating an unusual excess of such tumors. This may be an under-
estimate because of the possibilities that some soft-tissue sarcomas may
have been coded to categories other than ICD 171. Individually, none of the
reported case studies reported a significant excess of soft-tissue sarcomas.
*Department of Health, Education, and Welfare. U.S. Public Health Service.
National Center for Health Statistics of the United States, 1974. Vol. II.
Mortality, Part A.
11-61
-------
Cook (1981a) found an additional malignant fibrous h1st1ocytoma after a
later review of the medical records from his earlier cohort study. Cook,
who was familiar with the three earlier cases, noted that frank chloracne
occurred previously 1n two cases of the four having a diagnosis of malignant
fibrous hlstlocytoma. A third person diagnosed as having a flbrosarcoma
worked 1n a trlchlorophenol (TCP) process area contaminated with 2,3,7,8-
TCDD. This Individual exhibited facial dermatitis but there was no
diagnosis of chloracne. The fourth case (diagnosed as a Uposarcoma) was an
Individual who had been employed earlier 1n a plant producing 2,4,5-T. Cook
(1980) noted that although chloracne was not reported, 1t could not be
discounted. He also noted that all four were cigarette smokers and
suggested that smokers with chloracne caused by 2,3,7,8-TCDD exposure may be
subject to an Increased risk of fibrous soft-tissue sarcomas, although no
prior reports have shown soft tissue sarcomas associated with cigarette
smoking.
Hardell and Eriksson (1981) discounted this hypothesis by citing that
only one of Harden's seven cases exhibited chloracne before the appearance
of the soft-tissue saromcas, and that In their subsequent later case control
study, they found no difference 1n smoking habits between his cases and
controls.
Hoses and Sellkoff (1981) reported a fifth soft-tissue sarcoma 1n a
worker employed at the Monsanto Chemical Company at a time when trlchloro-
phenol and 2,4,5-T were being produced. The worker died of a retroperlto-
neal neurogenlc sarcoma (malignant schwanoma) 1n 1980 at the age of 58. The
employee, before his death, In a detailed occupational history said that he
believed he was exposed to these chemicals while he was a truck driver,
hauler and maintenance worker, but that he did not work 1n the production of
either chemical. He was a nonsmoker and had no history of chloracne.
11-62
-------
Johnson et al. (1981) treated a father and son with soft-tissue sarcomas
(the 33-year-old son was diagnosed as having a flbrosarcomatous mesothe-
Homa, while the 53-year-old father had a Uposarcoma). Both were exposed
to halogenated phenol derivatives. The author noted that 2,4-dlchlorophenol
can be a precursor of 2,4-D and 2,4,5-T. The father had had prolonged expo-
sure before his disease. The son supposedly had a shorter latency, accord-
Ing to the author. In neither case was the follow-up time given.
Sarma and Jacops (1981) reported three cases of thoracic soft-tissue
sarcoma 1n Individuals who were presumably exposed to Agent Orange while
serving 1n Vietnam. The diagnoses were fibrous hlstlocytoma, medlastlnal
flbrosarcoma, and a pleural/dlaphragmatic lelomyosarcoma. All three served
1n areas where defoliants were used at the time. One was drenched with the
material 1n one spraying.
Bishop and Jones (1981) found two cases of non-Hodgk1n's lymphomas of
the scalp 1n a related clinical study of 158 employees of a pentachloro-
phenol manufacturing plant 1n Wales. Homologues of 2,3,7,8-TCDO occurred as
contaminants at up to 300 ppm at Intermediate manufacturing stages and 5 ppm
1n the final products. H1ld, moderate and severe cases of chloracne were
seen In many employees. Including the two men who subsequently developed
lymphomas. Both men worked 1n processes where exposure to other chemicals
occurred, Including exposure to aromatic hydrocarbons. The authors reported
that only 0.28 tumors of this type could be expected to occur 1n a group of
158 workers (ICD 200 and 202), although the basis for the computation of
expected numbers 1s not stated.
Olsson and Brandt (1981) noted that of 123 male patients seen at their
clinic 1n Sweden with a recent diagnosis of non-Hodgk1n's lymphoma (NHL), 5
had cutaneous lesions as the only clinically detectable manifestation of
11-63
-------
NHL. Four of the five were reported to have repeatedly sprayed large areas
with phenoxy acid herbicides. In the remaining 118 NHL patients, only seven
had a similar occupational exposure to phenoxy acids. The authors reported
this to be significant at p<0.001. Olsson and Brandt suggested that a
relationship exists between cutaneous presentation of NHL and occupational
exposure to phenoxy adds, and believed their observations were similar to
those of Bishop and Jones (1981).
The total number of workers with these Illnesses who were exposed to
phenoxy adds and/or chlorophenols 1s small, but considering the rarity of
this cancer, 1t Is unusual that so many cases of soft-tissue sarcomas have
occurred. A Lancet editorial (Anonymous, 1982) calls this phenomenon
"disturbing."
11.2.2. Ep1dem1olog1c Studies.
11.2.2.1. SOFT-TISSUE SARCOMAS — Soft-tissue sarcomas (STS) consti-
tute a collection of heterologous lesions that Include both malignant and
nonmallgnant tumors. Not all of them have their origin In primordial mesen-
chymal cells. Some exceptions are tumors of peripheral nerves, and neuro-
ectodermal tumors that are classified as STS but are derived from nonmesen-
chymal cells. Classification, grading and staging of STSs Is difficult
because of the capacity of such cells to differentiate Into many different
tissues. Fairly precise hlstogenetlc classification of such tumors 1s
accomplished through consideration of growth patterns and cell morphology
and evaluation of IntraceTJular and extracellular products of tumor cells.
There are a dozen distinctly different classes of mesenchymal cells that
develop Into the following six well-defined tissue complexes: fibrous
tissue, tendosynovlal tissue, adipose tissue, muscle, vessels and bone.
11-64
-------
STSs can be Induced In any of these tissue types (Hajdu, 1983). The classi-
fication of STSs for cause of death coding 1n the ninth and latest revision
of the International Classification of Diseases (ICD, 1975} places STSs Into
one of several categories. But chiefly, they fall Into "malignant neoplasms
of connective and other soft-tissue" (ICD 171). Lymphosarcomas, retroperl-
toneal sarcomas and extra skeletal STSs of the bone are coded elsewhere. In
some Instances, 1f site 1s mentioned, 1t 1s coded to the site [I.e., Ie1o-
myosarcoma of the stomach (ICO 151.9), neuroflbroma of the chest wall
(215.4)].
Questions have been raised concerning the appropriateness of lumping
together malignant tumors of different sites and tumor types 1n order to
derive risk estimates. It may not be scientifically appropriate to do so
because an elevated risk cannot readily be ascribed to a particular site or
type as 1s usual with most carcinogenic chemicals and substances. Unfortu-
nately, with respect to STSs, tallies of deaths from STSs of particular
sites and types are not maintained separately by the vital statistics
offices because of their rarity; therefore, 1t 1s Impossible to derive risk
estimates for particular types at given sites. Altogether, -2000 deaths/
year can be attributed to STSs 1n the United States, most of which are coded
to ICO category 171 for purposes of developing Incidence and mortality rates
for this composite cause. Within ICD 171, Individual types that may be
correlated with exposure cannot be Identified.
A separate problem that potentially could arise from assigning STSs to
multiple ICD codes 1s that Incidence and death rates from STSs may be under-
estimated. Furthermore, risk estimates derived from dividing observed cases
(or deaths) by expected cases (or deaths) could be biased upward. This
could happen when observed STSs classified to ICO codes other than ICD 171
11-65
-------
are lumped together In ICD 171 while expected STSs are based upon STSs
classifiable to ICD 171 only. Thus, action of this sort, especially with
respect to cohort studies of Individuals exposed to d1ox1n-conta1n1ng herbi-
cides and/or chlorophenols, could lead to risk estimates that may be biased
upward by the Inclusion of STSs 1n the observed category for risk estimation
that should be coded to categories other than 171.
Prompted by clinical observations over a 7-year period of malignant
sarcomas 1n seven men with previous occupational exposure to phenoxyacetlc
add herbicides (Harden, 1977), researchers at the Department of Oncology,
University Hospital, Umea, Sweden, Initiated case-control ep1dem1olog1c
studies to test the hypothesis of an etlologlc association (Harden and
Sandstrom, 1979). Cases were defined as male patients with sarcomas of soft
connective tissue, such as smooth muscle (lelomyosarcoma) and fat (I1po-
sarcoma). The distribution of tumor types 1n the two studies Is shown 1n
Table 11-25. Sarcomas of tissues, such as bone and cartilage, were excluded
as cases. According to the authors, these tumors may have a different
etiology and there occurred a different age-distribution 1n patients with
these tumors as compared with that of STS (Harden, 1983).
Two case-control studies were conducted: the first 1n northern Sweden
(referred to below as Study A) and the second 1n the southern part of the
country (Study B). The exposures to the substances of primary Interest are
shown 1n Table 11-26. In the north (Study A), occupational exposure to
phenoxyacetlc adds took place 1n both forestry and agricultural work. In
the south (Study B), these exposures were predominantly agricultural. The
phenoxyacetlc adds to which exposure occurred consisted predominantly of
2,4,5-T and 2,4-D 1n both studies. Exposure to 2,4,5-T 1n the absence of
2,4-D was rarely reported 1n either study. Exposure to chlorophenols, which
11-66
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TABLE 11-25
Distribution of Tumor Types 1n Two Case-Controls Studies
of Soft-Tissue Sarcoma
Diagnosis
Tissue of Origin
Percent of Cases
Study Aa Study Bb
(n=52) (n=11Q)
Lelomyosarcoma
Fibrous hlstlocytoma
Llposarcoma
Neurogenlc sarcoma
Anglosarcoma
Hyxosarcoma
Flbrosarcoma
Other sarcomas
Total
Smooth muscle
Subcutaneous connective
tissue
Fat tissue
Nerve tissue
Blood vessels
Primitive connective
tissue
Fibrous tissue
30
17
14
10
8
6
4
11
100
23
25
6
4
2
8
8
24
100
aUnpub!1shed Information supplied by Harden to EPA {Harden and Sand-
strom, 1979)
bEr1ksson et al., 1979, 1981
11-67
-------
TABLE 11-26
Exposure Frequencies 1n Two Case-Control Studies of Soft-Tissue Sarcoma
Substance(s)
Phenoxyacetlc adds only
Chlorophenols only
Both
Total
Study
Cases
(n=52)
23.1
11.5
1.9
36.5
Percent
A
Controls
(n=206)
6.3
2.4
0.5
9.2
Exposed
Study
Cases
(n=110)
12.7
10.0
0
22.7
B
Controls
(n=219)
2.3
3.6
0
5.9
*Sources: Study A, Harden and Sandstrom, 1979; Study B, Eriksson et a!.,
1979, 1981
11-68
-------
contain chlorinated d1benzod1ox1n Impurities (Levin et a!., 1976), occurred
mostly 1n sawmill work and paper pulp production. Very few persons reported
exposure both to phenoxyacetlc add and chlorophenols 1n these studies. Of
the two predominant phenoxyacetlc adds, only 2,4,5-T 1s known to be contam-
inated with 2,3,7,8-TCDD. In Study B, a relative risk of 4.9 (90H confi-
dence Intervals 1.6-11.1) was found 1n relation to exposure to phenoxyacetlc
add herbicide other than 2,4,5-T (2,4-D, MCPA, mecbprop, dlchloroprop).
Relative risks 1n relation to the three major categories of exposure are
shown 1n Table 11-27.* Studies A and B Indicate a risk of developing STSs
among workers exposed to phenoxyacetlc adds only, chlorophenols only, or
phenoxyacetlc adds and/or chlorophenols several times higher than among
persons not exposed to these chemicals. In each comparison, the relative
risk 1s high and was thus unlikely to have resulted by chance alone.
Since IHtle 1s known of the etiology of STSs, the consideration of
confounding 1n these studies was largely a hypothetical matter. The authors
presented the effects of age, sex, and place of residence as possible
confounding factors In the selection of controls.f Because of the high
correlation between exposure to the substances of Interest and employment In
agriculture and forestry, a possible alternative hypothesis could be that
some other unknown factor present 1n these occupations was responsible for
the elevated relative risks.
*In the analyses considering phenoxyacetlc adds only and chlorophenols
only, persons exposed to the other categories of substances were excluded.
In Study A, the three persons exposed to both chlorophenols and phenoxy-
acetlc acids were Included In all comparisons.
tControls were matched Individually to cases on the basis of these factors.
Unmatched analyses are presented In Table 11-26 for the sake of simplicity.
The matched-method relative risks for exposure to phenoxyacetlc adds and/or
chlorophenols were 6.2 (p<0.001) In Study A and 5.1 (p<0.001) 1n Study B.
11-69
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TABLE 11-27
Relative Risks of Soft-Tissue Sarcoma In Relation to Exposure to
Phenoxyacetlc Adds and Chlorophenols 1n Two Case-Control Studies3
Phenoxyacetlc Acids
Only
Relative r1skb
90X Confidence Interval
Significance leveld
Study A
5.3
2.7-10.2
<0.001
Study B
6.8
3.1-14.9
<0.001
Chlorophenols
Only
Study A
6.6
2.8-15.6
<0.001
Study B
3.3
1.6-7.0
<0.005
Phenoxyacetlc Acids
and/or
Chlorophenols
Study A
5.7
3.2-10.2
<0.001
Study B
4.7
2.7-8.3
<0.001
aSource: Study A, Harden and Sandstrom, 1979; Study B, Eriksson et al., 1979, 1981
^Unmatched odds ratio
cTest-based method of Hlettlnen, 1976
square statistic, no continuity correction, one-tailed test
-------
To test this hypothesis, 1t 1s possible to calculate the relative risk
1n relation to the phenoxyacetlc add exposure 1n Study B, restricting the
analysis to workers within agriculture and forestry. The result 1s a rela-
tive risk of 6.1 (90% confidence Interval 2.4-15,4). This finding suggests
that a confounding risk factor for STS distributed throughout agriculture
and forestry work was not responsible for the overall Increase 1n risk found
1n relation to phenoxyacetlc add exposure.
Because exposure histories were obtained by means of questionnaires and
Interviews, the major potential source of bias 1n these studies stems from
the need to rely upon the personal recollection of cases and controls for
exposure histories. The published papers Indicate that the researchers paid
a great deal of attention to this potential problem and specific efforts
were made to avoid It during the conduct of the study.
In addition, the relative risk calculated by considering the agriculture
and forestry workers who did not report exposure to phenoxyacetlc adds or
chlorophenols and comparing them with unexposed persons 1n other occupations
was 0.9 (90K confidence Interval 0.3-2.4) 1n Study B. This suggests that
little recall bias was present (Axelson, 1980).
In an update of their earlier study, Eriksson et al. (1981) obtained
Information on the effects of phenoxy adds 1n the absence of the Impuri-
ties — polychlorlnated dlbenzodloxlns and dlbenzofurans. The risk ratio
given exposure to phenoxy adds free of polychlorlnated dlbenzodloxlns and
dlbenzofurans equaled 4.2 based upon 7 of 14 respondents who Indicated
exposure to phenoxy acid herbicides. When consideration was given to
persons exposed only to phenoxy adds that contain such Impurities, the
relative risk was 17.0. A description of the basis for the determination of
exposure or nonexposure to dloxlns 1s not well presented 1n this study.
11-71
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The author concluded that exposure to phenoxy adds and ehlorophenols
"might constitute a risk factor 1n the development of soft-tissue sarcomas."
This risk relates not only to 2,4,5-trlchlorophenoxy acids containing dloxln
Impurities but to other phenoxy acids as well. Some doubt was raised
concerning the possible m1sclass1f1cat1on of Individuals who were exposed to
phenoxy adds free of polychlorlnated d1benzod1ox1ns [I.e., 1n particular,
"dlchloroprop" 1n the Eriksson et al, (1981) study]. In a recent communica-
tion from Hardell (1983), Eriksson recalculated his risk estimates after
reclasslfylng his dlehloroprop-exposed cases and controls Into the category
of probable exposure to phenoxy adds contaminated with polychlorlnated
d1benzod1ox1ns and removing them from the nonexposed category. His new
estimates were 4.0 based upon 5 of 8 respondents who were exposed to phenoxy
adds allegedly free of contamination and 10.9 for those exposed to contami-
nated phenoxy add. The first estimate was of only borderline significance
utilizing the H1et1nen test based statistic, thus, weakening any finding
that the risk of STS extends to phenoxy adds free of dloxln.
In a cohort mortality Investigation Cook et al. (1980) studied 61 males
Involved 1n a 1964 exposure Incident who had absorbed 2,3,7,8-TCDD through
the skin and developed chloracne. The skin lesions characterizing chloracne
ranged from a few comedones on the back of one employee (predating his entry
Into the process area where exposure could occur) to severe cysts and
comedones over the faces, scalps, ears, necks and backs of the remaining
employees of the group. Since the main route of exposure was not through
the respiratory tract, no measurements of dloxln In the air were provided by
the author. On the other hand, the author divided the cohort of 61 males
Into potentially "high" vs. "low" exposure by place of work based upon
11-72
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dermal exposure, although not stated. Vital status was traced from the data
of the Incident through 1978, Altogether only 4 deaths were observed by the
end of the follow-up, vs. 7.8 expected. Of these, 3 were cancer deaths vs.
1.6 expected. The remaining death was hypersensitive heart disease vs. 3.8
expected. The hlstopathologlc causes of death of the three cancer victims
were 1) flbrosarcoma, 2) glloma with metastases, and 3) adenocardnoma. The
authors report that all three victims smoked a minimum of one pack of
cigarettes a day for "many years." Not enough Information 1s provided by
the authors to conclude that any of these four deaths were smoking related.
Site of tumor 1s not mentioned 1n the cancer deaths.
Cancer mortality 1s slightly elevated 1n this cohort. The study has low
sensitivity and lacks a sufficient latent period. This Increased mortality
was not attributable to any particular cause and no deaths were attributable
to Hver cancer. Additionally, the authors state that only one of the
cancer deaths possessed "documented" evidence of chloracne, although this
appears to be at variance with the definition of the cohort, which was
reported by the authors to consist of males who reported to the medical
department with skin conditions subsequently "diagnosed as chloracne." The
authors concluded that the latency period was sufficient to "allow the
Identification of a potent human carcinogen," since 1t "exceeded 14 years."
Orris (1981) noted that 1n the Harden and Sandstrom (1979) study the
authors stated that the latent period for soft-tissue tumors may be as long
as 27 years and for many, over 14 years. In any case, Hueper and Conway
(1964) noted that the latent period for the chemical Induction of solid
malignant tumors 1n man exceeds 15 years and 1s probably <30 years.
11-73
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Smith et al. (1982b) conducted an Initial case-control study of 102
males Identified from the New Zealand Cancer Registry as having STSs (ICO
171) between 1976 and 1980. For each case, three controls each with another
form of cancer were matched by age and year of registration. The selection
of cancer controls from the same registry was done to eliminate recall bias
and/or Interviewer bias. The distribution of hlstologlcal types 1n the
cases 1s given 1n Table 11-28. An Interview to elicit occupational history
Information was accomplished by telephone either with the next of kin to the
patient or the patient himself If he was well enough, although the Informa-
tion was not used 1n this preliminary analysis.
Comparisons between cases and controls were accomplished by use of
occupational groupings according to the Standard Classification System of
New Zealand focusing on those occupational groups with a potential for
exposure to phenoxy herbicides and chlorophenols. Expected cases for each
major occupational classification were derived based upon the occupational
distribution of the controls. The authors found no unusual excess of cases
of STS 1n any major occupational category. In agriculture, forestry and
fishing, 14 cases were observed vs. 14.0 expected. In laborers, production
and transport workers, 35 cases were observed vs. 37.0 expected. A further
breakdown of these two broad categories Into finer subcategorles within the
major occupational categories revealed no significant excesses. The study,
however, 1s not useful 1n assessing the risk of STS from exposure to phenoxy
adds and/or chlorophenols for several reasons. First, as was pointed out
by the authors but subsequently dismissed by them as having not much of an
Influence, 1s the possibility that movement from one major occupational
category to another over the time period Involved for latent conditions to
11-74
-------
TABLE 11-28
Distribution of Hlstologlcal Types of Soft-Tissue Sarcomas*
Cell Type
Flbrosarcoma
Uposarcoma
Rhabdomyosarcoma
Lei omyosar coma
Malignant H1st1ocytoma
Other
Unspecified
Total
Number of Cases
25
20
9
7
6
22
13
102
Percent
24
20
9
7
6
21
13
100
*Source: Smith et al., 1982b
11-75
-------
manifest themselves could Introduce a negative bias Into any estimates of
relative risks. The latency for STS was suggested to be a minimum of 15
years (Hueper and Conway, 1964).
The finding of no switching from one occupational category to another
that was noted 1n the "first 20 Interviews" 1n which a change could be noted
1s not necessarily Indicative of fidelity to the same job over long periods
1n all 408 cases and controls. Information Identifying a change may be
lacking 1n those cases and controls 1f 1n fact one did occur possibly
because of several reasons, for example, separation of the earlier work
history from the latter and purging of earlier employment records. Besides
the "first 20 Interviews" where a change could be noted 1s not necessarily
representative of the entire cohort 1n any case.
Furthermore, the authors do not know absolutely that any of their cases
and controls were exposed to phenoxy adds or chlorophenols or to both since
apparently no effort was made to confirm "potential" exposures. Only dif-
ferences 1n occupational classification were noted where "potentially" cases
or controls could have had exposure to the d1ox1n-conta1n1ng herbicides. It
was pointed out that the risk estimates noted do not "preclude" the possi-
bility that an association may be found 1n this study when the cases and
controls (or surviving kin) are Interviewed for chemical spraying at a later
time. The authors themselves concluded that the preliminary study results
"should not be taken as substantial evidence against the hypothesis that
phenoxy herbicides and chlorophenols may cause human cancer."
The distribution of tumor types differed considerably from the Harden
and Eriksson study to the Smith study. Lelomyosarcomas, malignant hlsto-
cytomas, neurogenlc sarcomas and myxosarcoma seem to predominate 1n the
Hardell and Eriksson study, whereas flbrosarcomas and llposarcomas appear
11-76
-------
prominently In the Smtth study. Moreattention should be devoted to the
study of the distributions of STS types 1n registry data everywhere 1n order
to determine 1f such variations 1n the reporting of STS types are random
occurrences. It 1s possible that the cancer effect of exposure to phenoxy
herbicides may be narrowed to just certain types of STSs, the predominant
ones 1n the Swedish studies.
In a later study of STSs, Smith et al. (1983a) conducted a case-control
study of STSs 1n males that were reported to the Mew Zealand Cancer Registry
by Public Hospitals between 1976 and 1980. The author matched one cancer
control randomly chosen from the registry with each case, Initially starting
with 112 of each. Controls were matched for year of registration and by
date of birth +_ 2 years. Inquiries were made by the authors with the hos-
pital consultant, family doctor, and finally the next-of-k1n or patient 1f
alive. Telephone Interviews were conducted by only one Interviewer, who had
no knowledge of the patient's cancer history, and were completed on 80 cases
and 92 controls. Because some 32 potential cases (14 Ineligible) and 20
controls were excluded or lost from the study for various reasons, 1t raises
a question whether control of confounding by age and year of registration
was maintained In the final group of 172 cases and control Included 1n the
analysis. Presumably the corresponding "matched" case or control to each of
the 52 lost members of the total study group were not excluded. However,
since the span of registration was only 5 years, not much age confounding
could occur.
Patients were classified as having had potential exposure to phenoxy-
acetlc adds 1f they had definite, probable or possible exposure to phenoxy-
acetlc add through spraying or hand contact. The actual chemical was
Identified only 1n some Instances. The authors concluded 1n all remaining
11-77
-------
situations that 1f the member sprayed "gorse" and/or "blackberries" this was
tantamount to potential exposure to phenoxyacetlc add. Smith (1983) calcu-
lated elevated but nonsignificant relative risks of exposure to phenoxy-
acetlc add ranging from 1.3 1n those Individuals who were "probably
exposed" for a minimum of 5 days not 1n the previous 10 years before cancer
registration to 1.6 1n Individuals "probably exposed" for a minimum of 1 day
not 1n the previous 5 years before cancer registration. When risk ratios
were calculated after stratifying by year of birth and whether or not the
patient or a relative was Interviewed, the rates Increased to 1.7 (from 1.6)
1n the latter and 1.4 (from 1.3) 1n the former calculation, although still
nonsignificant. If the numbers would allow, 1t would be of Interest to
repeat the above calculations excluding only those with potential exposure
occurring only within the 15-year period Just before cancer registration.
