United States
Environmental Protection
Agency
Office of Health and
Environmental Assessment
Washington DC 20460
EPA-600/8-84-014A
May 1984
External Review Draft
Research and Development
£EPA
Health Assessment
Document for
Polychlorinated
Dibenzo-p-Dioxins
Review
Draft
(Do Not
Cite or Quote)
Part 1 of 2
Notice
This document is a preliminary draft. It has not been formally
released by EPA and should not at this stage be construed to
represent Agency policy. It is being circulated for comment on its
technical accuracy and policy implications.
-------
EPA-600/8-84-OHA
May 1984
External Review Draft
DRAFT
Oo not cite or quote
HEALTH ASSESSMENT DOCUMENT
FOR
POLYCHLORINATED DIBENZO-£-DIOXINS
Part 1 of 2
Notice
This document 1s a preliminary draft. It has not been
formally released by EPA and should not at this stage be
construed to represent Agency policy. It 1s being circu-
lated for comment on Us technical accuracy and policy Im-
plications.
U.S. ENVIRONMENTAL PROTECTION AGENCY
Office of Research and Development
Office of Health and Environmental Assessment
Environmental Criteria and Assessment Office
Cincinnati, Ohio 45268
Project Manager: Dr. Debdas Mukerjee
Ciucsgo, iliiiiO's fcOG'J4
-------
DISCLAIMER
This report 1s an external draft for review purposes only and does not
constitute Agency policy. Mention of trade names or commercial products does
not constitute endorsement or recommendation for use.
NOTE
For Information concerning this document, please contact the project
manager, Debdas Mukerjee (513/684-7531), of the Environmental Criteria and
Assessment Office, Cincinnati, OH 45268.
11
-------
PREFACE
The Office of Health and Environmental Assessment has prepared this Health
Assessment Document on polychlorInated d1benzo-f)-diox1ns 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 dibenzo-p_-
dloxlns 1s qualitatively and whore possible, quantitatively, Identified.
Observed effect levels and dose-response relationships are discussed where
appropriate In order to Identify the critical effect and to place adverse
health responses 1n perspective with observed environmental levels.
This document has been reviewed by a panel of expert scientists during the
peer review workshop held at the Cincinnati Convention/Exposition Center,
Cincinnati, OH, on July 27, 28 and 29, 1983.
111
-------
AUTHORS, CONTRIBUTORS, AND REVIEWERS
The EPA Office of Health and Environmental Assessment (OHEA) was responsible
for the preparation of this draft health assessment document. The OHEA
Environmental Criteria and Assessment Office (ECAO-Cincinnati) had overall
responsibility for coordination and direction of the document preparation and
production effort (Debdas Mukerjee, Project Manager, Jerry F. Stara, Director,
ECAO-Cincinnati).
The participating members of the Environmental Criteria and Assessment
Office-Cincinnati, Ohio are:
D. Mukerjee, M.Sc., Ph.D.*
H. Ball, M.S.
C. DeRosa, Ph.D.
L. Erdreich, Ph.D.
R. Hertzberg, Ph.D.
R. Bruins, M.S.
M. Dourson, Ph.D.
B. Farren, B.S.
S. Lutkenhoff, B.S.
C. Mullin, M.S.
W.B. Peirano, M.S.
D.J. Reisman, M.S.
J.F. Stara, D.V.M.; D.S., Director*
M.W. Neal, Ph.D. (Syracuse Rsch. Corp.)*
S. Que Hee, Ph.D. (Univ. Cinc./Med.Ctr.)*
M.A. Schneiderman, Ph.D. (Env. Lawlnst.)*
D.K. Basu, Ph.D. (Syracuse Rsch. Corp.)*
J.R. Olson, Ph.D. (State Univ./New York)*
Safe, Ph.D. (Texas A&M Univ.)*
Dorough, Ph.D. (Univ. Kentucky)*
Mink, Ph.D.
Orme, M.S.
S
W
F
J
W. Pepelko, Ph.D
J. Risher, M.S.
The OHEA Carcinogen Assessment Group (CAG) was responsible for preparation
of the sections on carcinogenicity. Participating members of the CAG are listed
below:
Roy E. Albert, M.D. (Chairman) Charalingayya B. Hiremath, Ph.D.*
Elizabeth L. Anderson, Ph.D.
Larry D. Anderson, Ph.D.
Steven Bayard, Ph.D.*
David L. Bayliss, M.S.*
Chao W. Chen, Ph.D.
Herman J. Gibb, B.S., M.P.H.
Bernard H. Haberman, D.V.M., M.S.
James W. Holder, Ph.D.
Robert E. McGaughy, Ph.D.
Jean C. Parker, Ph.D.
Dharm V. Singh, D.V.M., Ph.D.
Todd W. Thorslund, Sc.D.
The OHEA Reproductive Effects Assessment Group (REAG) was responsible for
the preparation of the sections on mutagenicity. Participating members of
the REAG are listed below:
John R. Fowle III, Ph.D.
Ernest R. Jackson, M.S.
David Jacobson-Kram, Ph.D.
Casey Jason, M.D.
K. S. Lavappa, Ph.D.
Sheila L. Rosenthal, Ph.D.*
Carol N. Sakai, Ph.D.
Vicki Vaughan-Dellarco, Ph.D.
Peter E. Voytek, Ph.D. (Director)
^Authors
i v
-------
The following individuals were asked to review this document and
earlier drafts of this document:
Bernard H. Haberman EPA Carcinogen Assessment Group
Franklin L. Mink ECAO-Cincinnati
Charles H. Nauman EPA Exposure Assessment Group
William E. Pepelko ECAO-Cincinnati
David J. Reisman ECAO-Cincinnati
John L. Schaum EPA Exposure Assessment Group
The following members of the ECAO-Cincinnati Technical Services Staff
were responsible for document production:
Cynthia Cooper „ Karen Mann
Patricia Daunt Judith Olsen, B.A.
Erma Durden, M.A. Bette Zwayer
Cindy Fessler
-------
POLYCHLORINATED DIBENZO-p-DIOXINS PEER REVIEW PANEL MEMBERS
July 27, 28 and 29, 1983 Cincinnati, Ohio
Co-chairmen:
Debdas Mukerjee, ECAO-CIN
Jerry F. Stara, ECAO-CIN
MEMBERS
Roy Albert
Institute of Environmental Medicine
New York University Medical Center
Donald G. Barnes
Office of Pesticides and Toxic
Substances
U.S. Environmental Protection Agency
K. Diane Courtney
Health Effects Research Laboratory
Research Triangle Park
U.S. Environmental Protection Agency
Frederick Coulston
White Sands Research Center
David Firestone
Food and Drug Administration
S. Garattln!
Institute d1
Farmacologlc
Milan, Italy
Recerche
"Mario NegM"
Dolores Graham
Health Effects Research Laboratory
Research Triangle Park
U.S. Environmental Protection Agency
Richard Grelssmer
Oak Ridge National Laboratory
Lennart Hardell
University Hospital
Umea, Sweden
Robert Harless
Environmental Monitoring Systems
Laboratory, Research Triangle Park
U.S. Environmental Protection Agency
Rolf Hartung
University of Michigan
Allstalr W.M. Hay
University of Leeds
Leeds, United Kingdom
Otto Hutzlnger
University of Amsterdam
Amsterdam, The Netherlands
R.O. Klmbrough
Centers for Disease Control
Richard J. Koclba
Dow Chemical Company
Frederick Kopfler
Health Effects Research Laboratory
Cincinnati
U.S. Environmental Protection Agency
Marvin Legator
University of Texas Medical Branch
Ruth Lllls
Mt. S1na1 School of Medicine
Prab D. Lotllkar
Temple University School of Medicine
Benjamin Lyklns, Jr.
Municipal Environmental Research
Laboratory, Cincinnati
U.S. Environmental Protection Agency
Fumlo Matsumura
Michigan State University
E. McConnell
National Institute of Environmental
Health Sciences
W.P. McNulty
Oregon Regional Primate Research
Center
V I
-------
Robert Miller
National Cancer Institute
Ralph Nash
U.S. Department of Agriculture
James Olsen
State University of New York
Francesco Pocch1ar1
Institute SupeMore d1 Sanlta
Rome, Italy
Shane Que Hee
University of Cincinnati Medical
Center
Chrlstoffer Rappe
University of Umea, Sweden
Steven H. Safe
Texas A&M University
Marvin Schnelderman
Environmental Law Institute
Larry Sllbart
National Wildlife Federation
Ellen Sllbergeld
Environmental Defense Fund
David Stalling
Columbia National Fisheries Research
Laboratory
Lewis Thlbodeaux
University of Arkansas
Thomas Tlernan
Wright State University
v i i
-------
TABLE OF CONTENTS
Page
1. INTRODUCTION 1-1
2. SUMMARY AND CONCLUSIONS . . . 2-1
2.1. SUMMARY 2-1
2.2. CONCLUSIONS 2-5
2.3. NEEDS FOR FUTURE RESEARCH 2-6
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 4-16
4.3.5. Photochemical Process 4-16
4.4. RELATIONSHIP BETWEEN SOURCES AND CONTAMINATION IN
ENVIRONMENTAL MATRICES. 4-17
i x
-------
Page
4.5. ENVIRONMENTAL LEVELS 4-17
4.5.1. Water 4-19
4.5.2. Air 4-20
4.5.3. Soil 4-24
4.5.4. Foods and Biological Samples 4-27
4.6. EXPOSURE 4-30
4.7. SUMMARY 4-36
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 PHARMACOKINETICS 7-1
7.1. ABSORPTION 7-1
7.1.1. Absorption from the Gastrointestinal Tract 7-1
7.1.2. Absorption Through the Skin 7-4
7.2. DISTRIBUTION 7-5
7.3. METABOLISM 7-10
7.4. ELIMINATION 7-14
7.5. SUMMARY 7-18
-------
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 8-43
8.1.3. Chronic 8-50
8.2. HUMAN 8-59
8.2.1. Acute Exposure 8-59
8.2.2. Chronic Studies 8-64
8.3. MECHANISM OF TOXICITY 8-67
8.3.1. Receptor-Mediated Toxldty 8-68
8.3.2. MetaboTIsm 8-75
8.3.3. Vitamin A Depletion 8-76
8.3.4. L1p1d Perox1dat1on 8-77
8.3.5. EndocMn Imbalance 8-78
8.4. SUMMARY 8-80
8.4.1. Experimental Animal Data 8-80
8.4.2. Human Data 8-83
8.4.3. Mechanisms of Toxldty 8-84
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-KDD Studies 1n Mice 9-6
9.1.3. 2,3,7,8-TCDD Studies 1n Rats 9-12
9.1.4. 2,3,7,8-TCDD Studies 1n Rabbits and Ferrets .... 9-18
9.1.5. 2,3,7,8-TCDO Studies 1n Nonhuman Primates 9-19
9.1.6. Studies 1n Chickens 9-21
9.1.7. Studies of the Teratogenlc and Reproductive
Effects of HxCDD 9-22
9.2. STUDIES ON HUMAN POPULATIONS 9-23
9.3. OTHER REPRODUCTIVE EFFECTS 9-33
9.4. SUMMARY 9-34
10. MUTAGENICITY AND OTHER INDICATIONS OF GENOTOXICITY 10-1
10.1. RELEVANT STUDIES TO-1
10.1.1. Assays 1n 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 10-11
X I
-------
Page
11. CARCINOGENICITY 11-1
11.1. ANIMAL STUDIES 11-1
11.1.2. Van Miller et al. 2,3,7,8-TCDD Oral Rat
Study (1977a,b) 11-2
11.1.3. Kodba et al. (Oral) Rat Study (1978a) 11-4
11.1.4. National Toxicology Program (Oral) Rat
Study (1980a,b) 11-12
11.1.5. Toth et al. (Oral) Mouse Study (1979) 11-15
11.1.6. National Toxicology Program (Oral) Mouse
Study (1980a,b) 11-20
11.2. SUMMARY OF ANIMAL CARCINOGENICITY 11-49
11.3. EPIOEMIOLOGICAL STUDIES 11-58
11.3.1. Case Reports 11-58
11.3.2. Soft-Tissue Sarcomas 11-62
11.3.3. Malignant Lymphoma 11-83
11.3.4. Stomach Cancer 11-90
11.3.5. Summary of Ep1dem1olog1cal Studies 11-100
11.4. QUANTITATIVE ESTIMATION OF RISKS OF EXPOSURE 11-101
11.4.1. Introduction 11-101
11.4.2. Procedures for the Determination of Unit
Risk for Animals 11-102
11.4.3. Description of the Low-Dose Animal
Extrapolation Model 11-104
11.4.4. Selection of Data 11-106
11.5. ORAL 11-107
11.5.1. Calculation of the Unit Risk from Animal Studies. . 11-109
11.5.2. Interpretation of Quantitative Estimates 11-109
11.5.3. Alternative Methodological Approaches 11-111
11.5.4. Unit Risk Estimates for 2,3,7,8-TCDD via the
Oral and Inhalation Route 11-112
11.5.5. Unit Risk Estimate for HxCDDs (1,2,3,6,7,8 and
1,2,3,7,8,9) Via the Inhalation Route 11-115
11.5.6. Relative Potency 11-120
11.6. SUMMARY AND CONCLUSIONS 11-126
11.6.1. Qualitative Assessment-2,3,7,8-TCDD 11-126
11.6.2. Qualitative Assessment-HxCDD 11-129
11.6.3. Quantitative Assessment - 2,3,7,8-TCDD and HxCDD. . 11-129
11.6.4. Conclusion 11-130
X I I
-------
Page
12. SYNERGISM AND ANTAGONISM 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
13.1.1. Ambient Water 13-1
13.1.2. Drinking 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. Mutagenldty 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 Toxldty Data 14-10
14.4.2. Animal Carc1nogen1c1ty 14-11
15. REFERENCES 15-1
APPENDIX A A-l
APPENDIX B B-1
APPENDIX C C-l
XI I
-------
LIST OF TABLES
No.
3-1
3-2
3-3
3-4
3-5
3-6
4-1
4-2
4-3
4-4
4-5
5-1
6-1
6-2
6 3
6-4
THle
Physical Properties of a Few Selected PolychloMnated
Dloxlns
A Few Estimated Physical Parameters of Chlorinated
D1benzo-[)-d1ox1ns
Potential Interferences 1n the Determination of TCOOs
at m/e Values of 319.8966 and 321.8936
Some Packed and Capillary Columns Used for the Analysis
of PCDDs
The Detection Limit, Resolution and Ions Monitored by a Few
Mass Spectrometrlc Systems for the Determination of TCDOs . .
Some Published Method Validation Data for 2,3,7,8-TCDD
Recovered from Fortified Matrices and Determined by GC/MS . .
Levels of Tetra-, Penta- and Hexa-chlorod1benzo-p_-d1ox1ns
Reported 1n Chlorophenols and a Few Pesticides
Originating from Chlorophenols
Locations of Major Producers and Formulators of
Chlorophenols and Their Derivatives
Levels of TCDD 1n Soils and Sediments from Different
Locations .
Predicted BCFs from Calculated and Measured Values of Kow . .
Measured B1oaccumulat1on Factor for 2,3,7,8-TCDD 1n
Freshwater Aquatic Organisms
B1oconcentrat1on Factor of TCDD for Several Aquatic
Organisms ..
Effect of Acute Exposure to 2,3,7,8-TCDD on Aquatic Animals .
Effects of Chronic or Subchronlc Exposure to 2,3,7,8-TCDD
on Aquatic Animals.
Levels of 2 3 7 8 TCDDs 1n F1sh and Shellfish
KDD Levels 1n Wildlife
Page
3-4
3-6
3-12
3-14
3-17
3-31
4-7
4-10
4-25
4-34
4-35
5-16
6-2
6-5
6-10
6-15
XIV
-------
No. Title Page
7-1 Gastrointestinal Absorption of 2,3,7,8-TCOD 7-2
1-2 Distribution of 2,3,7,8-TCDD 7-6
7-3 Elimination of 2,3,7,8-ICDD 7-15
8-1 Lethal Doses of 2,3,7,8-TCOO Following Acute Exposure .... 8-2
8-2 loxlc Responses Following Exposure to 2,3,7,8-TCDO:
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 8-51
9-1 Studies on the Potential Teratogenlc Effects of 2,3,7,8-TCOD
Contaminated 2,4,5-T 9-2
9-2 Studies on the Potential Teratogenlc Effect of 2,3,7,8-TCOD . 9-7
10-1 The Results of MutagenlcHy Assays for 2,3,7,8-TCOD 1n
Salmonella typh1mur1um 10-2
11-1 2,3,7,8-ICDD Intake and Mortality In Male 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 In 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 In Male Rats Fed Diets Containing
2,3,7,8-TCOD 11-11
11-7 Dow 2,3,7,8-TCDD Oral Rat Study by Dr. Kodba, With
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. Kodba, With
Dr. Squire's Review (8/15/80) Sprague-Dawley Male Rats -
Spartan Substraln (2 years) 11-14
XV
-------
_ No. IlLle Page
11-9 Incidence of Primary Tumors In Male Rats Administered
?,3,7,8-TCDD by Gavage 11-16
11-10 Incidence of Primary Tumors 1n Female Rats Administered
2,3,/,8-TCOD by Gavage 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 Gavage 11-23
11-14 Promoting Effect of 2,3,7,8-TCDD on Hepatocarclnogenesls
by a Single Dose of Olethylnltrosamine (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 WHh
Subcutaneous Administration of 3-MC on Tumor1genes1s 1n
D2 Mice 11-32
11-18 Incidence of Tumors 1n Mice Treated WHh 3-MC and With
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 In Male and Female B6C3F1 Mice
Administered HxCDD by Gavage for 104 Weeks 11-44
11-22 Carclnogenldty Bloassays of 2,3,7,8-TCDD and HxCDD
by Dermal Application to Mice 11-46
11-23 Carclnogenldty Bloassays of PCDD Administration by the
Oral and Dermal Route 11-50
11-24 Distribution of Tumor Types 1n Two Case-Controls Studies
of Soft-Tissue Sarcoma 11-65
XVI
-------
No. Title Page
11-25 Exposure Frequencies In Two Case-Control Studies of
Soft-Tissue Sarcoma 11-66
11-26 Relative Risks of Soft-Tissue Sarcoma 1n Relation to
Exposure to Phenoxyacetlc Adds and Chlorophenols 1n
Two Case-Control Studies 11-68
11-27 Distribution of H1stolog1cal Types of Soft-Tissue
Sarcomas 11-72
11-28 Midland County Soft and Connective Tissue Cancer
Deaths 1960-1981 11-81
11-29 Other Occupations (Minus Forestry/Agriculture) 11-87
11-30 Other Occupations (Minus Forestry/Agrlculture/Woodworkers . . 11-88
11-31 Analysis of Stomach Cancer Mortality 1n a Group of West
German Factory Workers Exposed to 2,3,7,8-TCDD 11-93
11-32 Reanalysls of Stomach Cancer Mortality 1n a Group of
West German Factory Workers Exposed to 2,3,7,8-TCDD 11-95
11-33 Stomach Cancer Mortality In Three Studies of Workers
Exposed to Phenoxyacetlc Add Herbicides and/or
2,3,7,8-TCDD 11-97
11-34 NIP HxCDD (Gavage) Bloassay. Osborne-Mendel Rats
(2 years) Incidences of Neoplastlc Nodules and Hepato-
cellular Carcinomas 11-116
11-35 NIP HxCDD (Gavage) Bloassay. B6C3F1 Mice (104 weeks)
Incidences of Neoplastlc Nodules and Hepatocellular
Carcinomas 11-118
11-36 Relative Carcinogenic Potencies Among 54 Chemicals
Evaluated by the Carcinogen Assessment Group as Suspect
Human Carcinogens 11-122
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 ...... 14-3
14-2 No-Observed-Effect Levels and Low-Observed-Effect Levels
Obtained from Subchronlc and Chronic Oral Toxldty
Studies of HxCOD 14-5
14-3 Carclnogenldty Bloassays of 2,3,7,8-TCDD 14-12
14-4 Carclnogenldty Bloassays of a 1:2 Mixture of 1,2,3,6,7,8-
and 1,2,3,7,8,9-HxCDD 14-16
XV I I
-------
LIST OF FIGURES
No. Title Page
4-1 Ullmann Condensation Reactions 4-3
4-2 Possible potential relationship between various sources
of PCODs and the environmental matrices where PCDOs have
been detected 4-18
11-1 lime-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 54 suspect carcinogens evaluated by
the Carcinogen Assessment Group 11-121
XV II I
-------
LIST OF ABBREVIATIONS
ADI
AHH
bw
BCF
BromoPeCDD
OCOO
OMSO
DNA
EC/GC
"50
FEL
GC/MS
GC/SIM/MS
HPLC
HRGC
HRMS
HxCDDs
"50
LOAEL
LRMS
MFO
NICI
NOAEL
NOEL
Acceptable dally Intake
Aryl hydroxycarbon hydroxylase
Body weight
B1oconcentrat1on factor
Bromopeniachlorod1benzo-j)-d1ox1n
D1chlorod1benzo-j3-d1ox1n
D1methylsulfox1de
Deoxyr1bonucle1c acid
Electron capture/gas chromatography
Median effective dose
Frank effect level
Gas chrornatography/mass spectrometry
Gas chromatography/spedf 1c 1on monitoring/mass spectrom-
etry
High performance liquid chromatography
High resolution gas chromatography
High resolution mass spectrometry
Hexachloro derivatives of d1benzo-p_-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 1on chemical 1on1zat1on
No-observed-adverse-effect level
No-observed-effect level
X I X
-------
OCOD
PCOOs
PCP
PeCDOs
ppb
ppm
ppt
RBC
RNA
SA
ICDDs
1MCDD
2,4,5-T
1WA
UV
WCOT
Octachlorlnated d1benzo-j3-d1ox1ns
All polychlorlnated d1benzo-£-d1ox1ns
Pentachlorophenol
Pentachloro derivatives of d1benzo-£-d1ox1ns
Parts per billion
Parts per million
Parts per trillion
Red blood cells
R1bonucle1c add
Satellite association
Tetrachloro derivatives of d1benzo-p_-d1ox1ns
Tr1chlorod1benzo-{>-d1ox1n
2,4,5-TMchlorophenoxyacetlc add
Time-weighted average
Ultraviolet
Wall-coated open tubular
XX
-------
1. INTRODUCTION
Dloxlns are a class of compounds that contain the d1benzo-£-d1ox1n
nucleus. In chlorinated dloxlns, the d1benzo-p_-d1ox1n nucleus 1s 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. "fhls 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 1,2,3,7,8,9-hexachlorod1benzo-p_-d1ox1n.
Of these four congeners, the 2,3,7,8-tetrachlorod1benzo-p_-d1ox1n has been
studied extensively. This compound Is often described 1n 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-£-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 discussing the polychlorlnated
d1 benzo-£-dloxlns:
PCDDs Polychlorlnated d1benzo-£-d1ox1ns
2,3,7,8-KDD 2,3,7,8-Tetrachlorod1benzo-p_-d1ox1n
1,2,3,7,8-PeCOD 1,2,3,7,8-Pentachlorod1 benzo-p_-d1 ox1 n
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
1848A 1-1 02/28/84
-------
2. SUMMARY AND CONCLUSIONS
2.1. SUMMARY
At present, most of the relevant physical properties of the four PCODs
discussed 1n this document remain scientifically undetermined. The solubil-
ity of 2,3,7,8-TCDD 1n water 1s 0.2 pg/su 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 temperatures >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
manufacture of chlorophenols and their derivatives. The primary sources of
the PCDDs in the environment are industrial manufacture of chlorophenols and
their derivatives and the chemical disposal sites containing the wastes from
these industries. Municipal Incineration may also produce some environ-
mental emission of PCDDs. From the available data, 1t is difficult to
ascertain the comparative Importance of these three sources 1n contributing
1849A 2-1 04/13/84
-------
to environmental emissions. The 1,2,3,7,8-PeCDD found In environmental
samples has only been reported 1n emissions from Incinerators.
The monitoring data to date Indicate that the maximum level of PCDDs 1s
likely to be found In soil and drainage sediment samples near chlorophenol
manufacturing Industries and chemical waste disposal sites. WHh the excep-
tion of air near certain contaminated sites, very limited attempts have been
made to determine the level of PCDDs In United States air samples. In the
United Slates, the highest levels are reported 1n fish and wildlife 1n and
around the Great Lakes and rivers 1n Michigan.
The environmental fates of the four PCDDs are not known with certainty.
Most of the Investigations 1n this field have been conducted with 2,3,7,8-
1CDD, and the conclusions regarding the environmental fate of the other
three PCDDs have been drawn by analogy. Few data exist 1n 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 in determining the fates of these chemicals in var-
ious ambient media is not known with certainty, but they are susceptible to
photochemical reactions 1n the presence of hydrogen donors. In aquatic
media, a substantial proportion of the PCDDs may be present in the sediment-
sorbed state or in the biota. In the atmosphere, the PCDDs are expected to
be present in the vapor phase and particulate-sorbed states. The atmospher-
ic transport of these compounds can be predicted from dispersion modeling
equations. In the case of the accidental release of 2,3,7,8-TCDD at Seveso,
Italy, it 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
1849A 2-2 04/13/84
-------
PCDDs from soils are transport to atmosphere via contaminated dust parti-
cles, direct volatilization from the surface or near surface zones (<5 cm),
and transport to surface water via eroded soil.
Both the calculated and the experimental results show that the PCDDs
will concentrate In sediments and biota present 1n aquatic media. It has
been shown by static test procedures that, depending on the species, the 8CF
for 2,3,7,8-TCDD ranges from -2000-30,000. The U.S. EPA's best estimate of
the BCF for 2,3,7,8-TCDD Is 5000 (U.S. EPA, 1984).
In mammals, 2,3,7,8-TCDD 1s readily absorbed through the gastrointes-
tinal 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 Hpid 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 of
lactatlng rats.
Ihe PCDDs discussed 1n this document are among some of the most toxic
compounds known, with the LD level for guinea pigs being 0.6 yg/kg for
2,3,7,8-TCDD. The other congeners are somewhat less toxic; however, the
LD values are still In the yg/kg range. Although 2,3,7,8-TCDD 1s
highly toxic 1n all species tested, there are large species differences 1n
sensitivity, with the ID™ for hamsters being 1n the low mg/kg range. The
characteristic 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 produces an acute liver Injury which 1s
not observed 1n either monkeys, hamsters or guinea pigs. In mice, the
1849A 2-3 04/13/84
-------
Immune response 1s also suppressed. After subchronlc or chronic exposure to
2,3,7,8-TCDD 1n rats or mice, the liver appears to be the most severely
affected organ, although hemorrhage, 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 1n many
cases are similar to those observed 1n animals, with exposure leading to
altered liver function and I1p1d metabolism, porphyrla cutanea tarda, neuro-
toxldty 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 symptoms such as chloracne are attrib-
uted to the PCDDs, the other signs of toxldty may arise, at least In 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.