The small numbers that remain following the 15-year lapse probably precludes
such an analysis. Furthermore, the categories of exposure "probably or
definitely" exposed for >1 day or even 5 days raises a question whether any
of the cases or controls could really be said to have ever come In contact
with enough phenoxyacetlc add to justify such a designation. It could be
that, In fact, potentially exposed Individuals 1n New Zealand have had
little or no contact with the herbicide.
The authors did conclude that the finding of a relative risk of 1.7 1n
Individuals with >1 day exposure not 1n the last 5 years cannot be entirely
discounted. But then the authors stated that 1f length of exposure was >5
days prior to 10 years before cancer registration, they would expect an
Increase, and since they do not see an Increase, there 1s no evidence of a
"real causal link." One might ask whether this 1s a suitable criterion for
providing evidence of a causal association. Perhaps a more valid group for
11-78
-------
study would be one where the potential exposure was considerably longer than
"5 days" and >15 years before Initial cancer registration. As kind of a
subtle justification for the finding of no significant risk 1n workers
exposed In phenoxy acids, the author alluded to the fact that there were 500
full-time workers registered 1n New Zealand who did full time ground spray-
Ing and altogether some 2000 workers who were at some time professionally
Involved 1n phenoxyacetlc add herbicide spraying from the air or ground
with exposure "very much greater" than that of patients In this study. This
kind of argument has appeal 1f these workers could be shown to have had
their exposure sufficiently far In the past that latency considerations
could be adequately addressed. However, the real question again remains;
how much real exposure did those patients 1n the study really have 10-15
years earlier, and 1n what numbers. The author remarked that 1t was
surprising that he found no STS victims who had ever worked full-time 1n
phenoxyacetlc add herbicide spraying. Perhaps they have not yet been
observed for a long enough period. The time interval of 10 years and/or 5
years from exposure to registration may not have been long enough to allow
latent effects to become evident. However, as was pointed out by the
author, the findings do not support the hypothesis that exposure to phenoxy-
acetlc add herbicides causes STS. But neither do they support a negative
finding without better documentation regarding actual exposure and time of
actual exposure. Smith (1983), however, noted that his documentation of
exposure to 2,4,5-T (and 2,4-D) was at least as good as that 1n the Hardell
and Sandstrom (1979) study, and that although Harden and Sandstrom (1979)
noted higher relative risks of <30 days exposure, Smith (1983) did not.
Hence the paradox. Smith (1983) admitted the possibility that 2,3,7,8-TCDD
contaminations might be lower In New Zealand as opposed to 2,3,7,8-TCDD
contamination In the Swedish studies, although there 1s no evidence for 1t.
11-79
-------
He stm maintains that his study showed that exposure to phenoxyacetlc
adds may not be associated with STS.
Pazderova-Vejlupkova et al. (1981) studied 80 workers Involved In the
production of 2,4,5-sodlum trlchlorophenoxyacetate and butylester of tr1-
chlorophenoxyacetlc acid who subsequently became 111 from exposure to
2,3,7,8-TCDD during the period 1965-1968. Only 55 members of this group
were followed for 10 years. The remaining 25 either refused participation
or moved leaving no forwarding address. Host patients developed chloracne
while 11 developed porphyrla cutanea tarda. Chief chemical signs were
metabolic disturbances, pathologically elevated Uplds with abnormalities 1n
the llpoproteln spectrum, and "pathological" changes 1n glucose tolerance.
Other symptoms noted were biochemical deviations consistent with "a mild
liver lesion," light steatosls, perlportal flbrosls or activation of Kupffer
cells, or nervous system focal damage (peripheral neuron lesion 1n lower
extremities). Altogether six patients were reported to be deceased during
this 10-year period, 2 from bronchogenlc carcinoma, 1 from cirrhosis, 1
atherosclerosis preclpue cerebl and 2 1n auto accidents. No STSs or lympho-
mas were found. Since there was no comparison population with which to
estimate relative risk for cancer, the study must be classified at best as
clinical with respect to cancer. The 6 deaths (of 55) that occurred during
the 10-year observation period cannot be construed to be associated with
exposure to the 2,4,5-T. Because of the small number of cases and the short
follow-up period, nothing can be said concerning the association of exposure
with cancer, especially specific types of cancer such as STS or non-
Hodgkln's lymphoma.
R11h1mak1 et al. (1982, 1983) studied a cohort of 1926 herbicide appli-
cators formed In 1972 from personnel records of four Finnish employers
11-80
-------
(e.g., the Forestry Authority, Highway Authority, State Railways and a
state-owned electric power company). Chlorinated phenoxyadds had been used
since the 1950s 1n Finland for spraying. They constituted 2:1 mixtures of
emulsified esters of 2,4-D and 2,4,5-T dissolved 1n water. Analyses from
old herbicide formulations dating back to the 1960s revealed that these
mixtures contained 0.1-0.9 mg/kg of 2,3,7,8-TCDD.
This cohort of male workers was exposed a minimum of 2 weeks during at
least one growing season from 1955-1971. Follow-up continued 9 years
through 1980 for mortality but only until 1978 for morbidity. Fifteen
Individuals could not be traced by 1980. Expected deaths were generated
based upon cause- and age-specific national Finnish death rates for 1975.
Expected cases were similarly calculated based upon national Incidence rates
of 1975.
By 1980, 144 deaths had occurred vs. 184.0 expected, a deficit of 22% 1n
observed mortality. Only 26 cancer deaths had occurred vs. 36.5 expected, a
29% deficit. The authors separated out "natural" deaths from the total.
The observed residual deaths equaled 39 while the expected deaths equaled
28.7. This excess was of borderline significance. The authors also con-
sidered 10-year and 15-year latent periods. Even after 15 years, the defi-
cit of deaths continued to manifest Itself both 1n categories of all causes
and total cancers; 35 observed vs. 53.6 expected and 5 observed vs. 11.3
expected, respectively. Similarly, the 7-year follow-up of cancer morbidity
revealed 26 cases of cancer vs. 37.2 expected. After a 10-year latent
period, 16 cancer cases were observed vs. 20.1 expected. None of the 26
cancer deaths or 26 cancer cases were of the STS or lymphoma type. (How-
ever, only 0.1 STS and 0.5 lymphomas were expected.) In no Instance was
cancer of any site significantly elevated.
11-81
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The authors noted that this unusual deficit of mortality and morbidity
of between 70 and 82% (even after 15 years from Initial exposure) was prob-
ably a consequence of the "healthy worker effect" In that only able-bodied
and healthy Individuals were selected Into the Industry. The fact that the
cohort was assembled In 1972 from records of persons who were exposed as
early as 1955 (17 years prior) raises the likelihood that 1n 1972 a
"survivor" population remained (45 deaths before 1972 were eliminated from
the cohort) that was relatively healthy. Furthermore, the unusually large
number of not "natural" expected and observed deaths (probably accidents and
external causes) occurring to this cohort Indicate a relatively youthful
population was under scrutiny. The leading cause of death to persons under
35 years 1s from accidents, based on national *v1tal statistics.
The authors correctly noted that, because of limitations 1n the study
material, only powerful carcinogenic effects could be detected. Risk ratios
higher than 1.5 for all cancers, 4.0 for lymphomas and 10.0 for STS could be
excluded based on this data set from the authors' own calculations. More
follow-up 1s needed 1n order to provide a stable assessment of the relation-
ship between exposure and cancer. The authors concluded that this study
will allow no assessment of STS because "the number of persons having a suf-
ficiently long latency period 1s too small." It was suggested that more
valid conclusions could be made only with the passage of time (RUhlmakl et
a!., 1983).
Recently, the Michigan Department of Public Health (1983b), produced an
ecological study of soft and connective tissue cancer mortality rates 1n
Midland and other selected Michigan counties. They found that mortality
rates for this cause were 3.8-4.0 times the national average for the periods
1960-1969 and 1970-1978, respectively, for white females In Midland. These
11-82
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estimates are based upon 5 deaths and 7 deaths, respectively, and are listed
1n Table 11-29. No excess risk was reported among white males, however.
The Michigan Department of Health concluded that because of the occurrence
of these two successive elevated rates, 1t 1s unlikely to be a chance
happening. At the same time the age-adjusted male and female cancer mortal-
ity rates for Midland were below that of the State of Michigan for the
period 1970-1979. Midland County 1s the home of a major chemical company
that produced phenoxyacetlc add herbicides until recently. The authors
stated that a detailed review of death certificates, hospital records, resi-
dency and occupational histories of the 20 male and female cases revealed no
"commonalities" suggesting a "single causative agent," although a majority
or their spouses had worked at this chemical facility. They recommend that
a case-control study should be employed to evaluate possible Influences,
such as lifestyle, occupation or location of residence on the risk of STS.
In a series of reports prepared under the auspices of the U.S. A1r
Force, Col. William H. Wolfe and his associates just completed the first
phase of a study of A1r Force personnel Involved 1n the aerial dissemination
of TCDD-conta1n1ng herbicides 1n the Republic of Vietnam (RVN). During the
period of time beginning 1n 1962 and ending 1n 1971, -1278 male A1r Force
personnel {Ranch Handers) were Identified as having been Involved 1n the
effort to 1} defoliate vegetation In Vietnam 1n order to decrease the risk
of ambush and 2} destroy enemy crops (Wolfe et al», 198S). Based on an 1984
report of baseline mortality study results (Wolfe et a!., 1984), the cohort
Involved 1n the mortality study was smaller at 1256 because of the
11-83
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TABLE 11-29
Midland County Soft and Connective Tissue Cancer Deaths 1960-1981*
00
Identification
Year of
Death
1961
1963
1964
1968
1969
1970
1970
1974
1976
Sex
F
f
F
F
F
F
F
F
F
Age
24
75
51
37
45
59
56
1
77
Type, Site and Progression
Type
Hemanglosarcoma
Uposarcoma
Lelomyosarcoma
Uposarcoma
Flbrosarcoma
Lelomyosarcoma
Kaposl sarcoma
Flbrosarcoma
Lelomyosarcoma
Rhabdomyosarcoma
Llposarcoma
Primary Site
Face
Right gluteal
Uterus
Spine
Right thigh
Uterus
Right leg
Right thigh
Abdominal wall
Inguinal area
Right thigh
of Malignancy
Hetastases
Skull and upper lobe
of lung
Unknown
Widespread
Lungs, pelvis
Lung, liver
Adrenal gland and skin
Lymph nodes
Spine
Lung
Unknown
Buttock, lung, rib,
Month and Year
Diagnosed
5-58
Unknown
11-63
1-66
10-68
8-68
1960
1967
8-73
12-74
1978
64
Lelomyosarcoma
Left knee
lymph nodes
Liver, lymph nodes,
lung, bone
7-70
-------
TABLE 11-29 (cont.)
00
Identl
Year of
Death
1978
1978
1979
1962
1967
1967
1969
1971
1972
1976
flcatton
Sex
F
F
F
H
n
M
H
H
M
M
Age
26
88
27
63
77
20
32
76
89
53
Type. Site
Type
Rhabdomyosarcoma
Flbrosarcoma
Lelomyosarcoma
Rhabdomyosarcoma
Nesothelloma
Rhabdomyosarcoma
Uposarcoma
Lelomyosarcoma
Lelomyosarcoma
Flbrosarcoma
and Progression
Primary Site
Rectum
Right cheek
Left thigh
Left lower leg
Lung
Pharynx
Left arm
Small
Intestine
Retro-
perl tonal
region
Perltloneum
of Malignancy
Netastases
Lung, neck, Inguinal
region
Facial area
Lung
Lung and right outer
chest wall
Lung, peritoneum and
diaphragm
Perlorbltal area and
Hver
Perineum and buttock
Liver
Hepatic system
Lung, Hver
Month and Year
Diagnosed
6-76
6-78
3-78
8-61
6-67
1-67
6-64
10-69
7-72
3-75
'Source: Adapted from Michigan Department of Public Health, 19835
-------
exclusion of 22 killed 1n action and was divided Into three main occupa-
tional categories as follows;
1. Officers (pilots, navigators and others) 466
2. Enlisted (flight engineers) 206
3. Enlisted (others) 584
TOTAL 1216
The authors categorized the Ranch Handers as having had "exposure" to
the TCDD-conta1n1ng herbicides 1f they were Involved 1n the aerial spraying
of the herbicides. They were matched to 6171 cargo mission air crew members
and support personnel generally on a 5 to 1 basis according to similarity of
training and military background experiences, occupation and race. The
comparison population presumably had no exposure to TCDD. In an earlier
1983 report (Lathrop et a!., 1983), 50 deaths were Identified 1n the study
group versus 250 In the comparison population. Of these 50 deaths, 23 were
due to external causes, 4 were malignant neoplasms, 16 were circulatory
causes, 5 were digestive disorders and 1 was an endocrine disorder.
In the later December 1984 update, Wolfe et al. (1984) added 4 more
deaths to the study population for a total of 54 deaths occurring to Ranch
Hands while adding 15 to the 250 that had already occurred 1n the comparison
group through December 31, 1983. Altogether this update produced a total of
6 cancer deaths 1n the Ranch Hands versus 43 cancer deaths In the comparison
population. The greatest cause of death In both Ranch Handers and the
comparison population were accidents with 19 and 94, respectively. None of
the 6 cancer deaths and 1 of the 43 deaths In the comparison group were
STSs. Comparison of overall mortality 1n the Ranch Handers with other A1r
Force military personnel was nearly Identical (~4.3J£). Ranch Hand ground
11-86
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ifilfsted personnel suffered somewhat greater (although not significant)
mortality than did Ranch Hand officers. Comparison of mortality 1n the
Ranch Handers with other groups such as U.S. white males, Department of
Defense retired enlisted men, U.S. civil servants, active duty Air Force and
West Point officers from the class of 1956, were similar except for A1r
Force active duty officers who exhibited significantly less mortality. The
authors attribute this to higher health qualification standards.
There were few biological markers that might tend to support the assump-
tion that Ranch Hands were exposed to 2,3,7,8-TCDD. In the Banbury report,
Lathrop et al. (1984) reported that the dermatologlc evaluation revealed no
cases of chloracne through clinical diagnosis or bloassay. A questionnaire
analysis of acne In Ranch Handers and comparison groups showed no unusually
different Incidence, severity, duration or distribution of anatomical
locations In either group. Lathrop et al. (1984) said 1n fact that the
"historical occurrence of chloracne was highly unlikely In the Ranch
Handers".
This study suffers from several deficiencies that limit Its usefulness
1n a determination of human health effects, notably cancer, and especially
STS from exposure to 2,3,7,8-TCDD-contam1nated phenoxy herbicides. First,
It 1s mainly a study of basically young men who were Involved 1n the A1r
Force aerial spraying missions. This Is evidenced by the exceptionally
large number of accidents attributable to members of the cohort. It 1s the
largest single cause of death 1n these men. Because this 1s a young group
It 1s unlikely that substantial mortality will occur to the cohort until
many more years of follow-up have passed. In fact, even after 15 years
following Initial exposure <5% of the cohort have died. Since most cancers
11-87
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have a latency of >15 years following Initial exposure 1t 1s not likely that
a cancer risk from 2,3,7,8-TCDD, 1f any, will manifest Itself for some time.
Furthermore, the relatively rare STS, which 1s thought to have an even
longer latency period, may not appear as a risk 1n this cohort until well
after the 20th year. Additionally, this cohort exhibits Uttle evidence of
actual exposure to the herbicide 1n question, thus raising the possibility
of m1sclass1f1cat1on. In other small cohort studies (Cook et al., 1980; Ott
et al., 1980; Zack and Susklnd, 1980} substantial numbers of the study
cohorts exhibited evidence of exposure to 2,3,7,8-TCDD as Indicated by the
presence of chloracne, a clear biological marker. Few of the Ranch Handers
exhibited evidence of this condition (Lathrop et al., 1984). In fact, as
was suggested by the authors, the historical, occurrence of chloracne was
considered highly unlikely 1n the Ranch Hands. Neither do they present
convincing evidence of other conditions suggestive of an association with
exposure to the d1ox1n-conta1n1ng herbicide that cannot be explained by con-
founders, according to the authors. In fact Ranch Handers, who were heavily
populated with officers, pilots, navigators and flight engineers, may not
have been as heavily exposed to the phenoxy herbicides as other U.S. mili-
tary personnel 1n Southeast Asia. Perhaps Army combat foot soldiers or the
non-Ranch Hand personnel who did the spraying on the ground around the mili-
tary bases would constitute a more appropriate cohort for study. Lathrop et
al. (1984) concluded that the absence of any association of "clinical end-
points" with herbicide exposure must be viewed as Insufficient evidence
supporting a cause-and-effect relationship. But this absence of any "clini-
cal endpolnts" might also Indicate evidence of a lack of exposure to the
11-88
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ptienoxy herbicides In question by the Ranch Handers. This study must be
viewed as Inadequate 1n assessing the risk of cancer from exposure to
2,3,7,8-TCOD-conta1n1ng phenoxy herbicides.
In a separate review of the ep1dem1olog1cal evidence for STS from expo-
sure to 2,4,5-T-conta1n1ng herbicides, the United Kingdom Ministry of Agri-
culture, Fisheries and Food (1983) concluded that there was no evidence to
recommend altering their earlier conclusion that formulations of phenoxy
add herbicides and related wood preservatives as "presently cleared" are
safe and may continue to be used. This report readily discounts the posi-
tive studies of Harden and Eriksson (T979) as being biased, and 1t makes no
reference to the later validity study by Harden (1981) of his own work
utilizing colon cancer controls (see Section 11.2.2.2.). In this report
Harden answered these early criticisms that were reiterated by the British
1n their report. At the same time, the British report appears to put undue
emphasis on nonposltlve studies that do not demonstrate a risk, although
most of them have methodological limitations (e.g., low power, Insufficient
latency and Inappropriate study methods). In short, the British review
appears to be overly optimistic about the safety of 2,4,5-T herbicides.
Flngerhut et al. (1984) recently completed a review of medical and
available exposure records of seven U.S. chemical workers that have been
diagnosed as having STS and who were reported to have had possible exposure
to dloxln. These cases collectively produced a clustering effect of the
relatively rare STSs among former employees of a portion of the U.S. chemi-
cal Industry where exposure to compounds contaminated with 2,3,7,8-TCDD 1s
most likely to have occurred. Flngerhut et al. (1984) reported that a
subsequent review of the Armed Forces Institute of Pathology and a review of
one of the authors of the Flngerhut paper confirmed the diagnosis of 5 of
the 7 U.S. chemical workers as STSs.
i
11-89
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In terms of occupational exposure, Flngerhut et al. (1984) proposed a
strict definition of exposure as follows: a record must exist somewhere
that shows an assignment to either a 2,4,5-T department or to a trlchloro-
phenol department at some time In the past. If such a record did not exist,
then the Individual would not have been considered to have had a confirmed
exposure. Four of the seven who had a confirmed exposure 1n this manner
were also members of cohorts that had been studied previously, while the
remaining three could not be confirmed as having been assigned to any
2,4,5-T department or trlehlorophenol department. The latter three were not
Identified as having been part of any earlier study but were case reports of
Johnson et al. (1981) and Moses and Sellkoff (1981). Individuals who were
members of study cohorts of "exposed Individuals" might be expected to have
better documentation of exposure, based upon employment records, than would
cases turning up 1n a medical practice.
However, Flngerhut et al. (1984) pointed out that of these three cases,
one worked 32 years 1n production, clerical, truck driving and maintenance
Jobs 1n a chemical manufacturing site that produced trlchlorophenol and
2,4,5-T; the second worked 2.1 years as a production worker 1n a plant that
made 2,4,5-T; and the third was a production and maintenance worker for 29
years at the same facility as the second worker. It would seem that the
opportunity for exposure to 2,3,7,8-TCDD containing 2,4,5-T or trlchloro-
phenol must be considered a distinct possibility 1n the first two cases,
especially since both were Involved with maintenance for many years.
Johnson et al. (1981) pointed out that the second case could not have
satisfied a minimum latency requirement for exposure to TCDD since his 2.5
years as a production worker occurred Just before his diagnosis and death.
11-90
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However, this man's father was employed with this same plant almost as long
as his son was alive and 1t seems plausible that because of this connection
the son may have been exposed.
One must have reservations about the usefulness of a classification
scheme that relies on documentation of an assignment to a specific area of a
plant as proof of exposure to dloxln without real evidence substantiating
that exposure (I.e., either biological or physical measurements), while at
the same time assignment to all other areas of the same plant 1s considered
Insufficient evidence of exposure although nothing Is offered to substan-
tiate the presence or lack of exposure to 2,3,7,8-TCDD 1n either case. In
most occupational prospective cohort ep1dem1olog1c studies, employment at a
plant where the suspect agent 1s produced or found has been considered
sufficient enough to call such a person "exposed" and thus Included 1n a
cohort for study. On the other hand, 1f the Flngerhut et al. (1984) defini-
tion were retrospectively applied to the already small occupational cohorts
from which the first four STSs came, even two of these relatively rare STSs
might probably constitute an excessive risk 1n the much smaller cohorts
circumscribed by their definition. Flngerhut et al. (1984) agreed that an
excess risk of STS would remain even with just two confirmed cases, and
hence the possibility of a causal relationship between exposure to 2,3,7,8-
TCDD and the development of STSs cannot yet be ruled out.
In summary, the associations reported In the two Swedish soft-tissue
sarcoma studies are strong enough to make 1t unlikely that they have result-
ed entirely from random variation bias or confounding, even though the
possibility cannot be excluded. These studies provide a strong suggestion
that phenoxyacetlc acid herbicides, chlorophenols or their Impurities are
carcinogenic 1n humans.
11-91
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11.2.2.2. MALIGNANT LYHPHOHAS -- A separate series of clinical obser-
vations at the Department of Oncology In Umea, Sweden (Harden, 1979), led
the researchers to conduct a case-control study of malignant lymphoma 1n
relation to phenoxyacetlc add, chlorophenols, and other organic compounds
(Hardell et al., 1980, 1981). Approximately 33% of the cases 1n this study
were patients with Hodgkln's disease; the remainder of the cases were non-
Hodgkln's lymphomas.
This study employed essentially the same methods and produced results
comparable with those of the STS studies: statistically significant 5-fold
to 6-fold relative risks In relation to phenoxyacetlc adds and chloro-
phenols were confirmed. In addition, an elevated relative risk was found In
connection with exposure to organic solvents, such as benzene, trlchloro-
ethylene, and styrene. In the published report, the methods and results
were Incompletely documented, especially the possibility of confounding by
exposure to the organic solvents.
In the update of the earlier 1980 study, Harden et al. (1981), utiliz-
ing the same basic data source, found that 36.1% of the cases had been
exposed to phenoxy herbicides or chlorophenols, while only 9.6% of their
controls were so exposed. The estimated relative risk was 6.0 when matching
was considered and 5.3 when matching was eliminated. When cases and con-
trols that were exposed to chlorophenols only were excluded, the relative
risk of lymphoma from phenoxy adds alone was 4.8 (95% C.I. 2.9-8.1). On
the other hand, 1f exposures to phenoxy acids are excluded and consideration
Is given to just chlorophenols (which Includes combined exposure to phenoxy
adds and chlorophenols), then the relative risk equaled 4.3 (95% C.I.
2.7-6.9). The author further subdivided this group Into "low-grade" vs.
"high-grade" exposures to chlorophenols. A continuous exposure of not more
than 1 week or repeated Intermittent exposures totaling not more than 1
11-92
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month was classified as low-grade. The relative risk for high-grade expo-
sure was 8.4 (95% C.I. 4.2-16.9), while that for low-grade(exposure equaled
9.2 (95% C.I. 1.6-5.2). If exposure to organic solvents 1s examined, given
that cases and controls exposed to only phenoxy adds and/or chlorophenols
were excluded except for combined exposure to organic solvents, 1t 1s found
that high-grade and low-grade relative risks were 2.8 (95% C.I. 1.6-4.8) and
1.2 (95% C.I. 0.5-2.6), respectively. However, the author noted that expo-
sure to phenoxy adds and high-grade organic solvents (exposure to chloro-
phenols excluded) produced a relative risk of 11.2 (95% C.I. 3.2-39.7) based
upon a few cases and controls with exposure to both. The authors concluded
that "exposure to organic solvents, chlorophenols and/or phenoxy acids
constitutes a risk factor for malignant lymphoma."