Exposure to 2,3,7,8-lCDD 1n mice produces facial clefts, while exposure 1n
rats results 1n edema, hemorrhage and kidney anomalies; rabbits have a
higher Incidence of extra ribs. Certain human epidemiology studies have
shown positive associations with exposure to chemicals contaminated with
2,3,7,8-TCOD and birth defects and abortions, while others have not.
There 1s only limited and conflicting evidence that 2,3,7,8-TCDD Is a
mutagen.
A number of chronic animal bloassays show that the compound 1s an animal
carcinogen. In rats, oral exposure to 2,3,7,8-TCDD resulted 1n an Increased
Incidence of hepatocellular carcinomas, carcinomas of the tongue, hard
palate/nasal turblnates and lung. In both male and female mice, Increased
1849A 2-4 04/13/84
-------
Incidences of liver tumors were observed. A mixture of the two Isomers of
HxCDD, discussed 1n this document has also been tested for carclnogenldty
and shown to produce Increased Incidences of liver tumors 1n rats and mice.
Also, 2,3,7,8-TCDD has produced fibrosarcomas at the site of application
after dermal administration, although there was no significant Increase 1n
dermal tumors when the mixture of HxCDDs was tested. Since both compounds
produce Increased Incidences of tumors 1n two species of animals, there 1s
"sufficient" evidence, according to IARC criteria, to Indicate that these
compounds are animal carcinogens. Some evidence from human ep1dem1olog1c
studies associate exposure to herbicides contaminated with 2,3,7,8-TCDD with
soft tissue sarcomas and non-Hodgk1ns lymphomas; however, the exposures to
2,3,7,8-TCDD were always compounded with exposures to herbicide chemicals.
These epidemiologic studies are consistent with the position that 2,3,7,8-
KDD 1s probably carcinogenic for humans. Because 2,3,7,8-TCDD is almost
always found in association with other materials (e.g., chlorophenols,
phenoxyacetlc acids, combustion products, etc.), It is not presently pos-
sible to evaluate the carclnogenldty of 2,3,7,8-TCDD by itself 1n humans.
2.2. CONCLUSIONS
The PCDDs discussed 1n this document, 2,3,7,8-TCDD, 1,2,3,7,8-PeCDO,
1,2,3,6,7,8- and 1,2,3,7,8,9-HxCDD, are highly toxic following acute expo-
sure. All animal species administered high levels of these compounds
developed weight loss and thymic atrophy. In some species liver damage,
edema, hair loss and Immunosuppresslon were also observed. Chronic toxicity
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 nonneoplastic lesion was
fatty and necrotic change in the liver.
The fetus has been shown to be highly sensitive to the toxic effects of
2,3,7,8-TCDD. In rats the fetotoxicity observed included hemorrhage, edema
1849A 2-5 04/13/84
-------
and kidney anomalies, while 1n mice the predominant lesions were cleft
palate and kidney anomalies. The lowest reported exposure In rats, 1 ng/kg,
produced a significant (by some analyses but not others) effect on the
fetus, and was similar to the NOEL observed 1n chronic studies.
Evidence from animal bloassays 1s "sufficient" (according to IARC
criteria) to conclude that 2,3,7,8- TCDO and a mixture of the two Isomers of
HxCDD are animal carcinogens and therefore, probably carcinogenic 1n humans.
2,3,7,8-TCDD has Increased the Incidence of a variety of tumors, Including
hepatocellular tumors In rats and mice, while the mixture of HxCDD tested
Increased the Incidence of hepatocellular tumors 1n both sexes of rats and
mice. In terms of low dose potency, 2,3,7,8-TCDD and the HxCDD mixture are
the two most potent carcinogens evaluated by the U.S. EPA. Epidemiology
studies of workers exposed to chemicals contaminated with 2,3,7,8-TCOO such
as 2,4,5-tr1chlorophenoxyacet1c add and 2,4,5-trlchlorophenol have findings
which are consistent with the position that 2,3,7,8-TCDD 1s probably car-
cinogenic for humans. There were no chronic studies to determine the car-
cinogenic potential of 1,2,3,7,8-PeCOD.
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 1n 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.
Isotoplcally labeled Internal standard compounds (37C1 or
13C) should be prepared for PeCDDs and HxCDDs.
1849A 2-6 04/23/84
-------
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.
Pharmacok1net1c studies should be conducted to demonstrate
more clearly the degree of absorption of the PCOOs 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-PeCOD, 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 PCOOs might assist In determining which effects
observed In animals are also present 1n humans. In these
studies, careful quantltatlon of PCDO levels 1n humans and
Industrial hygiene samples might provide dose-response data
necessary for health assessment.
B1oava1lab1l1ty studies from contaminated soil, fly ash, etc.,
are needed.
Mechan1sm-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.
1849A 2-7 04/23/84
-------
3. PHYSICAL AND CHEMICAL PROPERTIES/ANALYTICAL METHODOLOGY
3.1. INTRODUCTION
01benzo-p-dloxln Is a derivative of the basic chemical structure
p-dloxane. The structure of d1benzo-j)-d1ox1n and the conventional numbering
system used for defining substltuent positions are shown below:
A number of substltuents Including nltro, amlno, alkyl, alkoxy and
halogen can be Introduced at the different positions of the two benzene
rings. Most environmental Interest In substituted d1benzo-p_-d1ox1ns and
most studies of this family of compounds have centered on chlorinated
dlbenzo-p-dloxlns that are loosely referred to as "dloxlns." Theoretically,
there are 7S different congeners of chlorinated d1benzo-p_-d1ox1ns. In this
document, only four polychlorInated d1benzo-£-d1ox1ns, namely 2,3,7,8-tetra-
chlorod1benzo-£-d1ox1n (2,3,7,8-KDD), 1,2,3,7,8-pentachlorod1benzo-p_-d1ox1n
(1,2,3,7,8-PeCDD), 1,2,3,6,7,8-hexachlorod1benzo-p_-d1ox1n (1,2,3,6,7,8-
HxCOO) and I,2,3,7,8,9-hexachlorod1benzo-p-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-Tetrachlorod1benzo-p_-d1ox1n (2,3,7 ,8-1CDD)
1850A
3-1
03/02/84
-------
Chem. Abstr. Name: 2,3,7,8-tetrachlorodlbenzo[b,e]{l,4)-dlox1n
Synonyms: Dloxin; TCDBD; KOD; 2,3,7,8-tetrachlorod1benzod1oxin, 2,3,7,8-
tetrachlorodlbenzo-1,4-d1ox1n.
1,2,3,7,8-Pentachlorod1benzo-£-d1ox1n (1,2,3,7,8-PeCDD)
Chem. Abstr. Name: 1,2,3,7,8-Pentachlorod1benzo[b,e](l,4)diox1n
Synonym: 1,2,3,7,8-Pentachlorod1benzod1ox1n
l,2,3f6,7,8-Hexachlorod1benzo-p_-dlox1n (1,2,3,6,7,8-HxCDD)
Cl
Cl
Chem. Abstr. Name: 1,2,3,6,7,8-Hexachlorod1benzo[b,e](l,4)d1ox1n
Synonym: 1,2,3,6,7,8-Hexachlorod1benzod1ox1n
(1,2,3,7,8,9-HxCDD)
Chem. Abstr. Name: 1,2,3,7,8,9-Hexachlorod1benzo[b,e](l,4}d1ox1n
Synonym: 1,2,3,7,8,9-Hexachlorodlbenzodloxln
1850A
3-2
02/28/84
-------
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-PeCDD, 1,2,3,6,7,8-HxCOD 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 I1poph1l1c, exhibiting a higher degree of
solubility In fats and oils than 1n water. The solubility of 2,3,7,8-TCOO
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 in the near UV region. The absorption coefficients due to
this transition at longer wavelengths are presented 1n 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, 1973). Values
1850A 3-3 03/29/84
-------
oo
-------
for other physical properties for these compounds have been estimated from
various correlation equations and are given 1n Table 3-2.
The Infrared, mass, phosphorescence, and nuclear magnetic spectra of
?,3,7,8-TCOO 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 PCDDs are. also available (Mahle and Shadoff, 1982; Gray
et al., 197S, 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, acids and alkalies, although heat treatment with alkali (under condi-
tions similar to alkaline extraction of tissue) completely destroys octa-COD
(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 in the presence of UV light. They also undergo photoreductlve
dechlor1nat1on 1n the presence of an effective hydrogen donor. Gamma
radiation degrades 2,3,7,8-TCDD 1n organic solvents (Fanelll et al., 1978).
3.3. ANALYTICAL METHODOLOGY
Several publications on the analytical methods for the determination of
PCDD levels in 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
1850A 3-5 03/29/84
-------
TABLE 3-2
A Few Estimated Physical Parameters of Chlorinated D1benzo-£-D1ox1nsa
Parameter
Vapor pressure (mm of Hg)
at 25°C and 1 atmosphere
Octanol/water partition
coefficient at 25°C
Sorption partition
coefficient (Koc)
Water solubility (ppb)
at 25°C
2,3,7,8-TCDD
1.7 x 10~6
1 x 1(T6C
1.4 x 10*
6.9 x 106C
1.9 x 107d
1.4 x 10se
9.9 x 10s
3.3 x 10*c
0.2f
PeCDDb HxCOOb
NA NA
7 x 10* 4.2 x 107
5 x 106 3 x 107
0.04 0.008
aSource: NRCC, 1981a (unless otherwise stated), based on vapor pressure
data (Firestone, 1977a) and the octanol/water partition coefficient value
(Kenaga, 1980)
blhese are estimated values for nonspecific Isomers
cMabey et al., 1981
dU.S. EPA, 1984
elhis is a measured value (Neely, 1979)
flh1s Is the experimental value (Crummett and Stehl, 1973)
NA = Not available
1850A
3-6
03/02/84
-------
specific Isomer or a mixture of Isomers was responsible for the observed
effect(s). In the case of TCODs, the specific Isomer 2,3,7,8-lCDD has been
more thoroughly studied than any of Us other Isomers because of Us high
toxlclty. It is not the purpose of this section to review the various
analytical methodologies 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 analyti-
cal methods available for PCDDs.
3.3.1. General Procedure for the Analysis of PCDDs. The analysis of
PCDDs can be broadly divided Into three basic steps (e.g., sample prepara-
tion, 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 Is homogen-
ized 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 al.,
1981; Harless et al., 1980; Van Ness et al., 1980). If the sample does not
contain water, as is the case with fly ash and atmospheric particulate
samples, either benzene or toluene appears to be the desirable solvent
(Kooke et al., 1981). Toluene should be preferred over benzene, however,
1850A 3-7 02/28/84
-------
because of Us lower toxlclty. For the extraction of PCDDs from aquatic
media, a solvent leading to high partition coefficient should be selected.
No systematic study, however, has been done on the extractabllHy 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 llpld (Albro, 1979).
In other sample matrices that contain high amounts of water, such as
tissues and food samples, the water may alter the extractabllHy 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 1n the presence of ?% water, resulting 1n non-
reproductible extraction (Albro, 1979).
Samples that may contain PCOOs 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
(Tosine, 1981; Harless et al., 1980). Kookc et al. (1981) reported highest
extraction efficiencies by acid treatment of fly ash before extraction. The
increase in efficiency was hypothesized to be due to opening of some of the
pores In the fly ash structure, thus making the solvent more accessible to
the sorbed PCDDs. Refluxing with alkaline potassium hydroxide, however, may
cause decomposition of the higher polychlorinated dioxlns and oxidation
1850A 3-8 03/29/84
-------
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., 1980).
Ihe extraction efficiency may also depend on the method of extraction.
Ihc 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 1ri extraction efficiencies of PCDDs from fly ash by
sonicatlon or soxhlet extraction, Kookc 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 H may be possible to extract liver in
a Teflon-glass homogenizer, 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 is required. The first
step in the cleanup procedure consists of the removal of Uplds from the
extracted sample matrix. The I1p1d cleanup can be achieved by two routes,
namely, solvent extraction or reaction with an acid or a base. The use of
solvents such as hexane, hexane-acetone, chloroform, chloroform-methanol and
petroleum ether (NRCC, 1981b) 1s common. The use of nonpolar solvents
(hexane or CC1 ) gives excellent results when Uplds consist primarily of
trlglycerides and/or phosphollpids. When the lipld consists of cholesterol
esters, however, sulfuric add treatment gives a better result than non-
polar solvent extraction (Albro, 1979). Similarly, base wash of the organic
phase may remove Interfering lipids and other materials through saponlflca-
tion, hydrolysis or degradation. However, add wash is more commonly used
1850A 3-9 03/29/84
-------
than base wash presumably because 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 Us less frequent use. It should be men-
tioned that some Investigators used chromatographlc columns such as silica
gel containing sulfurlc add for the add/base cleanup step Instead of wash-
ing off the liplds by simple shaking (Lamparskl et al., 1979; Fanelll et
al., 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 chromato-
graphy with alumina, Florlsll, silica, foam charcoal or carbon dispersed on
glass fibers has been used for this purpose (Harless et al., 1980; Mitchum
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 AgN03-1mpregnated silica gel columns (Lamparskl et al., 1979; Toslne,
1981; Langhorst and Shadoff, 1980). The AgN03/s1lica column system is
claimed to be effective in the removal of DDE, chlorinated allphatics and
sulfides.
There is 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, it has been
shown by Albro (1979) that acidic alumina may be better than basic alumina,
which in turn may be better than neutral alumina for the separation of
residual lipids from the PCDDs in the sample extracts.
1850A 3-10 03/12/84
-------
Ihe third step in 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; MHchum et al., 1980). The
removal of these additional Impurities has been obtained by using HPLC with
both normal and reversed phase packing materials. Recently, Phllllpson and
Puma (1980) reported that chlorinated methoxyblphenyls 1n fish extract could
coelute with TCDOs through an alum1na-Flor1s1l cleanup sequence and Inter-
fere with the determination of TCDDs. A few compounds that may Interfere
with the determination of 1CDO 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 PCODs
into several different fractions by means of chromatographic techniques.
Both liquid chromatography with alumina columns (Mass et al., 1978; Albro
and Corbett, 1977) and HPLC with normal and reverse phases have been used
(Tosine, 1981; Ryan and P1lon, 1980; Langhorst and Shadoff, 1980; MHchum et
al., 1980). The separation of PCODs using HRGC is necessary for the unequi-
vocal separation of 2,3,7,8-lCDD, 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 S1lar 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 TCOD
Isomers. Ihe various cleanup and analysis procedures have been compared by
Brumley et al. (1981).
1850A 3-11 03/29/84
-------
1ABLE 3-3
cc
o
3>
00
ro
03/29/
Potential Inter
Compound
Heptachloroblphenyls
Nonachloroblphenyls
Tetrachloromethoxy
blphenyls
Tetrachlorobenzyl-
phenyl ethers
Pentachlorobenzyl-
phenyl ethers
DDT (4 Isomers)
DDE (4 Isomers)
Hydroxytetrachloro-
dlbenzofurans
Tetrachlorophenyl-
benzoqulnones
Tetrachloroxanthenes
ferences 1n the Determination of TCDDs at m/e Values
Molecular Formula Interfering Ion
C,2H3»C17 H* -23.C!
C]2H 35Cl9 M+ -435C1
Ci2H 35C18 37C1 M+ -335C1 37C1
^13^8 35C^3 37C10 («j+
s*l|Acoin U4*
Cl3H8 35Cl40 M1"
C]3H8 35Cl3 37ClO fl*
Cl3H7 "Cl4 37ClO M+ _H35ci
C13H7 35C13 37C120 Mf _H3SC1
C]4Hg 35Cl3 37C12 H+ -H35C1
C]4Hg 35C12 37Cl3 ft+ -H35C1
C14H8 35C12 3?C12 Mf
Ci4H8 35C1 37C13 M+
C12H,C,,02
C12H4C,402
ClsHftO 35Cl3 37C1 Mt
C13H60 35C12 37C12 H+
of 319.8966 and
m/e
321.8678
319.8521
321 .8491
319.9329
321.9299
319.9329
321.9300
319.9143
321.91138
319.9321
321.92917
319.9321
321.92916
319.8966
321.8936
319.8966
321.8936
319.9143
321.9114
321 .8936*
Resolution for
Separation
12476
7189
7233
8805
8848
8813
8843
18043
18104
9006
9050
9011
9052
NR
NR
NR
NR
18043
18104
CO
*Source: NRCC, 1981b
NR = Not resolved by MS
-------
The cleanup of the samples through liquid chromatography with subsequent
quantification of PCDDs requires concentration of the sample solution.
Evaporation to dryness by an Inert gas stream appears to be an accepted
procedure for concentrating the TCDD solutions. If the concentration proce-
dure 1s not properly controlled, H can Introduce error In 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% H.SO, on
2 4
Bio-811 A, and Carbosieve 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 is done at temperatures >50°C.
3.3.1.3. SAMPLE ANALYSIS — The final analysis of PCDOs 1s almost
exclusively performed by GC/MS. Although some of the earlier Investigators
(Lamparski et al., 1978; Firestone, 1977b) used GC with electron capture
detection, it does not have the sensitivity for complex samples containing
low levels (<10 ng kg"1) of PCODs (Hass and FMesen, 1979).
Ihe final separation procedure for PCDD analysis uses GC with packed or
capillary columns. A typical list of packed and capillary columns used for
the analysis of PCDOs is given in 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
1850A 3-13 03/29/84
-------
TABLE 3-4
Some Packed and Capillary Columns Used for the Analysis of PCDOs
PACKED COLUMNS
1.8 m x 2 mm l.d., 3% Dexsil 300
0.6-2 m x 2.5 mm 1.d., 3% OV-1, 3% OV-17,
3% OV-61, 2% OV-101
1.8 m x 2 mm 1.d., 3% 0V -7
2 m x 2 mm l.d., 3% OV-210
2 m x 2 mm 1.d. specially packed 0.2% carbon
wax 20 M (Aue packing)
2 m x 2 mm 1.d., 0.6% OV-17/0.4% Poly SI79
2 m x 4 mm 1.d., 1.2% S1lar IOC
1.8 m x 2 mm 1.d., 5% SE-30
CAPILLARY COLUMNS
18 m x 0.3 mm l.d., OV-61 WCOT
22 m x 0.3 mm l.d., OV-17, 101, S1lar IOC
50 m x 0.36 mm 1.d., OV-17 WCOT
30 m x (l.d. not given), SE-30 WCOT
30 m x 0.25 mm 1.d., OV-101 WCOT
30 m x 0.25 mm l.d., SE-30 WC01
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., glass, 60/40 w/w OV-17/
Poly S-179
50 m x 0.25 mm 1.d., glass S1lar IOC
55 m x 0.37 mm 1.d., glas OV-17
55 m x 0.40 mm l.d., glass OV-101
60 m x 0.26 mm 1.d., Supelco SP-2330
50 m x 0.4 mm l.d., OV-101 fused silica
60 m OV-101 WC01 (1.d. unspecified)
Van Ness et al., 1980
DlDomenlco et al., 1980a
Tlernan et al., 1980
Parker et al., 1980
EUeman 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
MUchum 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
1850A
3-14
03/29/84
-------
1s the problem of easy overload 1n 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 1n 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 DOS column and a normal phase
silica column. D10omen1co et al. (1980a) also found low resolution GC
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 D1Domen1co et al. (1980a)
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 S1lar 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 Its 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 Sllov (Tlernan,
1983). A 50 m length of a S1lar 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.
1850A 3-15 03/12/84
-------
As previously mentioned, MS 1s used almost exclusively for the detection
and quantification of PCDOs. Basically, three MS techniques (e.g., LRMS,
HRMS and NIC!) have been used. A few different MS systems used for the
determination of ICDDs are shown in Table 3-5. It 1s obvious from Table 3-5
that electron Impact ionization 1n 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
(Mf). Fragmentation occurs through the loss of CO and Cl radicals. Major
ions are at M^-63 (M^-COCl) and Mf-126 (M*-2COC1). Doubly charged
molecular ions (M +) and minor fragmentation ions occur at M -35
(M*-C1), M+-70 (M*-2C1) and Mf-98 (Mf-COCl-Cl). The usual charac-
teristic ion clusterings because of the chlorine isotopes are also observed.
Based on molecular ions and fragmentation pattern, PCDOs 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 its chlorine Isotope clusters, that is, 320 (3SC14 CDD),
322 (3!>C1 3'C1 CDD) and 324 (3SC1?37C12 CDD). In some
Instances, fragment ions at 257 (320-C03SC1), 259 (322-C03SCl) and 194
(320-2C035C1) are also monitored. The Intensity ratios in the mass spec-
trometric peaks due to chlorine Isotope proportions In native TCOD 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 arc expected to be 77:100:49 (NRCC, 1981a). The response for
the ion at 257 is -30% of the response for the 1on at 322 (Glaser et al.,
1981). Sometimes internal standards containing (C. H 37C1 0 ) or
M 4 4 I
0) used for TCDD analysis give prominent ion peaks
1850A 3-16 03/29/84
-------
oo
tn
O
3>
TABLE 3-5
The Detection Limit, Resolution and Ions Monitored by a Few Mass Spectrometric Systems
for the Determination of TCDDs3
CO
o
IV)
r\j
oo
CO
lonization Method and
Reference
ELECTRON IMPACT
Baughman and Meselson, 1973
Crummett and Stehl, 1973
Hummel, 1977
Hummel, 1977
Mahle et al.( 1977
Adamoli et al . , 1978
Adamoli et al., 1978
O'Keefe et al., 1978
DIDomenico et al., 1980a
Buser and Rappe, 1980
Cavallaro et al., 1980a
Chess and Gross, 1980
Fanelli et al., 1980a
Harless et al., 1980
Langhorst and Shadoff, 1980
Lamparski and Nestrick. 1980
TCDD Limit
of Detection
(P9)
5
6
5-10
5-10
5
50
50
NR
20
40-80
50
250
5-10
5
40-60
M/AMb
10,000
600
400
3,000
NR
unit
unit
10,000
unit
unit
unit
2,000
400
9,000
1,000
unit
320
4-
4-
4
4-
4
4-
4-
4
4-
4-
4-
4
f
4-
4-
-k
m/e Values Monitored for TCDD
322 324 326 328 332 259 257 194
1 4- 4-
4- 4-
4- 4-
f 4-
4- 4-
4- 4-
4- 4-
4- 4-
4- 4-
f 4-
4-4- 4-
f f 4-
f
4- If 4-4-
4- 4-
4-4- 4-
-------
tn
0
TABLE 3-5 (cent.)
lonlzation
Method and
Reference
Norstrom et al
Toslne, 1981
Ryan and PHon
Tlernan et al .
Tlernan et al .
., 1982
, 1980
, 1980
, 1980
TCDD Limit
of Detection
(pg)
5-10C
10C
10C
ld
100C
m/e Values Monitored for TCDD
M/AMb
320 322 324 326 328 332 259 257 194
unit + 1-4 + t
unit * f «. +
1,000 +
350 + *
12,500 + + + +
03
CHEMICAL IONIZATION
Mass et al., 1978
MHchum et al., 1980
50-500
10
unit
NR
323 for MNCIe, 252 and 276 for MONCIf, 176 for
ONCI9
-176 from 320, -182 from 332 by ONIAPCIh
Source: NRCC, 1981a
resolution of mass
ng.kg~1
o
CO
PO
oo
methane negative chemical lonlzation
methane-oxygen negative 1on chemical lonlzation
^oxygen negative 1on chemical lonlzation
oxygen negative 1n atmospheric pressure chemical lonlzation
NR = Not reported
-------
at 328 and 332, respectively. The primary 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, HRMS with appropriate resolution
1n most cases may positively Identify 2,3,7,8-TCDD when the sample cleanup
Is not specific (Hummel and Shadoff, 1980). However, an unequivocal Identi-
fication and quantification of 2,3,7,8-TCDD 1n the presence of Us Isomers
will still require HPLC fractlonatlon or HR6C 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 37C1-TCDD and 2,3,7,8-TCDD 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 TCDD structure.
2. GC/MS peak-matching analysis of molecular Ions 320 and 322 1n
real time to confirm the 2,3,7,8-TCDD elemental composition.
Although the limit of detection for TCDD Is about the same on both HRMS
and LRMS (Crummett, 1983), the advantage of HRMS over LRMS for PCDD analysis
Is 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
1850A 3-19 02/28/84
-------
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 TCOD 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 > M, <- COC1) based on the
concept of MS-MS was used for the analysis of 1CDD (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 PCDDs 1n Specific Environmental Media. Although the
general procedure for the analysis of PCDDs 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 PCOOs 1n each medium have
been discussed individually.
3.3.2.1. WATER —
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
preconcentration of the samples during collection 1s made. Grab samples are
collected in clean (detergent washed, rinsed with acetone or methylene
chloride, and dried) amber glass bottles of 1 I or 1 quart capacity fitted
with screw caps lined with Teflon or aluminum foil (U.S. EPA, 1982a). If
aluminum foil is used as a liner, 1t should be washed with acetone and the
dull side should race 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
1850A 3-20 03/29/84
-------
containers must 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 fi. of water was allowed to pass
through a 12 cm x 1.5 cm l.d. XAD-2 column at a rate of 60 mil/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 In the literature
described 2,3,7,8-lCDD 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 PCODs should be established with added Internal standards.
An appropriate volume of water (depending on the desired detection
limit) with added Internal standard of either 13Cn^ or 37C1,
12 4
2,3,7,8-lCDD in 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;
DIDomenico 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 acid 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
1850A 3-21 02/28/84
-------
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-HRMS (Van Ness et al., 1980; Harless et al., 1980) or high
resolution GC-HRMS or LRMS (U.S. EPA, 1982a). If an unequivocal Identifica-
tion of 2,3,7,8-TCDO Is required, the U.S. EPA (1982a) method seems to be
most appropriate since H recommends using a 50 m SHar IOC capillary column
and multiple Ion monitoring MS mode that 1s known to unequivocally Identify
and quantify 2,3,7,8-TCDD in the presence of its other isomers (Buser and
Rappe, 1980). Harless et al. (1980) reported that 1CDD in 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 particulate 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,
1981b). 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 in a polyurethane foam or XAD-2 trap maintained at room
temperature. The sampling must be performed In an isokinetic manner to
ensure representative sampling. To permit evaluation, the efficiency of the
1850A 3-22 02/28/84
-------
collection method must be documented. The sampling methodology for point
sources Is In 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 {Santodonato et al., 1981) for collec-
tion 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 PCDDs 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 (DIDomenico et al., 1980b).