The Harden et al. (1981) study 1s stm subject to the same methodo-
logical criticisms to which the earlier study was subjected. Chief among
those 1s the possibility of observational and/or recall bias creeping Into
the responses that are elicited from self-administered questionnaires on
kind and length of exposure. Secondly, confounding by exposure to poten-
tially carcinogenic organic solvents and other agents could have had an
effect on the risk estimate, although Harden (1981) Insists that they did
not.
Other research has tentatively suggested that lumberjacks may be at
\
Increased risk of lymphoma (Edllng and Granstam, 1979). The Nltro study
found three deaths from cancers of the lymphatic and hematopoletlc system,
against only 0.88 expected (p=0.06, one-tailed Polsson test).
The lymphoma case-control study (Harden et al., 1980, 1981) 1s con-
sistent with the two STS studies discussed above. On the other hand, the
consistency could also reflect an (as yet) unidentified common flaw In all
these studies.
11-93
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The two Swedish case control studies on STSs and a later case control
study of malignant lymphoma (Hardell et al., 1981) were subjected to a
validity analysis with respect to the assessment of exposure by Hardell and
Eriksson (1981). To answer the question raised regarding the recall of
occupation 1n a forestry/agriculture job, secondary to the recall of expo-
sure to phenoxy adds and/or chlorophenols, the cases and controls were
divided Into three groups: those who worked their entire time since 1950 1n
an agriculture/forestry job; those who worked some time 1n an agriculture/
forestry job but not exclusively; and the remainder who never worked 1n a
forestry/agriculture job. The study found that the risk ratio was still 8.2
for STS 1n exclusively agriculture/forestry workers who were exposed to
phenoxy acids compared with workers found 1n other occupations having no
apparent exposure to phenoxy acids or chlorophenols. Even when comparing
phenoxy add- and/or chlorophenol-exposed agricultural/forestry workers
exclusively with nonexposed agricultural/forestry workers, the risk ratio
was still 7.1. This argument seems to answer effectively questions regard-
Ing recall of occupation secondary to exposure.
On the other hand, the relative risk remains 5.4 when comparing phenoxy
add and/or chlorophenol exposed workers exclusively 1n occupations other
than agriculture/forestry with nonexposed workers 1n those same occupations,
thus, suggesting the presence of either recall bias or still another
occupation with potential exposure to phenoxy adds and/or chlorophenols
(Table 11-30).
When woodworkers are separated out (possible exposure to chlorophenols
1n treatment of wood) the risk ratio becomes 9.7 (Table 11-31). These data
suggest the presence of some recall bias.
11-94
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TABLE 11-30
Other Occupations (Minus Forestry/Agriculture)*
Group Phenoxy Adds/Chlorophenols Non-exposed
Cases 11 68
Referents 5 167
RR = 5.4 X2 = 11.01 (P<0.01)
*Source: Hardell and Erlkkson, 1981
RR = Relative risk
11-95
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TABLE 11-31
Other Occupations (Minus Forestry/Agrlculture/Woodworkers)*
Group Phenoxy Adds/Chlorophenols Non-exposed
Cases 4 66
Referents 1 160
RR = 9.7 X2 - 5.98 (P<0.05)
*Source; Harden and Erlkkson, 1981
RR » Relative risk
11-96
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Another focus of the Harden and Erlkkson (1981) study was to determine
1f observational bias on the part of the Investigators could explain the
significantly high risk estimates. To answer the question, the study com-
pared the exposure data derived from the Interviewee's returned question-
naires only with the combined Information from both the phone Interviews and
questionnaires. The study found no substantial differences 1n the frequency
of reporting exposure.
Stm a third consideration of possible bias Involves recall of exposure
to phenoxy acids and/or chlorophenols because of subject knowledge of having
cancer 1n the cases versus no knowledge of cancer 1n the referent popula-
tion. The study chose as a referent group for the 52 STS cases (Harden and
Sandstrom, 1979} and the 169 malignant lymphomas (Harden et al., 1981} a
group of 154 colon cancer cases from the same population source and compared
their exposure to phenoxy adds and/or chlorophenols by broad age groupings,
and by rural vs. urban residence.
Utilizing a Mantel-Haenszel rate ratio, the study found the risk of
exposure to phenoxy adds remaining significantly high at 5.5 and to chloro-
phenols 5.4 1n the STS cases compared with the colon cancer controls. Simi-
larly, with the malignant lymphomas, the Identically derived risk ratios
remain significantly high at 4.5 with respect to phenoxy adds and/or
chlorophenol exposure 1n the cases, hence, the study concludes, no "sub-
stantial observational bias" exists. If 1t 1s assumed 1n this study that
recall bias was and 1s the same as observational bias, then such a conclu-
sion may not be entirely warranted from the comparison. Certainly, 1t
appears that no recall bias existed because of subject "knowledge of having
cancer" based on the authors' analysis. But 1t does not rule out the possi-
bility that recall bias can still be present 1n their data for other
11-97
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reasons. Harden et al. (1981) refers to an Intense "debate about phenoxy
adds and their presumptive risk" 1n Sweden at the time the colon cancer
study was conducted. But, there 1s no reason to think that colon cancer
victims would assume their disease was brought about from exposure to dloxln
containing chemicals 1f no connection was suggested.
It seems plausible that STS and non-Hodgk1n's lymphoma patients would
either learn at the time of their diagnosis that exposure to d1ox1n-conta1n-
1ng chemicals was the likely cause of this rare type of tumor or quickly
learn from other sources, such as the news media, that exposure to herbi-
cides containing dloxln could cause this rare form of cancer whereas colon
cancer victims (a rather common form of cancer) would not necessarily be led
to believe that exposure to the same d1ox1n-conta1n1ng chemicals caused
their disease. Hence, 1t 1s not difficult to Imagine that such unusual
victims of cancer could better "remember" exposure to such chemicals than
could colon cancer patients.
Therefore, although the Harden (1981) study may explain any biases
Introduced from secondary recall of occupation, observational bias Intro-
duced from the telephone Interviewer and recall bias based on subject
knowledge of cancer, 1t does not adequately answer questions of recall bias
Introduced through the acquired awareness on the part of the victim of STS
or non-Hodgk1n's lymphoma that his condition may have been caused by
exposure to d1ox1n-conta1n1ng herbicides.
11.2.2.3. STOMACH CANCER — Studies of two of the oldest cohorts of
workers known to have been exposed to 2,3,7,8-tcdd containing phenoxyacetlc
add herbicides report stomach cancer mortality rates significantly higher
than expected. The results 1n each study were based on small numbers of
deaths. In one study (Axelson et al., 1980), 348 Swedish railroad workers
11-98
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with at least 46 days of herbicide exposure between 1955 and 1972 were
followed through October 1978. The workers were grouped on the basis of
their primary herbicide exposures: those primarily exposed to phenoxyacetlc
acids (2,4-D and 2,4,5-T) only, to amltrole (amlnotrlazole) only, and to
both types of herbicides. After a 10-year latency was achieved, 3 stomach
cancer deaths were observed vs. 0.71 expected (p<0.05). None were attribut-
able to amltrol alone, but two were assigned to phenoxy acids alone while
the remaining stomach cancer death occurred In a worker exposed to both
amltrol and phenoxy acids. The excess was more pronounced (3 observed vs.
0.57 expected, p<0.05) among those with early exposure (1957-1961) to
phenoxy adds and/or amltrol. If persons who were exposed to just amltrol
alone are excluded, thus leaving Individuals exposed to phenoxy add alone
and amltrol In combination, the excess 1s enhanced further (3 observed vs.
0.41 expected, p<0.01).
Axelson et al. (1980) also noted an excess 1n total "tumors" after 10
years latency as well {15 observed vs. 6.87 expected, p<0.005). This Is
pronounced 1n those exposed early to phenoxy acids alone (6 observed vs.
2.60 expected, p<0.01) and phenoxy acids 1n combination with amltrol (5
observed vs. 1.34 expected, p<0.05). Presumably, "tumors" In Sweden are
analogous to malignant neoplasms In the United States. The author states
that no specific type of tumor predominates and no breakdown by tumor type
1s provided.
The other study showing Increased stomach cancer mortality 1s the
follow-up of 75 workers exposed to 2,3,7,8-TCDD during and after a 1953 run-
away reaction at a trlchlorophenol manufacturing facility 1n Ludwlgshafen,
Federal Republic of Germany (Thless and Frentzel-Beyme, 1977). Two sources
-------
were used to calculate expected deaths; national mortality rates for the
period 1971-1974, and .1972-1975 rates for Rhlnehessen-Palatlnate, the region
1n which Ludwlgshafen 1s located.*
The results, shown 1n Table 11-32, Indicate an Increased rate of stomach
cancer mortality that also 1s not likely to have been due to chance alone.
Two aspects of the methodology used could have Influenced these results.
First, the available report does not Include an analysis allowing for a
minimum period of cancer Induction. All three stomach cancer deaths 1n the
Ludwlgshafen cohort occurred more than 10 years after Initial exposure.
Employing a 10-year restriction to follow-up (as In the Swedish cohort
study) would result 1n a higher relative risk estimate by reducing the
number of expected deaths.
Secondly, national and regional mortality rates from the 1970s were used
to generate expected deaths to compare with observed mortality over a much
longer period (1953-1977). The substantial decline In stomach cancer
mortality 1n West Germany during the late 1950s and 1960s would likely make
these expected figures too large.
The researchers also used an Internal control group that does not raise
the second concern discussed above. This group consisted of 75 men, each
matched to study group members by age and date of entry Into employment, and
selected at random from a 11st of over 10,000 persons who had been Included
1n previous cohort studies by the same Investigators. No stomach cancer
deaths occurred 1n this control group during the follow-up period. Thus,
use of the Internal control groups also Indicates an excess of stomach can-
cers 1n the exposed workers.
*The report originally Included expected deaths using rates for the city of
Ludwlgshafen, which were later shown to be Inaccurate.
n-ioo
-------
TABLE 11-32
Analysis of Stomach Cancer Mortality In a Group of
West German Factory Workers Exposed to 2,3,7,8-TCDD*
Source for Stomach Cancer Deaths Relative Significance
Expected Deaths Risk Level
Observed Expected
Federal Republic
of Germany
1971-1974 3 0.559 5.4 0.02
Rhlnehessen-
Palatlnate
1972-1975 3 0.495 6.1 0.01
*Source: Thless and Frentzel-Beyme, 1977
11-101
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In an update of this earlier study, Thless et al. (1982) continued the
follow-up of his cohort through 1979 by adding 2 additional years of follow-
up and apparently reducing the size of his cohort from 75 to 74. Altogether
21 deaths (4 more than from the earlier study) occurred vs. 18 and 19 deaths
1n the 2 matched (1 to 1) Internal comparison groups. With respect to can-
cer deaths, the numbers were respectively 7, 5 and 5. The first control
group was manually matched from the total number of persons (5500 Included
1n the cohort until the end of 1976) and the second, at random, by computer
for some 8000 employees. In addition, 19 expected total deaths were esti-
mated based on 1970-1975 mortality statistics of Rh1nehess1n-Palat1nate, 18
expected deaths based on 1970-1975 mortality statistics of Ludwlgshafen, and
20 expected deaths based upon 1971-1974 mortality statistics of the Federal
Republic of Germany. Just as 1n the earlier study, the three stomach car-
cinomas noted earlier appear to be significantly elevated regardless of
which external comparison group 1s used (Table 11-33).
On the other hand, one stomach cancer appeared 1n the randomized Inter-
nal control group. None appeared 1n the manually matched Internal control.
No other elevated risks for any other cause were evident and no STSs
appeared. When latency was considered only, the risk of stomach cancer
remained significantly elevated after a lapse of 10 years (3 observed, 0.52
expected, p<0.016) and then after a lapse of 15 years (2 observed, 0.23 ex-
pected, p<0.02) based upon death rates of Rh1nehess1n-Palat1nate, 1970-1975.
Again, these study conclusions are limited by the small size of the
study group and the very few cancer deaths noted at any particular site.
Thus, 1t 1s Insensitive to the detection of a significantly elevated risk
for most causes of cancer, especially STS and lymphomas. Although, stomach
cancer 1s elevated significantly, 1t 1s based only upon three deaths and
11-102
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TABLE 11-33
Reanalysls of Stomach Cancer Mortality 1n a Group
of West German Factory Workers Exposed to 2,3,7,8-TCDD*
Source for
Expected Deaths
Stomach CancerDeaths
Observed Expected
Relative
Risk
Significance
Level
Federal Republic of
Germany 1971-1974
Rh1nehess1n-
Palatlnate
1970-1975
Ludwlgs-Shafen
1970-1975
3
3
0.7
0.64
0.61
4.3
4.7
4.9
0.034
0.027
0.024
*Source: Thless et a!., 1982
11-103
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since one stomach cancer death has been noted 1n an Internal control group
1n the updated version, It appears that this finding has been weakened some-
what. Furthermore, as was pointed out earlier, trends 1n stomach cancer
mortality during the 1950s, 1960s and 1970s could make the comparison of
stomach cancer mortality with expected deaths less valid based upon
1970-1975 rates.
In summary, the evidence that phenoxyacetlc adds and/or 2,3,7,8-TCDD
might Increase the risk of stomach cancer consists of two studies, each of
which reports a statistically significant excess that 1s based on only three
stomach cancer deaths. Further follow-up of these and similar cohorts 1s
warranted, but firm conclusions cannot yet be made.
Four additional cohort studies have reported results that do not show
Increased stomach cancer mortality rates 1n groups of workers exposed to
phenoxyacetlc adds and/or 2,3,7,8-TCDD. These are studies of 2,4,5-T pro-
duction workers 1n Midland, Michigan (Ott et al., 1980), Finnish phenoxy-
acetlc add herbicide applicators (R11h1mak1 et al., 1978), the NHro study
1n which workers were exposed to 2,3,7,8-TCDD (Zack and Susklnd, 1980) and
trlchlorophenol manufacturing workers (Cook et al., 1980).
As previously mentioned, the NHro study Included a single death from
STS and a weakly suggestive Increase 1n lymphatic and hematopoletlc system
cancer mortality. The Midland study of 204 workers Included only one cancer
death, a tumor 1n the respiratory system. In the Finnish study, hlstologlc
Information on tumor types was not provided; however, there were no deaths
from lymphoma.
The results pertinent to stomach cancer mortality 1n the three studies
are shown 1n Table 11-34. Results of neither the Midland study nor the
11-104
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TABLE 11-34
Stomach Cancer Mortality 1n Three Studies of Workers Exposed
to Phenoxyacetlc Add Herbicides and/or 2,3,7,8-TCOO
Stomach Cancer Deaths Relative
Risk
Observed Expected
0 0.14a 0
5 6.9a»D 0.7
0 0.5b 0
9554 Confidence
Interval
0-26.3
0.2-1.7
0-7.4
Reference
Ott et al., 1980
R11h1mak1 et al.,
1978
Zack and Susklnd,
1980
Estimated from total cancer expected deaths (see footnote 1n text).
^Entire follow-up period without regard for minimum time for cancer Induc-
tion (Ott et al., 1980 used a 10-year minimum Induction period).
11-105
-------
N1tro study contradict the findings of the Swedish and West German Investi-
gations previously discussed. This can be shown 1n two ways. First, the
upper 95% confidence limits for the relative risk estimates from these two
"negative" studies exceed even the highest point estimates of relative risk
(6.1) from the two "positive" studies (see Table 11-31).
This Indicates that the relative risk estimates from the Midland and
NHro studies, even though equal to zero, are nevertheless not significantly
different from the estimates of 6.1, given the sample sizes, follow-up per-
iods, age distribution and comparison group rates.
In addition, the smallest detectable relative risk In the Midland study
(a = 0.05,
-------
Statistically, the study of Finnish herbicide applicators Is Inconsis-
tent with the results of the Swedish and West German cohort studies. The
smallest reasonably detectable relative risk (a = 0.05, 1 spraying
season).
There are also certain Inconsistencies 1n the data from the Finnish
study that the authors note but find difficult to explain. In particular,
no cancer deaths occurred during the latter part of the study period among
Forestry Authority workers (1 of 4 groups Included In the cohort), even
though 9.0 deaths were expected. This finding strongly suggests some defi-
ciency In follow-up or 1n the source records from which vital status was
determined.
In summary, four cohort studies of workers exposed to phenoxyacetlc add
herbicides and/or 2,3,7,8-TCDD do not report Increased risks of stomach can-
cer. Only one of these, however, was statistically powerful enough to be
Inconsistent with the two studies that tentatively suggest an Increase 1n
stomach cancer risk. The available report of this study of Finnish herbi-
cide applicators contains methodologlc questions that require clarification.
*The expected stomach cancer deaths were estimated 1n the same manner as for
the Midland study. A proportion of 20% of all cancer deaths was applied
because Finnish male mortality rates are known to be very high.
11-107
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11.2.3. Summary of Case Reports and Ep1dem1olog1c Studies. By adding
together the number of workers exposed to phenoxy adds and/or chlorophenols
from all case studies, an unusually high number of STSs Is shown, consider-
ing the rarity of the disease. This excess 1s suggestive of an association
of cancer with exposure to phenoxy adds and/or chlorophenols, and conse-
quently, with the Impurities found 1n these herbicides, Including 2,3,7,8-
TCDD.
Two Swedish case-control studies report highly significant association
of STS with exposure to phenoxy add and/or chlorophenols. They do not pin-
point the risk to the dloxln contaminants, however. In fact, In one study,
the risk was found to extend to phenoxy acids free of dloxln Impurities. In
that study, the risk Increases to 17 when phenoxy adds known to contain
dloxln Impurities (polychlorlnated dlbenzodloxlns and dlbenzofurans) are
considered. The extent of possible observer bias and recall bias Introduced
Into these studies by using self-administered questionnaires 1s not of
sufficient magnitude to have produced the highly significant risks found In
the studies.
Later studies did not reveal a significant excess risk of STS. However,
methodology problems make these latter studies limited with respect to
evaluating the risk of STSs from exposure to phenoxy acids and/or chloro-
phenols and, consequently, 2,3,7,8-TCDD.
The Swedish case-control studies provide limited evidence for the
carclnogenldty of phenoxy adds and/or chlorophenols In humans. However,
with respect to the dloxln Impurities contained therein, the evidence for
the human carclnogenldty for 2,3,7,8-TCDD based on the epidemlologic
studies 1s only suggestive because of the difficulty of evaluating the risk
of 2,3,7,8-TCDD exposure 1n the presence of the confounding effects of
phenoxy adds and/or chlorophenol.
n-108
-------
There 1s less evidence Incriminating 2,4,5-T and/or 2,3,7,8-TCDD as the
cause of malignant lymphoma and stomach cancer 1n humans,
11.3. QUANTITATIVE ESTIMATION OF RISKS OF EXPOSURE TO 2,3,7,8-TCDD AND
HxCDOs
11.3.1. Introduction. This quantitative section deals with the Incre-
mental unit risk from exposure to 2,3,7,8-TCDD and HxCDDs by Inhalation and
oral routes, and their potencies relative to other carcinogens that the CA6
has evaluated. The Incremental unit risk estimate for an air pollutant
present 1n such small quantities as the dloxlns 1s defined as the Increased
lifetime cancer risk occurring to an Individual exposed continuously from
birth throughout lifetime to an air concentration of 1 pg/m3 of the agent.
The unit risk from oral exposure 1s similarly defined 1n terms of either
ng/kg bw/day or 1n terms of ng/a water. These calculations are done to
estimate 1n quantitative terms the Impact of the agent as a carcinogen.
Unit risk estimates are used for two purposes: 1) to compare the carcino-
genic potency of several agents with each other and 2) to give a crude
Indication of the population risk that might be associated with known (or
anticipated) air or water exposure to these agents.
The Incremental unit risks for both the Inhalation and oral routes will
be estimated from animal oral bloassays, since there are no animal Inhala-
tion studies, and none of the epidemiology studies provides sufficient expo-
sure Information for extrapolation purposes. The anlmal-to-man extrapola-
tions for the oral route will assume equivalent absorption In both species.
However, the unit risk for the ambient air concentration of 2,3,7,8-TCDD
must be considered 1n terms of both Us physical properties and Its sources.
It does not occur naturally but 1s emitted In small amounts from sources
Including the production of 2,4,5-T, trlchlorophenol, sllvex and hexachloro-
phene; the application of 2,3,7,8-TCDD-contam1nated herbicides or wood
11-109
-------
preservatives; the burning of municipal waste, wood and PCBs; and, possibly,
dust from 2,3,7,8-TCDD-contam1nated soil.
/
Physically, 2,3,7,8-TCDD has a very low vapor pressure and 1s not
normally airborne. At room temperature It 1s a crystalline solid, melting
at 305°C. When 2,3,7,8-TCDD 1s present 1n air, 1t 1s likely to be attached
to partlculates, to which 1t strongly binds. It has been measured 1n air
only 1n the vicinity of burning processes and 1n dust from contaminated
soil, and has not been found In the general air environment.
11.3.2. Procedures for the Determination of Incremental Unit Risk from
Animal Data and Description of the Low-Dose Animal Extrapolation Model.
Following 1s an abbreviated description of the procedures used 1n animal-to-
man extrapolation. A more complete description 1s given 1n Anderson et al.
(1983).
In the development of quantitative estimates of carcinogenic risk from
lifetime animal studies 1t 1s assumed, unless evidence exists to the
contrary, that If a carcinogenic response occurs at the dose levels used 1n
the study, then responses will also occur at all lower doses with an
Incidence determined by the dose as Indicated by the extrapolation model.
While both TCDD and HxCDD cause cancer 1n animals at lower doses than any
other known or suspect carcinogen, environmental levels are also extremely
low. Thus, an extrapolation methodology must be employed.
There 1s no solid scientific basis for any mathematical extrapolation
model that relates carcinogen exposure to cancer risks at the extremely low
concentrations that must be dealt with In evaluating environmental hazards.
Such low levels of risk cannot be measured directly either by animal experi-
ments or by ep1dem1olog1c studies.
11-110
-------
In the absence of any strongly suggestive evidence to the contrary for
TCDD or HxCDD, the linear nonthreshold model has been adopted as the primary
basis for risk extrapolation 1n the low-dose region of the dose-response
relationship. The risk estimates made with this model should be regarded as
conservative, representing the most plausible upper limit for the risk;
I.e., the true risk 1s not likely to be higher than the estimate, but 1t
could be lower.
The mathematical formulation chosen to describe the linear nonthreshold
dose-response relationship at low doses 1s the linearized multistage model.
It 1s called the linearized model because the procedure determines a linear
function, q ,*, consistent with the observed data 1n a statistical sense.
Thus, the multistage model procedure employs enough arbitrary constants to
be able to fit almost any monotonlcally Increasing dose-response data, and
then 1t Incorporates a procedure for estimating the largest possible linear
slope (1n the 95% upper confidence limit sense) at low extrapolated doses
that 1s consistent with the data at all dose levels of the experiment. The
multistage model has the form
P(d) = 1 - exp [-(q0 * q-id + q2d2 + ... + q^d )]
where
q1 > 0, 1 = 0, 1, 2, .... k
and P(d) = the lifetime risk (probability) of cancer at dose d.
Equlvalently,
P,(d) = 1 - exp [(q,d + q?d2 + ... + q,,dk)]
I* It. l\
where
- P(0)
1 - P{0)
1s the extra risk over background rate at dose d. The estimate q * Is the
95% upper-limit on q, at lower doses. A more complete description of the
model 1s given In Appendix B.
11-111
-------
11.3.3. Selection of Data. For some chemicals, several studies 1n
different animal species, strains and sexes, each run at several doses and
different routes of exposure may be available. A choice must be made as to
which of the data sets from several studies to use 1n the model. The proce-
dures used 1n evaluating these data are consistent with the approach of
making a maximum-likely risk estimate. They are listed below as follows:
1. The tumor Incidence data are separated according to organ sites
or tumor types. The set of data (I.e., dose and tumor Inci-
dence) used 1n the model Is the set where the Incidence 1s
statistically significantly higher than the control for at least
one test dose level and/or where the tumor Incidence rate shows
a statistically significant trend with respect to dose level.
The data set that gives the highest estimate of the lifetime
carcinogenic risk, q-j*, 1s selected 1n most cases. However,
efforts are made to exclude data sets that appear to have pro-
duced spuriously high risk estimates because of a small number
of animals. That 1s, 1f two sets of data show a similar dose-
response relationship, and one has a very small sample size, the
set of data having the larger sample size 1s selected for calcu-
lating the carcinogenic potency.
2, If there are two or more data sets of comparable size that are
Identical with respect to species, strain, sex and tumor sites,
the geometric mean of q-j*, estimated from each of these data
sets, 1s used for risk assessment.
In some cases one or more of these studies may be negative, but
the 95% upper limit q^* will still be greater than zero.
3. If two or more significantly Increased tumor sites are observed
1n the same study, and 1f the data are available, the number of
animals with at least one of the specific tumor sites under
consideration 1s used as Incidence data In the model. Alterna-
tively, the total number of significant tumors may also be used
1n some cases,
11.3.4. Calculation of Human Equivalent Dosages for An1mal-to-Man Extrapo-
lation. It 1s appropriate to correct for metabolism differences between
species and absorption factors through different routes of administration.
Following the suggestion of Mantel and Schnelderman (1977), 1t 1s
assumed that mg/surface area/day provides an equivalent dose between
species. To a close approximation, since the surface area 1s proportional
11-112
-------
to the 2/3 power of the weight, as would be the case for a perfect sphere,
the exposure 1n mg/day per 2/3 power of the weight 1s also considered to be
equivalent exposure. In an animal experiment, this equivalent dose 1s
computed 1n the following manner.
Let
Le = duration of experiment
le = duration of exposure
m = average dose/day 1n mg during administration of the agent
(I.e., during le) and
W = average weight of the experimental animal
Then, the lifetime average exposure 1s
d = 1e X "*
Le x W2/3
A more expanded discussion Is given 1n Anderson et al. (1983).
11.3.5. Alternative Methodological Approaches. The methods used by the
CAG for quantitative assessment are consistently conservative, I.e., tending
toward high estimates of risk. The most Important part of the methodology
contributing to this conservatism In this respect 1s the linear nonthreshold
extrapolation model. There are a variety of other extrapolation models that
could be used, most of which would give lower risk estimates. These alter-
native models have not been used by the CAG 1n the following analysis, but
three are Included for comparison 1n the appendix. The models presented
there are the one-hit, problt and Welbull models. The CAG feels that with
the limited data available from these animal bloassays, most of which are
conducted at high dosage levels, almost nothing Is known about the true
shape of the dose response curve at low environmental levels. The position
1s taken by the CAi that the risk estimates obtained by use of the linear
nonthreshold model are upper limits, and the true risk could be lower.
n-ns
-------
Another modification of the method described here Involves the choice of
the specific animal bloassay as the basis for extrapolation. The present
approach 1s to use the most sensitive responder. Alternatively, the average
responses of all of the adequately tested bloassay animals could be used,
and then some confidence limits placed on this estimate.
Extrapolations from animals to humans could also be done on the basis of
relative weights rather than surface areas. The latter approach, used here,
has more basis 1n human pharmacological responses; 1t 1s not clear which of
the two approaches Is more appropriate for carcinogens. In the absence of
Information on this point, 1t seems appropriate to use the most generally
accepted method, which also 1s more conservative. In the case of 2,3,7,8-
TCDD and HxCDD gavage studies, the use of extrapolation based on surface
area rather than weights Increases the Incremental unit risk estimates by a
factor of 5.8 for rats and about 13 for mice.
11,3.6. Interpretation of Quantitative Estimates. The Incremental unit
risk estimate based on animal bloassays 1s an approximation to the excess
risk 1n populations exposed to known carcinogen concentrations. This 1s
because there may be Important species differences 1n uptake, metabolism and
organ distribution of carcinogens, as well as species differences 1n target
site susceptibility, 1mmunolog1cal responses, hormone function, and dietary
factors and other diseases. The concept of equivalent doses for humans
compared with animals on a mg/surface area basis has Uttle experimental
verification regarding carcinogenic response. Human populations are more
variable than laboratory animals with respect to genetic constitution and
diet, living environment, activity patterns and other cultural factors.
11-114
-------
The unit risk estimate can give an Indication of the relative response
per unit dose ("potency") of a given agent compared with other carcinogens.
The comparative potency of different agents should be more reliable when the
comparison 1s based on studies 1n the same test species, strain and sex, and
by the same route of exposure.
The quantitative aspect of the carcinogen risk assessment 1s Included
here because 1t may be of use 1n the regulatory decision-making process, for
example, setting regulatory priorities and evaluating the adequacy of
technology-based controls. However, the estimation of cancer risks to
humans at low levels of exposure 1s uncertain. At best, the linear extrapo-
lation model used here provides a rough but plausible estimate of the upper
limit of risk; I.e., It Is not likely that the true risk would be much more
than the estimated risk, but It could very well be considerably lower. The
risk estimates presented 1n subsequent sections should not be regarded as an
accurate representation of the true cancer risks even when the exposures are
accurately defined. The estimates presented may be factored Into regulatory
decisions to the extent that the concept of upper risk limits 1s found to be
useful.
11.3.7. Incremental Unit Risk Estimates for 2,3,7,8-TCDD via the Oral and
Inhalation Routes. The positive animal cancer data available for calculat-
ing an Incremental unit risk estimate for 2,3,7,8-TCDD are presented 1n
Appendix B In Tables B-l through B-5. These are as follows:
1. The Dow (1978) diet study on Sprague-Daw!ey rats, Spartan
substraln. Significantly Increased cancers In the males Includ-
ed stratified squamous cell carcinomas of the tongue and squa-
mous cell carcinomas of the nasal turblnates and hard palate.
Both the original pathological analysis (Koclba) and that of an
Independent reviewer (Squire) are presented (Table B-l).
Significant cancers 1n the females Included lung, nasal turbl-
nate and hard palate cancers, and liver tumors (Table B-2). As
with the males, the total number of animals with at least one of
these significant tumors was recorded.
11-115
-------
2. The NCI gavage study In Osborne-Hendel rats and 86C3F1 mice.
a. 2,3,7,8-TCDD 1n male rats caused an Increase \n folUcular
cell adenomas and carcinomas combined of the thyroid. How-
ever, these tumors were not considered biologically signifi-
cant for risk assessment purposes. In females, the combined
neoplastlc nodules and hepatocellular carcinomas were consid-
ered significant (Table B-3), and these data were used. The
adrenal cortical adenomas or carcinomas were not considered
biologically significant.
b. 2,3,7,8-TCDD 1n male mice caused an Increase In hepatocellu-
lar carcinomas and 1n combined hepatocellular adenomas and
carcinomas (Table B-4). In female mice, 2,3,7,8-TCDD caused
an Increase 1n subcutaneous tissue Hbrosarcomas, lymphomas
or leukemlas of the hematopoletlc system, liver hepatocellu-
lar carcinomas and adenomas, and thyroid folUcular cell
adenomas (Table B-5).
The above data have been fit by the linearized multistage model
described 1n Section 11.3.2. These results are presented In Appendix B 1n
some detail *1n Tables B-6 through B-12, and summarized 1n Table 8-13. The
results of all estimates are within an order of magnitude, with the upper-
limit estimates lowest for the Dow male rats, higher for the NCI study, both
rats and mice, and highest for the combined tumor sites of the female rats
1n the Dow study. The data from which the steepest slope factor (q-,*)
(I.e., greatest potency) was calculated were from the Squire review of the
slides. A summary of Squire's review 1s presented 1n Table B-2 and the
results of the linearized multistage model extrapolation procedure are
presented 1n Table 6-9. An examination of Table B-9 shows that the high-
dose group 1n the study was eliminated because Us Inclusion resulted 1n a
poor fit of the model (p<0.01). A second analysis of the female rat data
adjusted for early Increased mortality 1n the high-dose group by eliminating
all animals that died during the first year, so that the first tumors con-
sidered were those detected during the 13th month of the study. The results
of the analysis from this adjustment are presented 1n Tables B-8A and B-9A.
11-116
-------
The results yield acceptable fits of the data without dropping the responses
at the highest dose levels, and these results were chosen for the final
Incremental unit risk estimates. The slope estimates for the Kodba (Table
B-8A) and Squire (Table B-9A) analyses, 1.51xlOs and 1.61xl05 (mg/kg/
day)"1, were averaged by taking the geometric mean, and the final estimate
thus becomes
q^ = [(1.51 x 10=) x (i.61 x io=)]1/2 = 1.56 x 10s (mg/kg/ day)'1.
This estimate 1s about one-third that derived from the Squire review 1n
Table B-8.
This upper-limit estimate represents a range of uncertainty that 1s
related as much to the fitting procedure as to the model Itself. The
dropping of the highest dose-response data and the resulting Increased 95%
upper-limit slope estimate based on the Squire analysis can be defended on
the basis that the highest dose data 1n this bloassay 1s 100 times that of
the lowest, and would therefore contain very little Information about the
shape of the dose-response curve at low dose levels. It could also be
argued on the basis of a saturation effect of either dose or response; the
data can partially support either hypothesis. An adjustment of the multi-
stage model needed to Incorporate such an effect or effects, however, 1s
felt to be unwarranted by the sparslty of the supporting evidence. As an
alternative, to Incorporate this uncertainty, a range of 95% upper-limit
estimates of q * = 9.0xl04 to 4.25xlOs (mg/kg/day)"* has been chosen
to accommodate this unusual data set.
In order to estimate an Incremental unit risk for a 1 ng/8, concentra-
tion In drinking water, the following conversion 1s used:
1 yg/kg/day x 70 kg x 103 ng/yg x 1 day/2 a = 3.5 x 10* ng/a.
11-117
-------
based on human consumption of 2 a water/day for a lifetime. Therefore,
the Incremental unit risk corresponding to 1 ng 2,3,7,8-TCDD/a water 1s
q!* = 1.56x10* (yg/kg/dayr1 x = 4 5xlo-3 (ng/i)-i
3.5xl04 ng/8,
Similarly, the lower and upper limits of the range vary from q * = 2.6xlO~a to
1.2xl(T2
This Incremental unit risk estimate from an oral study must be trans-
formed before an estimate can be made from exposure to 2,3,7,8-TCDD 1n the
ambient air. Exposure will be assumed to occur only through respiration of
2,3,7,8-TCDD-contam1nated participates. The amount of exposure depends on
the partlculate size distribution. Based on the report of the International
Commission on Radiological Protection (ICRP, 1959), 1t can be assumed that
100% of partlculates of <0.1 micron 1n size pass the nasopharyngeal (upper
respiratory tract) barrier and are deposited on the tracheobronchlal and
alveolar passages. For the larger-sized particles, the percentage deposi-
tion of 5-m1cron particles In the lower respiratory tract 1s not more than
30%. Even those larger particles retained by the upper respiratory tract,
however, may be swallowed and eventually absorbed by 1ngest1on. In the
absence of specific data on the size distribution and eventual fate of the
particles, the Information developed by the ICRP, Committee 2, will be used.
The Committee developed the following estimates for retention of partlculate
matter 1n the lungs. For compounds not readily soluble, 25% will be
exhaled, 5054 will be deposited 1n the upper respiratory passages and subse-
quently swallowed, and the final 25% will be deposited 1n the lungs (lower
respiratory passages). Of this final 25%, half 1s eliminated from the lungs
and swallowed 1n the first 24 hours, making a total of 62.5% swallowed; the
remaining 12.5% remains In the lung alveoli for long periods of time;
eventually some are transferred to pulmonary lymph nodes.
11-118
-------
If we take a worst-case estimate and assume that all of the swallowed
material 1s eventually absorbed Into the body, then 75% of the Inhaled mate-
rial will be absorbed. We further assume a breathing rate of 20 mVday
for a 70 kg man. Given these assumptions and the fact that one plcogram 1s
equal to 10~9 mg, the lifetime cancer risk for an ambient concentration of
1 pg/m3 of 2,3,7,8-TCOD Is 3.3 x 10~5, as calculated below;
q-|*(resp.) = 1.56 x 10s (mg/kg/day)"1 x 1 x 10~9 tng/pg x .75 x 20 ma/70 kg
or
q-|*(resp.) = 3.3 x 10~s (pg/m3)'1.
Slmllary, the range of estimates Is 1.9 x 10~s to 9.1 x 10~5 (pg/m3)'1.
11.3.8. Incremental Unit Risk Estimate for HxCODs (1,2,3,6,7,8 and
1,2,3,7,8,9) Via the Oral and Inhalation Routes. The results of the
National Toxicology Program (NTP) gavage study on a mixture of 1,2,3,6,7,8-
and 1,2,3,7,8,9-HxCDD showed positive results for male and female rats (com-
bined "liver neoplastlc nodules or hepatocellular carcinomas), the greater
response being !n the females. In the females, carcinomas appeared only 1n
the high-dose group. In the male rats, there was also a definite trend 1n
neoplastlc nodules and carcinomas combined, but this was only marginally
significant. These results are presented 1n Table 11-35, which Includes the
recent NTP reevaluatlon of the female rat liver slides. The review shows
responses 1n the range of 10% less than that of the original analysis. The
responses for neoplastlc nodules and combined nodules and carcinomas are
still statistically significant. These results have been detailed 1n the
qualitative section of this document.
11-119
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TABLE 11-35
NTP HxCDD (Gavage) Bloassay (NTP, 1980d)
Osborne-Hendel Rats {2 years)
Incidences of Neoplastlc Nodules and Hepatocellular Carcinomas
_J
1
rvj
O
Tumor
Number of animals examined
Hepatocellular carcinoma (HC)
Neoplastlc nodule (NN)
HC + NN combined
Human equivalent dose
Vehicle
Control
74
0
0
0
0
Untreated
Control
HALE
75
0
2(354)
2(354)
0
Low-Dose
1.25
(700 g)b
49
0
0
0
0.04
yg/kg/week
Mid-Dose
2.5
50
0
1(254)
1(254)
0.08
High-Dose Estimates3
5 of q-|*
(pg/kg/dayr*
48
1(254)
3(654) 5.6xlO~a
4(854)c 5.9X10"1
0.15
jig/kg/day
-------
TABLE 11-35 (cont.)
I
fSJ
Tumor
Number of animals examined
Hepatocellular carcinoma (HC)
Neoplastlc nodule (NN)
HC * NN combined
Human equivalent dose
tig/kg/day
Vehicle
Control
75
0
2(3%)
2(3%}
0
Untreated
Control
FEMALE
73
0
1(1%)
K1H)
0
Low-Dose
1.25
(450 g)d
50
0
5(10%)
5(10%)
0.03
wO/kg/week
Mid-Dose
2.5
50
0
7(14%)c
7{14%)c
0.06
High-Dose
5
50
2(4%)
16(32%)e
18(36%)e
0.12
Estimates3
of q-j*
(vg/kg/dayr1
—
3.2X10"1
3.3
3.5
—
a95% upper-limit estimate of linear term In the multistage model based on human equivalent dosages using
surface area correction.
Analysis by NTP (1980d)
cp<0.05 versus vehicle-control
Devaluation by Hlldebrandt (1983)
ep<0.001 versus vehicle-control
-------
In female mice, there was a dose-related trend In hepatocellular carci-
nomas, but only the combined adenomas and carcinomas were significant. In
male mice, there was a minor trend 1n hepatocellular adenomas, but no In-
crease, statistical or otherwise, 1n hepatocellular carcinomas (Table 11-36).
Although no statistically significant Increase In carcinomas occurred In
mice or rats of either sex, when neoplastlc nodules 1n the rats and hepato-
cellular adenomas In Ihe mice were Included 1n the data, the results became
significant for all groups. These combined results were then fitted to the
multistage model for all four groups. As shown In Tables 11-35 and 11-36,
the 95J4 upper-limit unit risk estimates are as follows:
Rat - male q-|* = 0.59 (vg/kg/dayr1
female q-|* = 3.5 (vg/kg/day)"1
House - male q-|* = 11.0
female qi* = 2.9 (vg/kg/day)"1
The usual CAG procedure Is to use the most sensitive sex-species for
estimating the 95% upper-limit unit risk. Under that procedure, which Is
based on the linearized multistage model with surface area correction for
animal-to-man extrapolation, the male mouse data base yielding a q * =
11.0 (vg/kg/day)"1 would be selected to provide the upper limit estimate
of potency. However, as examination of Tables 11-35 and 11-36 show, there
are several reasons to give weight to the female rat data base also. These
are as follows: 1) low spontaneous (control) rates In the rat vs. the male
mouse Hver; 2) statistically significant Increases 1n both the mid and high
level dose groups vs. control for the female rat; the male mouse response
was significant only at the high dose; 3) a more distinct dose response
trend 1n the female rat vs. the male mouse; and 4) the only hepatocellular
carcinomas 1n the female rat were In the high dose group. There were none
1n 148 control animals. By comparison, the male mouse showed no clear trend
In carcinomas.
11-122
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NTP HxCDD (Gavage) Bloassay (NTP, 1980d)
B6C3F1 Mice (104 weeks)
Incidences of Adenomas and Hepatocellular Carcinomas
r\3
CO
Tumor
Number of animals examined
Hepatocellular carcinoma
(HC)
Hepatocellular adenoma
(HA)
Combined HA and HC
Human equivalent dally
Vehicle
Control
73
8(11%)
7(1054)
15(21%)
0
Untreated
Control
75
12(16%)
15(20%)
27(36%)
0
Low-Dose
1.25
HALES
50
9(18%)
5(10%)
14(29%)
0.014
ug/kg/week
Mid-Dose
2.5
49
5(10%)
9(18%)
14(29%)
0.027
High-Dose
5
48
9(19%)
15(31%)b
24(50%)c
0.054
Estimates of q-|*a
(vg/kg/dayr*
—
3.71
6.99
11.00
_ —
dose (yg/kg/day)
-------
TABLE 11-36 (cent.)
Tumor
Number of animals examined
Hepatocellular carcinoma
(HC)
Hepatocellular adenoma
(HA)
Combined HA and HC
Human equivalent dally
dose (tig/kg/day)
Vehicle
Control
73
1(1%)
2(356)
3(4%)
0
Untreated
Control
74
0
2 (3%)
2(3%)
0
Lou-Dose
2.5
FEMALES
48
0
4(8%)
4(8%)
0.027
«q/k
-------
In addition to the above reasoning, we point to the uncertainty of the
surface area correction. Nearly all the quantitative Increase 1n the
estimate of the 95% upper limit risk of the male mouse vs. the female rat
(11.0/3.5 = 3.1) can be attributed to the surface area correction 1n the
extrapolation procedure, which 1s greater for mice than for rats by a faclor
of 2.5. The surface area correction 1s an assumption used 1n the HxCDD
analysis but neither supported nor contradicted by data.
Finally, for 2,3,7,8-TCDD, the female rat (different strain) has been
shown to be more sensitive than the mouse even with the surface area correc-
tion.
Based on the above qualifications, the CAG has decided to modify Us
procedure slightly and to take the geometric mean of the 95X upper-limit
estimates from the male mouse and the female rat. The final estimate 1s
q^ = (3.5xll.0)1/2 = 6.2 (vQ/kg/day)"1
In terms of exposure to 1 yg/fi. of HxCC contaminate and 2 8,/day for a
lifetime, we use the same assumptions as with 2,3,7,8-TCDD:
= 3.5x10* ng/l.
Thus, for 1 ng/l 1n the drinking water the estimate of Incremental risk 1s
-6.2/3.5x10-
p = ^
In terms of continuous lifetime exposure to ambient air containing 1 pg/rri3
HxCDD, the transformation as was done before with 2,3,7,8-TCDD, 1s
q.,*(HxCDD) (resp.) = 6.2xl03 (mg/kg/day)'1 x lxlO~9 mg/pg x 0.75x20 m'/70 kg
q.j*(HxCDD) (resp.) = 1.3 x 10~6 (pg/m3)'1.
11.3.9. Relative Potency. One of the uses of unit risk 1s to compare the
relative potencies of carcinogens. Potency 1s defined for this purpose as
the linear portion of the dose-response curve, which was used to calculate
the unit risk factors. To estimate the relative potency on a per-mole
11-125
-------
basis, the unit risk slope factor Is multiplied by the molecular weight, and
the resulting number 1s expressed 1n terms of (mMol/kg/day)'1. This Is
called the "relative potency Index."
Figure 11-2 Is a histogram representing the frequency distribution of
potency Indices of 55 chemicals evaluated by the CA6 as suspect carcinogens.
The actual data summarized by the histogram are presented 1n Table 11-37.
Where human data are available for a compound, they have been used to calcu-
late the Index. When no human data are available, animal oral studies have
been used In preference to animal Inhalation studies, since animal oral
studies have been conducted on the majority of these chemicals; this allows
potency comparisons by route.
The potency Index for 2,3,7,8-TCDD based on Hver, lung and nasal turbl-
nate and hard palate tumors In the female rat 1n the Dow 2,3,7,8-TCDD feed-
Ing study (Kodba et al. (1978a) 1s SxlO7 (mHol/kg/day)"1. This number
1s derived by multiplying as follows: the 9554 upper-limit slope estimate
from the Dow study using the geometric mean of the Squire and Kodba anal-
yses, q,* * 1.56xl05 (mg/kg/day)"1, by the molecular weight of 322.