Dustfall jars were constructed from 10 9. glass vessels topped with metal
gridded 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 pm to be collected. Samples were collected
for 1 month or the time required for the vessel to be filled with meteroic
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 mVminute
(DIDomenico 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. Particulate and vapor phase TCDD was also
1850A 3-23 02/28/84
-------
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 — Ihe analysis of PCDIK in 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 HC1
pretreated particles, followed by soxhlet extraction with benzene or
toluene. L1bert1 and Brocco (1981) found that xylene was a better solvent
than toluene, while Cutie (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 (Llberti and Brocco, 1981; EIceman et al., 1980; "Mernan,
1983; Buser et al., 1978)]. In one analytical procedure, Lamparskl and
Nestrick (1980) added Internal standards of 13C-2,3,7,8-TCDO, 13C-
1,2,3,4,7,8-HxCDD and 13C-OCDD to the partlculate extract. The extract
was cleaned with acid and base washes. Next, the extract was cleaned by
liquid chromatography with AgNO«/s1l1ca column and basic alumina column,
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-LRMS. 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.
1850A 3-24 03/29/84
-------
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
2,3,7,8-13C -1CDD, 2,3,7,8~37C1 -TCQF (tetrachlorodibenzofuran) and
37C10-OCDD and treated with 1 M hydrochloric acid. The PCODs and PCOFs
8
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 chloride-n-hexane (1:1)
fraction from the second column containing PCDDs and PCDFs was subjected to
HRGC/MS analysis. A 55 m x 0.26 mm i.d. Sllar column was found to be suit-
able for the Isomeric separation of all 22 isomers of TCDO.
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 due to fallout from a point source, sampling sites should be estab-
lished in a grid over a topographical map of the suspected area. Soil
samples may be collected by Inserting a 0.5 m long and 7 cm i.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 (DiDomenico et al., 1980c). The bags should be cooled to 4°C
during transportation.
To determine the distribution of PCDDs in 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 in this fashion should be
1850A 3-25 02/28/84
-------
stored 1n plastic bags and transported by storing them at 4°C. The details
of the soil sampling procedure have been described by D1Domen1co 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 1n 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 in 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-TCOO, 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.
1850A 3-26 03/12/84
-------
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 HRGC-HRMS (Harless et
al., 1980; Buser and Rappe, 1980). If an unequivocal Identification and
quantification of 2,3,7,8-TCDD 1s required, the 55 m S1lar IOC capillary
column used by Buser and Rappe (1980) or the 60 m SP-2330 fused silica
column (Rappe et al., 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 in soils.
3.3.2.4. BIOLOGICAL MEDIA — In this section, the sampling and
analysis of PCDDs in 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 in 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
in blood is -2-3 orders of magnitude lower than their concentrations in
adipose tissue (Firestone et al., 1979). There is also evidence in several
species that the accumulation of 1CDD in liver tissue is higher than in
adipose tissue (Section 7.2.). Liver is also preferable because Its lipid
content is lower than adipose tissue (samples with high lipld 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 is milk. Because of the high Hpid content of milk, PCDDs are
expected to be accumulated in this medium (Langhorst and Shadoff, 1980).
1850A 3-27 02/28/84
-------
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 In a freezer (-18°C) until
analyzed (Jensen et al., 1983). However, 1t has been reported that tissue
samples stored in linear polyethylene bottles sorbed -2% of added 1*C-DDT
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). Sample preparation
procedures using the acid/base procedure and neutral procedure are avail-
able. 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 consideration.
Fish 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 is 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 sulfuric acid in silica, and sodium hydroxide in silica,
followed by a second dual-column system of silver nitrate on silica and
1850A 3-?8 03/29/84
-------
basic alumina. The PCOD 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 Huckins et
al. (1978) and Lamparskl et al. (1979), and is expected to provide a better
method for the analysis of PCOOs in 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 in fish and other
aquatic species was carried out (Ryan et al., 1983). No significant differ-
ences in the determined concentration of 2,3,7,8-TCDD in these species
occurred from methods differing in the use of digestion or extraction
technique, HRMS or LRMS, and isomer specific or nonspecific separation. The
relative standard deviations In 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 Na SO /g fat) In 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 is a combination of procedures
employed by Mass et al. (1978) and Lamparskl et al. (1979).
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 in hexane and the clean-up procedure described
1850A 3-29 03/29/84
-------
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 Lamparski
et al. (1979) may be employed.
3.3.3. B1oanalys1s of PCDOs. There are currently three methods for the
bioanalysls of PCDOs, namely, radloimmunoassay (Albro et al., 1979; HcKlnney
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 PCDOs, 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 in the determination of PCDD levels in 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 due to sorptlon on container
walls or photodecomposition during transportation and storage, and contami-
nation of the sample by collection equipment or sample containers can all
cause errors, particularly 1n 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 in the analytical methods
as well. Although some validation data are available for the overall
recovery of 2,3,7,8-TCDD in fortified matrices, these data, as shown 1n
Table 3-6, may not represent the true recoveries, since It is difficult 1f
1850A 3-30 03/29/84
-------
CO
o
3>
OJ
1
00
0
rvs
CO
m/e
320.
320,
320,
320,
335
320,
320,
320,
320,
329
320,
320,
320,
329
320,
Values
322,
322,
322,
322,
322,
322,
322,
322.
322,
322,
322,
322,
335
324
324
324,
328
328
328
328,
328
328
328,
328
Some Published
Matrix
human milk
soil
soil '
soil
fish, liver
human milk
water,
sediment
water,
sediment
water,
sediment
water,
sediment
bovine feed
liver
1 ADI
Method Validation Data for 2,3,7,8-TCDD
TCDD Level of Fortification, nq/kq~J
Native Isotope
13C, ( 37C1 )
2.6 166
NA 100a
10 NA
50 +b
0-125 1000a
0-5 250a
0.01-1000 250a
0.7-65 66
2 NA
NA 625a
13-200 390-1000
20 1000
.t J-D
Recovered from Fortified
Number of
Replicates
8
6
28
8
17
13
14
12
3
4
16
9
Matrices
Mean %
Native
25 + 7
NA
87 + 17
99.2 + 5
+15C
+38C
+16C
85-100
(+8-+17)
83.3
NA
80-100
(+5-+18)
34 + 7
and Determined by GC/MS
Recovery with S.D.
Isotopes
37 + 19
87+15
NA
59.8
86 + 15
68
87
71-87
(+12-+21)
NA
64
77-105
(+9-+18)
27 i 5
Reference
Langhorst and
Shadoff. 1980
Hummel, 1977
DIDomenlco
et al . , 1980a
Lamparskl and
Nestrlck, 1980
Harless et al. ,
1980
Harless et al.,
1980
Harless et al. ,
1980
O'Keefe et al. ,
1978
Mahle et al..
1977
Mahle et al..
1977
O'Keefe et al.,
1978
Baughman and
Meselson, 1973
CO
-------
TABLE 3-6 (cont.
TCDD Level of Fortification, nq/kq"1
m/e Values Matrix Native Isotope
13C, (37C1)
320, 322, 324 carrots 0.5-1.0 NA
320, 322, 324 beets 0.5-1.0 NA
i
CO
^ 3?0, 322, 324 spinach 0.5-1.0 NA
Mean % Recovery
Number of Native
Replicates
20 64.5-66.6
(±18.9-i25.5)d
20 60.8-79.8
(+17-+17 .7)d
20 46.6-67.7
(il 4.2-t24. 7 )d
with S.O.
Isotopes Reference
NA Cavallaro
et al., 1980b
NA Cavallaro
et al., 1980b
NA Cavallaro
et al., 1980b
alnd1cates publishing author's recovery data was converted from ng to ppt or from ppt to X.
bPlus Indicates fortified with Isotope but amount not specified clearly.
cThese data Indicate the mean X accuracy for TCOD obtained with quality assurance samples.
dNumber 1n the bracket represents the X variation experienced; unclear as to how calculations were made.
NA = Not added; SD = Standard deviation
CD
\
CD
-------
not Impossible to Incorporate the Internal standard In the same physical/
chemical form In the sample matrix as the PCOOs. This situation weakens the
reliability of much of the analytical data on PCDD levels 1n various
matrices.
The recovery of the overall analytical procedures Is 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
TCOD 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
In 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-TCOD (assuming that the GC column
resolves 2,3,7,8-TCDD from other TCDOs), 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, 1980a). 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 In the field Is 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.
1850A 3-33 02/28/84
-------
3.4. SUMMARY
Most of the relevant physical properties of the four PCDDs discussed 1n
this document are unknown. The solubility of 2,3,7,8-TCDD In water 1s 0.2
vg/l. This congener and the other three PCDOs are more soluble In
aromatic solvents than aliphatic solvents. The PCDDs 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 1s finally analyzed for the PCDDs by a 6C/MS method.
All the possible GC/MS combinations, namely, HRGC-LRMS, LRGC-LRMS, LRGC-HRMS
and HRGC-HRMG, 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 S1lar IOC glass capillary or a 60 m SP-2330
fused silica column 1n combination with LRMS. Another method using RP-HPLC
and normal phase HPLC separation 1n combination with LRMS has been found to
be satisfactory.
1850A 3-34 04/05/84
-------
4. PRODUCTION, USE, SYNTHESIS, ENVIRONMENTAL SOURCES
AND ENVIRONMENTAL LEVELS
4.1. PRODUCTION AND USE
PCDDs Including the four compounds discussed In this document are not
commercially produced. Rather, these compounds are formed as trace amounts
of unwanted Impurities In the manufacture of other chemicals, primarily
chlorophenols and their derivatives. There Is 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 1,2,4,5-Tetrachlorobenzenes
1n DMSO. This general reaction has been used to synthesize 2,3,7,8-TCDD
according to the reaction scheme shown below:
reflux
The yield of 2,3,7,8-TCDD by this reaction is low (Kende et al., 1974).
A better method Is the reaction of o-dichlorocatechol with 3-nitro-2,5,6-
trlchlorobenzene as shown below (Gray et al., 1976):
DMSO
HO
1851A
4-1
03/02/84
-------
4.2.2. Substitution Reaction. The following substitution reactions have
been used for the synthesis of 2,3,7,8-TCDD:
CI2 + FeCI3
tri-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 unsubstHuted d1benzo-p_-
dloxln was conducted without the Fed , the yield of 2,3,7,8-TCDD was
O
reported to be 40-50% (U.S. EPA, 1980a). The substitution of d1benzo-p_-
dloxln with 2,3-d1chlorod1benzo-£-d1ox1n 1n the presence of FeCl_ and
O
Iodine, on the other hand, reportedly also produced a high yield (41%) 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-CDD.
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,
dechloMnatlon 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
1851A 4-2 03/12/84
-------
OK
CL /V ,CI
Cl \^ Cl
Cl
Cl
290 C
1-4 hr.
Cl
Cl
O
O
o
Cl
Cl
Cl
Cl
Cl
o
o
.O.
,CI
XI
OK
"6:
Y^
ci
.Cl
29O C
1-4 hr.
minor
OK
o:
290 C
1-4 hr.
cr ^v^ ci
ci
Cl
Cl
Cl
FIGURE 4-1
Ullmann Condensation Reactions
1851A
4-3
02/28/84
-------
(necessary to retain some precursor compounds 1n the liquid state to permit
dloxin formation), and the presence of some catalyst provide the most suit-
able conditions for dloxin formation (U.S. EPA, 1980a). However, some of
the catalysts, namely, Cu, Fe, Al-salts and I2> 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 (di-, tr1- and tetra-)
using a simple pyrolysis procedure (Buser and Rappe, 1980). Pyrolyses of
these Chlorophenates were carried out by placing 1 mg of the Chlorophenates
in a glass reaction tube plugged with glass wool and alumina. They were
heated for 30-60 minutes at 300°C. The yields of the TCODs have been
reported to be in the pg range (Buser and Rappe, 1980).
4.2.6. Conversion Through NHratlon. It has recently been shown by
Oliver and Ruth (1983) that 1,2,3,6,7,8-hexachlorodibenzo-£-diox1n can be
selectively prepared from two synthetic routes each consisting of dinitra-
tion of a tetrachlorod1benzo-£-diox1n, followed by reduction and a Sandmeyer
reaction as shown below:
Cl
NH,
trlfluoro-
acetlc
anhydride
Cl
The recovery of 1,2,3,6,7,8-HxCDD was excellent by this method.
1851A
4-4
02/28/84
-------
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 PCDD contamination 1n the environment. Each of these categories 1s dis-
cussed individually in 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 polychlori-
nated diphenyl ethers are incinerated.
4.3.1.1. PRODUCTION OF CHLOROPHENOLS — PCODs are formed as
by-products during the manufacture of chlorophenols. Chlorophenols are
produced by two processes, the chlorination 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) Cl.
cr ^^ ci ci
2,4,5-Tr1chlorophenol(phenolate)
2,3,7,8-TCDD
NaOH
1,2,4,5-Trlchlorobenzene
1851A
4-5
03/26/84
-------
Similarly, HxCODs are formed during the manufacture of tetrachlorophenols by
the above reaction process. PCDDs 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 TCODs, 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 In a ratio of 1:4:5 (Buser,
1979). However, the composition and quantities of PCDOs 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 (Rappc et al., 1979). The chlorophenols are used as fungicides, herbi-
cides, sUmaddes, bacterlddes and Intermediates 1n the production of
chlorinated phenoxy add herbicides 1n agriculture and forestry. The anti-
septic, hexachlorophene, 1s also prepared from 2,4,5-trlchlorophenol (Rappe
et al., 1979). Therefore, the use or presence of contaminated chlorophenols
In facilities such as chlorophenol and pesticide/herbicide plants, cooling
towers, pulp and paper Industry, Incinerators and disposal sites are
potential exposure areas for PCDOs (Josephson, 1983).
1851A 4-6 03/12/84
-------
TABLE 4-1
CO
in
3>
Compound
o-Chlorophenol
2,4-D1chlorophenol
2,6-D1chlorophenol
2,4,5-TCPc
2,4,6-TCP
f
^ 2,4,5-TCP (Na salt)
TCP (unspecified)
2,3,4,6-Tetrachlorophenol
Tetrachlorophenol
(unspecified)
PCpe
o
CO
oo
» *. i j u i icLia-, r en i.a - anu ncAa-LilluiuuiuciltU-u-U!u
and a Few Pesticides Originating from
Ch1orod1benzo-p-d1ox1n
Tetra-
ND
0.037b
ND
NO
ND-6.2 (2,3,7,8-)«J
NO-0.3 (1.3, 6, 8-)
49 (1.3.6. 8-)
1.40 (2.3.7,8-)
ND
ND
0.7
NR
ND
ND
ND
NR
ND
ND
ND
(-CDD) level.
Penta-
ND
NR
ND
ND
ND-1.5
ND
ND
NR
ND
5.2
NR
NR
ND
NR
NR
NR
NR
ND
i :ii Keponeo in u
thlorophenols
ppm
Hexa-
ND
NR
ND
ND
ND
ND
ND
ND-<10
ND-29
9.6
6
ND-<100
0.17-39
ND-<100
9
9-27
0.02-42
0.03-10
n loropneno is
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., 197?
Anonymous, 1979
Firestone
et al.. 1972
Firestone
et al.. 1972
Firestone
et al., 1972
Firestone
et al., 1972
Firestone
et al.. 1972
Woolson et al. ,
1972
Firestone
et al., 1972
Rappe et al. ,
1978
Buser, 1975
Woolson et al . ,
1972
Firestone
et al.. 1972
Woolson et al . ,
1972
Buser, 1975
AWPI, 1977
vnianeuva
et al., 1973
Buser and
Bosshardt. 1976
-------
1ABL F 4 -1 (cont
_, Chlorodlbenzo-p-d1ox1n (-CDD) level.
CD
Ln
3>
1
CO
Compound
PCP {cont.)
PCP (Na salt)
2,4-D (-08, -DP)f
2,4-D and 2,4,5-T mixtures
(formulated products)
2,4-D (add, esters, and
amines)
2,4-D (add, esters, and
amines)
2,4,5-Tf
2,4,5-T (acid, esters, and
formulated products)
S1lvex»
Agent Orange (1:1 mixture
of butyl esters of 2,4-D
and 2,4,5-T)
Agent Purple (5:3:2 mixture
of n-butyl 2,4-D, n-butyl
2,4,5-T and 1so-butyl 2,4,5-T)
Tetra-
NR
ND
0.06-0.4
ND
ND
ND-8.739 (1,3,6,8-7
1,3,7.9-)
D (1,3.6,8-)
ND-<100
0.010-0.080
(2,3,7,8-1
ND-<10
1.981
(2.3.7,8-)
32. 81
(2.3,7,8-)
Penta-
NR
ND
ND-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. Contain. a
No. Tested
several
samples
2/2
6/6
1/28
0/10
28/58
2/30
23/42
12/30
1/7
490/490
NR
Reference
Dow, 1978
F Ires tone
et al., 1972
Buser and
Bosshardt, 1976
Woolson et al . ,
1972
Norstrom
et al., 1979
Cochrane
et al., 1981
Thomas, 1980a;
Harless, 1981
Woolson et al. ,
1972
ACP, 1980
Woolson et al . ,
1972
Young, 1983
Young, 1983
CO
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
dThese Indicate specific dloxln concentrations.
ePCP: pentachlorophenol
AThese are dUhlorophenoxy-acetlc, -butyric add and -proplonlc add.
9The Isomers could not be separated.
"This 1s 2,4,5-tdchlorophenoxy acetic add.
1Th1s 1s an average value.
NO = Not detected; NR = Not reported; D = detected; NA = Not available
-------
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-TCOD (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 1n 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 mg/kg (Cochrane et al., 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-lCDD 1n quantities 1n the range of 0.1-47 pg/g
(Rappe et al., 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 (50% n-butyl 2,4-D, 30% n-butyl 2,4,5-T and 20%
Isobutyl 2,4,5-T) preparations used 1n the 1960's were shown to be 1.98 and
32.8 ppm, respectively (Young, 1983). Efforts were made during the 1970's
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 pg/g of
2,3,7,8-TCDD (Rappe et al., 1979).
1851A 4-9 03/26/84
-------
TABLE 4-2
Locations of 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
Miller Chemical, Whlteford, MD
Monsanto, Co., Sauget, IL
North American Phillips Corp., Kansas CUy, 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 CUy, KS
PBI-Gordon Corp., Kansas City, KS
Rhodla Inc., Portland, OR
St. Joseph, MO
*R1verdale Chemical Co., Chicago Heights, IL
Rorer-Amchem, Ambler, PA
Fremont, CA
1851A 4-10 02/28/84
-------
TABLE 4-2 (cont.)
Chemical
Producer
2,4,5-T (cont.)
2,4,5-7 derivatives
Sllvex esters and salts
Ronnel
Erbon
Hexachlorophene
2,4,5-lCP and salts
2,3,4,6-Tetrachlorophenol
PCP and salts
Rorer-Amchem, St. Joseph, MO
Jacksonville, AR
Thompson Chemical, St. Louis, MO
Union Carbide Corp., Fremont, CA
St. Joseph, MO
Ambler, PA
Vertac, Inc., Jacksonville, AR
Dow Chemical U.S.A., Midland, MI
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, MI
GAP 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., Nltro, 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 Mateo, 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
aSources: 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.
1851A
4-11
02/28/84
-------
As can be seen from Table 4-1, 2,4-D, 2,4,5-T and their formulated
products may contain other PCOOs In addition to ICDDs. 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-TCDO. Agent
Orange and European 2,4,5-T formulations from the 1960's, on the other hand,
may contain primarily 2,3,7,8-TCDO and only minor amounts of other PCDOs
(Rappe et al., 1979). The average 2,3,7,8-TCOD contents 1n Agent Orange and
Agent Purple given 1n Table 4-1 refer to these materials manufactured 1n the
1960's.
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 In this product has been reported to be
<0.03 pg/g (Rappe et al., 1979).
The locations of current and former producers of chlorophenol deriva-
tives have been shown In Table 4-2.
4.3.1.3. CONTAMINATED 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 2,4,5-1, and the equipment remained contami-
nated with 2,3,7,8-TCDO (Federal Register, 1980a).
1851A 4-12 03/26/84
-------
4.3.1.4. DIPHENYL ETHER HERBICIDES — The presence of KDOs, PeCDDs
and HxCDDs as contaminants 1n dlphenyl ether herbicides was reported by
Yamaglshi et al. (1981). The source of PCDDs'In these herbicides was specu-
lated to be the trlchlorophenol used In their production. The concentra-
tions of the two major Impurities, TCDDs and PeCDDs, in commercial formula-
tions were -150 and 30 ppm, respectively. The Isomeric distribution of
ICDDs 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 in 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 in
incinerators operating at 750-900°C; chlorophenols are probably the precur-
sors of PCDO formation. At temperatures >1200-1400°C and residence time of
<1 second, PCDDs are likely to decompose and these compounds are not
expected to form (Junk and Richard, 1981). From kinetic and thermodynamlcal
considerations, Shaub and Isang (1983) estimated that 99.99% 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 in the United States where PCP was known to have
been burned, Rappe et al. (1983b) reported -5 ppm PCDDs in the bottom and
baghouse ash. More than 90% 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 Binghamton, NY, In 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 in the
transformer oil (Buser, 1979). The analysis of wipe tests from a garage
1851A 4-13 02/28/84
-------
adjacent to this site did reveal the presence of PCDDs before cleaning the
garage. Following the cleanup, no contamination was found (Hernan et al.,
1982b; Tlernan, 1983). Therefore, It 1s Important to recognize the possi-
bility of production of PCDDs and PCDFs In 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 PCDDs than the precipitated material (fly ash) (Lustenhouwer
et al., 1980). The concentration of total TCDDs, PeCDDs and HxCDDs 1n the
fly ash from a variety of municipal Incinerators 1n Canada, Europe and the
United States have been studied by several authors (Elceman et al., 1979,
1980; Nestrlck et al., 1982; Karasek et al., 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). PCDDs have been detected 1n the emissions of some municipal waste
Incinerators in Europe (G1zz1 et al., 1982; Benfenati et al., 1983; Taylor
et al., 1983; Ol1e et al., 1982, 1983; Lustenhouwer et al., 1980; Barnes,
1851A 4-14 03/26/84
-------
1983). Observations on PCDO emissions from an Industrial boiler have been
discussed In 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 1n Europe, Lamparskl and NestMck (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 1n 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). Ihese Investigators detected PCDDs Including TCDDs and HxCDDs
1n particulate 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 it. 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.,
1982a, Hernan, 1983), production from the combustion of coal and hydro-
carbons (such as occurs in gas burners, and auto and truck engines) has not
been confirmed (NRCC, 1981a). For example, Rappe et al. (1979) concluded
1851A 4-15 03/26/84
-------
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 1n fireplaces 1s often unknown. Similarly, Klmble and Gross
(1980) and Junk and Richard (1981) have failed to detect even 1 ppt of TCDD
from their analysis of one fly ash sample from stack emissions of a low
sulfur and high-ash coal burning power plant. Recent Investigations (Halley
et al., 1983; Stanley et al., 1982) also failed to detect (detection I1ra1t:
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.
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 kkg/year of hazardous
wastes are produced 1n the manufacture of chlorophenols and -79,000 kkg/year
are produced in the manufacture of phenoxy compounds (Jett, 1982). Process
wastes from the manufacture of chlorophenols and phenoxy compounds are land-
filled, or injected into deep-well. Treatment wastes are frequently sub-
jected to on-s1te impoundment (Jett, 1982). Recent Canadian environmental
data indicates that 2,3,7,8-TCDO 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 dlmerization of chlorophenols to OCDO
has been studied by Crosby and Wong (1976). Lamparskl et al. (1980) also
reported that photolysis of PCP-treated woods may lead to the formation of
1851A 4-16 03/26/84
-------
PCDDs. Similarly, photochemical cycHzatlon of predloxlns (chlorinated
2-phenoxyphenols, precursors of PCDDs) can also produce PCDDs. Since pre-
dloxlns are common Impurities (1-5%) In commercial chlorophenols, exposure
of chlorophenols containing those Impurities to light may produce PCDDs
(Mlsson et al., 1974).
Another photochemical process of potential environmental Importance 1s
the formation of highly toxic TCDD and PeCDD congeners from the dechlorl -
nation of higher PCDDs. However, photolysis of 1,2,3,6,7,8-HxCDD and
l,2,3,7,8,9HxCOD produced only 13% of the toxic 1,2,3,7,8-PeCDO 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 PCDDs (Buser and Rappe, 1978).
4.4. RELATIONSHIP BETWEEN SOURCES AND CONTAMINATION IN ENVIRONMENTAL
MATRICES
The potential relationship between various sources of PCDDs and the
environmental matrices where these compounds have been detected (NRCC,
1981a) is depicted 1n Figure 4-2. Figure 4-2 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, in 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 1n the environment are extremely limited for sev-
eral reasons. The primary reasons are the nonavailability of standardized
1851A 4-17 03/26/84
-------
wood preservation plants
mammals
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
air
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
effluents
receiving waters
— biota
.skin scrapings fftfld .. 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
1851A
4-18
02/28/84
-------
sampling methods and the specialized analytical techniques that must be used
for the determination of traces of these difficult to separate compounds in
the presence of a large number of interfering compounds. Measurable quanti-
ties of these compounds have been detected in the environment under special
circumstances, that is, after accidents in factories producing chlorophenols
and their derivatives, in the environment after certain herbicide use, and
in the environment near certain dumpsites. In other words, the current
available data demonstrate that the major sources of PCDDs in the environ-
ment are those associated with the production, use and disposal of chloro-
phenols and their derivatives. It should also be recognized that most of
the environmental monitoring investigations measured 2,3,7,8-TCDD levels,
whereas monitoring data for other PCDDs are even more limited. With 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 1,2,3,7,8,9-HxCDD in various environmental media have
been presented in the following subsections.