Rounding off to the nearest order of magnitude gives a log 10 value of 8,
which Is the scale presented on the horizontal axis of Figure 11-2. The
Index of 5xl07 1s the most potent of 55 chemicals that the CAS has evalu-
ated as suspect carcinogens. It 1s 50 times more potent than the third most
potent chemical, bls(chloromethyl) ether, and 50,000,000 times as potent as
vinyl chloride. The potency Index of HxCDD, based on combined hepatocellu-
lar adenomas and carcinomas In male mice In the NTP gavage study (NTP,
1980d), and combined nodules and hepatocellular carcinomas In female rats by
gavage {NTP, 1980d) 1s 2.4xlO*6 (mHol/kg/dayF1. This 1s derived by
11-126
-------
4th 3rd 2nd 1st
QUARTlLf QUARTiLE QUARTIL1 QUARTiLE
I
1 MO'1
1 1
4 x 10*2 2 * 10*3
>
O
z
lu
O
UJ
20
18
16
14
10
6
4
2
0
-1
1234 5 C
LOG OF POTENCY INDEX
B
FIGURE 11-2
Histogram Representing the Frequency Distribution of the Potency Indices
of 55 Suspect Carcinogens Evaluated by the Carcinogen Assessment Group
11-127
-------
TABLE 11-37
Relative Carcinogenic Potencies Among 55 Chemicals Evaluated by the Carcinogen Assessment Group as Suspect Human Carcinogens
INJ
00
Compounds
Acrylonltrlle
Aflatoxln B-)
Aldrln
Allyl chloride
Arsenic
B[a]P
Benzene
Benzldene
Beryllium
1,3-Butadlene
Cadmium
Carbon tetrachlorlde
Chlordane
Chlorinated ethanes
1,2-Dlchloroethane
Hexachloroethane
1 ,1 ,2,2-Tetrachloroethane
1 ,1 ,2-Trlchloroethane
Chloroform
Chromium VI
DDT
Dlchlorobenzldlne
CAS Number
107-13-1
1162-65-8
309-00-2
107-05-1
7440-38-2
50-32-8
71-43-2
92-87-5
7440-41-7
106-99-0
7440-43-9
56-23-5
57-74-9
107-06-2
67-72-1
79-34-5
79-00-5
67-66-3
7440-47-3
50-29-3
91-94-1
Level of
Humans
L
L
I
S
I
S
S
L
I
L
I
I
I
I
I
I
I
S
I
I
Evidence3
Animals
S
S
L
I
S
S
S
S
S
S
S
L
S
L
L
L
S
S
S
S
Grouping
Based on
IARC
Criteria
2A
2A
3
1
28
1
1
2A
2B
2A
2B
3
2B
3
3
3
28
1
2B
2B
Slopeb
(mg/kg/day)-'
0.24 (W)
2900
11.4
1.19x10-'
15 (H)
11.5
2.9x10-' (H)
234 (W)
2.6 (W)
l.OxlO'i (I)
6.1 (W)
1.30X10"1
1.61
9.2x10-'
1.42x10-'
0.20
5.73x10-'
8.1x10-'
41 (H)
0.34
1.69
Molecular
Weight
53.1
312.3
369.4
76.5
149.8
252.3
78
184.2
9
54.1
112.4
153.8
409.8
98.9
236.7
167.9
133.4
119.4
100
354.5
253.1
Potency
Indext
IxlO*1
9x10*5
4x10*3
9x10-i
2x10*3
3x10*3
2x10°
4xlO*4
2x10*1
5x1 0»
7x10*2
2X10*1
7x10*2
9x10°
3x10°
3x1 O*1
8x10°
IxlOi
4x10*3
1x10*2
4x10*2
Order of
Magnitude
(logiQ Inde
.1
»6
»4
0
,3
*
0
,5
»1
*1
,3
»1
*3
0
»1
»4
»2
»3
-------
H-3/ (com.)
fSi
Compounds
1,1-Dlchloroethylene
(Vinyl Idene chloride)
Dlchloromethane
(Hethylene chloride)
Dleldrln
2,4-Dlnltrotoluene
D 1 pheny Ihydraz 1 ne
Eplchlorohydrln
B1s(2-chloroethy1)ether
B1s(ch1oromethy1)ether
Ethylene dl bromide (EDB)
Ethylene oxide
Heptachlor
Hexachlorobenzene
Hexachlorobutadldne
Hexachlorocyclohexane
technical grade
alpha Isomer
beta Isomer
gamma Isomer
Hexachlorodlbenzodloxln
1,2,3,6.7.8- and
1,2,3.7,8,9-
Nlckel refinery dust
Nickel subsulflde
CAS Number
75-35-4
75-09-2
60-57-1
121-14-2
122-66-7
106-89-8
111-44-4
542-88-1
106-93-4
75-21-8
76-44-8
118-74-1
87-68-3
319-84-6
319-85-7
58-89-9
34465-46-8
0120-35-722
Level of
Humans
I
1
I
I
I
I
I
S
I
L
I
I
I
I
I
I
I
S
S
Evidence
Animals
L
S
S
S
S
S
S
S
S
S
S
S
L
S
L
L
S
S
S
Grouping
Based on
I ARC
Criteria
3
?B
2B
2B
2B
2B
2B
1
2B
2A
2B
2B
3
2B
3
3
?B
1
1
Slopeb
(mg/kg/day)-'
1.16 (I)
1.4xlO'» (I)
30.4
0.31
0.77
9.9xlfl-»
1.14
9300 (I)
41
3.5X10"1 (I)
3.37
1.67
7.75xlO~»
4.75
11.12
1.84
1.33
6.2xlOf3
1.05 (H)
2.1 (M)
Molecular
Weight
97
84.9
380.9
182
180
92.5
143
115
187.9
44.1
373.3
284.4
261
290.9
290.9
290.9
290.9
391
240.2
240.2
Potency Order of
Indexc Magnitude
(logig lnde>
Ixl0f2 »2
1x10° 0
Ixl0f4 »4
6xlOf1 »2
Ixl0f2 »2
j
2xlOf2 *2
IxlO*6 »6
8x10*3 f4
2xlOf1 »1
Ixl0f3 »3
5xlOf2 »3
2xlOfl »1
Ixl0f3 f3
3xlOf3 »3
5xlOf2 »3
4xlOf2 »3
2x1 Of& »6
2.5xlOf2 »2
5.0xlOf2 v3
-------
TABLE 11-37 (cont.J
I
««J
o
Compounds
HHrosaiilnes
Dlnethylnltrasamlne
Dlethylnltrosamlne
Dlbutylnltrosamlne
N-nHrosopyrrol 1 dine
N-nltroso-N-ethylurea
H-nl troso-H-nethyl urea
N-n 1 troso-dl pheny lamlne
PCBs
Phenols
2,4,6-Trlchlerophenol
2,3,7,8-Tetrachlorodlbenzo-p-
dloxln (TCDD)
Tetrachloroethylene
Toxaphene
Trlchloroethylene
Vinyl chloride
CAS Number
62-75-9
55-18-5
924-16-3
930-55-2
759-73-9
684-93-5
86-30-6
1336-36-3
88-06-2
1746-01-6
127-18-4
8001-35-2
79-01-6
75-01-4
Level of
Humans
I
I
I
I
I
I
S
Evidence3
Animals
S
S
S
S
S
S
S
S
S
S
L
S
L/S
S
Grouping
Based on
1ARC
Criteria
28
28
2B
2B
2B
2B
2B
28
28
2B
3
28
3/2B
1
Slopeb
lug/kg/day}"*
25.9 (not by qj*)
43.5 (not by qi*)
5.43
2.13
32.9
302.6
4.92xlO"«
4.34
1 .99x10"*
1.56xlO*5
S.lxlO"'
1.13
1.1x10"'
1.7SxlO"» (I)
Molecular
Weight
74.1
102.1
158.2
100.2
117.1
103.1
198
324
197.4
322
165.8
414
131.4
62.5
Potency
Indexc
2x10*3
4xlO*3
9xlOfZ
2x10*2
4x10*3
3x10**
1x10°
1x10*3
4x10°
5x1 O*7
8x10°
5x10*2
1x10°
1x10°
Order of
Magnitude
{1og10 Index)
*3
f4
t3
t2
»4
M
0
f3
»1,
»8
»1
*3
0
0
aS = Sufficient evidence; L * Limited evidence; I = Inadequate evidence
''Animal slopes are 95X upper-bound slopes based on the linearized multistage model. They are calculated based on animal oral studies,
except for those Indicated by I (animal Inhalation), U (human occupational exposure) and H (human drinking water exposure). Human slopes are
point estimates based on the linear nonthreshold model. Not all of the carcinogenic potencies presented In this table represent the same
degree of certainty. All are subject to change as new evidence becomes available. The slope value Is an upper bound In the sense that the
true value (which Is unknown) Is not likely to exceed the upper bound and may be much lower, with a lower bound approaching zero. Thus, the
use of the slope estimate In risk evaluations requires an appreciation for the Implication of the upper bound concept as well as the "weight
of evidence* for the likelihood that the substance Is a human carcinogen.
cThe potency Index Is a rounded-off slope In (mmol/kg/day)"1 and 1s calculated by multiplying the slopes In (mg/kg/day}~* by the molecu-
lar weight of the conpound.
-------
multiplying the mean 95% upper-limit slope factor q.j* = 6.2x103 (mg/kg/
day)""1 by the molecular weight, 391. This potency Is about one-twentieth
that of 2,3,7,8-TCDD, making U the second most potent of 55 chemicals that
the CAG has evaluated as suspect carcinogens.
The ranking of relative potency Indices 1s subject to the uncertainties
Involved In comparing a number of potency estimates for different chemicals
based on varying routes of exposure In different species, using data from
studies whose quality varies widely. Furthermore, all the Indices are based
on estimates of low-dose risk using linear extrapolation from the observa-
tional range. These Indices are, therefore, not valid for the comparison of
potencies 1n the experimental or observation range If linearity does not
exist there. Nevertheless, the potency rankings of one and two for these
dloxlns cannot be easily dismissed.
11.4. SUMMARY AND CONCLUSIONS
11.4.1. Summary.
11.4.1.1. QUALITATIVE ASSESSMENT OF 2,3,7,8-TCDD —There are several
chronic animal cancer bloassay studies of 2,3,7,8-TCDD: 1) a Dow Chemical
Company (Kodba et a!., 1977, 1978a) study 1n male and female Sprague-Dawley
(Spartan substraln) rats; 2) the Van Miller et al. (1977a,b) study 1n male
Sprague-Dawley rats; 3) the Toth et al. (1979) study 1n Swiss mice; 4) the
National Toxicology Program (1980a,b) studies 1n rats and mice; 5) the P1tot
et al. (1980) promotion study 1n rats; and 6) the Kourl et al. (1978)
cocardnogenldty study 1n mice.
The 1978 study by the Dow Chemical Company of male and female Sprague-
Dawley rats fed 2,3,7,8-TCDD In doses of 22, 210 and 2200 ppt showed a
highly statistically significant excess of hepatocellular carcinomas 1n
female rats at the highest dose level and hepatocellular carcinomas and
11-131
-------
hepatocellular hyperplastlc nodules In female rats at both the middle and
high dose levels, as compared with the controls. In addition, at the high
dose there were significant Increases In carcinomas of the hard palate/nasal
turblnates 1n both males and females, of the tongue 1n males, and of the
lungs 1n females. The Van Miller et al. (1977a,b) study also showed some
evidence of a carcinogenic response 1n the liver and lungs of male Sprague-
Dawley rats at dosages of 1000 and 5000 ppt 1n the diet, even though the
study used a relatively small number of animals. The Toth et al. (1979)
study provides suggestive evidence that 2,3,7,8-TCDD Induced an Increased
Incidence of liver tumors 1n male mice (females were not tested) receiving
0.7 yg/kg/week by gavage.
In the National Cancer Institute rat study (NTP, 1980a), male and female
Osborne-Mendel rats were administered 2,3,7,8-TCDD by gavage at three dose
levels: 0.01, 0.05 and 0.5 vg/kg/week. 2,3,7,8-TCDD Induced statistic-
ally significant Increases of hepatocellular carcinomas, subcutaneous flbro-
sarcomas and adrenal cortical adenomas In high-dose female rats. 2,3,7,8-
TCDD also Induced significant Increases of thyroid tumors In male rats at
all dose levels.
In a companion mouse study by the National Cancer Institute (NTP,
1980a), male and female B6C3F1 mice were given 2,3,7,8-TCDD by gavage at
dose levels of 0.01, 0.05 and 0.5 vg/kg/week for males and 0.04, 0.2 and
2.0 ng/kg/week for females. 2,3,7,8-TCDD Induced statistically slgnlfl-
/'
cant Increases of hepatocellular carcinomas In the high-dose males and
females, and thyroid tumors, subcutaneous flbrosarcomas and hlstlocytlc
lymphomas 1n females.
In the study by Pltot et al. (1980), 2,3,7,8-TCDD has been shown to be a
potent liver cancer promoter after Initiation with d1ethyln1trosam1ne.
11-132
-------
Several tests of 2,3,7,8-TCDD as a promoter on mouse skin were negative, but
Poland et al. (1982) showed that 2,3,7,8-TCDD can promote In one mouse
strain. In the study by Kourl et al. (1978), 2,3,7,8-TCDD has been shown to
be a potent cocardnogen with 3-methyl chloranthrene.
2,3,7,8-TCDD 1s a potent Inducer of arylhydrocarbon hydroxylase (AHH) 1n
mammals. The AHH contains enzyme epoxldase that 1s known to mediate the
formation of epoxldes, that are potentially active carcinogenic metabolites.
2,3,7,8-TCDD may be metabolized 1n mammalian species by the reactive epoxlde
Intermediate to dlhydrodlol and further conjugated. 2,3,7,8-TCDD was found
1n Hver and fat at the end of the 2-year rat feeding study. Significant
covalent binding of 2,3,7,8-TCDD (14C or tritium) derived radioactivity
with protein has been demonstrated. Covalent binding of 2,3,7,8-TCDO (14C
or tritium) derived radioactivity with DNA 1s not significant In liver cells.
Currently available studies on the mutagenlclty of 2,3,7,8-TCDD are
Inconclusive. Two bacterial systems, Escherlchla coll and S. typhlmurlum
(without metabolic activation), exhibited positive mutagenlc activity.
However, 1n another study of Si, typhlmurlum (with and without metabolic
activation), the results were negative.
Several ep1dem1olog1cal studies have been conducted that are relevant to
the cardnogenlclty assessment of 2,3,7,8-TCDD. Two Swedish epidemlologic
case-control studies (Hardell and Sandstrom, 1979; Eriksson et al., 1979,
1981) reported a significant association between STSs and occupational
exposure to phenoxyacetlc add herbicides and/or chlorophenols that contain
2,3,7,8-TCDD as an Impurity. These studies Indicated ~5-fold to 7-fold
Increases 1n the risk of developing soft-tissue sarcomas among people
exposed only to phenoxyacetlc acids and/or chlorophenols In comparison with
people not exposed to these chemicals. The associations are high enough to
11-133
-------
make 1t unlikely that they have resulted entirely from random variation bias
or confounding, although the possibility exists that recall bias may account
for a small part of the excess; but not enough to account for the exces-
sively high risks. When an attempt was made to separate exposures Into two
categories based on expected presence or absence of polychlorlnated dlbenzo-
£-d1ox1n Impurities, the relative risks were 17 and 4.2, respectively. This
Indicates that agents themselves, without the dloxln Impurities, may be con-
tributing to the risk of STSs as well. The nonposltlve studies that seem-
ingly do not support the finding of an elevated risk of cancer, specifically
STS, suffer from a variety of methodological problems that will make such a
risk Impossible to detect In some and difficult to detect In others. Sev-
eral of these require many more years of follow-up before a significant
elevated risk of the relatively rare STS Is found. Within this group of
nonposltlve studies are several where evidence of exposure to 2,3,7,8-TCDD
Is questionable at best and as such no elevated risk of STS will ever be
found. On the other hand, several small-scale cohort studies with proven
evidence of exposure to chemicals containing 2,3,7,8-TCOD have produced a
small number of the relatively rare STS that certainly would not have been
expected at the time. However, several epldemlologlc studies are now In
progress, the results of which are not yet available, that will provide
additional epldemlologlc evidence that may Influence our conclusions at a
later time. Another Swedish case-control study (Harden et al., 1980, 1981}
provides suggestive evidence of an Increased risk of developing lymphomas
resulting from occupational exposure to phenoxyacetlc adds.
Two cohort studies, one by Axelson et al. {1980) and the other by Thless
and Frentzel-Beyme (1978) provide suggestive evidence that phenoxyacetlc
adds and/or 2,3,7,8-TCDD Increase the risk of stomach cancer In humans.
11-134
-------
Four other cohort studies by Ott et al. (1980). R11h1mak1 et al. (1978),
Cbok et al. (1980) and Zack and Susklnd (1980) Indicated no significantly
Increased risk of stomach cancer 1n people exposed to phenoxyacetlc acids
and/or chlorophenols, but two of these studies were of relatively low
statistical power, and another study has certain Inconsistencies requiring
clarification.
11.4.1.2. QUALITATIVE ASSESSMENT OF HxCDD — Hexachlorod1benzo-£-
dloxln has been tested for cardnogenldty 1n rats and mice by gavage (NTP,
1980d) and by dermal application to mice (NTP, 1980b,c). In these studies,
a 1:2 mixture of 1,2,3,6,7,8- and 1,2,3,7,8,9-HxCDD was tested. In the oral
study, animals received HxCDD at doses of 0.0, 1.25, 2.5 or 5.0 yg/kg/
week, except for female mice, which received 0.0, 2.5, 5.0 and 10.0 yg/kg/
week. In both species and both sexes, only tumors of the liver occurred at
a significantly greater Incidence than 1n controls. In male rats and male
and female mice, the liver tumor Incidence was significantly Increased over
control values only 1n the high-dose groups, while 1n female rats the Inci-
dence was significantly greater at both the medium- and high-dose levels.
At the request of EPA this study was audited during May-August 1985 by
several scientists as to the pathologic evaluation and conduct of the study.
The scientists have reconfirmed the NTP conclusions that the study provides
carcinogenic evidence 1n both rats and mice. In the study of HxCDD carclno-
genldty 1n mouse skin conducted by NTP (1980c), there were no treatment-
related tumors In either the carclnogenlclty bloassay or the tumor promotion
assay using DMBA as an Initiator. There are no available epidemlologic
carclnogenlclty studies 1n the published literature for HxCDD as the sole
compound of concern. The mutagenlc potential for HxCDD 1s unknown since no
tests are reported 1n the available literature.
11-135
-------
11.4.1.3. QUANTITATIVE ASSESSMENT OF 2,3,7,8-TGDD AND HxCDD — Quanti-
tative estimates of the potential carcinogenic Impact on humans, due to both
oral and Inhalation exposure to both 2,3,7,8-TCDD and HxCDD, have been
calculated. These estimates are all based on animal-to-human extrapolation
procedures. The animal gavage and feeding studies provide the only data
base for estimating the carcinogenic potency (unit risk) for 2,3,7,8-TCDD
and HxCDD. While the epidemiology studies provide positive, although
limited, evidence for carclnogenldty, the population exposures are unknown
and the findings cannot be attributed to exposure to 2,3,7,8-TCDD alone.
Thus the 1ngest1on unit risks as well as the estimates for Inhalation unit
risk are derived from the gavage and feeding studies.
There 1s Insufficient metabolism and pharmacoklnetlc Information to
alter the typically used assumptions regarding dose extrapolation. The
reported 1ntragastr1c absorption for 2,3,7,8-TCDD 1n rats varies from
52-8654; there are no absorption data for HxCDD. The assumptions used 1n
both the TCDD and HxCDD unit risk estimates assume that human absorption by
oral exposure 1s equal to that of the rat. Information regarding absorption
by Inhalation 1s totally lacking and 1s assumed to be 75% based on an ICRP
(1959) lung uptake model. The upper limit unit risks were calculated using
a multistage extrapolation model that 1s linear at low doses as programmed
1n GLOBAL 79.
For cancer risk due to oral exposures, the upper-limit quantitative
Incremental unit risk estimate Is q,* = l.S&xlO"1 (ng/kg/day)"1,
derived from the Koclba et al. (1977, 1978a) 2,3,7,8-TCDD feeding study 1n
female rats that Induced a statistically significant Increased Incidence of
tumors 1n the liver, lungs, hard palate and nasal turblnates. Based on
continuous lifetime exposure to 1 ng/8, 2,3,7,8-TCDD 1n drinking water, the
11-136
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95% upper limit estimate of Individual Incremental cancer risk 1s 4.5xl0~9
with a range of upper limit values of 2.6xlO~3 to 1.2xlQ~2, depending
upon pathological Interpretation and mortality correction. Based on contin-
uous lifetime exposure to 1 pg/m3 2,3,7,8-TCDD 1n ambient air, the 95%
Upper-limit estimate of Individual Incremental cancer risk 1s 3.3xlO~s,
with a range of upper-limit estimates of 1.9xlO~5 to 9.1xlO~5 depending
upon pathologic Interpretation and mortality correction. Since the Inhala-
tion unit risk .values are based upon the observed Incidence 1n the feeding
study, an Implicit assumption 1s made that 2,3,7,8-TCDD 1s as potent by
Inhalation as by 1ngest1on exposure.
An upper-limit Incremental unit risk estimate for a mixture of HxCDDs
has been calculated from the NCI gavage study (NTP, 1980d). Based on com-
bined liver heptacellular carcinomas and neoplastlc nodules 1n female rats,
and hepatocellular adenomas and carcinomas In male mice, q,* = 6.2xlO~3
(ng/kg/day)"1. A continuous lifetime exposure to 1 ng/fc of HxCDD 1n
drinking water 1s estimated to result 1n an upper limit Incremental unit
risk of 1.8xlO~4. Similarly, for ambient air, a continuous lifetime
exposure to 1 pg/m3 of HxCDD 1s estimated to yield an upper-limit unit
risk of 1.3xl(r6.
The cancer potency of 2,3,7,8-TCDD as represented by a potency Index 1s
also estimated relative to 54 other chemicals which the CAG has evaluated as
carcinogens. The relative potency Index 1s 5xlQ? (mMol/kg/day)"1,
making 2,3,7,8-TCDD the most potent animal carcinogen evaluated by the CAG.
It 1s about 50 times more potent than the third most potent chemical, b1s-
(chloromethyl)ether and ~50,000,OOQ times more potent than vinyl chloride.
The relative potency Index for HxCDD 1s 2xl06 {mHo1/kg/day)"*, making 1t
the second most potent carcinogen, about one-twentieth the low dose potency
of 2,3,7,8-TCDD.
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11.4.2. Conclusions. There 1s evidence from chronic animal cancer bio-
assay studies that 2,3,7,8-TCDD and HxCOD are probable human carcinogens.
There are no chronic animal cancer bloassay studies available that evaluate
the carcinogenic potential for other polychlorlnated d1benzo-p-d1ox1n com-
pounds. The available data for 2,3,7,8-TCDD and HxCDD come from gavage and
feeding studies, there being no studies available for Inhalation exposure.
The ep1dem1olog1c evidence for the cardnogenlcHy of 2,3,7,8-TCDD alone 1s
Inadequate, and there have been no ep1dem1olog1c studies, as yet, for HxCDD
as the sole compound of concern.
2,3,7,8-TCDD has Induced hepatocellular carcinomas In two strains of
female rats and both sexes of one mouse strain, along with the Induction of
thyroid tumors, subcutaneous flbrosarcomas and tumors of the lung, nasal
turblnates/hard palate 1n male rats, and tongue tumors 1n female rats.
These effects notably occur at extremely low doses. There 1s evidence that
2,3,7,8-TCDD Is also a promoter and a cocardnogen. The evidence of
carc1nogen1c1ty for 2,3,7,8-TCDD 1n animals Is regarded as "sufficient"
using the EPA Interim we1ght-of-ev1dence classification system for carcino-
gens (U.S. EPA, 1984).
The human evidence for the carc1nogen1c1ty of 2,3,7,8-TCDD alone 1s
regarded as "Inadequate" using the EPA classification criteria, because of
the difficulty of attributing the observed effects solely to the presence of
2,3,7,8-TCDD that occurs as an Impurity 1n the phenoxyacetlc acids and
chlorophenols. However, the human evidence for the cardnogenlcHy of
chlorinated phenoxy acetic herbicides and/or chlorophenols with chlorinated
d1benzod1ox1n Impurities 1s judged to be "limited" according to the EPA
criteria.
11-138
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The overall evidence for cardnogenldty, considering both animal and
human studies, would place 2,3,7,8-TCDO alone In the B2 category of EPA's
classification scheme, and 2,3,7,8-TCOO 1n association with the phenoxy
herbicides and/or chlorophenols 1n the Bl category. Chemicals 1n category B
are regarded as being "probably" carcinogenic 1n humans.
The EPA has, 1n the past, used an IARC we1ght-of-ev1dence classification
scheme for evaluating cardnogenldty data. Using IARC classification
criteria, the positive evidence In the rat and mouse studies, together with
Inadequate evidence 1n humans for 2,3,7,8-TCDD alone, 1s equivalent to an
IARC 2B category, meaning that 2,3,7,8-TCDD 1s "probably" carcinogenic 1n
humans. However, the overall welght-of-evidence for 2,3,7,8-TCDD In combi-
nation with chlorinated phenoxyacetlc add herbicides and/or chlorophenols
would be classified as IARC 2A, meaning that chlorophenoxyacetlc add and/or
chlorophenols containing 2,3,7,8-TCDD are "probably" carcinogenic 1n humans.
Hepatocellular tumors have been Induced 1n mice and rats of both sexes
following administration of a 1:2 mixture of 1,2,3,6,7,8- and 1,2,3,7,8,9-
HxCDD. This level of carcinogenic evidence 1n animals would be regarded as
"sufficient" according to the EPA classification scheme. Based on animal
evidence and the lack of ep1dem1olog1c data,.HxCDD would be placed 1n EPA's
B2 category, which characterizes HxCDD as "probably" carcinogenic 1n humans.
Using the IARC classification scheme, based on animal evidence and no
epidemiology data, HxCDD would be considered to be 1n a 2B category meaning
that HxCOD 1s "probably" carcinogenic 1n humans.
Assuming that 2,3,7,8-TCDD and HxCDO are carcinogenic 1n humans, upper
bound Incremental unit cancer risks have been estimated for both 1ngest1on
and Inhalation exposure. The development of these unit risk estimates 1s
for the purpose of evaluating the magnitude of the possible health Impact
11-139
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from exposure to these compounds. The upper bound nature of these risk
estimates 1s such that the true risk 1s not likely to be exceeded and may be
lower.
Using the data from a feeding study with female rats, the cancer potency
(unit risk per mg/kg/day) for 2,3,7,8-TCDD 1s 1.56X1CT1 (ng/kg/day)"1.
The upper limit estimate of Incremental cancer risk Is 4.5xlO~3 for a
continuous lifetime exposure to 1 ng/8, of 2,3,7,8-TCDD 1n drinking water.
The upper limit estimate of Incremental cancer risk Is 3.3xlO~5 for a
continuous lifetime exposure to 1 pg/m3 of 2,3,7,8-TCDD 1n ambient air.
Using data from a gavage study with female rats and male mice the cancer
potency for HxCDD 1s 6.2xlO~3 (ng/kg/day)""1. The upper limit estimate
of Incremental cancer risk Is 1.8xlO~4 for a lifetime exposure to 1 ng/i
of HxCDD 1n drinking water. For ambient air a lifetime exposure to 1
pg/m3 of HxCDD 1s estimated to have an upper limit risk of 1.3xlO~6.
In terms of low dose response, 2,3,7,8-TCDD and the 1:2 mixture of
1,2,3,6,7,8- and 1,2,3,7,8,9-HxCDD rank as the most potent and second most
potent, respectively, carcinogens evaluated by EPA's CA6.