4.5.1. Water. NAS (1977) reported that no 2,3,7,8-TCDD has ever been
detected In drinking water using methods with limits of detection in the ppt
range. Other PCDDs Including PeCDD and HxCDD have not been detected in
drinking water. However, TCDD, including the 2,3,7,8-isomer, has been
reported in aqueous Industrial effluent samples and leachates from hazardous
waste disposal sites. For example, Van Ness et al. (1980) analyzed eight
effluents from a trichlorophenol manufacturing plant site and detected 1CDO
in two of these effluents (detection limit 10-30 pg/g). The concentrations
of TCDD in the two samples with detectable KDD 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-isomer.
1851A 4-19 03/26/84
-------
The analysis of leachate samples from two waste disposal sites for the
analysis of TCDD 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-TCDD 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-TCOO.
The treated leachate (samples taken after remedial steps were Installed to
minimize PCDD-leach1ng 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 2,4,5-1. These locations were an Impoundment from the
drainage of a watershed 1n 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-TCDD at a detection
limit of 0.1-0.2 ppt.
4.5.2. A1r. One possible source of PCDDs In 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 Beall (1980) determined
1851A 4-20 03/26/84
-------
the atmospheric concentration of 2,3,7,8-TCDD at various times after the
application of emulsified and granular Sllvex (1.3-2.0 kg/ha Sllvex) con-
taining 44 ppb to 15 ppm 1CDD 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 (emulslf1able formulation) 1n chambers. The emuls1f1able formulation
resulted 1n considerably higher 1CDD concentrations (-1000-fold or more) 1n
air than In granular formulation Initially, but with time (200 days)
approached the concentrations in air similar to the granular formulation (10
fg/m3; fg : 10"1S g). In a small field trial, with a nonshaded plot,
1CDD concentrations In air from the application of 2 kg/ha of emulsifiable
Sllvex containing 15 ppm KDD 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.
Air filter samples collected from Elizabeth, NJ, after an industrial
fire on April 22, 1980, were analyzed for KDD by Harvan et al. (1981).
Collision-induced-dlssociation 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 in the air cannot be given for these samples because
the air volumes corresponding to the filters analyzed were not specified by
the investigators.
1851A 4-21 03/26/84
-------
The atmospheric concentrations of TCDD near two hazardous waste sites
have been monitored. In one study, U.S. EPA (1982b) failed to detect
(detection limit 1-20 ppt) any 2,3,7,8-TCDO 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 TCDD 1n two air partlcu-
late samples collected near the disposal site was reported (Thlbodeaux,
1983).
Ihe levels of 2,3,7,8-TCDD 1n atmospheric dust were monitored 1n the
Seveso, Italy, area between 1977 and 1979. The concentrations of 2,3,7,8-
1CDD 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 (D10omen1co et al., 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 (Glzzl
et al., 198?; Benfenatl et al., 1983; Taylor et al., 1983; Ol1e 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 1CDD 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 NestMck et
al. (1982). Recently, Karasek et al. (1982) also determined the total TCDD,
1851A 4-22 03/26/84
-------
PeCDD and HxCDD levels In 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 pm
vs. >850 pm).
In another study, Bumb et al. (1980) studied the PCDD level In fly ash
from a municipal Incinerator 1n Nashville, TN, several European municipal
Incinerators, and the Industrial Incinerators of the Dow Chemical Co. facil-
ity In Midland, MI. The 1CDD 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 in 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-
1CDD, 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 dloxins found. In another study (Buser and
Rappe, 1983), the presence of TCDDs (3 ppb), PeCDDs (20 ppb) and HxCDDs (50
ppb) was indicated in the fly ash from a municipal incinerator in 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-HxCOO 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
1851A 4-23 03/26/84
-------
levels of PCDOs In comparison with the Swiss fly ash sample, 1t showed
similar proportions of 2,3,7,8-substHuted PCDOs (4, 12 and 27% of the total
TCODs, PeCDOs and HxCDDs, respectively). It 1s not yet known whether the
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 TCDO Isomers,
but 2,3,7,8-TCDD was found to be a minor product (U.S. EPA, 1980a).
The U.S. EPA evaluated the magnitude and significance of TCDD 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 (CtQ, 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-TCDD (0.4-2 ppt) In eplgeal
parts of a large number of plants grown In the proximity of municipal
Incinerators, and the toxlcologlcal evaluation of 1CDD In 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.
4.5.3. Soil. The levels of PCDDs 1n soil, sediment and dust samples are
presented 1n this subsection. In general, the PCDDs 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 PCDDs besides TCDD. Even 1n the case of
1CDD, the specific Isomer Identification was not performed 1n many cases.
Ihe levels of TCDD in different soil, sediment and dust samples are shown in
Table 4-3.
1851A 4-24 03/26/84
-------
TABLE 4-3
oo
3>
Sample Type
Soils
Se'f'Tients
Soils
Sediments
i
PO
LTt
Sediments
Soils
Soils
Soils
Sediments
Soils
CD
CO
PO
\ Soils
oo
^-
Levels of TCDD In Soils and
Sampling SHe Sample History
Love Canal, NY waste disposal
site
Love Canal, NY sediments from
storm sewers and
creeks near water
disposal site
Love Canal, NY soils collected
away from source
of contamination
Love Canal, NY sediments from
storm sewers
Love Canal, NY sediments from
sump
NR sample originated
from an Industrial
site
Eastern Missouri, sample originated
U.S.A. from contaminated
horse arena
Seveso, Italy sample originated
from ICMESA plant
accident site
canal north of sample originated
Amsterdam from a dump site
Seveso, Italy sample originated
from ICEMSA plant
accident site
Jacksonville, AR waste disposal
site
Sediments from Different Locations
Concentration 1n Sample
Total TCDD 2,3,7,8-TCDD
<0. 0025-6. 7 ppb NR
NR 0.9-312 ppb
NR ND (1-20 ppt)a
NR ND (1-20 ppt)a-
672 ppb
NR ND (1-20 ppt)a-
9570 ppb
ND (20-2300 ppt)a- NR
559 ppb
NR detected6
NR detected6
NR 55-5062 ppt
NR <5-20,000 vg/m2
NR 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
Buser and
Rappe, 1980
Helda, 1983
DIDomenUo
et al., 1980c
Thlbodeaux,
1983
-------
CD
Ul
TABLE 4-3 (cont. )
o
to
CD
Concentration 1n Sample
Sample Type
Sediments
Soil/sludge
Soils
Soil/dust
Soil/dust
Soils
Soils
Sampling SHe
Jacksonville, AR
Love Canal, NY
unspecified
Midwestern
community In
U.S. A
Midland, MI
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 TCDD
NR
0.3-199 ppb
ND (<3 ppt)a-
0.021 ppb
1-1 20C ppb
l-4d ppb
NO (1-10 ppt)a-
0.03 ppbc
ND (1-10 ppt)a-
0.04 ppbd
0.010-0.70 ppbf
12.3 ppb9
31 .8-33.0 ppm
Reference
2,3,7,8-TCDO
ND-22.1 ppb Thlbodeaux,
1983
NR Tlernan, 1982
NR Hryhorczuk
et al., 1981
0.3-100C ppb Bumb et al.,
0.7-3d ppb 1980
NRe Bumb et al.,
1980
NR Cockerham
et al., 1980
NR Carter et al.,
1975
Not detected and the detection limit Indicated within parentheses
Value not quantified
GValue for soil
dValue for dust
eDust sample from St. Louis, MO, area showed 0.12 ppb 2,3,7,8-TCDO.
fTh1s 1s the soil residue after 10 years of periodic aerial spraying of 2,4-D and 2,4,5-T.
"This 1s the soil residue Immediately after spraying.
NR = Not reported; ND = 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 1n the Love Canal, NY, area. This Is
reflected by the high level of 2,3,7,8-TCDD found 1n sediments from sump and
In 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-contamlnated herbicides, such as Sllvex 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 1n the proximity
of municipal Incinerators. Besides determining the PCDD levels 1n food
chains, this subsection will discuss the levels of these compounds in wild-
life and in 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 in Seveso, Italy, 1n 1976
(Fanelll et al., 1982; Cocucd et al., 1979; Pocch1ar1 et al., 1983; Wipf et
al., 1981). 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
1851A 4-27 03/26/84
-------
spraying nor taken up from the soil 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
(400-1000 vg/m* of 2,3,7,8-TCDD 1n soil) Seveso area 1n Italy, Cocucd
et al. (1979) concluded that 2,3,7,8-TCDD 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 1n 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-TCDD 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 1n retail
stores throughout the United States. Contamination of fruits, vegetables or
grains 1n the United States with TCOO has never been reported.
The contamination of a large number of vegetables grown In the proxim-
ity of municipal Incinerators has been reported by Pocchlarl 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
1851A 4-28 03/26/84
-------
to the ICMtSA plant accident. This finding suggests the possibility of
human exposure of TCDD from edible vegetables grown 1n areas close to muni-
cipal 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
(Meselson et al., 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-lCDD 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 1n 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
Fanelll 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 HxCDO (detection limit 25 ppt) In 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).
1851A 4-29 03/26/84
-------
TCODs Including 2,3,7,8-TCDD, PeCODs Including 1,2,3,7,8-PeCDD and
HxCDDs Including 1,2,3,6,7,8-HxCDD have been detected In fish from a few
PCOO-contam1nated areas. This 1s discussed 1n detail 1n Section 6.2.
PCDDs have been detected 1n gelatin samples obtained from supermarkets
and In bulk gelatin (Firestone et al., 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 In
any sample. The origin of PCDDs 1n gelatin was speculated to be PCP and
tMchlorophenol 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
1950's (Firestone, 1973).
Dumb 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 HxCDD (detection limit 10-50 ppt)
in 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 In areas
within the United States that were sprayed with 2,4,5-T revealed no TCDD at
a detection limit of 1-4 ppt (U.S. EPA, 1980a).
The monitoring of urine samples from two personnel 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 PCDDs
cannot be estimated because the levels of these compounds in air, drinking
1851A 4-30 03/26/84
-------
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-
sive study 1s 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 Is 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 in 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 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 further away from the
dump site. This study demonstrates the possibility of TCDD contamination
near dump sites.
1851A 4-31 03/26/84
-------
Ihe results of analysis for ?,3,7,8-TCDD and HxCOD 1n human milk
samples were reported by Langhorst and Shadoff (1980). About 6 of the 9
samples showed 2,3,7,8~1CDD at levels slightly higher than the detection
limits (0.2-0.7 ppt). All nine samples showed HxCOD at levels slightly
higher than the detection limit (0.2-0.5 ppt). However, these results
remain unconfirmed because of the lack of validation of the precision and
accuracy of data. Investigations of 103 breast milk samples from mothers
living 1n areas In the United States sprayed with ?,4,5-T could not detect
any TCDD at a detection limit of 1-4 ppt (U.S. EPA, 1980a).
Ihe monitoring of urine samples from two personnel 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).
In one Polish study (Gorskl, 1981), 1,2,3,6,7,8-HxCDD was detected 1n
latex nipples at a concentration of 20-400 ppt. However, no TCOD or PeCDO
was detected. The origin of PCDDs in the latex was speculated to be the
result of Y-irrad1at1on of latex (for crosslinklng) containing PCP during
its manufacturing process.
A BCF relates the concentration of a chemical in aquatic species to the
concentration in water. The steady-state BCFs for a lipld-soluble compound
1n the tissues of various aquatic species seem to be proportional to the
percent lipid in the tissue. Thus, the per capita ingestion of a lipld-
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 In 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
estuarine fish and shellfish in the United States is 6.5 g/day (Stephan,
1851A 4-32 03/26/84
-------
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.OX.
Several equations have been developed for predicting the steady-state
BCf for an organic compound from Us octanol-water partition coefficient
(Kenaga and Goring, 1980; Velth et al., 1980; Velth and Kosian, 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-
TCOO. Use of six equations with four values for the partition coefficient,
K , results In 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-TCOD (Table 4-5),
but none can be considered definitive values. Many were determined In model
ecosystems In which the concentrations In 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 1s 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 7.6% I1p1ds on which the equation 1s based
to the 3.0% llplds 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-lCDD In the edible portion of all freshwater and estuarlne
aquatic organisms consumed by Americans 1s calculated to be 5000x0.395-1975.
1851A 4-33 03/26/84
-------
TABLE 4-4
Predicted BCFs from Calculated and Measured Values of Kow a
log
log
log
log
log
BCF
BCF
BCF
BCF
BCF
BCF
= 0
- 0
-- 0
- 0
-- 0
- 0
.048
Equation
.542 log Kow + 0.124
.76 log Kow - 0.23
.79 log Kow - 0.40
.635 log Kow + 0.7285
.85 log Kow - 0.70
Kow
log
Kow
Calculated
6.84
6
93
101
118
130
332
,780
,000
,000
,000
,000
,000
7.1
9,
157,
174,
183,
234,
663,
4
860
000
000
000
000
000
7.28
11
201
224
225
308
915
,700
,000
,000
,000
,000
,000
Measured*'
6
2
27
28
43
33
67
.15
,870
,800
,740
,000
,700
,800
aSources: Kenaga and Goring, 1980; VeHh et aU, 1980; VeHh and Koslan,
1983
blh1s measured value has been reported by Neely, 1979
1851A 4-34 02/28/84
-------
CD
en
i
CO
en
ro
oo
CO
TABLE 4-5
Measured Bioaccumulation Factor for 2,3,7,8-TCDD In Freshwater Aquatic Organisms
Species Tissue
Alga, NR
Oedogonlum cardiacum
Alga, NR
Oedogonium cardiacum
Snail , whole body
Physa sp.
Snail , whole body
Physa sp.
Cladoceran, whole body
Daphnia magna
Cladoceran, whole body
Daphnia magna
Catfish, whole body
Ictalurus punctatus
Mosqul tof ish , whole body
Gambusia affinis
__
--
Percent
Lipid
NR
NR
NR
NR
NR
NR
NR
NR
NR
NR
Duration
(days)
33
32
33
32
30
32
28
14
-
Bioconcentration
Factor
3094a
2075b
2083
5471a
2095b
3731
3895a
7070b
7125
2000
4850b
4875
9080C
5400
Reference
Isensee, 1978
Isensee, 1978
Yockim et al . ,
Isensee, 1978
Isensee, 1978
Yockim et al. ,
Isensee, 1978
Isensee, 1978
Yockim et al . ,
U.S. EPA, 1983a
Thomas, 1983
Isensee, 1978
Yockim et al . ,
Neely, 1979
Kenaga, 1980
1978
1978
1978
>
1978
These are arithmatic mean of several values given
These are values at equilibrium tissue concentrations
""Calculated as ratio of uptake and clearance rate constants
NR = Not reported
-------
Uptake by fish from lower tropic levels may add to uptake from water, so
this BCF may underestimate concentrations 1n wild aquatic organisms.
The BCF for 2,3,7,8 TCOO 1n the earthworm, Allobophora cal1q1nosa or
rosea, from soil with Initial 2,3,7,8-TCDD concentration In the range of
0.06-9.2 ppb has been determined to be about 10 (Fanelll 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 1n 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 2,4,5-1 and Sllvex. At the present time, there 1s no
known manufacturer of trlchlorophenol 1n the United States. Its derivatives
distributed in the market before banning, however, continue to be used as
pesticides in the United States. The level of 2,3,7,8-TCDD contaminants 1n
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 In 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 in environmental samples has
only been reported in emissions from incinerators.
1851A 4-36 03/26/84
-------
PCDDs, particularly TCDD and Us specific Isomer 2,3,7,8-TCOO, have
been monitored 1n a number of environmental media, Including air, water,
soil, food and biological media. The monitoring data to date Indicate that
the maximum level of PCDDs 1s likely to be found 1n soil and drainage sedi-
ment 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 In fish and
wildlife 1n the United States 1n areas around chlorophenol manufacturing
Industries and chemical waste disposal sites.
1851A 4-37 03/26/84
-------
5. ENVIRONMENTAL FATE AND TRANSPORT PROCESSES
5.1. FATE
5.1.1. Water.
5.1.1.1. BIODEGRADATION — 2,3.7.8-TCDD exhibits relatively strong
resistance to blodegradatlon. Only 5 of -100 mlcroblal 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-TCDO
in 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. (198?) and Matsumura et al. (1983) estimated
the apparent half-life of 2,3,7,8-TCDD to be ~1 year. Although blodegrada-
tion may have been responsible for part of the degradation, 1t 1s 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; Camonl 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 biodegradation 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
1852A 5-1 03/26/84
-------
constant of 1x10 10 mfc cell * hour"1 (Mabcy et al., 1981) and the
concentration of microorganisms capable of degrading KDD as 5xlOs cell
ml"1 (Burns et al., 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 PCOOs 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 H"1 cm"1 (NRCC,
1981a). 2,3,7,8-TCDD In a pure state 1s photochemically stable but 1t will
photolyze In sunlight 1n the presence of a hydrogen atom donating substrate
(Crosby and Wong, 1977). For example, Pllmmer 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-KDD on dry and wet soils showed negligible photodegrada-
tion after Irradiation with sunlamps (Crosby et al., 1971). In contrast,
2,3,7,8-lCDD In methanol solution or benzene solution of 2,3,7,8-TCDD 1n
water stabilized by surfactant underwent substantial photodegradatlon under
sunlamp or sunlight Irradiation (Pllmmer 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-TCDO in a model laboratory ecosystem than In an outdoor
pond, Matsumura 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 photosensitlzatlon of 2,3,7,8-TCDD may cause some photode-
composltlon of this compound. Nestrlck et al. (1980) estimated the photo-
1852A 5-2 03/26/84
-------
lytic half-life of 2,3,7,8-TCDD 1n n-hexadecane under sunlamp Irradiation to
be -57 minutes. From the available Information, H Is difficult to predict
the fate of 2,3,7,8-TCDD In 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 1n chlorine substitution Is expected to decrease the rate of
photodegradation (NestMck et al., 1980; Helling et al., 1973). For
example, Crosby et al. (1971) showed that although complete decomposition of
2,3,7,8-TCDD in methanol occurred 1n 24 hours under UV Irradiation, >80%
OCDD in methanol remained unreacted during the same period under similar
irradiation conditions.
Although the degree of photolysis may be related to the extent of
chlorination, positional 1somer1zat1on also plays a critical and perhaps
dominant part In the photolysis of higher PCDDs. In higher PCDDs, there
appears to be preferential loss of chlorine from the 2, 3, 7 and 8 positions
(Nestrick et al., 1980; Buser and Rappe, 1978). Thus, PCDD compounds with
chlorine substitutions in positions 2, 3, 7 and 8 are likely to photodegrade
faster than compounds not having these positional substitutions. According
to such a predicted rule, it is not likely that photodegradation of OCDD and
other higher PCDDs will yield 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 In n-hexadecane solution under sunlamp Irradiation (Nestrick 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 in hexane solutions under sunlight
irradiation have been determined to be 5.4, 17 and 47 hours, respectively
(Dobbs and Grant, 1979). Nestrick et al. (1980) reported a half-life value
1852A 5-3 03/26/84
-------
of 6.8 hours for 1,2,3,6,7,8-HxCDD In n-hexadecane under sunlamp Irradia-
tion. The Intermediates of the photodegradatlon of higher PCDDs are prob-
ably lower chlorinated dloxlns, but the pathways of degradation are not
known with certainty (NRCC, 1981a).
From the preceding discussions of the photolysis of PCDDs In the
presence of organic hydrogen donating substrates, 1t Is 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 (RO •,
R0«, etc.) 1n 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 Matsumura, 1977; Matsumura et al., 1983; Huetter and
Ph1lipp1, 1982). 2,3,7,8-KDD may undergo some water-mediated evaporation
1n aquatic media (Matsumura et al.f 1983). Using the formulas of Liss and
Slater (1974), a vapor pressure value of 1.7xlO~6 torr (0.2 m Pa) and a
1852A 5-4 03/26/84
-------
solubility value of 6.2xlO~10 mole/a, 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 in 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; soe NRCC, 1981a, for definitions) and the Input
parameters for 2,3,7,8-TCDD given In NRCC (1981a), volatilization has been
estimated to account for 100% of the fraction lost; blodegradatlon has been
calculated to be 0%. 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 m2), the calculated volatilization rate was 15-16 mg/year.
Pertinent data regarding the volatilization of PeCODs and HxCDDs from
aquatic media could not be found In 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 Matsumura, 1978). More than 90% of 2,3,7,8-TCDD In an
aquatic medium may be present In the adsorbed state (Ward and Matsumura,
1852A 5-5 03/26/84
-------
1978; Matsumura et al., 1983). Considering the low water solubility and the
high octanol/water partition coefficient, this 1s not surprising. In fact,
the equation of Karickhoff et al. (1979) predicts a sorptlon partition co-
efficient value of 10" for 2,3,7,8-TCDO In sediments containing 2% organic
carbon. Similarly, the higher PCODs are likely to be present predominantly
in the sediment-sorbed state in aquatic media.
5.1.2. Air. A number of PCODs, including TCOOs, PeCODs and HxCOOs, have
been detected In the dust and fly ash from municipal incinerators (Cavallaro
et al., 1980b; Clement and Karasek, 1982; Elceman et al., 1981). Size
fractionations of fly ash from municipal incinerators have shown that larger
concentrations of 2,3,7,8-TCOD and PeCDDs occurred on the larger (550 ym)
particles, while the 30 v»m particles had greater relative concentrations
of OCDD (Clement and Karasek, 1982). Tiernan et al. (1982b) also reported
higher concentrations of ICDDs on larger particles (3-10 ym) 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 particulate matter (Nestrlck et al., 1980).
Atmospheric PCDDs originating from other noncombustlon sources, such as
herbicide-treated soils and vaporized PCDDs from aquatic media (Thibodeaux,
1983), are also likely to be associated with air particulate matter. CupHt
(1980) presented mathematical descriptions of physical removal mechanisms
for the fate of toxic and hazardous materials in 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
1Q~7 torr (mm) or less. 2,3,7,8 TCDD has an estimated vapor pressure of
1.7x10~6 torr.
1852A 5-6 03/26/84
-------
Photodegradatlon and wet and dry deposition of particulate-bound PCDDs
are probably the most Important fate-determining processes for the atmo-
spheric 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 al., 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, photodecomposltlon of
TCDD 1n 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-lCDD to light of wavelength >290 nm and observed 92% decomposition
1n 7 days. The half-life for photodegradation of 2,3,7,8-TCDD film on glass
surfaces has been estimated to be 5.8 days under Irradiation with sunlamps
(Nestrick et al., 1980). It is not known whether a similar photodegradatlon
of particle-bound 2,3,7,8-TCDD will occur in the atmosphere since the state
of sorption 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 in the
*3 X
atmosphere 1s unknown.
5.1.3. Soil.
5.1.3.1. SORPTION — From the empirical correlation of Karlckhoff et
al. (1979), It 1s possible to predict a soil/water partition coefficient of
4.8xl04 for a soil containing 10% organic matter.
1852A 5-7 03/26/84
-------
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-TCOO (and presumably other PCDOs) 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
Ph1l1ppi, 1982).
Several authors have shown that vertical movement of 2,3,7,8-TCOO 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 In soil Increases. In areas of heavy rainfall
and sandy soil, vertical migration of 2,3,7,8-TCDD and Its 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 (DIDomenico 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 In 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
air/soil Interface or solvatlon of 2,3,7,8-7CDD 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-TCOD on soil.
1852A 5-8 03/26/84
-------
5.1.3.2. PHOTOTRANSFORMATION — The photodecomposHlon of 2,3,7,8-
7CDD on wet or dry soil under artificial and natural sunlight was studied by
Crosby et al. (1971). The photodecomposHlon was found to be negligible 1n
soils. Similarly, PUmmer et al. (1973) determined that photodecomposHlon
of TCDD on soils was too slow to be detected. In a later experiment,
PUmmer (1978) found that although TCOD 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 1CDD 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/cm3),
rubber plant, Hevea brasH1ens1s (6.7 mg/cm2), and on the surface of
sieved Sacramento loam soil (10 mg/cm2) and exposed to sunlight, TCDO was
found to photodecompose. The loss of TCDD In 6 hours was >50% from glass
plate, -100% from the surface of leaves and -10% from the surface of soil.
The rapid photolysis of 1CDD from these surfaces Indicates that the herbi-
cide formulation provided a hydrogen donor which 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. BIODEGRADATION — Polger and Schlatter (1980) noted that
2,3,7,8-TCDD absorbs strongly onto soil particles, thereby reducing Its
bloavailabllHy. 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 In soil 1s likely to
be >1 year.
1852A 5-9 03/26/84
-------
The overall half-life of 2,3,7,8-KDD 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
1CDD. The half-life of this chemical in soils under relatively dry condi-
tions (Utah test area) was found to be -330 days and In 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 TCOO
determined by Commoner and Scott from the soil 1n rural Missouri after the
accidental spraying of TCOD-contam1nated oil (IARC, 1977). However, these
half-life estimates may greatly underestimate the true value, since 1t has
recently been shown that radiolabeled TCOO adsorbed to soil becomes progres-
sively more resistant to extraction (Phlllppl et al., 1981; Huetter and
Ph1l1ppi, 1982).
The rate of disappearance of 2,3,7,8-TCOD following an accidental
2,3,7,8-TCOO release from a trichlorophenol manufacturing plant at Seveso,
Italy, was studied by DIDomenico 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 in soil 1s about 10-12
years. Since most of the other PCOOs are no more susceptible to transforma-
tion/degradation than ICDDs, their half-lives 1n soil are presumed to be
similar to that postulated for TCDDs.
1852A 5-10 03/26/84
-------
5.1.4. Food. Isensce 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 In soil contami-
nated with 0.06 ppm 2,3,7,8-TCDD showed <1 ppb of 2,3,7,8-TCDD In the seeds.
Cocucci 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 In 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.
Wipf et al. (1982), however, failed to detect any measurable 2,3,7,8-TCDD in
the flesh of fruits and vegetables collected from the contaminated area in
Seveso during 1977-1979, although the soil 2,3,7,8-TCOD 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 Pocchiarl et
al. (1983) from their uptake experiments with plants. It can be concluded
from these studies that 2,3,7,8-TCDD is not likely to concentrate 1n plants
grown In 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, photodecomposition was negligible.
However, in 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 1CDD was found not to persist on foliage (Sundstrom et
al., 1979; Crosby and Wong, 1977) after application with other pesticides
1852A 5-11 03/26/84
-------
(2,4,5-1, 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 al., 1983).
Cattle fed rations fortified with a maximum of 90 ppt 7CDD were monitored
for KOO content 1n the body fat. TCDD 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-TCOO showed a maximum
level of 90 ppt of 2,3,7,8-TCDD In 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. Water. The two likely transport processes for PCDDs In 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 m1crob1al
studies (Ward and Matsumura, 1977; Huetter and Ph1l1pp1, 1982). There Is a
very wide difference in the calculated values of half-life of volatilization
for 2,3,7,8-lCDD. For example, calculation based on the Llss 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~], 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-lCDD in pond, river and lake water, respectively.
These wide variations are conceivable when examined with the volatilization
1852A 5-12 03/26/84
-------
models for half-life (Ihlbodeaux, 1979). Evaporation half-life Is 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 TCDO 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 TCDO, the evaporation rate 1s reduced significantly. A
comparison of calculated transport rates from an Industrial site Indicates
that evaporation of TCDO from a contaminated cooling water pond sediment 1s
negligible 1n 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 PCOOs from aquatic media.
It has already been shown (see Section 5.1.1.5.) that 2,3,7,8-TCDD 1s
highly sorbed to sediments and biota (Isensee and Jones, 1975) and >90% of
2,3,7,8-lCDD In aquatic media may be present In the sorbed state (Ward and
Matsumura, 1978). 1h1s 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 1n aquatic media.
5.2.2. A1r. All the PCDOs are believed to be transported In the vapor-
phase and 1n partlculate bound form In 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 (Josephson, 1983). Although such
1852A 5-13 03/26/84
-------
dispersion modeling has been performed for 2,3,/,8-lCDD (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 ROD at Sevcso, Italy, Cavallaro et al. (1982) determined the
transport pattern and the ground deposition of the TCDD from the cloud.
They determined that the TCDD 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 (yg/m2) should be Y =- 2900 e~2-3x for x<2 km and y = 45
-0 5x
e ' for 2 km
-------
between 120 and 1200 g/year of 7CDD 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 soil and downward migration caused by
soil 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. BIOACCUMULATION/BIOCONCENTRATION
The bloconcentratlon of TCDO 1n various aquatic species has been studied
under controlled laboratory conditions using static test chambers. The
results of these Investigations have been discussed 1n Section 4.6. and are
given In Table 5-1. In all these experiments, the total amounts accumulated
were found to be related to the Initial TCDD concentrations In aquatic
phase. The Investigation of Ph1l1pp1 et al. (1981) made 1t clear that
bloaccumulatlon would be significantly affected by the physical form (sorbed
or In solution) in which ICDD occurs 1n the environment. Isensee (1978)
reported that the concentration in the tissues of the tested species reached
equilibrium in 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 (Fanelll et al., 1982).
1852A 5-15 03/26/84
-------
oo
tn
PO
*" B1oconcentrat1on
Species
Algae, Oedogonlum cardlacum
Algae, Oedoqonium cardlacum
Algae, Oedogonlum cardlacum
Ostracod
i
Duckweed, Lemna minor
Snail, Physa sp.
Snail, Physa sp.
Snail, Helosoma sp.
Daphnlds, Daphnla magna
Daphnids, Daphnla magna
lABLt b-l
Factor of TCDD for Several
Initial Aquatic
Concentration (ppt)
0.05-1300
0.05-1300
2.6
0.05-1300
0.05-1300
0.05-1300
0.05-1300
0.05-1300
Aquatic Organisms3
Bioconcentratlon
Factor
2,075
9,000b
2,080
110
3,625b
2,095
20,000b
2,080
7,070
26,000b
Reference
Isensee, 1978
Isensee and Jones,
1975
Yockim et al . ,
1978
Matsumura and
Benezet, 1973
Isensee and Jones,
1975
Isensee, 1978
Isensee and Jones,
1975
Yockim et al . ,
1978
Isensee, 1978
Isensee and Jones,
o
CO
OO
Daphnlds, Daphnla magna
0.4
2,200
1975
Matsumura and
Benezet, 1973
-------
CO
en
r>o
3>
1ABLE 5-1 (cont.)
o
CO
r\j
co
-P.
Mosquito
Mosquito
Mosquito
Mosquito
Brine shr
Catfish,
Catfish,
Brook Sil
Pond Weed
Species
fish, Gambusia affinis
fish, Gambusia affinis
fish, Gambusia affinis
larvae, Aedes aeqypti
imp, Artimia salina
Ictalurus punctatus
Ictalurus punctatus
verside, Laludesthes sicculus
, Elodea nuttali and
Ceratophyllon demersum
Initial Aquatic
Concentration (ppt)
0.05-1300
0.05-1300
O.lc
0.45
0.1^
0.05-1300
0.05-1300
1.3
53.7
Bioconcentration
Factor
4,850
26,000°
4,875
9,200
1,570
9,000b
4,875
545d
30,300
Reference
Isensee, 1978
Isensee and Jones ,
1975
Yockim et al . ,
1978
Matsumura and
Benezet, 1973
Matsumura and
Benezet, 1973
Isensee and Jones ,
1975
Yockim et al. ,
1978
Matsumura and
Benezet, 1973
Tsushimoto 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.
dError in the original publication corrected in the value reported here.
-------
5.4. SUMMARY
The four transformation processes {photoreactlon, blotransformatlon,
hydrolysis and radical oxidation) that control the fate of a chemical 1n
aquatic media do not appreciably transform TCDO and possibly other PCDOs 1n
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 In 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 1n aquatic media.
The potential for oxidation of PCDDs by tropospherlc free radicals 1s
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 parUcle-bound PCDOs may be
theoretically predicted from dispersion modeling equations. In the case of
accidental release of toxic clouds containing TCDD at Seveso, Italy, 1t 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 In soils may be >10 years. These
compounds are likely to be transported from soil through movement of
partkulate matter containing sorbed PCDDs. The most probable transport
mechanisms are transport of these compounds to the atmosphere via contami-
nated airborne dust particles, evaporation, and transport to surface water
1852A 5-18 03/26/84
-------
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 In 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-
tion factors for these compounds 1n different aquatic species.
1852A 5-19 03/26/84
-------
6. ECOLOGICAL EFFECTS
6.1. EFFECTS ON ORGANISMS
6.1.1. Aquatic Life Toxicology. Almost all of the available Information
concerning the toxlcity of PCDDs 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-TCOO.
6.1.1.1. ACUTE TOXICITY — The effects of acute exposure to 2,3,7,8-
TCOO 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-lCDD concentrations for up to 96 hours. Concentrations were
expressed as ng/g wet bw and as ng/«. of. water, based on the amount of
2,3,7,8-TCDD added to the water 1n 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 In 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 yg/kg bw (0.0056
pg/8,), but not at 0.54 Pg/kg bw (0.00056 vq/l) or lower. Growth
and survival of larger fish were measured until 114 days after exposure and
were significantly reduced at 5.4 pg/kg bw (0.0105 pg/8.) but not at
0.54 yg/kg bw (0.00105 yg/t) 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.
1853A 6-1 03/26/84
-------
TABLE 6-1
CXI
01
00
Species
Coho Salmon,
Oncorhynchus klsutch
Coho Salmon,
Oncorhynchus klsutch
Rainbow Trout,
Sal mo galrdnerl
Rainbow Trout,
Sal mo galrdnerl
Rainbow Trout,
Salmo galrdnerl
i
r\j
Guppy,
Poedlla retlculata
Guppy.
Poedlla retlculata
Northern P1ke,
Esox ludus
Northern P1ke,
Esox ludus
Frog,
Rana catesblana
: Frog,
Rana catesblana
0
ro
E
Life Stage, Duration
Height or of Exposure
Length (hours)
3.5 g 96
6.6 g 96
eggs and 96
larvae
eggs and 96
larvae
0.85 g 96
9-40 mm 120
8-12 mm 24
eggs and 96
larvae
eggs and 96
larvae
larvae 1.p.
Injection
adults 1.p.
(150-250 g) Injection
ffect of Acute Exposure to 2,3,7,8-TCDD on Aquatic Animals
Duration LCso LT50a Lowest Effect No Effect
of Test (wgA) (days) Concentration Concentration Effect
(days) Ug/i) (yg/t)
64 0.0056 60 0.0056 0.00056 reduced growth, food
consumption, survival
114 0.0105° 114 0.0105 0.00105 reduced growth, food
consumption, survival
72 NR NR 0.0001 NO temporary growth
Inhibition
164 NR NR 0.001 0.0001 teratologlc effects,
decreased survival
and growth
72 NR NR 0.010 NR decreased survival
and growth, hlsto-
loglcal effects
37 NR 21.70.1 ND 100X mortality by
3.7 days after
beginning exposure
69 NR NR 0.0001 0.00001 higher Incidence
of fin necrosis
23 NR NR 0.0001 ND temporary Inhibition
of egg development
23 0.001 23 0.001 0.0001 decreased survival
and growth
50 NR NR ND 1000 Mg/kg bw no effect on survival
metamorphosis,
histology
35 NR NR 500 yg/kg bw 250 yg/kg bw 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, 1980
Helder, 1980
Beatty et al., 1976
Beatty et al., 1976
rv
t-O
CO
aLT5Q = median lethal time 1n days after beginning exposure
b47X mortality
NR = Not reported; ND = Not determined
-------
Acute exposure experiments were also conducted by these researchers
(Miller et al., 1973, 1979; Norrls and Miller, 1974) with gupples, PoedHa
retlculata. Miller et al. (1973) and Norris and Miller (1974) reported the
effects of exposing gupples to nominal concentrations of 0.1, 1.0 and 10.0
yg/8. for 120 hours followed by transfer to clean water. Some fish
(8-18%) died In each test concentration during the exposure period. All
treated fish died by 37 days after beginning exposure, and smaller fish
generally died first. F1n necrosis was observed In all fish surviving more
than 10 days. In a later study, Miller et al. (1979) measured the Incidence
of fin necrosis 1n gupples exposed for 24 hours to much lower nominal con-
centrations 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 yg/kg bw
(0.0001 yg/a.) than In controls or In fish exposed to 0.08 yg/kg bw
(0.00001 yg/l).
The effects of static acute exposure to 2,3,7,8-TCDD on eggs and larvae
of northern pike, Esox luclus, 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-lCDD concentrations (0.0001, 0.0010, 0.010 yg/8.) followed by
transfer to clean water. There was no significant Increase in egg mortality
up to the highest nominal test concentration of 0.010 yg/a. for either
species. Significantly greater mortality occurred after hatching and during
yolk sac absorption in both species at concentrations as low as 0.0010
yg/ft.. Total mortality of pike fry reached 99% at 0.010 yg/a. and 50%
at 0.0010 yg/a. by 23 days after fertilization. Total mortality of trout
fry was 26% at 0.010 yg/a. and 12% at 0.0010 yg/j.. Although cumula-
tive mortality was not significantly Increased at the lowest test concentra-
1853A 6-3 02/29/84
-------
tion (0.0001 pg/S,), sublethal effects occurred In both species. At this
concentration, growth was significantly, but temporarily, retarded In both
species.
Helder (1981) also exposed juvenile trout to nominal concentrations of
0.100 and 0.010 vg/s, for 96 hours and followed growth and survival for
72 days. Growth was significantly reduced In both groups. Mortality
reached 100% by 27 days at the highest concentration, but was only 7% at the
lowest concentration.
The only other study regarding the effects of acute exposure on aquatic
animals is that of Beatty et al. (1976), who Investigated the effects of
single Intraperltoneal Injections of 2,3,7,8-TCDD 1n larval and adult frogs,
Rana catesblana. Groups of 15 tadpoles and 5 adults were Injected with
2,3,7,8-TCDD In olive oil at maximum nominal dosages of 1000 and 500 pg/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. Histopathologlcal examination revealed no
significant lesions in metamorphoslzed or adult frogs. The lack of toxlclty
in this amphibian species Is in sharp contrast to the results previously
described with fish. Although the difference may be due, 1n part, to the
different routes of exposure, H is 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 (Miller et al., 1973, 1979).
6.1.1.2. CHRONIC TOXICITY -- The effects of chronic or subchronic
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 oligochoete worms, Paran-
aVs_ sp., and mosquito larvae Aedes aegypti to a nominal Initial concentra-
1853A 6-4 02/29/84
-------
CO
TABLE 6-2
Effects of Chronic or Subchronlc Exposure to 2,3,7,8-TCDD on Aquatic Animals
1
0
rv>
LD
CD
-fa.
Life Stage,
Species Weight or
Length
Mosquito, larvae
Aedes aegyptl
OUgochaete Worm, adult
Paranals sp.
Snail, adult
Physa sp.
Snail, adult
Helosoma sp.
Waterflea, adult
Daphnla magna
Mosqu1tof1sh, NR
Gambusla aff 1n1s
Channel Catfish, flngerllngs
Ictalurus punctatus
Rainbow Trout, 7.8 cm
Sal mo galrdnerl
NO = Not determined
Duration Duration Lowest Effect No Effect
of Exposure of Test Concentration Concentration Effect
(days) (days) (yg/l) (jjg/l)
17 30 ND 0.2 no effect on pupation
55 55 0.2 ND reduced reproduction
36 48 0.2 ND reduced reproduction
32 46 ND 0.003 no apparent effects
32 32 ND 0.003 no apparent effects
15 15 0.003 ND 100% mortality
20 20 0.003 ND 100% mortality
105 105 2300 yg/kg 2.30 yg/kg reduced survival,
1n diet In diet food consumption and
growth, Increased
fin erosion
Reference
Miller et al.,
1973
Miller et al.,
1973
Miller et al.,
1973
Yocklm et al. ,
1978
Yocklm et al.,
1978
Yocklm et al.,
1978
Yocklm et al. ,
1978
Hawkes and NorMs,
1977
-------
tlon of 0.20 pg/s. 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 yg/8. 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 -30% 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 yg/a. exerted Its principal effect on reproduction rather than
Individual worm growth.
Miller et al. (1973) also conducted chronic feeding studies with rainbow
trout. The results of this study were also reported by Hawkes and NorMs
(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-TCDD/kg freeze-dry bw/day.
Consumption of food containing 0.0023 and 2.3 yg/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, mosqultof 1sh, 6ambus1a aff1n1s, waterfleas, Daphnla
1853A 6-6 02/29/84
-------
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/a) In 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 3?. The mean concentra-
tion through the 32-day period was 0.0031 yg/8.. 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
mosqu1tof1sh died by day 14, with a mean tissue concentration of 7.2 yg/kg
bw. A second group of mosqu1tof1sh 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/S. 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 In three places with 1 yg
of 2,3,7,8-TCOO. 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 motlllty of the algae filaments outward from the disks was
measured over a 3-hour period with a photoelectric cell. Relative to
controls, 1 pg of 2,3,7,8-TCDD caused a significant Inhibition of
1853A 6-7 03/26/84
-------
motllity. Although the exposure concentration Is 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 In the African blood
lily, 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/5L, 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 In the test chamber were
actually lower than reported. It was estimated that the higher concentra-
tion may possibly be approaching 0.2 pg/S,, the solubility of 2,3,7,8-
TCDD In water.
6.2. TISSUE RESIDUES
Levels of 2,3,7,8-TCDD In several species of commercial fish taken from
eastern Lake Ontario, Lake Erie and the Welland Canal ranged from 0.002-
0.039 pg/kg In those fish with positive test results (Josephson, 1983).
Rock bass showed no detectable levels. Highest concentrations generally
occurred in eels (0.006-0.039 yg/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-TCDO
1n 46 muscle {fillet) samples of Lake Ontario fish that ranged from
0.002-0.162 yg/kg 1n 45 samples and were undetectable 1n one sample (NRCC,
1981a). The fish that were sampled Included smallmouth bass, lake trout,
1853A 6-8 02/29/84
-------
white sucker, brown bullhead, rainbow trout, coho and chlnook salmon, and
brown trout. The Ontario Ministry of the Environment (NRCC, 1981a) reported
concentrations of 2,3,7,8-TCDD ranging between 0.010 and 0.019 yg/kg 1n
fillet samples of lake trout, brown trout, white bass, white perch and smelt
In Lake Ontario, but no detectable (<0.010 yg/kg) levels in 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 in
positive samples ranging from 0.020-0.230 pg/kg In THtabawassee River,
Saglnaw Bay and other locations near Midland, MI; 0.015-0.480 v"9/kg In the
Arkansas River; and 0.019-0.102 ng/kg In Lake Ontario and Niagara River.
OCDD concentrations In fish ranged from 0.040-0.150 pg/kg near Midland,
MI, and from 0.004-0.078 vg/kg 1n the Honesatonlc River. The levels of
2,3,7,8-lCDD in fish and shellfish as determined by various authors are
given in lable 6-3.
Levels ranging from 0.004-0.695 vg/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 Saginaw
Rivers, Lake Michigan and Saglnaw Bay. The highest concentrations were
detected in bottom-feeding catfish and carp, and the lowest concentrations
were detected In bass, perch and suckers (Harless and Lewis, 1980b).
Young et al. (1976) measured 2,3,7,8-TCDD residue levels in terrestrial
and aquatic animals from contaminated areas of Eglin Air Force Base, FL,
which had received massive amounts of herbicides, one of which (2,4,5-T) was
contaminated with 2,3,7,8-lCDD. Beach mice from contaminated areas
contained 0.540-1.30 pg/kg in the liver and 0.130-0.140 yg/kg in pelts.
Residues In racerunner lizards trapped from the most highly contaminated
1853A 6-9 02/29/84
-------
oo
U1
GJ
I
o
o
00
CD
TABLE 6-3
Levels of 2,3,7,8-TCDDs in Fish and Shellfish
Type/Section
of Fish
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
Sampling Site
Bayou Meto/Arkansas River
Bayou Meto/Arkansas River
Bayou Meto/Arkansas River
Bayou Meto/Arkansas River
Tone River, Japan
Lake Ontario/Lake Erie/
Welland Canal
Lake Ontario/Lake Erie/
Welland Canal
Bergholtz Creek, Love Canal
2,4,5-T contaminated
watershed in Arkansas and
Texas; Tittabawassee and
Saginaw Rivers
Lake Ontario
Lake Ontario
Lake Ontario
Lake Ontario
Lake Ontario
Lake Ontario
Lake Ontario
Concentration
(ppt)
480
NO (7 ppt)a-50
ND (7-13 ppt)a
77
200
ND (<2 ppt)a
2-39
3.7
ND (5-10 ppt)a
51-107
26-39
20-26
17-32
8-162
17-26
ND (3.2)-10
Reference
MHchum et al., 1980
Mitchum et al., 1980
Mitchum et al. , 1980
MHchum et al., 1980
Yamagishi 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
-------
oo
LTI
OJ
TABLE 6-3 (cont.)
CO
Type/Section
of Fish
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
Saginaw Bay
Saginaw Bay
Saginaw Bay
Concentration
(ppt)
5.9
3.6
87
32
31
12
1.4-0.5
2.6
1.6-<2.4
NO (2.6)
21
26
20
25
NO (8.7)
NO (3.8)
1.0
ND (3.8)-25
14-37
23-47
Reference
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.,
O'Keefe et al.,
Niemann et al . ,
Niemann et al . ,
Niemann et al . ,
1983
1983
1983
1983
1983
1983
1983
1983
1983
1983
1983
1983
1983
1983
1983
1983
1983
1983
1983
1983
-------
09
tn
TABLE 6-3 (cont.)
Type/Section
of F1sh
Sampling Site
Concentration
(ppt)
Reference
FNJ
CD
Catfish
Bottom feeders
Lake trout
Rainbow trout
Ocean haddock
Carp
Carp
Carp
Carp
Lake trout
Brown trout
Yellow perch
Channel catfish
Carp
Yellow perch
Small mouth bass
Bayon Meto/Arkansas River
Bayon Meto/Arkansas River
Lake Ontario
Lake Ontario
Atlantic Ocean
Lake Huron
Saglnaw Bay
Bay Port
TUtabawassee River
Lake Michigan
Lake Ontario
Woods Pond, MA
Tlttabawassee River,
Saglnaw River and
Grand River
Tlttabawassee River,
Saglnaw River and
Grand River
Tlttabawassee River
and Saglnaw River
Grand River
ND (3.8)
NO (6.7J-12
34-54
43
ND (4.6)
3-28
94
27
81
5
33
26
157 (13)c
55 (7)C
13 (5)C
8 (6)C
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
-------
cc
on
CO
TABLE 6-3 (cont.)
Type/Section
of Fish
Sampling SHe
Concentration
(ppt)
Reference
Sucker
Trout
Trout
Trout
Trout
THtabawassee River
and Saginaw Bay
Lake Michigan
Lake Ontario at
Burlington, Canada
Lake Ontario at Toronto
Harbor, Canada
Lake Huron at
Burnt Island, Canada
10 (4)c
NO (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
o
IX)
Not detected and the detection limit is indicated within the parentheses.
Only 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,
NO = Not detected
CD
•*»•
-------
areas contained 0.36-0.37 pg/kg 1n the visceral mass and trunk, respec-
tively. Residues were also found In three fish species taken from a stream
and pond In the contaminated area. Residue levels of 0.012 pg/kg were
found 1n the viscera of sallfln shiners and In the bodies (heads and tails
removed) of mosqultof1sh. Samples of skin, muscle, gonad and gut of spotted
sunflsh contained 0.004, 0.004, 0.018 and 0.085 pg/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 pg/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 is near a major 2,4,5-T manufac-
turing plant on the Saginaw/T 1 ttabawassee 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 Eglin Air
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-lCDD levels of 0.010-0.710 pg/kg. Large numbers of beach
mice were trapped from contaminated and control sites and evaluated for
differences 1n organ weights and histopathology. The only significant
differences in organ weight were Increased liver weight 1n females and
1853A 6-14 03/26/84
-------
03
TABLE 6-4
TCDD Levels In Wildlife
O
1X5
no
to
2.3.7.8-TCDD Concentration (ppb)
Type of
Animal
Rabbit
Field mouse
Hare
Toad
Snake
Snake
Earthworm
Eagle
Herring quill
Tissue Sampling Sit«
Average3
liver Seveso, Italy 31
whole body Seveso, Italy 4.5
liver Seveso, Italy 7.7
whole body Seveso, Italy 0.2
liver Seveso, Italy 2.7
adipose tissue Seveso, Italy 16
whole body Seveso, Italy 12
carcass throughout U.S. <50 ppb
eqq Saginaw Bay, NR
Reference
Range
1-<1024 Fanelli
et al.,
0.07-49 Fanelli
et al.,
2.7-13 Fanelli
et al.,
LS Fanelli
et al.,
LS Fanelli
et al.,
LS Fanelli
et al.,
LS Fanelli
et al.,
NR Helling
et al.,
0.043-0.093 Ogilvie
1980a
1980c
1980c
1980c
1980c
1980c
1980c
1973
, 1981
Lake Ontario
-------
TABLE 6-4 (cont.)
CO
en
CO
3>
2,3,7,8-TCDD Concentration (ppb)
Type of Tissue
Animal
Herring gull egg
Herring gull egg
Herring gull egg
0-1 Herring gull egg
01
Herring gull egg
Herring gull egg
Herring gull egg
Turtle egg and liver
Snake liver and
o muscle
ro
S> Muskrat liver
CD
Sampling Site
Lake Superior
Lake Michigan
Lake Huron
(main body)
Lake Huron,
Saginaw Bay, N.
Lake Huron,
Saginaw Bay, S.
Lake Erie
Lake Ontario
Bayou Meto/
Arkansas River
Bayou Meto/
Arkansas River
Bayou Meto/
Arkansas River
Average3
0.011
0.009
0.009
0.043
0.086
0.011
0.059
0.15
0.060
NO (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
Mitchum
et al., 1980
Mitchum
et al., 1980
Mitchum
et al., 1980
-------
oo
Ul
CO
TABLE 6-4 (cont.)
2,3,7,8-TCDD Concentration (ppb)
Type of
Animal
Racoon
Frog
Horse
Tissue Sampling Site
Average3
liver Bayou Meto/ NO (10 ppt)b
Arkansas River
liver and Bayou Meto/ >10
muscle Arkansas River
fat Midwest wire 0.045
Reference
Range
LS Mitchum
et al.,
LS Mitchum
et al.,
LS Hryhorc2
1980
1980
:uk
Horse
1 i ver
reclamation
incinerator
Midwest wire
reclamation
incinerator
NO (<6 ppt)b
LS
et al., 1981
Hryhorczuk
et al., 1981
o
X.
INJ
X.
co
These are averages of samples that had above detectable levels of TCDO,
Not reported and the limit of detection indicated in parentheses
NR = Not reported; LS = Limited samples
-------
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 In 1971 and 1973 Indicated that there was a signifi-
cant Increase In the number of families and total number of Insects In the
contaminated test site, which was correlated with the Increase 1n vegetation
after herbicide spraying. Aquatic species diversity studies were conducted
in 1969, 1970, 1973 and 1974 on a stream 1n the contaminated area and a con-
trol stream. As mentioned before, 2,3,7,8-TCDD was detected 1n sediments
and fish from the contaminated stream; however, there was no significant
difference in ichthyofauna diversity in the two streams, and no significant
change in diversity through time in either stream. As a result, the only
effects that can be attributed to 2,3,7,8-KDD contamination were increased
liver and spleen weight 1n beach mice. The ecological significance of this
effect Is unknown, especially since no obvious detrimental effects were
observed 1n this or other species from contaminated sites.
The only other information pertinent to ecosystem level effects was
provided by Bollen and Morris (1979), who Investigated the effects of
2,3,7,8-TCDD on respiration (C0? production) in forest litter and soil
samples. Litter and soil samples were air dried, placed in blometer flasks,
moistened and treated with 2,3,7,8-TCDD. Concentrations as high as 0.031
vg/kg dry weight in litter had no effect on respiration. Concentrations
as high as 0.052 yg/kg dry weight in soil caused a slight but significant
stimulation of CO production. Because higher concentrations were not
tested, it is unknown whether 2,3,7,8-TCDD would have inhibitory effects on
1853A 6-18 02/29/84
-------
son mlcroblal populations, carbon metabolism or nutrient cycling at the
higher levels of soil contamination found 1n such contaminated areas as the
Eglln Air Force Base test site.
6.4. SUMMARY
Almost all of the available Information concerning the toxlclty of PCODs
to wildlife deals with aquatic species. Acute exposure to Initial nominal
2,3,7,8-TCDD concentrations as low as 0.0001 yg/l 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 vg/l.
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, 1t Is possible that
constant acute or chronic exposure to dissolved concentrations <0.0001
pg/j, would produce toxic effects 1n sensitive aquatic organisms.
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 pg/8. had
no effect on mosquito population and caused a 30-50% decrease In reproduc-
tion of snails and ollgochoete worms (Miller et al., 1973). In contrast,
acute exposure to 0.1 yg/a caused 100% delayed mortality In gupples
%
(Morris and Miller, 1974) and juvenile rainbow trout (Helder 1981).