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12. SYNERGISH AND ANTAGONISM
The Interactions of 2,3,7,8-TCDD with other toxic substances are pre-
dominately mediated through Its potent enzyme Induction. 2,3,7,8-TCDD pre-
treatment significantly alters the metabolism of many other compounds,
resulting 1n either potentlatlon or Inhibition of their biological effects.
12.1. CHEMICAL CARCINOGENS
Synerg1st1c and antagonistic activities of 2,3,7,8-TCDD with chemical
carcinogens have been discussed 1n depth 1n Chapter 11 of this document.
12.2. NONCARCINOGENIC CHEMICALS
2,3,7,8-TCDD pretreatment has been observed to modify the effects of
anesthetics (Grelg, 1972). Adult male Porten rats were given a single oral
dose of 200 vg 2,3,7,8-TCDD/kg bw 1-3 days preceding treatment with 100
mg/kg zoxazolamlne hydrochlorlde or 150 mg/kg hexabarbltone sodium.
2,3,7,8-TCDD pretreatment resulted 1n a 54% decrease 1n the duration of the
paralysis Induced by zoxazolamlne and a 2-fold Increase In the sleeping time
produced by hexabarbltone. A recent report compares the 1iwnunotox1c1ty of
2,3,7,8-TCDD, 2,3,7,8-TCDF and 2,3,7,8-TCDF plus 2,3,7,8-TCDD (coadmln-
Istered) (R1zzard1n1 et al., 1983). Seven days after administration of 1.2
vg/kg of 2,3,7,8-TCDD to C57B1/6J mice, sheep red blood cells were
Injected 1ntraper1toneally and plaque-forming cells (RFC) 1n the spleen were
counted 5 days later. 2,3,7,8-TCDD Inhibited antibody production by 80%.
In a parallel study, a dose of 2,3,7,8-TCDF was administered (10 jig/kg)
and no significant 1mmunotox1c effects were observed. Coadm1n1strat1on of
2,3,7,8-TCDD (1.2 ug/kg) plus 2,3,7,8-TCDF (10 vg/kg) resulted 1n 50%
reduction 1n antibody production and demonstrates a significant antagonistic
effect by 2,3,7,8-TCDF. Coadm1n1strat1on of these two Isostereomers
resulted In antagonistic effects with respect to the Induction of hepatic
12-1
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mlcrosomal eytochrome P-450 and 7-ethoxycoumar1n 0-deethylase. Sweeney et
al. (1979) found that Iron deficiency protected mice against the development
of hepatocellular damage (Including porphyrla) normally caused by 2,3,7,8-
TCDO exposure.
12.3. SUMMARY
Exposure to 2,3,7,8-TCDD has been observed to alter the biological
response of many species to some compounds. This altered response 1s pre-
sumed to be the result of altered enzyme activities In tissue 1n which
2,3,7,8-TCDD exerts an Inductive effect (vide ante, see Section 8.1.1.5.},
although other mechanisms are possible (see Section 8.3.).
2,3,7,8-TCDD pretreatment Increases the conversion of some chemical car-
cinogens to mutagens by hepatic S-9 preparations 1n in. vitro test systems;
however, exposure to 2,3,7,8-TCDD often has an antlcardnogenlc effect jji
vivo (see Section 11.1.1.1.). This antlcardnogenlc effect may be the
result of Increased detoxification or an Increased cytotoxldty following
Increased production of metabolites. 2,3,7,8-TCDD pretreatment has the
potential of altering the biological effects of many compounds that are not
chemical carcinogens. This modification may reduce the effectiveness, as 1n
the case of zoxazolamlne, or Increase the effectiveness, as 1n the case of
hexabarbltone (Grelg, 1972). The direction and extent of the alteration
depends both on the effect of 2,3,7,8-TCDD on the particular enzyme system
Involved and on whether metabolism Is an activating or deactivating process.
12-2
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13. REGULATIONS AND STANDARDS
13.1. WATER
Previous release of PCDD-conta1n1ng herbicides has been one mechanism by
which these agents enter the environment. Their high environmental stabil-
ity and low water solubility (0.2 ppb) make the 2,3,7,8- TCDD tend to settle
1n the bottom sludge of waterways. The major risk to humans comes from
eating bottom-feeding fish 1n which 2,3,7,8-TCDD has bloaccumulated. The
U.S. EPA has set criteria of 1.3xlO~*, 1.3xlO~» or l.SxlO"9 yg
2,3,7,8-TCDD/l based on estimated human lifetime cancer risks of 10~5,
1(T6 and 10~7, respectively. These criteria are based on the assumption
of a dally consumption of 6.5 g contaminated fish and shellfish with the
additional dally consumption of 2 ft of contaminated drinking water (U.S.
EPA, 1984). No Information 1s available regarding concentration limits of
1,2,3,7,8-PeCDD, 1,2,3,7,8,9-HxCDD or 1,2,3,6,7,8- HxCDD 1n ambient water.
13.2. AIR
Many normal combustion processes are suspected of releasing dloxlns to
the atmosphere. However, the effect on human health from this source 1s
unknown, and no criteria exist regarding concentration limits.
13.3. FOOD
According to the FDA (Cordle, 1981, 1983; FDA, 1981, 1983) and the Code
of Federal Regulations (41 CFR 321), fish with a 2,3,7,8-TCDD content
averaging <25 ppt pose no serious health concern. Federal legal limits for
Great Lakes fish distributed 1n Interstate commerce are deemed unnecessary
because most of the samples analyzed by the FDA contained <25 ppt. Canada
has established a 20 ppt concentration limit for 2,3,7,8-TCDD 1n Lake
Ontario commercial fish Imported Into the United States to comply with the
levels believed by the FDA to be safe (NRCC, 1981a).
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A tolerance for hexachlorophene methylenebls (2,3,6-trlchlorophenol) 1n
or on feedstock cottenseeds has been established at 0.05 ppm, with the con-
dition that It not contain >0.1 ppm of 2,3,7,8-TCDD (U.S. EPA, 1982c).
No Information regarding concentration limits of other dloxln Isomers 1s
available.
13.4. SUMMARY
The regulation of dloxln by-products In substances such as chlorophenols
and 2,4»5-tr1chlorophenoxyacet1c acid 1s apparently expected to eliminate
dloxln releases to the environment. The Canadian concentration limit for
2,3,7,8-TCDD 1n fish 1s the only known criterion, and 1t agrees with levels
regarded by the FDA as being protective of human health. In the absence of
specific guidelines and standards regarding concentration limits of 2,3,7,8-
TCDD, the FDA examines Individual contamination situations separately, and
gives only general guidance regarding relative risk to humans (Delgado,
1983). No Information 1s available regarding concentration limits for other
PCDDs.
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14. EFFECTS OF MAJOR CONCERN AND HEALTH HAZARD ASSISSHENT
Of the four congeners of PCDDs discussed In this report (2,3,7,8-TCDD,
1,2,3,7,8-PeCDD, 1,2,3,7,8,9- and 1,2,3,6,7,8-HxCDD), the majority of toxl-
cologlc data are on 2,3,7,8-TCDD, The limited data on the other congeners
Indicate that they are qualitatively similar 1n their toxic action to
2,3,7,8-TCDD when comparisons are made 1n a single species; however, they
are less toxic than the 2,3,7,8-TCDD congener. This 1s Illustrated 1n mice,
1n which 2,3,7,8-TCDD has an LDgo value of 0.88 ymol/kg and 1,2,3,7,8-
PeCDD; 1,2,3,6,7,8- and 1,2,3,7,8,9-HxCDD have LDgo values of 0.94, 3.19
and 3.67 jimol/kf, respectively (McConnell et al., 1978b). This suggests
that either the position or the number of chlorine effects the toxldty of
the PCDDs.
In more recent studies using biochemical endpolnts, Poland et al.
(1979), Bradlaw and Casterllne (1979) and Bradlaw et al. (1980) supported
the contention that the position and number of chlorines on TCDD, PeCDD and
HxCDD are critical for the biologic activity of the compound. In this
study, the ED5Q for the Induction of AHH activity 1n hepatoma cells 1n
culture was used to establish a range of potency for congeners of PCDDs.
Although acute toxldty and Induction of AHH activity have been used to
quantify the difference 1n the biologic activity of the congeners 2,3,7,8-
TCDD, 1,2,3,7,8-PeCDD and 1,2,3,7,8,9-HxCDD, the extrapolation of this data
to estimate quantitative dose-response relationships for the chronic tox-
ldty of Individual congeners 1s not sufficiently supported at the" present
time. From the following data described, 1t 1s clear that sufficient Infor-
mation for quantitative hazard assessment 1s available only for 2,3,7,8-TCDD
and a mixture of the two HxCDD congeners.
14-1
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14.1. PRINCIPAL EFFECTS
14.1.1. Toxldty. The principal effect observed 1n all species after
acute exposure to 2,3,7,8-TCDD 1s weight loss and thymlc atrophy (see Table
8-1). The decrease 1n weight proceeds over a protracted length of time even
after a single exposure to a lethal dose. By the time of death, an almost
complete absence of body fat stores was often observed. At death, severe
deterioration of the animal was observed; however, there was no specific
lesion to associate with the cause of death. This was particularly evident
1n the guinea pig, the most sensitive species to 2,3,7,8-TCDD toxlclty.
Necropsy revealed no remarkable alteration 1n any Internal organ except for
thymlc atrophy (Gupta et a!., 1973). Although liver damage was observed 1n
rats, rabbits and mice (Schwetz et al., 1973), there are Insufficient data
to Indicate that this effect 1s the underlying cause of mortality after
acute exposure to 2,3,7,8-TCOD. Also, 1n the guinea pig and monkey, which
have the same general progression of gross signs of toxlclty as do rats,
rabbits and mice, there 1s only mild liver damage (see Section 8.1.). In
addition, 2,3,7,8-TCDD 1s an Immunosuppressant 1n mice (see Section
8.1.1.4.).
As a result of the long time necessary for the development of toxic
symptoms In animals, subchronlc and chronic studies are better able to
define dose and effect relationships than are acute studies. Subchronlc and
chronic animal studies that define NOELs and LOELs are summarized 1n Table
14-1 for orally administered 2,3,7,8-TCDD. The NOEL for subchronlc exposure
1s ~10 times higher than that observed for chronic exposures, suggesting
that the cumulative dose might be an Important factor In 2,3,7,8-TCDD
toxlclty. There are only limited data on the NOEL and LOEL for HxCDO
14-2
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TABLE 14-1
No-Observed-Effect Levels and Low-Observed-Effect Levels Obtained from Subchronlc and
Chronic Oral Toxlclty Studies of 2,3,7,8-TCDD
I
co
wQ/kg/day
Species/Strain
Rat/Sprague-Dawley
Rat/Osborne-Mendel
Ra t/Spr ague-Dawl ey
Rat/Sprague-Dawley
M1ce/B6C3Fl
Monkey/Rhesus
Rat/Sprague-Dawley
Rat/Osborne-Mendel
Mice/Swiss
NOEL
0.01
0.07
0.0014
ND
ND
ND
0.001
0.0014
ND
LOEL
0.1
0.14
0.014
0.014
0.014
<0.02
0.01
0.007
0.001
Duration
of
Exposure
13 weeks
13 weeks
16 weeks
28 weeks
13 weeks
36 weeks
104 weeks
104 weeks
52 weeks
Duration
of Study
26 weeks
13 weeks
40 weeks
40 weeks
13 weeks
52 weeks
104 weeks
107 weeks
life
Reported Effect
decreased bw
toxic hepatitis
elevated porphyrln
levels
fatty changes In
the liver,
decreased bw
toxic hepatitis
pancytopenla
degenerative and
necrotlc changes
1n the liver
toxic hepatitis
dermatitis and
amyloldosls
Reference
Koclba et al.
NTP, 1980a
Goldstein et
1982b
King and
Roesler, 1974
NTP, 1980a
Allen et al..
Koclba et al.
1978a, 1979
NTP, 1980a
Toth et al.,
, 1976
al.,
1977
»
1979
ND = Not determined
-------
(Table 14-2) and these were obtained from studies using a 1:2 mixture of
1,2,3,6,7,8- and 1,2,3,7,8,9-HxCDD. As observed with 2,3,7,8-TCDD, there 1s
a suggestion that the cumulative dose of this mixture 1s an Important
consideration 1n defining a NOEL. For both 2,3,7,8-TCDD and the mixture of
HxCDD, the liver appeared to be a target organ.
2,3,7,8-TCDD has been shown to produce fetal anomalies 1n rats, mice,
rabbits, ferrets and chickens (see Table 9-2). In mice fetuses, 2,3,7,8-
TCDD Induces cleft palate and kidney malformations, while 1n rat fetuses,
hemorrhage, edema and a number of anomalies were observed. There was only
one study available assessing the teratogenldty of 2,3,7,8-TCDD 1n rabbits
reported by 61av1n1 et al. (1982b) 1n which Increases 1n extra ribs and
total soft-tissue anomalies were observed. In mice, 1 yg/kg/day given for
9-10 days during the middle of gestation was the minimum dose necessary to
elicit a teratogenlc response (Smith et al., 1976; Moore et al., 1973),
while dilated renal pelvis and decreased fetal weight were observed 1n the
rat fetuses of dams receiving doses of 2,3,7,8-TCDD as low as 0.001
vg/kg/day throughout gestation. The statistical and biological signifi-
cance of effects at this later dose, however, 1s argued (Murray et al.,
1979; Nlsbet and Paxton, 1982; U.S. EPA, 1979c). The fetuses of rats appear
to be very sensitive to the effects of 2,3,7,8-TCDD, with adverse effects
occurring at maternal exposures that were similar to the NOEL observed 1n
chronic studies (see Table 14-1). Also, Schwetz et al. (1973) demonstrated
that HxCDD (Isomers not specified) was both fetotoxlc and teratogenlc when
administered to pregnant rats at 100 jig/kg on days 6-15 of gestation.
Some epidemiology studies have shown a positive association between
exposure to 2,4,5-T, of which 2,3,7,8-TCDD 1s a known contaminant, and birth
14-4
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TABLE 14-2
No-Observed-Effect Levels and Low-Observed-Effect Levels
Obtained from Subchronlc and Chronic Oral Toxldty Studies of HxCDDa»b
Species/Strain
Rat/Osborne-Mendel
M1ce/B6C3Fl
Rat/Osborne-Hendel
M1ce/B6C3Fl
vg/kg/day
MOEL LOEL
0.35
0.7
ND
ND
0.7
1.4
0.18
0.18
Duration
of
Exposure
13 weeks
13 weeks
104 weeks
104 weeks
Duration
of Study
13 weeks
13 weeks
107 weeks
107 weeks
Reported Effects
hepatotoxldty
hepatotoxlclty
toxic hepatitis
toxic hepatitis
aSource: NTP, 1980b
bThe HxCDD was a 1:2 mixture of 1,2,3,6,7,8- and 1,2,3,7,8,9-HxCDD.
ND = Not determined
14-5
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defects or abortions. Other studies have failed to demonstrate an associa-
tion (see Section 9.2.). These studies 1n humans can neither support nor
refute the animal teratogenldty data, since among many other difficulties
1n Interpreting human data the exposures were always mixed, and there were
Inadequate data concerning the levels of 2,3,7,8-TCDD to which the popula-
tions were exposed.
Animal studies also demonstrate that 2,3,7,8-TCDD 1s a carcinogen (see
Table 11-1). The limited studies by Van Miller et al. (1977a,b) and Toth et
al. (1978, 1979) Indicated that 2,3,7,8-TCDD caused a variety of tumors 1n
rats and mice, and the more Intensive studies by Kodba et al. (1978a) and
NTP (1980a) support these early findings. Also, paplllomas have been re-
ported 1n female mice after dermal application of 2,3,7,8-TCDD {NTP, 1980b),
and using the skin tumorlgenesls model, 1t has been shown that 2,3,7,8-TCDD
may affect the carcinogenic potential of other chemical carcinogens (see
Section 11.1.1.2.). Human exposure to 2,3,7,8-TCDD has resulted from
contamination of other polychlorlnated compounds with 2,3,7,8-TCDD (see
Section 11.1.3.).
A 1:2 mixture of 1,2,3,6,7,8- and 1,2,3,7,8,9-HxCDD also has been tested
for carclnogenlclty 1n rats and mice treated by gavage and by dermal appli-
cation 1n mice {NTP, 1980c,d). In both species, this mixture produced Hver
tumors when administered by gavage, while 1n the dermal study there was no
Increase 1n the Incidence of skin tumors.
Ep1dem1olog1cal studies of workers exposed to chemicals contaminated
with 2,3,7,8-TCDD such as 2,4,5-trlchlorophenoxyacetlc add and 2,4,5-tr1-
chlorophenol are consistent with the position that 2,3,7,8-TCDD 1s probably
carcinogenic for humans; the available evidence Indicates an excess Inci-
dence of soft tissue sarcoma. Because 2,3,7,8-TCDD 1s almost always found
14-6
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Jn association with the materials (chlorophenols, combustion products, etc.)
It may never be possible to evaluate the cardnogenldty of 2,3,7,8-TCDD by
Itself In humans.
14.1.2. Hutagen1c1ty. There have been many studies of the mutagenlc
potential of 2,3,7,8-TCDD (see Chapter 10). in vitro assays using bacteria
and yeast have generally Indicated that 2,3,7,8-TCDD Is not a mutagen.
These negative results were obtained both In the presence and absence^of a
mammalian metabolic activation system. A few studies have reported positive
results (Hussaln et al., 1972; Seller, 1973; Bronzettl et al., 1980); how-
ever, these positive studies had deficiencies 1n either experimental design,
or were reported only qualitatively with Inadequate description of experi-
mental detail for evaluation. With the available data, 1t 1s Impossible to
assert whether or not 2,3,7,8-TCDD 1s devoid of mutagenlc potential. There
are also some conflicting data from humans and animal studies that Indicate
that 2,3,7,8-TCDD causes chromosomal aberrations. Because the human data
are derived from populations 1n which exposure to other biologically active
compounds 1s possible, and because the Increases observed In animal studies
were small, 1t Is stm not substantiated that 2,3,7,8-TCDO produces clasto-
genlc changes.
Pertinent data regarding the mutagenlc potential of 1,2,3,7,8-PeGDD,
1,2,3,7,8,9-HxCDD or 1,2,3,6,7,8-HxCDD could not be found 1n the available
literature.
14.2. SENSITIVE POPULATIONS
Although there are no data from human studies to Indicate the presence
of sensitive populations, the data from animal studies suggest that the
fetus and newborn may be at greater risk. Studies 1n chickens, rats, mice,
14-7
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rabbits, ferrets and monkeys have shown that jji utero exposure to 2,3,7,8-
TCDD can result In malformations, fetal toxldty and abortions (see Table
9-2). The lowest dose reported to adversely affect the fetus Jin. utero was
0.001 jjg/kg/day administered to the dams throughout gestation (from Hurray
et a!., 1979, according to Nlsbet and Paxton, 1982); this dose 1s similar to
the NOEL reported for chronic exposure of adult rats (see Table 14-1).
Hoore et al. (1973) observed that the nursing of pups on mothers exposed to
2,3,7,8-TCDD could also result 1n kidney anomalies detected at the time of
weaning. These data suggest that both the fetus and the newborn may be more
sensitive than the adult to the adverse effects of exposure to 2,3,7,8-TCDD.
In addition, 2,3,7,8-TCDD 1s known to be a powerful Inducer of the MFO
system. There 1s Information to Indicate that MFO Induction by 2,3,7,8-TCDD
can affect the biologic activity of other xenoblotlcs that require metabolic
activation (see Chapter 12). ScarpelH et al. (1980), for example, demon-
strated that pretreatment of hamsters with 2,3,7,8-TCDD resulted 1n greater
activation of mutagenlc nltrosamlnes when assayed jji vitro with Isolated
mlcrosomes. Individuals exposed to chemicals that are activated by the MFO
may experience a synerglstlc effect and be at greater risk. In a similar
manner, 1f the MFO detoxifies a xenoblotlc, pretreatment with 2,3,7,8-TCDD
may antagonize the action of other compounds.
14.3. FACTORS INFLUENCING HEALTH HAZARD ASSESSMENT
It 1s expected that the PCDDs discussed here would be highly persistent
compounds In the environment, and that human exposure may occur through
Ingestlon of contaminated food and water, by Inhalation of the compound
absorbed to resplrable partlculates, or through dermal contact. Although
potential exposure may occur by all routes, most of the toxlcologlc Informa-
tion 1s from studies of oral exposure. The limited observation of toxic
14-8
-------
effects 1n humans and animals after dermal contact with 2,3,7,8-TCDD \n
organic solvents Indicates that dermal absorption occurs. Polger and
Schlatter (I960) have shown In rats that both dermal and GI absorption Is
dependent on the vehicle. Greatest absorption after oral exposure occurred
when 2,3,7,8-TCDD was administered 1n organic solvent followed by aqueous
suspension, with little absorption occurring 1f the 2,3,7,8-TCDD was
adsorbed onto activated carbon. In a similar manner, dermal absorption was
poor 1f the 2,3,7,8-TCDD was applied 1n a soil and water paste. Inhalation
exposure 1s likely to occur through airborne partlculate matter containing
absorbed 2,3,7,8-TCDD; however, 1t 1s not possible with the available data
to predict how efficiently absorption will occur through the respiratory
tract. The use of standard respiratory absorption assumptions 1n risk
assessment are most likely to provide conservative criteria levels.
14.4. QUALITATIVE HEALTH HAZARD ASSESSHENT
The data available from animal studies are sufficient to provide some
assessment of the human health hazards associated with exposure to 2,3,7,8-
TCDD and a mixture of 1,2,3,7,8,9- and 1,2,3,6,7,8-HxCDD. The only data
available on 1,2,3,7,8-PeCDD are an acute LDKn value and studies of Indue-
DU
tlon of AHH activity. Although both types of data Indicate that 1,2,3,7,8-
PeCDD might have slightly less biological activity than 2,3,7,8-TCDD, the
data are Insufficient to adequately predict the risk associated with a
particular dose of 1,2,3,7,8-PeCDD. This would be the case 1f attempts were
made to use these data from acute exposure to extrapolate the effects of
chronic exposure whether these effects are toxic or carcinogenic. For! the
other PCDDs discussed, the hazard assessment can be based on toxldty,
teratogenldty or cardnogenlcHy.
14-9
-------
Although there have been human epidemiology studies Investigating the
toxic, reproductive and carcinogenic effect of exposure to 2,3,7,8-TCDD,
these studies have major deficiencies for use 1n health assessment.
2,3,7,8-TCDD 1s a contaminant of the chemicals 2,4,5-T and TCP, and all
human data are derived from populations exposed to mixtures. In these
studies, 1t 1s not possible to attribute with certainty any observed effect
to exposure to 2,3,7,8-TCDD. Also, exposure data of sufficient quality are
not available to define a dose-response relationship 1n human population.
Without adequate exposure data, health assessments cannot be made.
14.4.1. Animal Toxlclty Data. Animal studies that are useful for hazard
assessment are studies with adequate experimental design to define the
levels of exposure that produce threshold effects. Tables 14-1 and 14-2
summarize these studies, providing data on NOEL (or NOAEL) and LOEL (or
LOAEL). Since there Is suggestive evidence that the cumulative dose 1s
Important to the toxldty of 2,3,7,8-TCDD and the mixture of HxCDD tested,
the chronic toxldty studies would be more appropriately used for hazard
assessment. The NOEL from the two studies In rats (Koclba et al., 1978a,
1979; NTP, 1980a) are 0.001 and 0.0014 vg/kg/day; however, 1n the mouse
(NTP, 1980a), the dose of 0.07 vg/kg/day was a PEL, as Indicated by fatty
changes 1n the liver, and 0.007 was a NOEL.
In addition, 1t may be Inappropriate to derive a tox1c1ty-based hazard
assessment for 2,3,7,8-TCDD from these chronic studies, since a 3-generat1on
study by Hurray et al. (1979) Indicates that exposure of pregnant rats to
this dose of 2,3,7,8-TCDD (0.001 vg/kg/day) throughout gestation resulted
1n the observation of dilated renal pelvis 1n the fetuses. Hurray et al.
{1979) and U.S. EPA (1979c) consider this effect not to be treatment-related
because 1t occurred 1n only one generation at this dose and not at higher
14-10
-------
doses. Hence, 0.001 wg/kg/day represented a NOAIL. However, a reevalua-
tlon of these data by different statistical methods (Nlsbet and Paxton,
1982) Indicated a statistically significant Increase of dilated renal pelvis
at higher doses, as well as the lowest one, and lower fetal weight 1n the
0.001 yg/kg group. With these data, 0.001 yg/kg could be considered a
LOAEL. No other studies are available regarding the effects of 2,3,7,8-TCOD
at even lower doses.