Similarly, exposure to relatively constant, measured, dissolved concentra-
tions of ~0.002-0.004pg/a In aquatic model ecosytems killed all exposed
mosqu1tof1sh and channel catfish In 15-20 days, but had no discernible
effects on snails and waterfleas over a total test period of 32-46 days
1853A 6-19 03/26/84
-------
(Yocklm et al., 1978). Ihe dying mosqultoflsh and catfish had mean whole-
body 2,3,7,8-lCDD concentrations of 1.2 and 4.4 pg/kg, respectively. In
contrast, single Intraperltoneal Injections of 2,3,7,8-TCOD at maximum doses
of 600 or 1000 pg/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 pg/kg 1n the diet was lethal to all but two fish (88%) 1n
71 days, but no significant effects were seen In fish fed dally a diet
containing 2.3 pg/kg for 105 days (Hawkes and Morris, 1977). Residue
analysis of single fish sampled at the end of the tests showed 2,3,7,8-TCDD
levels of 1380 pg/kg bw In one high dose fish and 1.573 pg/kg In one low
dose fish.
Although only limited Information was found concerning the effects of
2,3,7,8-TCOD on aquatic plants, It 1s probable that they are less sensitive
than fish. Using model ecosystems, Yockim et al. (1978) observed no obvious
effects on algae at concentrations (0.002-0.004 g) that killed fish. Zullel
and Benecke (1978) observed contact Inhibition of filamentous algae placed
In contact with 1 pg quantities of 2,3,7,8-TCDQ spotted on filter paper.
Ihe 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 Eglin A1r Force Base, FL. The concentrations of 2\3,7,8-TCDD In contami-
nated soils were 0.010-0.710 pg/kg. Mice trapped from the contaminated
site contained 0.540-1.30 pg/kg In the liver and had significantly higher
spleen and liver weights than mice from control sites. No other differences
(hlstopathology, weights of other organs, Incidence of abnormal fetuses,
1853A, 6-20 02/29/84
-------
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
in body weight or hlstopathology compared with lizards from control sites.
Residues of 2,3,7,8-ICDD 1n three fish species taken from a pond and stream
adjacent to the contaminated site ranged from 0.004-0.085 yg/kg. Sedi-
ments derived from the erosion taken from the contaminated site contained
localized concentrations of 0.010-0.035 pg/kg. PCDD residues have been
reported for numerous other fish species and water bodies (NRCC, 1981a).
The PCDD concentrations (primarily 2,3,7,8-TCDD) in positive fish tests
ranged from 0.002-0.695 V9/kg.
1853A 6-21 02/29/84
-------
7. COMPOUND DISPOSITION AND RELEVANT PHARMACOKINETICS
7.1. ABSORPTION
Data are available regarding the absorption of 2,3,7,8-KDD through the
gastrointestinal (GI) 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-TCDD when mixed with other chlori-
nated compounds, which Is presumably the case for human exposures.
7.1.1. Absorption from the Gastrointestinal Tract. Data on the GI
absorption of 2,3,7,8-TCDD are summarized in Table 7-1. The GI absorption
of 2,3,7,8-TCDD has been Investigated more extensively 1n the rat than in
other species. When 2,3,7,8-TCDD was administered in 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 in acetone:corn oil (1:25
or 1:9) as a single dose or as repeated doses (5 days/week x 7 weeks)
resulted in absorption of a larger percentage (70-86%) of the dose (Rose et
al., 1976; Piper et al., 1973). It would appear, therefore, that the GI
absorption of 2,3,7,8-lCDD may vary, depending upon the vehicle used. The
Influence of vehicle or adsorbent on GI absorption has been investigated by
Poiger 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 in 50% ethanol (for
doses of 12-280 ng, equivalent to 0.06-1.4 yg/kg) and the percentage of
the dose in 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 In the hepatic levels of 2,3,7,8-
lCDD as compared with hepatic levels resulting from administration of
1854A 7-1 02/29/84
-------
TABLE 7-1
Gastrointestinal Absorption of 2,3,7,8-TCDD
Species
Guinea
pig
Rat
Rat
Rat
Rat
Rat
Hamster
Vehicle
NR
7 ppb,
in diet
20 ppb,
in diet
A:C,
1:25
A:C,
1:25
A:C,
1:9
olive
oil
Dose Schedule % Absorption Reference
(vg/kg) Mean +_ SO
NR 50 Nolan et al., 1979
single dose
0.5 pg/kg/day x 50-60 Fries and Marrow, 1975
42 days
1.4 pg/kg/day x 50-60 Fries and Marrow, 1975
42 days
1.0 pg/kg, 84 * 11* Rose et al., 1976
single dose
0.1 or 1.0 86 i 12* Rose et al., 1976
pg/kg/day,
5 days/week x
7 weeks
50.0 pg/kg, 70 Piper et al., 1973
single dose
650 pg/kg, 74 f 23* Olson et al., 1980a
single dose
*Mean ^ standard deviation
NR - Not reported; A:C = Acetone:corn oil, v:v
1854A
7-2
02/29/84
-------
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. 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 In 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 In the LDcn value for female
DU
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 In different formulations, Including accident-contaminated Seveso soil,
was carried out by Bonaccorsl et al. (1983). On the whole, the results
Indicated that soil-borne 2,3,7,8-TCDD had a b1oava1lab1lHy lower than that
of free (solvent-borne) 2,3,7,8-TCDD.
The feeding of fly ash containing PCDDs to rats in 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 differ-
ence in hepatic levels noted between fly ash-treated and extract-treated
rats was greater for the more highly chlorinated Isomers than it was for
2,3,7,8-TCDD.
The GI absorption of 2,3,7,8-TCDD was also examined in the hamster, the
species most resistant to the acute toxidty of this toxin. Olson et al.
(1980a) administered a single, sublethal, oral dose of [1,6-3H]-2,3,7,8-
TCDD in olive oil (650 yg/kg) to hamsters and reported that 74% of the
1854A 7-3 03/26/84
-------
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-lCDD through the skin 1s extremely limited. Polger and Schlatter
(1980) administered 26 ng 2,3,7,8-lCDD in 50 vl methanol to the skin of
six rats. After 24 hours, the liver contained 14.8 ± 2.6% of the dose. By
comparing with hepatic levels obtained (1n the same study) after oral
administration in 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 is 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 in 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-lCDD required to Induce skin lesions
(-1 yg) 1n the rabbit ear assay (Polger and Schlatter, 1980). Application
of 2,3,7,8-TCDD In 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
lesions to 2-3 pg in 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 vg.
1854A 7-4 03/26/84
-------
7.2. DISTRIBUTION
The tissue distribution of 2,3,7,8-lCDD in a number of species is
summarized in Table 7-2. As would be predicted from the lipophillc nature
of this compound, accumulation tends to occur in tissues with a high lipid
content. In rats and mice, 2,3,7,8-lCDD residues are localized in 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; Poiger and Schlatter, 1979). The latter dose is within the LO
range for rats. Similar results were obtained 7 days following administra-
tion of a single intraperitoneal dose of 400 yg/kg of [3H]2,3,7,8-TCDO
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 admini-
stered dose of 2,3,7,8-TCDD 1 day after oral or intraperitoneal administra-
tion (Manara et al., 1982). In both species, 1-22 days after single-dose
oral or Intraperitoneal administration, levels of 2,3,7,8-TCDD In adipose
tissue were similar to or slightly lower than levels in the liver, and were
considerably higher than concentrations in 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 Miller et al., 1976).
In a 7-week gavage study and a 2-year dietary study of 2,3,7,8-TCDD in
rats, 2,3,7,8-TCDD was present in the liver at 3-5 times the concentration
in adipose tissue when the daily dose or intake of the compound was >0.01
yg/kg/day (Rose et al., 1976; Koclba et al., 1976) and was present at
about the same concentration as in adipose tissue when the daily Intake was
0.001 yg/kg/day (Koclba et al., 1976). As in the single-dose studies,
2,3,7,8-TCDD levels were considerably lower In other tissues, Including the
thymus, than 1n liver or adipose tissue (Rose et al., 1976).
1854A 7-5 02/29/84
-------
oo
on
TABLE 7-2
Distribution of 2,3,7,8-TCDD
Species
Route of
Administration
Principal Organ Depots
Reference
Rat
Rat
Rat
Rat
Rat
Rat
Mouse
Mouse
Rhesus monkey
Golden Syrian
hamster
Guinea pig
Guinea pig
oral
oral
oral
oral
oral
i.p.
oral
i.p.
i.p.
i.p. or oral
oral
i.p.
1 i ver
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., 1976
Piper et al., 1973
Kociba et al., 1978a
Allen et al., 1975
Van Miller et al., 1976
Manara et al., 1982
Manara et al., 1982
Van Miller et al., 1976
Olson et al., 1980a
Nolan et al., 1979
Gasiewicz and Neal, 1979
as
-is.
i.p. = intraperitoneal
-------
There Is some evidence of sex differences 1n tissue distribution 1n
rats. During 42 days of administration of 2,3,7,8-TCDD at 7 or 20 ppb In
the diet, -85% of the total body residue of male rats was located In the
liver, as compared with 70% 1n females (Fries and Marrow, 1975). This small
difference in distribution patterns may have resulted from sex differences
in relative adipose tissue content.
The ability of mouse liver to sequester 2,3,7,8-TCDD Increases with
prolonged exposure (Teltelbaum and Poland, 1978). The hepatic uptake of
[3H]2,3,7,8-1COD in Swiss-Webster mice was maximal 12 hours after 1ntra-
peritoneal 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 is consistent with other data which indicate that 2,3,7,8-
1CDD is a potent inducer of hepatic mlcrosomal mixed-function oxidase
(Section 8.1.1.5.) and that >90% of the hepatic 2,3,7,8-TCDD is localized in
the mlcrosomes (Allen et al., 1975). The toxlcity of 2,3,7,8-TCDD 1n mice
has been demonstrated to correlate with the affinity of the receptor that
controls this induction 1n mice (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 in adult rhesus monkeys, infant
rhesus monkeys, and Sprague-Dawley rats 7 days after a single 1ntraper1ton-
eal Injection of 400 Pg 2,3,7,8-TCDD/kg bw. They found that while 43% of
the administered dose was localized in the livers of the rats, only 10.4%
was found in the livers of adult monkeys and 4.5% In the livers of Infant
monkeys. This difference cannot be explained by differences 1n absorption
1854A 7-7 03/26/84
-------
or excretion, since these parameters were observed to be similar In both
species. In monkeys, larger percentages of the dose were found In adipose
tissue, skin and muscle than was the case for rats,
McNulty et al. (1982) reported that 2 years after administration of a
single oral dose of 1 ng/kg of 2,3,7,8-lCDD to an adult rhesus macaque
monkey, tissue levels of the compound were 1000 ppt In adipose tissue and 15
ppt 1n the liver. These results Indicate that prolonged retention of
2,3,7,8-lCDD may occur 1n this species. The tissue distribution of 2,3,7,8-
lCDD In the guinea pig appears to be similar to the monkey, with the highest
concentration of the toxin being found 1n adipose tissue (Gaslewlcz and
Neal, 1979; Nolan et al., 1979). The Interspecles difference In 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
In doubt. For example, the hepatotoxlcity 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-
lCDD 1n humans. Facchettl et al. (1980) reported tissue concentrations of
2,3,7,8-lCDD at levels of 1-2 ng/g 1n adipose tissue and pancreas, 0.1-0.2
ng/g In liver and <0.1 ng/g 1n 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.
1854A 7-8 03/26/84
-------
In addition, Young et al. (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 Air 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-ICOD (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
measurable 2,3,7,8-TCDD levels that did not exceed 3 ppt. In the 10 control
veterans, 4 had 2,3,7,8-lCOO levels between 6 and 14 ppt. Levels of
2,3,7,8-lCDD in adipose tissue did not appear to be associated in this study
with ill health or any particular symptom; however, it was considered that
information on background levels of 2,3,7,8-TCDD in adipose tissue was too
limited to draw any firm conclusions.
2,3,7,8-TCDD has been demonstrated to be fetotoxlc in the rat (Section
9.1.). The ability of 2,3,7,8-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-lCDD In the fetus at gestation days 14, 18 or 21. The radioactivity
appeared to be evenly distributed throughout the fetus on days 14 and 18;
however, Increased levels of radioactivity were detected in fetal liver on
day 21.
Nau and Bass (1981) (more recently reported by Nau et al., 1982)
Investigated the fetal uptake of 2,3,7,8-ICDD in NMRI mice following oral,
intraperltoneal or subcutaneous administration of 5, 12.5 or 25 pg/kg in
1854A 7-9 03/26/84
-------
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- 7CDD 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 In 2,3,7,8-TCDO concentration coincides with placentatlon.
2,3,7,8-TCDD concentrations In the placenta were an order of magnitude
greater than In the fetus Itself. The affinity of fetal liver for 2,3,7,8-
TCDD was relatively low, as compared with maternal liver; however, 2,3,7,0-
TCDO levels 1n fetal livers were 2-4 times higher than the levels In 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).
Autoradlographlc studies of tissue localization following Intravenous
administration of [14C]2,3,7,8-TCDD In DMSO to three strains of mice Indi-
cated that the liver had the highest concentration and longest retention of
radioactivity in the body, followed by the nasal mucosa (Appelgren et al.,
1983). In pregnant mice, the concentration of radioactivity In 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 in the
liver. Labelling of the thymus, lymph nodes, bone marrow and prostate were
low at all observation times.
7.3. METABOLISM
Vinopal and Casida (1973) found no evidence of water soluble metabolites
of 2,3,7,8-TCDD following incubation with mammalian liver mlcrosomes or
1854A 7-10 03/26/84
-------
IntraperItoneal 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 [14C]2,3,7,8-TCDD with rat hepatic
mlcrosomes resulted 1n the formation of bound radioactivity which, In
contrast to free 2,3,7,8-TCDD, was not ethyl acetate extractable. This
binding was found to result from oxldatlve metabolism, as Indicated by a
requirement for NADPH, and could be Induced by phenobarbHal 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 In the
bile of rats given dally oral doses of 15 vg [14C]2,3,7,8-TCDD. Incuba-
tion of the bile with
-------
Ihe ability of 1,6-3H-2,3,7,8-TCDI) derived radioactivity to bind to
rat hepatic macromolecules \n vivo was Investigated by Poland and Glover
(1979). They found maximum levels of 60 pmol 2,3,7,8-TCDD/mole of amlno
adds In 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 In 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.
lulp and Hutzlnger (1978) studied the metabolism of a variety of PCDDs,
Including 1,2,3,4-KDD, In 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 exposition, 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 In Isolated rat hepato-
cytes. The major product was deconjugated with tp-glucuronldase, deMvat-
1zed with dlazomethane, and separated Into two compounds by HPLC. These
metabolites were subsequently Identified as 1-hydroxy-2,3,7,8-TCDD and
2-hydroxy-3,7,8-tr1chlorod1benzo-£-d1ox1n.
Polger et al. (1982a) Identified six metabolites 1n the bile of dogs
that were given [3H]2,3,7,8-1CDD. The major metabolite was 1,3,7,8-tetra-
chloro-2-hydroxyd1benzo-£-d1ox1n. 2-Hydroxy-3,7,8-tr1chlorod1benzo-£-d1ox1n
1854A 7-12 03/26/84
-------
and 1,?-d1chloro-4,5-d1hydroxybenzene were also Identified as minor metabo-
lites. Ihe structures of the three remaining metabolites were not deter-
mined; however, two appeared to be tr1chloro-d1hydroxyd1benzo-j>-d1ox1ns and
the third was apparently a chlorinated 2-hydroxyd1phenyl ether. The pres-
ence of these metabolites Is consistent with a 1,2-arene oxide Intermediate.
Isolated rat nepatocytes In suspension have been used as an jm vitro
system for assessing 2,3,7,8-KDD metabolism under various conditions. Data
Indicate that the rate of 2,3,7,8-TCDD metabolism In rat hepatocytes
correlates directly with drug Induced changes In hepatic cytochrome P-450
monooxygenase activity, suggesting that 2,3,7,8-TCDD Is metabolized by this
enzyme (Olson et al., 1981).
Beatty et al. (1978) found a correlation between hepatic mixed-function
oxldase (MFO) activity and the toxldty of 2,3,7,8-lCDO in rats. Both in
naturally occurring age- and sex-related differences in MFO activity and
following the administration of Inducers and Inhibitors of MFO enzyme
systems, hepatic MFO activity was Inversely related to toxldty which corre-
sponds to direct relationship between the 20-day LD,. and MFO activity.
Ihe fate of 2,3,7,8-lCDD metabolites from dogs has been examined 1n rats
by Weber et al. (1982). 2,3,7,8-TCDD metabolites were extracted from the
bile of 2,3,7,8-lCDD-treated dogs and administered by gavage to female
Sprague-Dawley rats. Ihe 2,3,7,8-TCDD metabolites were rapidly cleared from
the bodies of bile-duct-cannulated rats, with >85% of the dose recovered In
the feces, bile and urine within 24 hours. In Intact rats, only 13% 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.
1854A 7-13 03/26/84
-------
Polger et al. (1982a) investigated the toxlcity 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 jag of
parent compound/kg bw. Other groups of guinea pigs received bile extract
from untreated dogs or 2,3,7,8-TCOO Itself. A comparison of the mortality
data at 5 weeks after dosing Indicated that the acute toxldty of 2,3,7,8-
1CDD to guinea pigs was at least 100 times higher than was the acute
toxlclty of Its metabolites.
Olson and BUtner (1983) reported that the rate of metabolite formation
\n vitro was considerably higher 1n hepatocytes from the hamster than 1n
hepatocytes from the rat. Qualitative evaluation of _1n vivo and \jn vitro
metabolites by HPLC also suggested major Interspedes variability. The
authors suggested that such differences 1n metabolism may partially explain
the differences 1n toxldty among species.
7.4. ELIMINATION
The following discussion assumes that elimination 1s a first order
process. With the exception of the guinea pig, which may follow zero order
kinetics (Gaslewicz and Neal, 1979), elimination data yield a straight line
on a semilogarlthmlc plot, Indicating a first order process. H1les and
Bruce (1976) have 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 is slowly excreted from the bodies of all species tested
(Table 7-3), with a half-life in the body of 10-43 days. In the Golden
Syrian hamster, the least sensitive mammalian species to the acute toxldty
of 2,3,7,8-TCDD, excretion occurs readily through both the urine (41%) and
1854A 7-14 02/29/84
-------
CO
o
t\J
10
CO
-p»
TABLE 7-3
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/2J
B6D2F-|/J*
Hamster
Hamster
Single Treatment
yg/kg (route)
2 (i.p.)
1.45 (oral)
1.0 (oral)
50 (oral)
50 (oral)
400 (i.p.)
400 (i.p.)
400 (i.p.)
1 (oral)
10 (i.p.)
10 (i.p.)
10 (i.p.)
650 (i.p.)
650 (oral)
Half-Life for
Elimination
(days)
30.2 +_ 5.8
22 - 43
31+6
17.4 + 5.6
21.3 + 2.9
NT
NT
NT
365
11.0 t 1.2
24.4 t 1.0
12.6 +. 0.8
10.8 ± 2.4
15.0 + 2.5
Relative % of TCDO-Derived
Radioactivity
Feces
94.0
NT
>99
80.0
95.5
91.0
78.0
39.0
NR
72.0
54.0
V2.0
59.0
NT
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
Urine
Gasiewicz 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 Miller et al., 1976
Van Miller et al., 1976
McNulty et al., 1982
Gasiewicz et al. , 1983a,b
Gasiewicz et al. , 1983a,b
Gasiewicz et al. , 1983a,b
Olson et al., 1980a
Olson et al ., 1980a
Offspring of C57BL/6J and DBA/2J which are heterozygous at the Ah locus
NT = Not tested; NR = Not reported
-------
feces (59%) (Olson et al., 1980a). The high levels found In 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-lCDD metabolites found 1n the urine (Piper et al.. 1973; Allen et
al., 1975; Rose et al., 1976; GaslewUz and Neal, 1979).
Rose et al. (1976) have Investigated the elimination of [1AC]2,3,7,8-
TCDD In rats given repeated oral doses of 0.01, 0.1 or 1.0 yQ/kg/day
Monday through Friday for 7 weeks, or a single dose of 1.0 v9/kg. In
these studies, no 14C was excreted In the urine following a single dose;
however, the urine contained 3-18% of the cumulative dose by 7 weeks. This
study Indicated that steady-state concentrations will be reached In the
bodies of rats In -13 weeks. The rate constant defining the approach to
steady-state concentrations was Independent of the dosage of 2,3,7,8-TCDO
over the range studied. This Is consistent with the observations of Fries
and Marrow (1975), who found that the total retention In the bodies of rats
was proportional to total Intake. When rats were maintained on a diet
containing either 7 or 20 ppb TCDO, the amount of 1CDD retained 1n the body
was 5.5 times the daily Intake of TCDD at 14 days, 7.5 times the dally
Intake at 28 days, and 10.0 times the daily intake at 42 days.
The data in Table 7-3 suggest some Interspecies differences in the half-
life for elimination (t 1/2) of 2,3,7,8-TCDD. In the hamster, the least
sensitive species to the acute toxlclty of 2,3,7,8-TCDO, a mean t 1/2 of
10.8 days was observed (Olson et al., 1980a,b), and in the guinea pig, the
most sensitive species to the acute toxldty of 2,3,7,8-TCDD, the mean t 1/2
was 30.2 days (Gaslewicz and Neal, 1979). The observed Interspecies dlffer-
1854A 7-16 02/29/84
-------
ences 1n the t 1/2 of 2,3,7,8-TCDD may 1n part be related to the relative
sensitivity of a given species to the acute toxldty of 2,3,7,8-TfJDD.
The 1ntrastra1n differences 1n the t 1/2 of 2,3,7,8-TCDD 1n three mouse
strains may be due to the finding that the DBA/2J strain possesses ~2-fold
greater adipose tissue stores than the C57B1/6J and B6D2F /J strains
(Gas1ew1cz et al., 1983b). The sequestering of the Upophlllc toxin 1n
adipose tissue stores of the DBA/2J mouse may contribute to the greater
persistence of 2,3,7,8-TCDD 1n this strain.
In all of the rat studies shown 1n Table 7-3, 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 BUtner (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 wg 3H-2,3,7,8-TCDD/kg. They
observed first order kinetics for elimination, with a fast component having
a t 1/2 of 7 days (represents 13% of total elimination) and a slow component
having a t 1/2 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). The apparent
absence of 2,3,7,8-TCDD metabolites 1n liver and fat suggests that once
1854A 7-17 03/26/84
-------
formed, the metabolites of 2,3,7,8-lCDD are readily excreted. Thus, urinary
and biliary elimination of 2,3,7,8-TCDD 1s apparently dependent upon metabo-
lism of the toxin. Although urine and bile appear to be free of unmetabo-
lized 2,3,7,8-TCDD, data from the hamster and rat Indicate that a signifi-
cant amount (10-40%) of unchanged 2,3,7,8-lCDD may be excreted Into the
feces. Unmetabolized 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. Ihese data suggest that the \n v 1 yo half-life for elimination of
2,3,7,8-lCDD may not directly reflect the rate of 2,3,7,8-TCOD metabolism In
a given animal (Neal et al., 1982). These data are consistent with the
observation of Manara et al. (1982) that the lethal effects of 2,3,7,8-TCDD
were decreased in C5/B1/6J mice regardless of whether the compound was
administered by gavage or Intraperitoneal injection if the animals were
given diets containing activated carbon.
7.5. SUMMARY
Exposure to 2,3,7,8-"ICDO occurs by inhalation, dermal or 61 absorption.
Inhalation exposure to detectable levels of 2,3,7,8-TCDD is less likely due
to the 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
(DiDomenico 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-lCDD have been performed, so the significance of this route of
exposure in heavily contaminated areas cannot be assessed.
1854A 7-18 03/26/84
-------
2,3,7,8-lCDD is readily absorbed under experimental conditions (vide
ante) and following environmental contamination (Cockerham et al., 1980;
Fanelli et al., 1980c; Walsh, 197/). After being absorbed, 2,3,7,8-TCDD Is
rapidly distributed to tissues with a high lipid content (fat, skin,
adrenals). In most species studied, the major storage site for 2,3,7,8-TCDD
is the liver (see Table 7-2). 2,3,7,8-TCDD exposure results in Induction of
MFO 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-lCDD to produce this effect has been correlated with the sensi-
tivity of various strains of mice to 2,3,7,8-lCDD toxlcity (Van Miller et
al., 1976; Poland and Glover, 1980).
2,3,7,8-lCDD appears to be distributed throughout the body and stored
largely as the parent compound (Olson et al., 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). Studies have also Indicated
that 2,3,7,8-TCDD was metabolized by the hepatic cytochrome P-450 monooxy-
genase 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 [1,6-3H]-2,3,7,8-
ICDD-derlved radioactivity was capable of binding covalently to cellular
macromoleculcs (Guenthner et al., 1979b; Nelson et al., 1977; Poland and
Glover, 1979), metabolism of 2,3,7,8-TCDD seems to be predominantly a detox-
ification process (Beatty et al., 1978; Polger et al., 1982a).
1854A 7-19 03/26/84
-------
2,3,7,8-lCDD and Us metabolites arc excreted from the body by a variety
of mechanisms. Lactatlng rats excrete 2,3,7,8-lCDD In the milk (Moore et
al., 1976). Piper et al. (1973) reported the excretion of [14C]2,3,7,8-
ICOD-derived radioactivity in 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 administered 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-ICDD-derived radioactivity in the urine (Olson et
al., 1980a). In all species so far studied, metabolism and excretion are
relatively slow processes, with the observed Initial half-lives in experi-
mental animals on the order of a few weeks (see Table 7-3).
1854A 7-?0 03/26/84
-------
8. TOXICOLOGY: ACUTE. SUBCHRONIC AND CHRONIC
8.1. EXPERIMENTAL ANIMALS
8.1.1. Acute
8.1.1.1. LETHAL EFFECTS — There have been studies In a variety of
species defining the doses necessary to cause death after acute exposure to
?,3,7,8-!CDD. A summary of the single dose LD data for 2,3,7,8-TCDD is
presented in Table 8-1. The dose that results in death varies extensively
with species, with the male guinea pig the most sensitive species tested
(ID of 0.6 pg/kg) (Schwetz et al., 1973), and the male hamster the
least sensitive species tested (LD of 5051 pg/kg) (Henck et al.,
1981). The rat and monkey appear to be the second most sensitive species,
with LD s between 22 and 70 pg/kg (Schwetz et al., 1973; McConnell et
al., 1978a), while other species tested (rabbit and mouse) had LD s
between 114 and 283 pg/kg (Schwetz et al., 1973; McConnell et al., 1978b;
Vos et al., 1974). Schwetz et al. (1973) found male rats more sensitive to
2,3,7,8-lCDD, 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 toxicity of 2,3,7,8-TCDD than adult females. Thus, data on sex
differences in sensitivity to the acute toxicity of 2,3,7,8-TCDD are con-
flicting and may depend on the species examined.