A toxldty-based hazard assessment 1s also possible for the mixture of
HxCDD tested by NTP (1980b). As 1s shown 1n Table 14-2, however, the
description of the hlstologlc observations was not sufficiently detailed to
determine whether the low dose represented a NOAEL or a LOAEL. These data
could be used for hazard assessment 1n either case with an additional
uncertainty factor for a LOAEL (Federal Register, 1980b).
14.4.2. Animal Carc1nogen1c1ty. In addition to the Inadequate data base
for a toxldty-based hazard assessment, the strong evidence of cardnogenl-
clty In animals for 2,3,7,8-TCDD would justify a carc1nogen1c1ty-based
assessment. That two adequate cancer bloassays used sufficiently large
groups of animals exposed for an appreciable portion of their Hfespan
Indicates that 2,3,7,8-TCDD 1s an animal carcinogen (NTP, 1980a; Koclba et
a!., 1978a) {Table 14-3). In the NTP (1980a) study, male rats developed
folUcular-cell adenomas or carcinomas of the thyroid. Female rats and mice
of both sexes had Increased Incidences of folUcular-cell adenomas of the
thyroid. In the study by Koclba et al. (1978a), rats maintained on diets
that provided doses of 0.0, 0.001, 0.01 and 0.1 jig/kg/day had elevated
Incidences of carcinomas of the hard palate and tongue, and adenoma of the
adrenal cortex 1n males of the high dose group, and carcinomas of the liver,
tongue and lungs 1n females of the high-dose group. The evidence 1s suffi-
cient to Indicate that 2,3,7,8-TCDD 1s an animal carcinogen.
14-11
-------
TABLE 14-3
Carclnogenlclty Bloassays of 2,3,7,8-TCDD
Exposure
Route
Gavage
Gavage
Species/Strain Sex Dost or
Exposure
rats/ . H 0.0 tig/kg/week
Osborne-Hendel
0.01 yg/kg/week
0.05 ug/kg/week
0.5 jig/kg/week
rats/ F 0.0 vS/kg/week
Osborne-Hendel
0.1 yg/kg/week
0.05 tig/kg/week
O.S jig/kg/week
Duration
of
Treatment
104 weeks
104 weeks
104 weeks
104 weeks
104 weeks
104 weeks
104 weeks
104 weeks
Duration
of Study
105 weeks
107 weeks
107 weeks
107 weeks
105 weeks
107 weeks
107 weeks
107 weeks
Vehicle
corn oil-
acetone
19:1}
corn oil-
acetone
(9:1)
corn oil-
acetone
(9:1)
corn o11-
acetone
(9:1)
corn oil-
acetone
(9:1)
corn oil-
acetone
(9:1)
corn oil-
acetone
(9:1)
corn oil-
acetone
(9:1)
Tumor Type
foll1cu1ar-cell adenomas
or carcinoma of the
thyroid
folllcular-cell adenomas
or carcinoma of the
thyroid
folllcular-cell adenomas
or carcinoma of the
thyroid
folllcular-cell adenomas
or carcinoma of the
thyroid
neoplastlc nodule or
hepatocellular carcinoma
of the liver
neoplastlc nodule or
hepatocellular carcinoma
of the liver
neoplastlc nodule or
hepatocellular carcinoma
of the Hver
neoplastlc nodule or
hepatocellular carcinoma
of the liver
Tumor
Incidence
1/69
5/48
8/50
11/50
5/75
1/49
3/50
14/49
Reference
NTP, 1980a
NTP, 1980a
-------
TABLE 14-3 (cont.)
4*
i
Exposure Species/Strain
Route
Savage ro1ce/B6C3Fl
Savage m1ce/B6C3Fl
Oral rat/
Sprague-Dawley
Sex Dose or
Exposure
H 0.0 pg/kg/week
0.01 pg/kg/week
0.05 pg/kg/week
0.5 pg/kg/week
F 0.0 pg/kg/week
0.04 pg/kg/week
0.2 pg/kg/week
2.0 vg/kg/week
N 0.0 pg/kg/day
Duration
of
Treatment
104 weeks
104 weeks
104 weeks
104 weeks
104 weeks
104 weeks
104 weeks
104 weeks
105 weeks
Duration
of Study
105 weeks
107 weeks
10? weeks
10? weeks
105 weeks
107 weeks
107 weeks
10? weeks
105 weeks
Vehicle
corn oil-
acetone
(9:1)
corn oil-
acetone
(9:1)
corn oil-
acetone
corn oil-
acetone
(9:1)
corn oil-
acetone
(9:1)
corn oil-
acetone
corn oil-
acetone
(9:1)
corn oll-
acetone
19:1)
In diet
Tumor Type
hepatocellular carcinoma
hepatocellular carcinoma
hepatocellular carcinoma
hepatocellular carcinoma
hepatocellular carcinoma,
folllcular-cell adenomas
of the thyroid
hepatocellular carcinoma,
folllcular-cell adenomas
of the thyroid
hepatocellular carcinoma,
folllcular-cell adenomas
of the thyroid
hepatocellular carcinoma,
folllcular-cell adenomas
of the thyroid
squamous cell carcinoma
of the hard palate,
squamous cell carcinoma
of the tongue,
adenoma of the adrenal
Tumor
Incidence
8/73
9/49
8/49
17/50
1/73
0/69
2/50
3/50
2/48
1/47
6/4?
5/46
0/85
0/85
0/85
Reference
NTP, 1980a
NTP, 19801
Kodtu et
al.. 19781
cortex
-------
TABLE 14-3 (cont.)
Exposure
Route
Oral
(cont.)
Oral
Duration
Species/Strain Sex Dose or of Duration Vehicle
Exposure Treatment of Study
rat/ 0.001 pg/kg/day 105 weeks 105 weeks In diet
Sprague-Dawley
0.01 vg/kg/day 105 weeks 105 weeks In diet
0.1 vg/kg/day 105 weeks 105 weeks 1n diet
rat/ F 0.0 vg/kg/day 105 weeks 105 weeks In diet
Sprague-Dawley
0.001 pg/kg/day 105 weeks 105 weeks In diet
0.01 vg/kg/day 105 weeks 105 weeks In diet
0.1 vg/kg/day 105 weeks 105 weeks In diet
Tunor Type
squamous cell carcinoma
of the hard palate,
squamous cell carcinoma
of the tongue,
adenoma of the adrenal
cortex
squamous cell carcinoma
of the hard palate,
squamous cell carcinoma
of the tongue,
adenoma of the adrenal
cortex
squamous cell carcinoma
of the hard palate,
squamous cell carcinoma
of the tongue.
adenoma of the adrenal
cortex
hepatocellular carcinoma,
squamous cell carcinoma
of the hard palate.
squamous cell carcinoma
of the lung
hepatocellular carcinoma,
squamous cell carcinoma
of the hard palate.
squamous cell carcinoma
of the lung
hepatocellular carcinoma,
squamous cell carcinoma
of the hard palate,
squamous cell carcinoma
of the lung
hepatocellular carcinoma.
squamous cell carcinoma
of the hard palate.
squamous cell carcinoma
of the lung
Timor
Incidence
0/50
1/50
0/50
0/50
1/50
2/50
4/50
3/50
5/50
1/86
0/86
0/86
0/50
0/50
0/50
2/50
1/50
0/50
11/49
4/49
7/49
Reference
Koclba et
al., 1978a
Koclba et
a!., 1378a
-------
A single bloassay tested a mixture of the two congeners of HxCDD for
carclnogenldty (NTP, 1980b). The results summarized 1n Table 14-4 show
that male and female rats and mice exposed to this mixture of HxCDD had
Increased Incidences of neoplastlc nodules or carcinomas of the liver.
Increased Incidence of tumors 1n two species 1s sufficient to Indicate that
this mixture was carcinogenic to animals; however, caution 1s required 1n
Interpreting these data for hazard evaluation since the NTP (1980a) study
used a mixture containing two Isomers, 1,2,3,6,7,8- and 1,2,3,7,8,9-, of
HxCDD and the HxCDD mixture used for this bloassay was found to be contami-
nated with other PCDDs Including 0.09% (±0.03%) of TCDD. The specific
Isomer of PCDDs was not Identified. There 1s Insufficient evidence to
confirm whether both Isomers are Independently carcinogenic or whether only
one Isomer or this specific mixture 1s needed to elicit a carcinogenic
response. Since the position of the chlorines may be extremely Important
for the toxic/carcinogenic properties of HxCDD, Information obtained from
this combined exposure may not be applicable to the Individual congeners.
14-15
-------
TABLE 14-4
CareInogenlcity lloassays of a 1:2 Mixture of 1,2,3,6,7,8- and 1,2,3,7,8,9-HxCDD
Duration
Exposure Species/Strain Sex Dose or Exposure of Duration
Route Treatment of Study
Vehicle
Tumor Type
Tumor Reference
Incidence
4k
I
Gavage rats/
Osborne-Mendel
Gavage rats/
Osborne-Mendel
H 0.0 pg/kg/week
H 1.25 pg/kg/week
2.5 pg/kg/week
5.0 pg/kg/week
104 weeks 105 weeks corn oil-
acetone (9:1)
104 weeks 107 weeks corn oil-
acetone (9:1)
104 weeks 107 weeks corn oil-
acetone (9:1)
104 weeks 107 weeks corn oil-
acetone (9:1)
liver neoplastlc nodules
or hepatocellular
carcinoma
liver neoplastlc nodules
or hepatocellular
carcinoma
liver neoplastlc nodules
or hepatocellular
carcinoma
liver neoplastlc nodules
or hepatocellular
carcinoma
0/74 NTP, 19BOI
0/49 NTP, 19iOe
1/50
4/46
fiavage rats/
Osborne-Hendel
F 0.0 pg/kg/week
1.25 pg/kg/week
2.5 jig/kg/week
5.0 pg/kg/week
104 weeks 105 weeks corn oil-
acetone (9:1)
104 weeks 107 weeks corn oll-
acetone (9:1)
104 weeks 107 weeks corn oil-
acetone (9:1)
104 weeks 107 weeks corn oll-
acetone (9:1)
liver neoplastlc nodules
or hepatocellular
carcinoma
liver neoplastlc nodules
or hepatocellular
carcinoma
liver neoplastlc nodules
or hepatocellular
carcinoma
liver neoplastlc nodules
or hepatocellular
carcinoma
5/75 NTP, 1980C
10/50
12/50
30/50
-------
TABLE 14-4 (cont.)
Duration
Exposure Species/Strain Sex Dose or Exposure of Duration
Route Treatment of Study
Vehicle
Tumor Type
Tumor
Incidence
Reference
Gavage rats/
Osborne-Mendel
F 0.0 ug/kg/week 104 weeks 105 weeks corn oil- hepatocellular adenomas
acetone (9:1) or carcinomas
11/73 MTP, 1980d
1.25 ng/kg/week 104 weeks 108 weeks corn oil- 'hepatocellular adenomas 14/50
acetone (9:1) or carcinomas
4k
1
2.5 pg/kg/week 104 weeks 107 weeks corn oil- hepatocellular adenomas 14/49
acetone (9:1) or carcinomas
Gavage m1ce/86C3F1
5.0 |ig/kg/week
0.0 pg/kg/week
2.5 ng/kg/week
5.0 iig/kg/week
10.0 pg/kg/week
104 weeks 108 weeks corn oil-
acetone (9:1)
104 weeks 106 weeks corn oil-
acetone (9:1)
104 weeks 108 weeks corn oil-
acetone (9:1)
104 weeks 108 weeks corn oil-
acetone (9:1)
104 weeks 107 weeks corn oil-
acetone (9:1)
hepatocellular adenomas
or carcinomas
hepatocellular adenomas
or carcinomas
hepatocellular adenomas
or carcinomas
hepatocellular adenomas
or carcinomas
hepatocellular adenomas
or carcinomas
24/4B
3/75 DTP, 1980d
4/48
6/47
10/47
-------
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15-111
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APPENDIX A
A-l
-------
TABLE A-l
Cumulative Mortality of Hale Ratsa
Time
(end of 30-day period)
N=
1-7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
Controls
(86)
0.0
0.0
0.0
0.0
2.3
5.8
7.0
10.5
12.8
16.3
18.6
24.4
31.4
41.9
48.8
58.1
69.8
77.9
82.6
ug/kg/day
0.1
(50)
0.0
2.0
4.0
4.0
4.0
8.0
12.0
18.0
18.0
20.0
28.0
34.0
44.0
46.0
62. P
74.0b
78.0
84.0
90.0
2.3.7
0.01
(50)
0.0
0.0
0.0
0.0
0.0
0.0
0.0
4.0
14.0
22.0
28.0
34.0
46.0
54.0
68.0
76. Ob
84.0
88.0
92.0
.8-TCDD
0.001
(50)
2.0
2.0
2.0
2.0
2.0
2.0
2.0
4.0
14.0
14.0
24.0
44. Ob
iO.O
56.0
60.0
68.0
74.0
76.6
78.0
aSource: Koclba et a!., 1977
^Interval of greatest difference, D, 1n cumulative mortality curves of
controls and treatment group. None of the differences were statistically
significant (Kolmogorov-Smlrnov test, p>0.05).
A-2
-------
TABLE A-2
Cumulative Mortality of Female Rats3
Time
(end of 30-day period)
N»
0-5
6-8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
Controls
(86)
0.0
1.2
1.2
1.2
1.2
1.2
3.5
3.5
7.0
12.8
15.1
18.6
25.6
34.9
40.7
58.1
64.0
70.9
70.9
0.1
(50)
0.0
0.0
2.0
4.0
8.0
16.0
20.0
26.0
28.0
32.0
38.0
44.0
56.0
60.0
66.0
82.0
86.0
88.0
92.0
ua/kg/day 2,3,7.8-
0.01
(50)
0.0
0.0
0.0
2.0
2.0
4.0
4.0
8.0
12.0
18.0
18.0
20.0
b 30.0
36.0
46. Ob
60.0
66.0
72.0
72.0
-TCDD
0.001
(50)
0.0
0.0
0.0
0.0
0.0
4.0
4.0
6.0
10.0
12.0
18.0
22.0
34.0b
36.0
44.0
52.0
58.0
66.0
68.0
aSource: Koclba et al., 1977
^Interval of greatest difference, D, 1n cumulative mortality curves of
controls and treatment group. The mortality curve for the rats fed 0.1
wg/kg/day differed significantly from that for controls (0 = 30.4, p<0.01,
Kolmogorov-Smlrov test). The other two groups did not differ significantly
from, controls (p>0.05).
A-3
-------
TABLE A-3
Hales: Interval Mortality Rates
Control
Days
40-30
31-210
211-240
241-270
271-300
301-330
331-360
391-420
421-450
451-480
481-510
511-540
541-570
571-600
601-630
631-660
661-690
691-720
721-726
Terminal
Kill
d/1
0/86
0/86
0/86
0/86
0/86
2/86
3/84
3/80
2/77
3/75
2/72
5/70
6/65
9/59
6/50
8/44
10/36
7/26
4/19
15
Rate
0.000
0.000
0.000
0.000
0.000
0.023
0.036
0.038
0.026
0.040
0.028
0.071
0.092
0.153
0.120
0.182
0.278
0.269
0.211
0.1 uq/kq/dav
d/1
0/50
0/50
1/50
1/49
0/48
0/48
2/48
3/44
0/41
1/41
4/40
3/36
5/33
1/28
8/27
6/19
2/13
3/11
3/8
5
Rate
0.000
0.000
0.020
0.020
0.000
0.000
0.042
0.068
0.000
0.024
0.100
0.083
0.152
0.036
0.296
0.316
0.154
0.273
0.375
0.01 uq/kq/dav
d/1
0/50
0/50
0/50
0/50
0/50
0/50
0/50
2/50
5/48
4/43
3/39
3/36
6/33
4/27
7/23
4/16
4/12
2/8
2/6
4
Rate
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.040
0.104
0.093
0.077
0.083
0.182
0.148
0.304
0.250
0.333
0.250
0.333
0.001 uq/kq/dav
d/1
1/50
0/49
0/49
0/49
0/49
0/49
0/49
1/49
5/48
0/43
5/43
10/38
3/28
3/25
2/22
4/20
3/16
1/13
1/12
11
Rate
0.020
0.000
0.000
0.000
0.000
0.000
0.000
0.020
0.104
0.000
0.116
0.263
0.107
0.120
0.091
0.200
0.188
0.077
0.083
Corrected for continuity for combined Interval:
421-510 7/77 vs. 5/41(X2=0.04, n.s.) 12/48(X2= 4.63, p<0.05)
10/48 (Xa=2.54, n.s.
451-540 10/72 vs. 8/41(X2=0.37. n.s.) 10/43(X*=1.27, n.s.)
15/43 (X2=6.37, p<0.025)
481-570 13/72 vs. 12/40(X2=1.48, n.s.) 12/39(X2=1.67, n.s.)
18/43 (Xa=6.59, p<0.025)
511-600 20/70 vs. 9/36(X2=0.03, n.s.) 13/36(X2=0.32, n.s.)
16/38 (X2= 1/47, n.s.)
A-4
-------
TABLE A-4
Females: Interval Mortality Rates
Control
Days
0-150
151-180
181-240
241-270
271-300
301-330
331-360
361-390
391-420
421-450
451-480
481-510
511-540
541-570
571-600
601-630
631-660
661-690
691-720
721-726
Terminal
Kill
d/1
0/86
1/86
0/85
0/85
0/85
0/85
0/85
2/85
0/83
3/83
5/80
2/75
3/73
6/70
8/64
5/56
15/51
5/36
6/31
0/25
25
Rate
0.000
0.012
0.000
0.000
0.000
0.000
0.000
0.024
0.000
0.036
0.063
0.027
0.041
0.086
0.125
0.089
0.294
0.139
0.194
0.000
0.1 ud/kq/day
d/1
0/50
0/50
0/50
1/50
1/49
2/48
4/46
2/42
3/40
1/37
2/36
3/34
3/31
6/28
2/22
3/20
8/17
2/9
1/7
2/6
4
Rate
0.000
0.000
0.000
0.020
0.020
0.042
0.087
0.048
0.075
0.027
0.056
0.088
0.097
0.214
0.091
0.150
0.471
0.222
0.143
0.333
0.01 uq/kq/dav
d/1
0/50
0/50
0/50
0/50
1/50
0/49
1/49
0/48
2/48
2/46
3/44
0/41
1/41
5/40
3/35
5/32
7/27
3/20
3/17
0/14
14
Rate
0.000
0.000
0.000
0.000
0.020
0.000
0.020
0.000
0.042
0.044
0.068
0.000
0.024
0.125
0.086
0.156
0.259
0.150
0.177
0.000
0.001 uq/kq/dav
d/1
0/50
0/50
0/50
0/50
0/50
0/50
2/50
0/48
1/48
2/47
1/45
3/44
2/41
6/39
1/33
4/32
4/28
3/24
4/21
1/17
16
Rate
0.000
0.000
0.000
0.000
0.000
0.000
0.040
0.000
0.021
0.043
0.022
0.068
0.049
0.154
0.030
0.125
0.143
0.125
0.191
0.059
Corrected for continuity for combined Interval:
421-510 10/83 vs. 6/37(X2=l.131 n.s.) 5/46(X2=0.0, n.s.)
6/47 (X2=0.01, n.s.)
451-540 10/80 vs. 8/36(X2=l.13, n.s.) 4/44(X2=0.8, n.s.)
6/45 (X2=0.01, n.s.)
481-570 11/75 vs. 12/34(X2=4.80, p<0.05) 6/41(X2=0.0, n.s.)
11/44 (X2=1.34, n.s.)
510-600 17/73 vs. 11/31(X2=l.08, n.s.) 9/41(X2=0.0, n.s.)
9/41 (X2=0.0, n.s.)
A-5
-------
APPENDIX B
Tables for 2,3,7,8-TCDO Quantitative Incremental
Unit Cancer Risk Estimates
B-l
-------
Tables for 2,3,7,8-TCDD Quantitative Incremental
Unit Cancer Risk Estimates
Tables B-l through B-5 are the 2,3,7,8-TCDD bloassay results judged
suitable for quantitative estimates of Incremental unit risk. Tables B-l
and B-2 show the results of the Dow rat feeding study for both males and
females. The results Include both the original (Kodba) analysis and the
(Squire) review. Individual organ sites where significantly Increased
tumors occurred are tabulated separately, then the total number of animals
with at least one of these tumors 1s compiled. Tables B-3, B-4 and B-5
compile similar data for the NCI bloassay. Table B-6 uses the data from
Table B-l to estimate the parameters of the linearized multistage model.
The X2 test for goodness-of-f1t of the model to the data determines
whether or not the highest dose group 1s retained 1n the fit. The 95%
upper-limit on the linear term q,* 1s then adjusted by the surface area
a i
1/3
constant (70/W,) to derive the final extrapolated animal-to-human 95%
d
upper-limit Incremental unit cancer risk estimate. Tables B-7 through B-12
present the extrapolation procedure for the remaining data sets, with Tables
B-8A and B-9A adjusting for high early mortality 1n the female rat high-dose
group. Table B-13 summarizes the estimates derived 1n Tables B-6 through
B-12. The q,* estimates from the female rat data of the Dow feeding study
using both the Koclba and Squire readings are averaged to derive the final
estimate q-j* = l.S&xlO5 (mg/kg/day)5.
Description of the An1mal-to-Human Extrapolation Procedure Using the
Linearized Multistage Model
Let P(d) represent the lifetime risk (probability) of cancer at dose d.
The multistage model has the form
P(d) = 1 - exp [-(q0
B-2
-------
wfiere
q1 > 0, 1 . 0, 1, 2, .... k
Equlvalently,
Pt(d) = 1 - exp [(q.,d + q2d* + ... * qkdk)]
where
Pt(d) = P(d) - P(0)
1 - P(0)
1s the extra risk over background rate at dose d.
The point estimate of the coefficients q., 1 =0, 1, 2, ..., k. and
consequently, the extra risk function, Pt(d), at any given dose d, 1s
calculated by maximizing the likelihood function of the data.
The point estimate and the 95% upper confidence limit of the extra
risk, P*.(d), are calculated by using the computer program GLOBAL 79
developed by Crump and Watson (1979). At low doses, upper 95% confidence
limits on the extra risk and lower 95% confidence limits on the dose
producing a given risk are determined from a 9554 upper confidence limit,
q,* on parameter q,. Whenever q,>0, at low doses the extra risk
P.(d) has approximately the form Pt(d) = q..xd. Therefore, q,* x d
1s a 95% upper confidence limit on the extra risk and Pt/cu* 1s a 95%
lower confidence limit on the dose producing an extra risk of P.. Let
LQ be the maximum value of the log-likelihood function. The upper limit,
q,*, Is calculated by Increasing q, to a value q,* such that when the
log-likelihood 1s remaximlzed subject to this fixed value q,* for the
linear coefficient, the resulting maximum value of the log-likelihood L,
satisfies the equation
2 (LQ - L.J) = 2.70554
B-3
-------
where 2.70554 1s the cumulative 90% point of the ch1-square distribution
with one degree of freedom, which corresponds to a 95% upper limit (one-
sided). This approach of computing the upper confidence limit for the extra
risk, Pt(d)» 1s an Improvement on the Crump et al. (1977) model. The
upper confidence limit for the extra risk calculated at low doses 1s always
linear. This 1s conceptually consistent with the linear nonthreshold. The
slope, q,*, 1s taken as a plausible upper bound of the potency of the
chemical In Inducing cancer at low doses. (In the section calculating the
risk estimates, ?t(d) 1s abbreviated as P.)
In fitting the dose-response model, the number of terms 1n the poly-
nomial 1s chosen equal to (h-1), were h 1s the number of dose groups 1n the
experiment, Including the control group.
Whenever the multistage model does not fit the data sufficiently well,
data at the highest dose are deleted and the model Is refit to the rest of
the data. This Is continued until an acceptable fit to the data 1s ob-
tained. To determine whether or not a fit 1s acceptable, the ch1-square
h
1 = i
statistic 1s calculated where N, is the number of animals 1n the 1
dose group, R, 1s the number of animals 1n the 1 dose group with a
tumor response, P.. 1s the probability of a response 1n the 1 dose
group estimated by fitting the multistage model to the data, and h Is the
number of remaining groups. The fit Is determined to be unacceptable when-
ever X2 1s larger than the cumulative 99% point of the ch1-square dis-
tribution with f degrees of freedom, where f equals the number of dose
groups minus the number of nonzero multistage coefficients.