Harris et al. (1973) have studied the toxic effects of 2,3,7,8-TCDD in
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 in
the single exposure. As Illustrated most clearly 1n rats, a single dose of
25 pg/kg, 6 weekly doses of 5 yg/kg, or 30 dally doses of 1 pg/kg were
1855A 8-1 02/29/84
-------
TABLE 8-1
CD
3>
Species/Strain Sex/No. /Group
Guinea pigs/ M/NR
Hartley
Guinea pigs/ M/NR
Hartley
Guinea pigs/ M/9
Hartley
Guinea pigs/ F/6
oo Hartley
i
Guinea pigs/ F/6
Hartley
Rats/ M/5-10
Sherman
Rats/ F/NR
Sherman
Rats/Sprague- M/6
Dawley
o
\ Rats/Sprague- F/6
tv> Dawley
CO
Lethal Doses of 2,3,7,8-TCDD Following Acute Exposure
Route/
Vehicle
gavage/corn
o1 1-acetone
(9:1)
gavage/corn
o1 1-acetone
(9:1)
gavage/
corn o1 1
gavage/
corn oil
gavage/
methyl
eel lulose
gavage/corn
o1 1-acetone
(9:1)
gavage/corn
oil-acetone
(9:1)
1 .p. /olive
oil
1 .p. /o live
oil
Dose Tested Duration of
(wg/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
LD5Q Comments
(wg/kg)
0.6 Time to death was 5-34 days, the
(0.4-0.9)* 2,3,7,8-TCDD was 91X pure
2.1 Time to death was 9-42 days, the
(1.5-3)* 2,3,7,8-TCDD was 99X pure
2 Median time to death was 17-20 days.
marked weight loss, thymus atrophy,
Intestinal hemorrhage, no porphyrla
and only mild liver Injury
2.5 Time to first death was 32 days 1n
(1.2-5.4, 95X the 2.5 wg/kg group, with SOX
confidence) mortality by day 42
19 Time to first death was 12 days 1n
(15-23, 95X the 20.0 wg/kg group, with 67X
confidence) mortality by day 42
22 Time to death was 9-27 days, the
2,3,7,8-TCDD was 91X pure
45 Time to death was 13-43 days, the
(30-66)* 2,3,7,8-TCDD was 91X pure
60 LDjQ (yg/kg, mean * SE) adult
male, 60.2 + 7.8; weanling male,
25.2 * 1.4
25 Adult female had a mean + SE of
24.6 * 2.0 wg/kg
Reference
Schwetz et al
Schwetz et al
1973
McConnell et
1978b
SUkworth et
1982
SUkworth et
1982
Schwetz et al
1973
Schwetz et al
1973
Beatty et al .
Beatty et al.
al.,
al.,
al.,
, 1978
, 1978
CO
-------
CO
en
01
TABLE 8-1 (cont. )
Species/Strain
Sex/No. /Group
Route/
Vehicle
Dose Tested
(yg/kg)
Duration of
Observation
1050 Comments
(yg/kg)
Reference
Monkey/rhesus F/3
H1ce/C57Bl
M/14
M1ce/C57Bl
M/9
oo
i
00
gavage/
corn oil
gavage/corn
o11-acetone
(9:1)
gavage/
corn oil
0
70
350
0
100
150
200
NR
>35 days
60 days
<70
114
30 days
283.7
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 1n
25X of treated animals, severe
thymlc and spleen atrophy, hemor-
rhage 1n the region of the eye and
small Intestine, liver necrosis In
the centr1lobular region
Median time to death was 22-25
days, dose-related bw loss, thymlc
atrophy. Increased liver weight
and porphyMa, gross and historic
liver alterations, subcutaneous
edema, Intestinal hemorrhage
McConnell et al.,
1978a
Vos et al., 1974
McConnell et al.,
1978b
M1ce/C57Bl/lO M/5
M1ce/C57Bl/lO F/5
H1ce/C57Bl/6J M/NR
o M1ce/DBA/2J M/NR
rsj
\
PO
if M1ce/B6D2F-j/J M/NR
^^
oo
-£=•
gauge/
arachls oil
gauge/
arachls oil
1 .p. /olive
oil
1 .p. /olive
oil
1 .p. /olive
oil
85 45 days 146
107
135
170
213
85 45 days >450
107
135
170
213
269
338
426
536
NR 30 days 132
NR 30 days 620
NR 30 days 300
95% confidence limits of 111-211
yg/kg. Most deaths occurred from
22-26 days after dosing. Signs
of porphyrla, edema, hemorrhage.
1 of 4 animals died at dose of
426 yg/kg
BGD2Fi/J mice are the offspring
of C57B1/6J and DBA/2J.
The BGD2-|/J mice are heterozygous
at the Ah locus.
No comment
Smith et al .
Smith et al.
Gas1ew1cz et
1983a,b
Gas1ew1cz et
1983a,b
Gaslewlcz et
1983a,b
. 1981
, 1981
al.,
al.,
al..
-------
TABLE 8-1 (cont.;
CO
Species/Strain Sex/No. /Group
Rabbits/ M&F/NR
New Zealand
Rabbits/ MiF/5
New Zealand
Rabbits/ M&F/NR
New Zealand
Hamster/ M/6
OP golden Syrian
j^
Hamster/ M&F/5-6
golden Syrian
Hamster/ M/5
golden Syrian
Dogs/Beagle M/2
Dogs/Beagle F/2
o
Route/
Vehicle
gavage/corn
o1 1 -acetone
(9:1)
corn oil
dermal/
acetone
gavage/corn
o1 1-acetone
(9:1 )
ol 1 ve oil
gavage/
ol 1ve oil
gavage/corn
o1 1-acetone
(9:1)
gavage/corn
o1 1-acetone
(9:1)
Dose Tested
(yg/kg)
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 1059
Observation (yg/kg)
2-8 weeks 115
(38-345)*
4 weeks NR
3 weeks 275
(142-531)*
55 days 5051
(3876-18,487,
95X confidence)
50 days >3000
50 days 1157
2-8 weeks NA
2-8 weeks NA
Comments Reference
Time to death was 6-39 days, the Schuptz et al
2,3,7,8-TCDO was 91X pure 1973
Time to death was 6-23 days, Schwetz et al.,
2-3 animals/group died 1n all 1973
but the low exposure group
Time to death was 12-22 days Schwetz et al.,
1973
Time to death was 26-43 days, the Henck et al., 1981
liver and thymus appeared to be the
primary target organs, only 1 death
occurred In the 300 and 3000 yg/kg
group
Significant, dose-related decrease Olson et al., 1980b
1n thymus weight starting at
500 yg/kg, only 2 deaths occurred
out of 11 hamsters 1n the 3000 yg/kg
group.
Death generally occurred between Olson et al., 1980b
24 and 45 days, decrease 1n bw above
2000 yg/kg, proHferatlve 1le1t1s
with mild to severe Inflammation
All animals died Schwetz et al..
1973
All animals survived Schwetz et al..
1973
CO
*The number 1n parentheses appears to Indicate the range of lethal doses; however, the article did not specify what these numbers represented.
1.p. = IntrapeMtoneal; NR = Not reported; NA = Not applicable
-------
all the threshold dose for observing a decrease In body weight. Other end-
points, Including lethality, decrease In thymus weight, and a no effect
level for body weight change In rats, mice and guinea pigs 1n general
appeared likewise to require 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-TCDD has over a 103-fold difference 1n toxlclty
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-lCDD was the protracted time between
exposure and death (see fable 8-1). In determining the LO In 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 In all other species tested Including the most sensitive
species, the guinea pig, in 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 and appeared to be "wasting away." In
female Wlstar rats Intubated with 2,3,7,8-lCDD. at a dose of 100 pg/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 associ-
ated 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 followed by a second, more gradual, decline In food and
water intake and weight until death. Providing animals with an adequately
nutritious liquid diet by Intubation did not appreciably alter the pattern
of weight loss nor affect survival. In contrast, Gas1ew1cz et al. (1980)
I855A 8-5 02/29/84
-------
observed that providing rats with total parenteral nutrition would prevent
some of the weight loss Induced by 2,3,7,8-lCDO; however, there was no pro-
tection from the lethal effects of 2,3,7,8-KDD. In yet another study,
Seefeld and Peterson (1983) suggest that a reduction In food Intake caused
by 2,3,7,8-lCDD Is 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-TCDD-
treated partners (25 or 50 Pg/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 mortality was 0 and 15%. The authors proposed a hypoth-
esis that 2,3,7,8-lCDD lowers a regulated level or "set-point" for body
weight control In the rat. The ensuing change in food Intake was thought to
occur secondarily to the change In set-point.
Also, severe thymlc atrophy Is universally observed In all species given
lethal doses of 2,3,7,8-lCDD, 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 toxidty 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 (3.5%), normal (26%) or high (55%) protein diets maintained
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 in the respective
control rats. Dietary protein also had no effect on preventing or enhancing
1855A 8-6 02/29/84
-------
the 2,3,7,8-lCDD induced thymlc atrophy. Although weight loss and thymic
atrophy wore present In most species tested, there were other symptoms which
were characteristic of toxldty 1n only some species.
In the guinea pig, besides thymlc atrophy, no gross changes were
observed In internal organs after a lethal oral or 1.p. dose of 2,3,7,8-TCDO
(Greig et a!., 1973, Gupta et al., 1973). Hemorrhages were observed In a
number of organs including the adrenal gland, urinary bladder, GI tract and
mesenterlc lymph nodes; however, these were considered unremarkable changes
by Gupta et al. (1973). Histologic examination confirmed the gross observa-
tions with atrophy and lymphoid cell depletion in the thymus, spleen and
lymph nodes, and hemorrhages observed In many organs. In addition, marked
hyperplasia of the urinary bladder was observed. Of particular Interest was
the absence of severe toxic effects on the liver. 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) in guinea pigs administered lethal doses of
?,3,7,8-Kl)D, with the additional observation that the sternal bone marrow
was hypocellular in all types of blood-forming cells.
Turner and Collins (1983) described some histologlc changes in the liver
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-TCOD at doses of 0.0, 0.1,
0.5, 2.5, 12.5 or 20 pg/kg, and 1 male guinea pig each was treated with a
dose of 0.1 or 0.5 pg/kg. The 2,3,7,8-lCDD was administered by gavage as
an aqueous suspension 1n 0.75% 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,
1855A 8-7 02/29/84
-------
100 and SOO mg/kg). The histologic observations as described were applied
In 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 hyalln-llke cytoplasmic Inclusion bodies were observed. Even though
there was no dose-response relationship for these liver lesions, the doses
spanned a range that resulted In the lowest dose being nonlethal (none of
the 4 female guinea pigs died during the study), while In the high dose
group 4 of 6 animals died before 42 days post-treatment. The LD for
female guinea pigs was determined 1n this study to be 2.5 or 19 ng/kg bw
depending on whether the compound was administered by gavage 1n corn oil or
in aqueous methyl cellulose (Sllkworth et al., 1982).
The greatest difference at necropsy 1n the gross and histologlc effects
in rats and mice of exposure to lethal doses of 2,3,7,8-TCDD was pathologic
alterations In the liver, as compared with guinea pigs. An early report by
Buu-Hol et al. (1972) described alterations in the architecture of the liver
of rats within 5 days of receiving a low dose of 2,3,7,8-TCDD (10 yg/kg by
i.p. injection). At higher oral doses of 100 or 50 pg/kg, which killed 43
and 7% of the animals, respectively, Gupta et al. (1973) also observed
marked distortion of liver architecture in rats; however, only mild regener-
ative changes of the liver were observed at the sublethal dose of 5 v>g/kg
administered weekly for 6 weeks. Liver toxidty appeared to develop slowly
in the rat with no change in liver function, as Indicated by plasma protein
and bilirubin levels, or alkaline phosphatase, glutamic-oxalacetic trans-
aminase (GOT) and glutamic-pyruvic transaminase (GPT) activity being
detected 3 days after intubation with 2,3,7,8-TCDD at a dose of 200 yg/kg
(Greig et al., 1973). BiHrubin levels were, however, markedly elevated
1855A 8-8 03/29/84
-------
from 0.33 ^g/100 m«. In control animals to 10.97 yg/100 ms. 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 al., 1974); however, Jones and Grieg (1975) reported that the centMlobu-
lar necrosis, bile duct proliferation and I1p1d accumulation were more
extreme 1n mice than In rats. Examination of mouse livers using long wave
UV light showed fluorescence suggestive of excess porphyrln accumulation
(McConnell et al., 1978b). Although excess porphyrlns may be present 1n the
livers from 2,3,7,8-KDD-exposed rats, fluorescence 1s not usually observed.
Besides effects on the liver, 2,3,7,8-lCDD exposure produced other toxic
effects In rats and mice that were not observed or were observed to a lesser
extent In guinea pigs. In rats that died from 2,3,7,8-KDD exposure, there
were extensive hemorrhages of the heart, liver, brain, adrenal gland and GI
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 (McConnell et al., 1978b; Vos et al.,
1974). Death In mice was frequently attributed to terminal hermorrhages
(Vos et al., 1974).
In monkeys exposed to lethal levals of 2,3,7,8-KDD, McConnell et al.
(1978a) reported clinical and histologlc signs of toxiclty, some of which
were similar to those already described for other species. Severe thymic
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 GOT and aldolase
18b5A 8-9 02/29/84
-------
activity and decreased albumin levels Indicative of liver pathology occurred
near the time of death. As observed 1n mice, the bone marrow of monkeys was
hypocellular. In addition to the above signs of toxlclty, which were
observed In 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 dysplasia. As with most other species, a
specific cause of death could not be determined for monkeys. Poland and
Knutson (1982) have summarized the toxic response of various species to
?,3,7,8-KDD (Table 8-2).
There was very little Information on the lethal effects of PCDD con-
geners other than 2,3,7,8-TCDD. McConnell et al. (1978b) determined the
LD for nine congeners of PCDD following a single treatment by gavage in
mice and guinea pigs. A comparison of the LD expressed as pmol/kg
body weight is presented in 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 in 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 in a decrease 1n lethality. Although the congeners vary In
effective dose between mice and guinea pigs, the relative order of toxlcity
of these congeners did not change. Also, similar effects of toxlclty were
observed for all congeners as described above for 2,3,7,8-TCDD when the com-
parison was made within a single species.
8.1.1.2. EFFECTS ON THE LIVER — The hlstologicaT and uHrastructuraT
changes in the liver Induced by oral exposure to 2,3,7,8-TCDD 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
18S5A 8-10 02/29/84
-------
co
CD
I
O
CD
CO
TABLE 8-2
Toxic Responses Following Exposure to 2,3,7,8-TCDD: Species Differences3
Monkey
Hyperplasia and/or metaplasia
Gastric mucus 4-4-c
Intestinal mucosa f
Urinary tract 4-4-
Bile duct and/or gall bladder 44-
Lung: focal alveolar
Skin 4-4-
Hypoplasia, Atrophy or Necrosis
Thymus 4-
Bone marrow 4-
Testicle 4-
Other
Liver lesions +•
Porphyria 0
Edema +•
Guinea
Pig
0
4 f
0
0
4-
f
4-
4-
0
0
Cowb Rat
4- 0
f4- 0
f
t 4-
*d o
4- 4-
f
1 4-
1
0
Mouse
0
0
1 4-
0
4-
t
4-
4-
1 4-
f
Rabbitb Chickenb Hamster
0
4-4-
0
44. 0
4- 4-
4-
4-
44- 4- 4-
f 0
H4- 4-
References: monkey (McConnell et al., 1978b; Norback and Allen, 1973; Allen et al., 1977); guinea pig
(McConnell et al., 1978b; McConnell, 1980; Moore et al., 1979; Turner and Collins, 1983); cow
(McConnell, 1980); rat (McConnell, 1980; Kociba et al., 1978a; Kociba et al., 1979); mouse
(Schwetz et al., 1973; McConnell et al., 1978b; Vos et al., 1973); rabbit (Kimmig 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).
bResponses followed exposure to 2,3,7,8-TCDD or structurally related chlorinated aromatic hydrocarbons.
cSymbols: 0, lesion not observed; 4-, lesion observed (number of "f" denote severity); ±, lesion
observed to a very limited extent; blank, no evidence reported in literature.
Skin lesions in cattle are observed, but they differ from the skin lesions observed in other species.
Adapted from Poland and Knutson, 1982.
-------
TABLE 8-3
Estimated Single Oral LQ^Q - 30 Values for PCDDs3
Chlorlnati
1,
1,
1,2,
1,2,
1,2,
1,2,3,
on of PCDDs
2,8
2,3,7
2,3,7,8
2,3,7,8
2,4,7,8
3,4,7,8
3,6,7,8
3,7,8,9
4,6,7,8
Guinea P1gs
(Pmol/kg)b
>1180
120.41
0.006
0.009
3.15
0.185
0. 178-0. 255C
0. 153-0. 255C
>1.400
Mice
(ymol/kg)b
NR
>10
0.88
0.94
>14
2.11
3.19
>3.67
NR
aSource: McConnell et al., 1978b
bSpearman-Karber method
cEst1mated range due to variability 1n replicates
NR - Not reported
1855A
8-12
02/29/84
-------
2,3,7,8-lCDD at 0.0, 5 and 25 iag/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 hlstologic examination. The major ultrastructural change
observed was a dose-related Increase In the smooth and rough endoplasmlc
reticulum (ER) In cells near the bile canal1cul1. 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 parenchymal cells, although
large amounts of rough ER were still present. By day 28 the cells had
returned to normal appearance. These changes In liver cells following
2,3,7,8-KDO 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-lCDD in Sprague-Dawley male rats
produces necrotizlng hepatic lesions which 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 proliferatlve changes In the liver 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
the necrotic changes were hyperplasia of the viable cells with multinucle-
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-lCDD also revealed Increases 1n smooth ER; however, the most
striking effect was degeneration of the plasma membrane with the resulting
185bA 8-13 03/29/84
-------
fusion of parenchyma! cells. In a study of similar design, Jones (1975)
followed the distribution with time after treatment of membrane associated
AfPase activity by h1stochem1cal techniques. At 3 days after treatment, the
first changes in ATPase patterns were observed, with loss of activity along
the canalicular borders and some Increased activity In the sinusoids. The
midzonal and periportal zones had normal activity at this time. The loss of
ATPase activity persisted for 34-42 days, and paralleled the histologic
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 (LSM) isolated from male Holtzman rats 2, 10, 20 or 40 days after
intubation with 2,3,7,8-lCDD at 0.0, 10 or 25 vg/kg were used for deter-
mination of Na , K -ATPase and Mg -AlPase activity. The activity of
Na , K -AlPase was depressed to the same extent for both doses of
2,3,7,8-lCDO from day 2 40 after treatment, while a similar depression of
the Mg -AlPase activity was observed only in the high dose group. In the
low dose group, there was a decrease in 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-lCOD on ATPase activity was not the
result of 2,3,7,8-TCDD induced food deprivation and In vi_tno studies indi-
cated that the loss of activity was not due to the direct interference of
2,3,7,8-KDD with 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 i.p. injection of 2,3,7,8-TCDD, indicating that exposure
was actually affecting membrane components (Brewster et al., 1982).
1855A 8-14 03/29/84
-------
Peterson et al. (1979a) did observe a positive correlation between the
levels of LSM AlPase activity and both \n vivo cumulative biliary excretion
of ouabain and bile flow (pl/m1n/g liver). Using perfused liver, however,
Peterson et al. (1979b) reported a segregation between tSM ATPase activity
and biliary excretion of ouabain when 2,3,7,8-TCDD rats were exposed to the
protective agents pregneno1one-!6a-carbonitr1le or splrenolactone. It was
concluded that LSM ATPase did not directly participate in ouabain transport.
Additional studies have described the effect of 2,3,7,8-TCDD on the bil-
iary excretion of a variety of xenobiotics. Early studies by Hwang (1973)
Investigated 2,3,7,8-lCDD inhibition of biliary excretion in male CD rats
given a single dose of 2,3,7,8-TCDD at 25 or 5 pg/kg by gavage. Animals
were examined for indocyanine green (ICG) 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 in the 25 yg/kg dose group at 16 days. Even with this
increased bile flow, however, the cumulative biliary excretion of IC(i was
decreased )n 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 in the
plasma and liver was higher in treated animals than in control animals,
while the concentration in the bile was lower, reflecting the decrease in
total excretion of ICG.
Yang and Peterson (1977) compared the effect of 2,3,7,8-TCDD on the bil-
iary excretion of the organic neutral compound, ouabain, with that of the
organic anions phenol-3,6-dibromophthalein (D8SP) and sulfobromophthalein
(BSP) in male Holtzman rats. Animals were Intubated with 2,3,7,8-TCDD at
doses of 10 or 25 pg/kg and excretion was evaluated periodically between
2-4 days postexposure. The biliary excretion of ouabain was depressed 1n a
1855A 8-15 03/29/84
-------
dose-related manner starting on the second day post-treatment, with maximum
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 ouabdln. The pattern of biliary excretion was different for DBSP and
BSP 1n 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 ICG, In
which a dose-related decrease 1n biliary excretion was observed. The
authors concluded that the effects of 2,3,7,8-TCDD on the multiple pathways
Involved In 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 little change 1n ICG
blood clearance rates, while 1n the rabbit, which develops 2,3,7,8-TCDD-
Induced liver damage similar to the rat, there was reduced blood clearance
of ICG (Seefeld et al., 1979, 1980). In the rabbit, there were Increases In
serum sorbltol dehydrogenase and glutamlc pyruvlc transamlnase activity as
further Indications of 2,3,7,8-TCDO-produced liver damage. In the monkey,
which received 2,3,7,8-KDD by gavage at doses of 5, 25 or 75 yg/kg, there
was an Initial slight Increase 1n the blood clearance of ICG at 2 days post-
treatment, followed in 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 histopathology of the liver was within normal limits. It appears that
major effects on biliary excretion occur only 1n species that are sensitive
to the hepatotoxic effects of 2,3,7,8-TCDD.
1855A 8-16 03/29/84
-------
Other gross signs of the hepatotoxlc effects of 2,3,7,8-7CDD observed in
some species Included fatty degeneration and porphyrla. Early observations
by Cunningham and Williams (1972) described a decrease In In v1_vo (1 hour
pulse) Incorporation of 3H sodium acetate Into liver llplds 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 pg/kg followed in either 3 or 7 days by
the assessment of I1p1d synthesis. At 3 days Incorporation decreased from
258 to 98 dpm/mg lipld In the control and treated animals, respectively.
There was an approximately similar decrease observed 7 days postexposure.
When individual classes of llplds were examined, there was a decrease In the
synthesis of triglycerides, diglycerldes and phospholipids. Although
Cunningham and Williams (1972) observed that 2,3,7,8-TCDD decreased llpid
synthesis, Albro et al. (1978) reported an Increase in total lipids in 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 lipids there was an Increase In free
fatty acids and cholesterol esters, while no change occurred In the content
of phospholipids, free cholesterol or trlglycerides. The fatty changes in
the liver were confirmed by ultrastructural examination of liver specimens.
At a sublethal dose of 10 pg/kg there was a different pattern of lipid
accumulation, with triglycerides and fatty acids Increased and cholesterol
esters decreased. The changes 1n the Hpid profile of the liver was attrib-
uted to 2,3,7,8-TCDD induced mobilization of body fat, a decrease in lyso-
somal add lipase (74% decline In this enzyme 10 days after a 50 v»9/kg
dose of 2,3,7,8-KDD) and an increase in llpid peroxidation as indicated by
a sharp increase in the production of lipofuscin pigments.
Porphyrla was Initially characterized quantitatively in mice by
Goldstein et al. (1978). Groups of 12 male C5781 mice received 4 weekly
1855A 8-17 03/29/84
-------
intubations of 2,3,7 ,8-KDD 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 in the 25 and
150 pg/kg groups where the levels were Increased 2000- and 4000-fold,
respectively. The difference in responsiveness to the development of por-
phyria was studied by Smith et al. (1981) in C57B1 mice which were sensitive
to, and DBA/2 mice which were insensitive to, the toxiclty of 2,3,7,8-TCDD.
Male and female C57B1 mice had a dose-related increase 1n hepatic porphyrlns
in the two high dose groups 3 weeks after a single exposure to 2,3,7,8-TCDO
at 0.0, 5, 15, 50 or 75 pg/kg, while only minimal nondose-related changes
In hepatic porphyrin were observed in DBA/2 mice exposed to up to 1200
pg/kg. In the sensitive C5/B1 mice there was only a small difference in
hepatic porphyrin between the sexes even though males were >3 times as sen-
sitive to the toxic effects of 2,3,7,8-TCOD than females (see Table 8-1).
Results similar to those above were reported for urinary porphyrin levels in
male C5/B1 and DBA/2 mice given 6 weekly doses of 2,3,7,8-lCDD at 25 yg/kg
(Jones and Sweeney, 1980). In the sensitive strain, the initial elevation
of porphyrin occurred 1n the second week.
In rats increased urinary porphyrin was observed only after subchronic
exposure to 2,3,7,8-TCDD (Cantoni et al., 1981). Female CD rats were orally
administered weekly dose of 2,3,7,8-lCDO at levels of 0.01, 0.1 and 1.0
pg/kg for 45 weeks. The initial Increase was observed in 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 porphyrin increase, but
the relative distribution also changed to compounds containing more carboxyl
groups. Only in the high dose group did the livers, at the terminal
necropsy, show signs of excess porphyrin under examination by UV light.