B-4
-------
TABLE B-l
CO
I
ui
DOW (Dr. Koclba) 2,3,7,8-TCDD Oral Rat Study (1978) with Dr. R. Squire's Review
Hale Sprague-Dawley Rats - Spartan Substraln (2 yrs)*
Dow
1.
2.
Tissue and Diagnosis 0
(control)
(Koclba) Analysis
Tongue
Stratified squamous cell 0/76 (0%)
carcinoma
Nasal turblnates/hard palate
Squamous cell carcinoma 0/51 (OX)
Total 0/76 (0%)
Dose Levels (pg/kg/day)
0.001 0.01
1/49 (2%) 1/49 (2X)
1/34 (3X) 0/27 (OX)
2/49 (4%) 1/49 (4%)
0.1
3/42 (7%)
(p=0.043)
4/30 (13X)
(p=0.016)
7/42 (17%)
(p=5. 12x10"*)
R. Squire's Review
1. Tongue
Squamous cell carcinoma
2. Nasal turblnates/hard palate
Squamous cell carcinoma
0/77 (OX)
0/55 (OX)
1/44 (2X)
1/34 (3X)
1/49 (2X)
0/26 (OX)
3/44 (7X)
(p=4.60x!0~*)
6/30 (20X)
(p=1.36x!0"a)
Total (1 or 2 above)
(each rat had at least
one tumor above)
0/77 (OX)
2/44 (5X)
1/49 (2X)
9/44 (20X)
(p=6.28x!0~5)
*Average body weight of male rat = 600 g
-------
TABLE B-2
DOH (Dr. Koclba) 2,3,7,8-TCDD Oral Rat Study (1978) with Dr. R. Squire's Review
Female Sprague-Daw!ey Rats - Spartan Substraln (2 yrs)*
Tissue and Diagnosis
(control)
Dose levels (yg/kg/day)
0.001
0.01
0.1
Dow (Koclba) Analysis
1. Lung
Keratinizing squamous
cell carcinoma
2. Nasal turblnates/hard palate
Stratified squamous cell
carcinoma (revised diagnoses
2/19/79)
3. Liver
Hepatocellular hyperplastlc
nodules/hepatocellular
carcinoma
0/86 (0%) 0/50 (0%)
1/54 (2%)
0/30 (0%)
9/86 (1054) 3/50 (6%)
0/49 (OX)
1/27 (4%)
18/50 (3654)
(2 had both)
(p=4.37x10"*)
7/49 (1454)
(p=6.21x10"*)
5/24 (2154)
(p=9.46x!0~3)
34/48 (7154)
(p=9.53x!0~ia)
Total (1, 2, or 3 above)
(each rat had at least one
tumor above)
9/86 (10%) 3/50 (654)
18/50 (3654)
(p=4.37x10'*)
34/49 (6954)
(p=2.13x!0"12)
-------
TABLE B-2 (cont.)
Tissue and Diagnosis
(control)
Dose Levels (ug/kq/day)
0.001
0.01
0.1
CO
I
R. Squire's Review
1. Lung
Squamous cell carcinoma
Nasal turblnate/hard palate
Squamous cell carcinoma
Liver
Neoplastlc nodules/hepato-
cellular carcinoma
0/86 (OK)
16/86 (0%)
0/50 (054)
0/54 (OX) 0/30 (OX)
8/50 (16%)
0/49 (OX)
1/27 (4X)
27/50 (54X)
(p=2.42x!0~s)
8/47 (17X)
(p=1.61x!0~«)
5/22 (23X)
(p=1.43x!0"3)
33/47 (70X)
(p=4.92xlO"9)
Total combined (1, 2 or 3 above)
(each animal had at least
one tumor above)
16/86 (19X) 8/50 (16%)
27/50 (54X)
(p=2.42x!0~5)
34/47 (72X)
(p=1.20x!0~9)
*Average body weight of female rat = 450 g
-------
TABLE B-3
NCI 2,3,7,8-TCDD (Gavage) Bloassay (No. 80-1765)
Osborne-Mendel Female Rats (2 years; weight = 450 g)
CD
05
Dose Levels { vq/kq/week )
Tissue and Diagnosis Vehicle Control
0
1. Liver
Neoplastlc nodule or 5/75 (1%)
hepatocellular carcinoma
2. Adrenal*
Cortical adenoma, or 11/73 (1554)
carcinoma
Lou
0.01
1/49 (2%)
9/49 (18X)
Medium
0.05
3/50 (6X)
5/49 (1054)
High
0.5
14/49 (28%)
(p^ 0.001)
14/46 (3054)
(p=0.038)
*The biological significance of this tumor In old rats Is questionable, since It Is commonly observed In
control rats and associated with the aging process.
-------
TABLE B-4
NCI 2,3,7,8-TCDD (Gavage) Bloassay (No. 80-1765)
B6C3F1 Hale Mice (2 years; weight = 48 g)
OD
I
(£>
Tissue and Diagnosis
Liver
Hepatocellular adenoma
or carcinoma
Hepatocellular carcinoma^
Vehicle Control
0
15,>73 (21%)
(p<0.001)a
8/73 (11%)
(p<0.001)a
Dose Levels
Low
0.01
12/49 (24%)
9/49 (18%)
(yq/kq/week)
Medium
0.05
13/49 (26%)
8/49 (16%)
High
0.5
27/50 (54%)
(p=1.31xlO~«)
17/50 (34%)
(p=0.002)
aCochran-Arm1tage test for linear trend
bUsed for Unit Risk Estimate
-------
TABLE B-5
NCI 2,3,7,8-TCDD (Gavage) Bloassay (No. 80-1765)
B6C3F1 Female Mice (2 years)3
Dose Levels ( uq/kq/week )
Tissue and Diagnosis
1. Subcutaneous tissue
Flbrosarcoma
2. Heroatopoletlc system
Lymphoma or leukemia
» 3. Liver
j:j Hepatocellular adenoma
or carcinoma
Hepatocellular carcinoma
4. Thyroid
Folllcular cell adenoma
Total (1, 2, 3 or 4 above)
(each mouse had at least
one tumor above)
Vehicle Control
0
1/74 (IX)
18/74 (24%)
3/73 (4%)
(p=0.0050)
1/73 (1%J
(p=0. 008)&
0/69
22/74 (30%)
Lou
0.04
1/50 (2%)
12/50 (24%)
6/50 (12%)
2/50 (4%)
3/50 (6%)
20/50 (40%)
Hedluni
0.2
1/48 (2%)
13/48 (27%)
6/48 (12%)
2/48 (4%)
1/47 (2%)
19/48 (40%)
High
2.0
5/47 (11%)
(p=0.032)
20/47 (43%)
(p=0.028)
11/47 (23%)
(p=l .84xlO-3)
6/47 (13%)
(p=0.014)
5/46 (11%)
(p=8.93xlO~3)
31/47 (66%)
(p=8.94x!0~5)
aAverage body weight of female mouse = 40 g
DCochran-Arm1tage test for trend
-------
TABLE B-6
Curve fit of the Multistage Model Parameters to Experimental Data by Study and Pathologist
Linear Parameter q^, Maximized to Give Upper 95% Limit q-j*
Compound 2,3,7,8-TCDD
Study Koclba - Dow
Sex-spedes Male rat
Height (wa) 600 g
Tumor sites (one or more) Tongue - squamous cell carcinomas
Nasal turblnates/hard palate - stratified squamous cell carcinoma
(ref. Table B-l)
Pathologist - Koclba
Exposure level (mg/kg/day) 0 1 x 10~* 1 x 10~s 1 x 10~*
DO tr/n 0/76 2/49 1/49 7/42
+r = number of animals with one or more of the tumors
n = total number of animals examined
Estimated Goodness of fit
multistage parameters q0 q-j q2 ^3 afll* *2
When all dose groups
are used 1.40 x 10~2 1.10 x 103 0 5.86 x 1010 3.01 x 103 3.34 (d.f. = 2)
When the highest dose
group Is not used Above fit 1s satisfactory
aq-|* = the maximum linear component from the model with adequate goodness of fit (p>0.01) = 3.01x10®
(mg/kg/day)"1
qi* = a^l* (70/wa)1/3 = 1.47x10* (mg/kg/day)"1, the upper 95% limit slope factor associated
with human dose response.
-------
TABLE B-7
Curve FH of the Hultl stage Hodel Parameters to Experimental Data by Study and Pathologist
Linear Parameter q-j, Maximized to Give Upper 9554 L1ra1t q-j*
*
Compound ..................... 2,3,7 ,8-TCDD
Study ........................ Dow
Sex -species ......... ; . . . . ---- Hale rat
Weight (wa) .................. 600 g /
Tumor sites (one or more).... Nasal turblnates/hard palate - squamous cell carcinoma
Tongue - squamous cell carcinoma (ref. Table B-l)
Pathologist - Squire
Exposure level (mg/kg/day) 0 1 x 10~* 1 x 10~5 1 x 10~*
+r/n 0/77 2/44 1/49 9/44
+r = number of animals with one or more of the tumors
n = total number of animals examined
Estimated Goodness of fit
multistage parameters qp q-j qg qa
When all dose groups
are used 0.015 1.05 x 103 0 109.40x10* 3.53 x 103 3.90 (d.f. =1)
When the highest dose
group 1s not used Above fit 1s satisfactory
aq-|* = the maximum linear component from the model with adequate goodness of fit (p>0.01) = 3.53xlOa
(mg/kg/day)*1
fll* = afll* (70/wa)l/3 = 1.73x10* (mg/kg/day)"1, the upper 95X limit slope factor associated
with human dose response.
-------
TABLE B-8
Curve Fit of the Multistage Model Parameters to Experimental Data by Study and Pathologist
Linear Parameter q^, Maximized to Give Upper 95% Limit q^*
Compound .2,3,7,8-TCDD
Study Dow
Sex-species Female rat
/Weight (wa) 450 g
Tumor sites (one or more)....Liver, lung, hard palate, or nasal turblnates (ref. Table B-2)
Pathologist - Koclba
Exposure level (mg/kg/day) 0 1 x 10~* 1 x 10"s
+r/n 9/86 3/50 18/50
1 x 10"
34/49
4
+r = number of animals with one or more of the tumors
n = total number of animals examined
Estimated
multistage parameters qp q-j q2 qs a^l*
When all dose groups 0.12 1.23 x 10* 0 0 1.67 x 10*
are used
When the highest dose Above fit Is satisfactory
group Is not used 0.09 0 3.5 x 109 0 4.69 x 10*
Goodness of
X2
6.67 (d.f. =
0.025 < p<0.
0.92 (d.f. =
p>0.25
fit
2)
05
1)
When the two highest dose
groups are not used Above fit Is satisfactory
afll* =
qi* =
the maximum linear component from the model with adequate goodness of fit (p>0.01) = 1.67x10*
- 4.69x10* (mg/kg/day)'1
aq-|* (70/wa)1/3 = 8.98x10* - 2.52xl05 (mg/kg/day)"1, the upper 95% limit slope
factor associated with human dose response depending on Inclusion or exclusion of the highest dose
data.
-------
TABLE B-8A
Curve FH of the Multistage Model Parameters to Experimental Data
by Study and Pathologist
Linear Parameter q-j, Maximized to 61ve Upper 95% Limit q-j*
Compound 2,3,7,8-TCDD
Study Dow
Sex-species Female rat
Height (wa) 450 g
Tumor sites (one or more) Liver, lung, hard palate, or nasal turblnates
(ref. Table B-2)
Pathologist - Kodba (Eliminating first year's data to adjust for high early
mortality 1n the high-dose group.)
Exposure level (mg/kg/day) 0 1 x 10~« 1 x 1Q~5 1 x 10~4
+r/n 9/85 3/48 18/48 34/40
+r = number of animals with one or more of the tumors
n « total number of animals examined
Estimated Goodness of fit
multistage parameters q0 q-j q2 qa
When all dose groups 0.11 2.08 x 104 0 0 2.82 x 10* 3.38 (d.f. = 2)
are used 0.25 < p < 0.10
When the highest dose
group 1s not used Above fit 1s satisfactory p > 0.25
aq-|* = the maximum linear component from the model with adequate good-
ness of fit (p>0.01) = 2.82xl04 ( mg/kg/day )"*
qi* « aqi* (70/wa)1/3 = 1.51xl05 (mg/kg/day)'1, the upper 95%
limit slope factor associated with human dose response.
B-14
-------
Curve Fit of the Multistage Model Parameters to Experimental Data by Study and Pathologist
Linear Parameter q-|, Maximized to Give Upper 95% Limit q-|*
Compound 2,3,7,8-TCDD
Study Koclba - Dow
Sex-species Female rat
Weight (wa) 450 g
Tumor sites (one or more) Liver, lung, hard palate, or nasal turblnates (ref. Table B-2)
Pathologist - Squire
Exposure level (mg/kg/day)
1 x 10'6
1 x 1(TS
1 x 10~«
+r/n
16/86
8/50
27/50
34/47
+r = number of animals with one or more of the tumors
n = total number of animals examined
*« Estimated
multistage parameters
0.01) = 7.90x10*
(mg/kg/day)'1
Ql* = a
-------
TABLE B-9A
Curve Fit of the Multistage Model Parameters to Experimental Data
by Study and Pathologist
Linear Parameter q-|, Maximized to Give Upper 95% Limit q-j*
Compound 2,3,7,8-TCDD
Study Kodba - Dow
Sex-species Female rat
Weight (wa) 450 g
Tumor sites (one or more)....Liver, lung, hard palate, or nasal turblnates
(ref. Table B-2)
Pathologist - Squire (Eliminating first year's data to adjust for high early
mortality 1n the high-dose group.)
Exposure level (mg/kg/day) 0 1 x 10~* 1 x 10~5 1 x 10~4
+r/n 16/85 8/48 27/48 34/40
+r = number of animals with one or more of the tumors
n = total number of animals examined
Estimated Goodness of fit
multistage parameters qg q-| qg q3
When all dose groups
are used 0.24 2.12 x 10* 0 0 3.00 x 10* 6.41 (d.f.= 2)
0.025 < p < 0.05
When the highest dose
group 1s not used Above fit 1s satisfactory
$•' .s
aq-|* = the maximum linear component from the model with adequate good-
ness of fit (p>0.01) = S.OOxlO4 (mg/kg/day)'1
qi* « a
-------
TABLE B-10
Curve Fit of the Multistage Model Parameters to Experimental Data by Study and Pathologist
Linear Parameter q-j, Maximized to Give Upper 95% Limit qi*
Compound 2,3,7,8-TCDD
Study NCI
Sex-species Female rat
Weight (wa) 450 g ,
Tumor sites (one or more) Liver neoplastlc nodules or hepatocellular carcinoma (ref. Table B-3)
Pathologist - NCI Reviewed
CO
Exposure level (mg/kg/day) 0 1.43 x 10~6 7.14 x 10 *
+r/n 5/75 1/49 3/50
7.14 x 10"5
14/49
+r = number of animals with one or more of the tumors
n = total number of animals examined
Estimated
multistage parameters qg q-| ^ qs a^l*
When all dose groups
are used 0.05 0 5.65 x 107 0 6.09 x 103
Goodness of fit
X2
1.44 (d.f. = 2)
When the highest dose
group 1s not used Above fit Is satisfactory
aqi* = the maximum linear component from the model with adequate goodness of fit (p>0.01) = 6.09xl03
(mg/kg/day)~a
-------
TABLE 8-11
Curve Fit of the Multistage Model Parameters to Experimental Data by Study and Pathologist
Linear Parameter q-|, Maximized to Give Upper 95% Limit q-|*
Compound 2,3,7,8-TCDD
Study . NCI
Sex-species Male mice
Height (wa) 48 g
Tumor sites (one or raore)....Hepatocellular carcinomas (ref. Table B-4)
Pathologist - NCI Review
CD
CO
Exposure level (mg/kg/day) 0 1.43 x 10~« 7.14 x 10~*
*r/n 8/73 9/49 8/49
7.14 x 10"s
17/50
+r = number of animals with one or more of the tumors
n = total number of animals examined
Estimated
multistage parameters qp q-j q2 qs a^l*
When all dose groups
are used 0.15 3.80 x 10* 0 0 6.63 x 103
Goodness of fit
X2
2.43 (d.f. « 2)
When the highest dose
group 1s not used Above fit Is satisfactory
= the maximum linear component from the model with adequate goodness of fit (p>0.01) = 6.63x103
(mg/kg/day)"1
fll* = a^l* (70/wa)1/3 = 7.52x10* (mg/kg/dayJ"1, the upper 9554 limit slope factor associated
with human dose response.
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TABLE B-12
Curve Fit of the Multistage Model Parameters to Experimental Data by Study and Pathologist
Linear Parameter q-j, Maximized to Give Upper 95% Limit q-|*
Compound 2,3,7,8-TCDD
Study NCI
Sex-species Female mice
Height (wa) 40 g
Tumor sites (one or more) Subcutaneous tissue - flbrosarcoma, hematopoletlc system lymphoma, or leukemia
Liver - hepatocellular adenoma or carcinoma (ref. Table B-5)
Pathologist - NCI Reviewed
Exposure level (mg/kg/day) 0 5.71 x 10~* 2.86 x 10~5
+r/n 22/74 20/50 19/48
2.86 x 10~*
31/47
+r = number of animals with one or more of the tumors
n = total number of animals examined
Estimated
multistage parameters qg q-| q2 qa a^l*
When all dose groups
are used 0.41 2.38 x 103 0 0 3.78 x 103
Goodness of fit
Xa
1.20 (d.f. = 2)
When the highest dose
group Is not used Above fit Is satisfactory
aqi* = the maximum linear component from the model with adequate goodness of fit (p>0.01) = 3.78xl03
(mg/kg/day)"1
Ql* = aQl* (?°/wa)1/3 = 4.56x10* (mg/kg/day)"1, the upper 95% limit slope factor associated
with human dose response.
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TABLE B-13
Summary of Hunan Slope Estimates for 2,3,7,8-TCDD
CO
1
rvj
o
Species Study
Rat Dow
Rat
Rat
Rat
Rat NCI
Mice NCI
Mice
Sex
Male
Female
Female
Female
Male
Female
Pathologist
Koclba
Squire
Koclba
Squire
NCI -
NCI -
NCI -
- unadjusted
- adjusted for
early deaths
- unadjusted
- adjusted for
early deaths
Reviewed
Reviewed
Reviewed
Human Slope Estimate qi*
In (mg/kg/day)"1
1.47
1.73
8.98 x 10*
1.51
4.25
1.61
3.28
7.52
4.56
x 10*
x 10*
- 2.52 x 10s
x 105t
x IQs
x lost
x 10*
x 10*
x 10*
Ref. Table
No.
B6
B7
B8
B8A
B9
B9A
BIO
Bll
B12
tvalues used to determine geometric mean of 1.56 x 10s (mg/kg/day)"1
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APPENDIX C
COMPARISON OF RESULTS BY VARIOUS EXTRAPOLATION MODELS
The estimate of unit risk from animals presented 1n the body of this
document 1s calculated by the use of the linearized multistage model, for
the reasons given herein. The use of this nonthreshold model 1s part of a
methodology that estimates a conservative linear slope at low extrapolation
doses that Is usually consistent with the data at all dose levels In an
experiment. The model holds that the most plausible upper limits of risk
are those predicted by linear extrapolation to low levels of the dose-
response relationship.
Other nonthreshold models that have been used for risk extrapolation are
the one-hit, the Iog-Prob1t, and the Welbull models. The one-hit model 1s
characterized by a continuous downward curvature, but 1s linear at low
doses. Because of Us functional form, the one-hit model can be considered
the linear form or first stage of the multistage model. This fact, together
with the downward curvature of the one-hit model, means that 1t will always
yield low-level risk estimates which are at least as large as those of the
multistage model. In addition, whenever the data can be fitted adequately
by the one-hit model, estimates based on the one-hit model and the multi-
stage model will be comparable.
The log-Prob1t and the WelbulJ models, because of their general "S"
curvature, are often used for the Interpretation of toxlcologlcal data 1n
the observable range. The low-dose upward curvatures of these two models
usually yield lower low-dose risk estimates than those of the one-hit or
multistage models. The Iog-Prob1t model was originally used 1n biological
assay problems such as potency assessments of toxicants and drugs, and 1s
C-l
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generally used to estimate such values as percentHe lethal dose or percen-
tlle effective dose. The development of the model occurred along strictly
empirical lines. I.e., 1t was observed 1n these studies that several log
dose-response relationships followed the cumulative normal probability
distribution function, $. In fitting the cancer bloassay data, assuming
an Independent background, this becomes
P(D;a,b,c) = c + (1-c) $ (a-f-blog.jp 0) a»b > 0 < c < 1
where P 1s the proportion responding at dose D, c 1s an estimate of the
background rate, a Is an estimate of the standardized mean of Individual
tolerances, and b Is an estimate of the log dose-Problt response slope.
The one-hit model arises from the theory that a single molecule of a
carcinogen has a probability of transforming a single normal cell Into a
cancer cell. It has the probability distribution function
P(D;a,b) = l-exp-(a4-bd) a,b > 0
where a and b are the parameter estimates. The estimate a represents the
background or zero dose rate, and the parameter estimated by b represents
the linear component or slope of the dose-response model. In discussing the
added risk over background, Incorporation of Abbott's correction leads to
P(D;b) = l-exp-(bd) b > 0
Finally, a model from the theory of cardnogenesls arises from the multlhlt
model applied to multiple target cells. This model has been termed here the
Welbull model. It 1s of the form
P(D;b,k) . l-exp-(bdk) b.k > 0
For the power of dose only, the restriction k > 0 has been placed on this
model. When k > 1, this model yields low-dose estimates of risks usually
significantly lower than either the multistage or one-hit models, which are
C-2
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linear at low doses. When 0 < k < 1, the model yields low-dose estimates of
risk that are greater than the one-hit and multistage models; this 1s
generally regarded as biologically Implausible. All three of these models
usually project risk estimates that are significantly higher at low exposure
levels than those projected by the Iog-Prob1t model.
The Dow Chemical Company data for female Sprague-Dawley rats were fitted
to the above models, after adjusting for early mortality by eliminating all
animals dying before 1 year. The results are Identical for the multistage
and one-hit models, as shown In Tables C-l and C-2. The Iog-Prob1t model
yielded by far the lowest estimates at low doses. The Welbull model yielded
estimates higher (by two orders of magnitude) at low levels than either the
one-hit or the multistage model, since k, determined by best fit to the
data, 1s <1. As discussed 1n the text and shown 1n Tables 8-8 and B-B,
dropping the highest dose resulted 1n a larger upper-limit slope estimate
for the multistage model. However, without the highest dose points, neither
the Iog-Prob1t nor the Welbull models could be fitted to the data, for the.
reason that the control group response was higher than that of the lowest
dose group.
C-3
-------
A toxicHy-based criterion has b6en 'calculated for comparlsort with the
cancer-based criterion 1n accordance, with public comments. Since the data
from the limited sturdy by Schantz ,et' al. (1979) are supportive of the find-
Ings by Hurray et al. (1979), It seems reasonable to "determine an ADI based
on the LOAEL. If one selects an uncertainty factor of 100 based on the
existence of. lifetime animal studies and knowledge of effects 1n man, as per
U.S. EPA methodologies (Federal Register, 1980b), and then an additional 10
because a LOAEL 1s used as the basis of this calculation,* then the ADI for
a 70 kg man would be:
-NT", pg/kg/day (LOAEL) . '
ADI = = 7.0 x 10-= pg/kg/day.
100 x 10
However, this concentration may not be sufficiently protective of human
health since 1t does not take Into account the demonstrated carcinogenic
effects of 2,3r7,8-TCDD 1n an1ma=ls and the probability that 2,3,7,8-TCDD 1s
a human carcinogen as dlscuss'ed 1n Section 11.6.1.
*Accord1ng to the methods published by U.S. EPA (Federal Register, 1980b),
an additional uncertainty factor between 1 and 10 must be used because the
calculation .1s, Jiased on a LOAEL.;. An uncertainty factor of 10 was chosen
because of the adverse effects seen 1n rhesus monkeys at 0.0015
,pg/kg/day, despite the equivocal nature of the effects 1n rats seen at
'.the 0.001 pg/kg/day dose level.
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