1855A 8-18 03/29/84
-------
In attempts to understand the mechanism of 2,3,7,8-TCDD Induced por-
phyria, the effects of 2,3,7,8-TCDD on the enzymes Involved In the synthesis
and catabolism of porphyrln have been studied. Goldstein et al. (1978)
showed that 6-am1nolevul1n1c add synthetase, a rate-limiting enzyme In
porphyrin synthesis, was slightly. Increased (2-fold) In male C5781 mice
given 4 weekly doses of 2,3,7,8-TCOD at 25 pg/kg. This dose of 2,3,7,8-
TCDD Increased liver porphyrin levels 2000-fold. Catabolism of porphyrln by
uroporphyrInogen decarboxylase (UD) also appeared to be decreased In
2,3,7,8-TCDD treated mice. Smith et al. (1981) reported a decrease In UO
activity from -25-7 n moles/hr/g liver In male and female C57B1 mice 3 weeks
after a single oral exposure to 2,3,7,8-KDD at a dose of 75 pg/kg. No
effect of 2,3,7,8-lCDD on UD activity was observed in DBA/2 mice which were
Insensitive to the induction of porphyria. A time course of changes in UD
activity with length of time after exposure to 2,3,7,8-KDD indicated a
steady decline in activity starting 3 days after exposure to 2,3,7,8-lCDD,
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 ng/kg. In this study the UD activity declined -48% in C57B1 mice
and only 4% in DBA/2 mice. Other factors besides the increase in 6-amino-
levulinic acid synthetase and the decrease in UD activity may also partici-
pate in the dramatic Increase in liver porphyrln 1n 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-
lCDD and the development of toxic effects, as well as the reported terato-
genic and carcinogenic potential of 2,3,7,8-TCDD, investigations have been
conducted to determine the Influence of 2,3,7,8-lCDD on DNA synthesis 1n the
1855A 8-19 03/29/84
-------
liver. Greig et al. (1974) measured the jn vivo Incorporation of 3H-thy-
mldlne (1 hour pulse) Into liver DNA of male and female Porten strain rats
after a single exposure to 2,3,7,8-TCDD at doses of 10 and 200 ^g/kg.
When the 2,3,7,8-lCDD 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-lCDD had no effect on jn vivo DNA synthesis, similar
studies by Conway and Matsumura (1975) and Dickens et al. (1981) demon-
strated an Increase 1n thymldlne Incorporation when determined jjn vitro.
Conway and Matsumura (1975) administered male Sprague-Dawley rats 2,3,7,8-
TCDD at a dose of 5 yg/kg followed In 10 days by removal of the liver and
tne Jn- vVtro determination of DNA synthesis in liver slices. Incorporation
of thymidine into the nuclei increased from 29 cpm/mg in 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 in rats treated 5 days earlier with 2,3,7,8-
lCDD as compared with hepatectomized controls. The onset of DNA synthesis
after partial hepatectomy (-20 hours) was the same 1n both 2,3,7,8-TCDD
treated and control animals; however, the treated animals had a more rapid
and extensive increase in 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 in both the treated and control animals was predomi-
nantly semiconservative. Further studies are needed to determine the reason
for the difference observed between in vitro and In vivo measurements of DNA
synthesis in the liver after exposure to 2,3,7,8-TCDD.
1855A 8-20 03/29/84
-------
Extensive hepatic necrosis In the rabbH may be responsible for death In
this species (Poland and Knutson, 1982).
Besides the effects on the liver of 2,3,7,8-TCOD exposure described
above, It is known that 2,3,7,8-TCDD 1s a potent Inducer of mlcrosomal
enzymes. Ihese 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-lCDD toxlcity Is 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-arglnlne and L-h1st1d1ne using
the everted intestinal sac technique 1n CD-I mice exposed to 2,3,7,8-TCOD.
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 in control animals. The two low doses produced a dose-related
decrease in absorption; however, at doses of >75 yg/kg the decrease was
uniform. At a dose of 150 yg/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. Ireatment with
1855A 8-21 03/29/84
-------
2,3,7,8-TCDD had no effect on the absorption of the other compounds Investi-
gated. In a similar experiment in Sprague -Dawley rats, Ball and Chhabra
(1981) also observed malabsorptlon of D-glucose. In this study, however,
absorption of leudne was also decreased. Ihe decrease 1n leudne absorp-
tion took longer to manifest Itself, with a significant decrease only
observed 2 weeks after treatment with 2,3,7,8-lCDD.
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
Kim (1979a) examined the effect of prior treatment of male Sprague-Oawley
rats on the 30-m1nute transport of S9Fe out of a duodenal loop created by
I1gat1ng d section of the Intestine In situ. At single 2,3,7,8-TCDD doses
of between 22 and 84 yg/kg there was Increased serosal transfer of 59Fe
measured 48 hours after treatment. At doses >42 pg/kg the Increase was
-100%. 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 In Inducing Iron
transport than 1.p. Injection. In similar experiments calcium transport was
decreased, and glulactose and prollne transport were unaffected by prior
exposure to 2,3,7,8-lCDD. Manls 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-lCDD exposure observed in the adjacent distal segment of the
Intestine. Increased Iron transport was also observed by Manls and Kim
(1979a) In an unidentified strain of mice. Increased Iron transport may be
one of the earliest effects of 2,3,7,8-lCDD; however, at present the toxlco-
loglc relevance of this transient disturbance in Iron transport Is unknown.
1855A 8-22 03/29/84
-------
One of the common gross observations of 2,3,7,8-lCOO toxlclty Is 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
il 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
ug/kg. (These results were also described by Hook et al., 1977). Anion
and cation transport were measured by the respective accumulation of
p-am1noh1ppur1c acid and N-methyln1cot1nam1de Into the cortical slices.
Anion accumulation was lower In the high dose group, while cation transport
was lower at both dose levels tested. The decrease 1n anlon transport was
confirmed in an In vivo study. Ammonlogenesls and gluconeogenesls were not
affected in 2,3,7,8-TCDD treated rats, even when the animals were made
addotlc, Indicative of no effect on the kidneys' ability to maintain add
base balance. Also, sodium reabsorptlon was shown jn v1vo 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 effects of 2,3,7,8-TCDD were
merely a general decrease In kidney functon reflecting the poor condition of
the treated animals (animals in 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-KDD, Grelg et al. (1974) demonstrated that pre-exposure to 2,3,7,8-
1CDD could reduce the ability of the rat kidney to respond to stimuli of DNA
sythesls. Folate-stlmulated DNA synthesis measured Iji v1yo 1n Porten strain
rats was decreased between 67 and 25% 1n animals receiving 2,3,7,8-TCDD at a
1855A 8-23 03/29/84
-------
dose of 10 pg/kg on day 0-9 before administration of folic acid. No sig-
nificant difference in folate-stlmulated DNA synthesis was observed if
2,3,7,8-lCDO was given 23 hours after follc acid. The lack of effectiveness
of administering 2,3,7,8-lCDD shortly after treatment with follc acid sug-
gested that ?,3,7,8-KDD 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 proliferative
stimuli is not known, although it was demonstrated that another agent
capable of Inducing microsomal enzymes, 3-methylcholanthrene (3-MC), had
similar effects on the kidney.
Additionally a number a hematologic and clinical chemistry changes have
been observed in the blood of laboratory animals after exposure to 2,3,7,8-
TCDD-. Many of these changes, as described by Zinkl et al. (1973), reflect
damage to previously described organ systems. In female CD rats given 30
daily doses of 2,3,7,8-TCDD at levels of 0.1, 1.0 or 10 pg/kg, the clini-
cal chemistry of the serum reflected liver damage. In the high dose group,
serum GO! and serum GPT were elevated starting 13-17 days after initial
treatment. There was a marginal change in GPT in the mid-dose group and
lactic dc-hydrogenase (LDH) in the high group, but the Increases were only
transitory. Serum cholesterol was increased in the high dose animals start-
ing at day 10, with a transitory increase again observed in the mid-dose
group. Conversely, there was a decrease in serum protein from day 24 on in
the high dose animals. Along with these clinical chemistry changes indica-
tive of liver damage, the only other major effect observed in the blood was
thrombocytopenia. The decrease in platelet count was detected early, by day
185bA 8-24 03/29/84
-------
3, in the 10 and 1 pg/kg groups, while In the 0.1 yg/kg group a signifi-
cant decrease was not observed until day 17. Thrombocytopenla was also
observed in female guinea pigs after 8 weekly oral doses of 2,3,7,8-TCDD at
0.2 pg/kg, and In mice (administered a single dose of 1.0, 10 or 50
ug/kg). In guinea pigs lymphopenla was also observed. Other hematologlc
changes were attributed to hemoconcentratlon.
In a more extensive Investigation of 2,3,7,8-TCDD-lnduced hyper!1p1dem1a
in male Sprague-Dawley rats, Poll et al. (1980) treated animals with a
single i.p. injection of 2,3,7,8-TCDD at 2 doses of 2.5, 5, 10 and 20
ug/kg. At day 21 after treatment there was a dose-related Increase in
total plasma cholesterol and high density lipoprotein cholesterol, while no
change was observed in trlglycerldes or very low and low density llpopro-
teins (VLOL and LOL, respectively). At a dose of 20 pg/kg the maximum
increase in 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 apoprotein
of HDL from 2,3,7,8-TCDD rats and control rats were Indicative of new apo-
protein synthesis. Although the Increases in HDL cholesterol may be in
response to eliminating excess llpids, the exact function has not been
clearly shown. There Is some evidence from studies of workers exposed to
2,3,7,8-KDD 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 I.p.
injection of 2,3,7,8-TCDD at a dose of 2 pg/kg had Increased hyperllpide-
mia characterized by Increases in VLDL and LDL (Swift et al., 1981). In
animals sacrificed 7 days after exposure to 2,3,7,8-TCDD, there was an
1855A 8-25 03/29/84
-------
Increase in total serum 11p1d, cholesterol esters, trIglycerldes and phos-
pholipids, when comparison was made with pair-fed, weight-paired or ad
11b1turn fed control groups. Serum-free fatty acids were not changed quanti-
tatively; however, some qualitative changes occurred, reflecting an Increase
1n the types of fatty adds that were abundant 1n the adipose tissue of
guinea pigs. Anaylsls of llpoprotelns revealed a 19-fold Increase in VLDL
and a 4-fold Increase 1n LDL, with no change observed In the levels of HDL.
The VLOL was also qualitatively different 1n the 2,3,7,8-TCDD treated
animals, containing less cholesterol ester and an altered C apoproteln. The
Importance of these qualitative changes Is unclear. The hyperHpldemla may
result from the 2,3,7,8-TCDD mobilization of free fatty acids, which are
then used In the synthesis of VLDL and are subsequently formed into LOL.
Ihe relationship of the changes 1n serum llpld levels to the mechanism of
2,3,7,8-lCDD toxicity needs further study.
Elovaara et al. (1977) observed some changes in blochemlcals of the
brain of male Wistar 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 in both the protein and RNA content of the Wistar rats, while levels
of acid proteinase and Ol-d1aphorase (an enzyme Induced by 2,3,7,8-TCDD 1n
the liver) had a small but significant increase in the heterozygous Gunn
rats. There were no significant changes observed in homozygous rats given
2,3,7,8-lCDD at 20 pg/kg. The authors noted that acid proteinase may
participate in chemically induced degeneration of the brain.
8.1.1.4. IMMUNOLOGICAL EFFECTS — During acute toxicity studies with
2,3,7,8-lCDD, thymlc atrophy was noted as a consistent effect in all species
that have been Investigated. This finding suggested that 2,3,7,8-TCDD may
1855A 8-26 03/29/84
-------
alter the Immune response, and Initiated immunotoxicity studies In 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 histological
examination, 2,3,7,8-lCDD-exposed rats had a severe depletion of lymphocytes
from the thymic cortex (Vos and Moore, 1974). Hematological changes were
noted in rats exposed to 10 and 14 dally doses of 10 yg/kg 2,3,7,8-TCDD
(Weissberg and Zinkl, 1973). Increased red blood cell count, decreased
platelet count, Increased neutrophil count and increased packed cell volumes
were reported in 2,3,7,8-TCDD-exposed rats. A summary of the data available
on the immunotoxlc effects of 2,3,7,8-TCDD in animals Is presented in Table
8-4. A review of 1mmunotoxic1ty and immunosuppression was reported by Vos
(1977).
Vos et al. (1973) investigated the humoral and cell-mediated immune
response in Hartley guinea pigs, CD rats and B6D2F1 mice. The humoral
Immune response was tested in 2,3,7,8-lCDD-treated hamsters by Injecting
tetanus toxoid (subcutaneously) into the footpad and later testing for the
concentration of tetanus antitoxin from the serum by an immunodiffuslon
technique. Cell-mediated immunity was tested by injecting Hycobacterium
tuberculosis (subcutaneously) into guinea pigs on day 35 of 2,3,7,8-TCDD
treatment (during a schedule of 8 weekly doses). Intradermal tuberculin
hypersensitlvlty was determined by measurements of skin thickening on days
47 and 54. Decreased skin hypersensitlvity was noted In hamsters treated
with 0.04 g 2,3,7,8-KDD/kg and higher doses. Decreased tetanus antitoxin
levels were evident in guinea pigs treated with 0.2 pg 2,3,7,8-TCDD/kg,
1855A 8-27 03/29/84
-------
TABLE 8-4
CD
U-l
c_n
Species/ Sex
Strain
M1ce/B6D2Fl M
M1ce/C57Bl/6 F.M
Mice/ M
C57Bl/6Jfh
CD
1
ro
CD
Mice/Swiss M
M1ce/B6C3Fl F
Mice/Swiss- F.M
Webster
Mice/CD M
o
\ Mice/CD M
rsj
in
Exposure Route
gavage
maternal ly
administered
(gavage)
gavage
gavage
1n vitro
(spleen cells)
maternal ly
administered
(diet)
gavage
1n vitro
Immunologlcal Effects of 2,
Dose(s) Duration of Exposure
0, 0.2, 1 .0, 5.0, 4 weeks
25.0 yg/kg bw/week
0, 1.0, 2.0, 5.0, 4 or 6 weeks (3 or 5
25.0 yg/kg administrations)
0, 0.5, 1, 5, 10, 4 weeks
20 yg/kg bw/week
0, 1.5, 5, 15, 4 weeks
50 yg/kg bw/week
0.5, 5.0, 50 ng/mi 5-60 seconds
0, 1, 2.5, 5, 10, 10 weeks (pre-gestatlon
20 ppb (dietary) and 3 weeks post-
parturition)
0, 0.01, 0.1, 1.0, up to 8 weeks
10.0 yg/kg bw/week
1(T4-10~» M single
3,7,8-TCDD 1n Animals
Minimum
Effective Dose
NA
5.0 yg/kg bw/week
5.0 yg/kg bw/week
1 .0 yg/kg bw/week
25.0 yg/kg bw/week
2.0 yg/kg bw/week
1 .0 yg/kg bw/week
1 .5 yg/kg bw/week
50 yg/ml
2.5 ppb
2.5 ppb
5 ppb
1 ppb
NA
0.01 yg/kg bw/week
1 .0 vig/kg bw/week
10"» M
Parameter
bw
thymus weight
graf t-versus-
host response
thymus weight
PHA response
skin graft
rejection
Salmonel la
1nf ec tlon
endotoxln (E . col! )
susceptibility
protein, DNA, and
RNA synthesis
^intlnonlr DRf*
a II I tytrll 11 HDL.
reaction
thymlc cortex
contact sensitivity
to DNFB
endotoxln
(Salmonella)
susceptibility
Lister la Infection
serum Immunoglobln
level
serum Immunoglobln
level
lymphocyte blasto-
genlc transforma-
Effect
no change
decreased
decreaseo
decreased
decreased
prolonged
1 ncreased
mortality
and
decreased
time to
death
Increased
mortal 1 ty
dec r ea sed
decreased
atrophy
decreased
Increased
mortality
no change
Increased
decreased
1 nc reas ed
Reference
Vos et al. ,
1973
Vos and
Moore, 1974
Tn 1 Qpen
et al., 1975
Vos et al..
1978a
Luster et al
1979a,b
Thomas and
H1nsd1ll, 1979
Sharma and
GehMng. 1979
Sharfna and
GehMng, 1979
CO
tion
-------
CC IftBLt B-1 (COni. )
u-
Spedes/ Sex Exposure Route Dose(s)
Strain
Mice/Swiss- F oral (diet) 0, 10, 100 ppb
Webster
Mice/ M 1.p. 0, 1, 2, 6,
C57B1/6J 30 yg/kg bw
CD
i
tO
M1ce/B6C3Fl M,F maternally 0, 1.0, 5.0,
administered 15.0 yg/kg bw/day
M1ce/C57Bl/6 M l.p. 0, 0.4, 4.0,
40 yg/kg bw/week
M1ce/C57Bl/6 M 1.p. 0, 0.004, 0.04,
0.4 yg/kg bw/week
Rat/CD F oral 0, 0.2, 1.0,
5.0 yg/kg bw/week
Duration of Exposure Minimum
Effective Dose
5 weeks (or more) 10 ppb
10 ppb
10 ppb
10 ppb
10 ppb
single Injection 1 yg/kg
1 yg/kg
4 days during gestation 1.0 yg/kg bw/day
and lactation
1 .0 yg/kg bw/day
5.0 yg/kg bw/day
4 weeks 4.0 yg/kg bw/week
0.4 yg/kg bw/week
4 weeks 0.004 yg/kg bw/week
6 weeks 5.0 yg/kg bw/week
5.0 yg/kg bw/week
NA
Parameter
tetanus response
antlgenlc RBC
response
sensl tlzatlon to
DNFB
resistance to
Salmonella
resistance 1n
L1ster1a
macrophage and
natural killer
cell activity
macrophage and
natural killer
cell number
antibody
production
L. monocytoqenes
susceptibility
PYB6-tumor suscep-
tibility
bone marrow hypo-
eel lularHy
thymus atrophy
cytotoxlc T-cell
response
1n vitro genera-
tion of cytotoxtc
T-cells
bw
thymus weight
tuberculin hyper-
Effect Reference
decreased H1nsd1ll,
decreased et al . , 1980
decreased
Increased
mortality
Increased
mortality
no change Mantovanl
et al. , 1980
decreased
decreased
Increased Luster et al . ,
1980
Increased
Increased
Increased Clark et al . ,
decreased 1981
decreased Clark et al . ,
1981
decreased Vos et al. ,
decreased 1973
no change
Rat/CD
F oral
0, 10 yg/kg bw/day 10, 14 days
CSS
10 yg/kg bw/day
10 yg/kg bw/day
10 yg/kg bw/day
sens! tlvlty
erythrocyte count
platelet count
neutrophll count
Increased Welssberg and
decreased Zlnkl. 1973
Increased
-------
TABLE 8-4 (cont. )
Species/ Sex Exposure Route Dose(s)
Strain
Rat/F-344 F,M maternally 0, 1.0, 5.0 yg/kg
administered bw/dose
Rat/Fischer F,M maternally NR
administered
(NR)
ID
1
£§ Rat/F1scher- F,H maternally 0, 5 yg/kg bw/dose
Wlstar administered
(NR)
Rat/Sprague- M 1.v. 0, 1 yg/kg bw
Oawley
Guinea pig/ F gavage 0, 0.008, 0.04,
Hartley 0.2, 1.0 yg/kg bw
Duration of Exposure Minimum
Effective Dose
4 or 6 weeks (3 or 5 1.0 yg/kg bw/dose
administrations)
5.0 yg/kg bw/dose
5.0 yg/kg bw/dose
5,0 yg/kg bw/dose
5.0 yg/kg bw/dose
NA
4-6 weeks (during ges- NR
tatlon and neonatally)
NR
3 or 4 applications 5 yg/kg bw/dose
during gestation and
neonatally
5 yg/kg bw/dose
5 yg/kg bw/dose
single Injection 1 yg/kg bw
B weeks 0.04 yg/kg bw/week
0.04 yg/kg bw/week
0.04 yg/kg bw/week
0.2 yg/kg bw/week
Parameter
bw and thymus
weight
spleen weight
PHA response
graf t-versus-host
response
skin graft
rejection
pseudorables
virus Infection
Con A and PHA
response
oxazolone skin
hyper sensitivity
antibody production
to bovine gamma
globulin
PHA and Con A
response
thymus and bw
thymlc RNA
synthesis
thymlc RNA
polymerase
activity
bw
thyraus weight
tuberculin hyper-
sensH1v1ty
tetanus antitoxin
o
\ M = male; F = female; l.p. = IntraperHoneal l.v. = Intravenous; PHA = Phytohemagglut1n1n; Con A = Conconavalln A; RBC = red blood
o 1-f luorobenzene; NA = Not applicable; NR = Not reported
co
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
cell; DNFB = 2,4-d1n1tro,
co
-------
but not at lower dose levels. Vos et al. (1973) also tested the cell-
mediated Immunity In rats exposed to 2,3,7,8-TCDD (0, 0.2, 1.0 or 5.0
ug/kg, once weekly for 6 weeks). M. tuberculosis was Injected Into rats;
by day ?8 of the treatment period, followed by Intradermal hypersens1t1v1ty
testing on day 42. No changes In the thickness of skin were noted In
2,3,7,8-KOO treated rats when compared with controls.
Mice were used to test the effect of 2,3,7,8-lCDD 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-TCDO-exposed mice (0, 0.2, 1.0 or
5.0 pg/kg once weekly for 4 weeks) of the C57B1/6 strain were Injected
Into the right footpad of a hybrid recipient mouse (C57B1/6 x DBA-2). Donor
cells possessing sufficient activity will respond to the DBA-2 antigen on
the host cells, resulting In 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-KDD-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) in hybrid recipient mice receiving spleen cells from mice treated
with 5 pg 2,3,7,8-KDD/kg/week than from donor cells of untreated mice.
Studies continued in an attempt to identify the mechanism of 2,3,7,8-
lCDD-1nduced Immunodeficiency. Rats (F-344) exposed pre- and postnatally by
maternal dosing (1 or 5 Pg 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 phytohemagglutinin (PHA) (Vos and Moore, 1974;
Moore and Vos, 1974). Response to conconavalin A (Con A), a humoral Immune
response, was actually Increased.
1855A 8-31 03/29/84
-------
Since thymus-derived lymphocytes (1-cells) play a central role In 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/6Jfh). 2,3,7,8-lCDD 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-
TCOD administration. Mortality rates and "time until Infection" were used
to determine the 1mmunolog1cal effect of 2,3,7,8-TCOO. A significant
(p<0.05) Increase 1n mortality and decrease 1n time of Infection were noted
1n groups treated with 1 pg/kg or higher doses of 2,3,7,8-lCDD when com-
pared with controls. 2,3,7,8-7CDD at 0.5 pg/kg did not alter these param-
eters and was regarded as a no effect level. The Immune-resistance of mice
to S. bern 1s therefore reduced by treatment with 1 pg 2,3,7,8-TCDD/kg/
week (for 4 weeks).
Pretreatment with 2,3,7,8-lCDD greatly enhances the susceptibility of
mice to E. coll endotoxln (Vos et al., 1978a). Injection of 250 pg of
endotoxln to mice pretreated with 0, 1.5, 5 and 15 pg 2,3,7,8-TCDD/kg
resulted in 0/5, 1/5, 6/6 and 6/6 deaths, respectively. Mice pretreated
with 15 and 50 pg 2,3,7,8-TCDD/kg and Injected with 10 pg 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) In a control group was noted only when 500 pg of endotoxin
was administered, while 10 pg of endotoxln was sufficient to cause similar
mortality (2/5) 1n mice treated with 50 pg 2,3,7,8-lCDD/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
1855A 8-32 03/29/84
-------
means (Thomas and Hlnsdill, 1979). The number of cells reactive to anti-
genie RBC, differential white blood cell counts, organ weights, hlstopathol-
ogles, hypersens1t1v1 ty to 2,4-d1n1tro-l-fluorobenzene (DNFB) and the
resistance to E.. colj 1 IpopolysacchaMde (IPS), Usterla monocytogenes and
Salmonella typhlmurlum IPS were all measured for mice exposed to different
levels of 2,3,7,8-TCOO. Adult female mice were exposed to 2,3,7,8-TCDD for
4 weeks before mating, throughout gestation and for 3 weeks postparturHlon.
Young mice being tested for 1mmunotox1c1ty were therefore exposed to
2,3,7,8-lCDD only jn utero and through lactation. The typical decrease In
thymus weight was noted In 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
(PFC) reactive to sheep RBCs was significantly reduced 1n the 2.5 and 5.0
ppb 2,3,7,8-lCDD-exposed groups. (Because of the poor survival of young In
the 10 and ?0 ppb 2,3,7,8-lCDD-exposed groups, results and comparisons were
usually reported for the three lower dose groups). The humoral content of
anti-RCD antibodies, however, was not lower 1n 2,3,7,8-TCDD-exposed groups
when compared with controls. A decrease In the skin hypersens1t1v1ty to
DNFB following sensltlzatlon was noted 1n all 2,3,7,8-TCOD-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 S.
typhlmurlum 1n a dose-related fashion. The response to £. 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) jn vitro to Con A, nor
was mltogen-induced lymphocyte proliferation affected (Thomas and Hlnsdill,
1979).
Similar findings were reported in F1scher/W1star rats exposed to
2,3,7,8-lCDD during gestation (18th day) and neonatally, or neonatally alone
1855A 8-33 03/29/84
-------
(on days 0, 7 and 14) (Faith and Luster, 1979). Dams were treated with 5
g/kg 2,3,7,8-lCDD on each dose day. Typically, body weight and thymlc
weights were decreased In progeny, which lasted until 135 days of age. The
thymlc- and splenic-cell response to PHA and Con A was decreased In all
2,3,7,8-TCOD-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 gTobulIn, which requires
T-heTper cell function, was not affected by 2,3,7,8-lCDD 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 yg/kg 2,3,7,8-lCDD, were studied for Immunotoxlc effects and host
susceptibility (Luster et al., 1980). At the 15.0 Pg 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-
ceTluTarlty and depressed macrophages-granulocyte progenitor cells and
pleuripotent stem cells were associated with 2,3,7,8-lCDD exposure at the
5.0 and 15.0 pg/kg dose levels. Hematological changes, such as decreased
RBC count, hematocrit and hemoglobin, and lymphocyte count showed a dose-
related response. Host susceptibility to L. monocytogenes and PYB6-tumor
cells was tested in the 2,3,7,8-TCDO-exposed neonates. Death occurred 1n 73
and 40% of the L. monocytogenes Inoculated (1.2xl06 viable organisms) mice
in the 5.0 and 1.0 yg/kg dose groups, respectively, compared with 28% of
controls. Tumor development occurred in 44, 60 and 22% of the neonates
InocuTated with 5x10" tumor cells from the 5.0 yg 2,3,7,8-TCDD/kg, 1.0
ng 2,3,7,8-TCOD/kg and control groups, respectively.
1855A 8-34 03/29/84
-------
Hlnsdlll et al. (1980) reported that ?,3,7,8-lCDD 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 In
6-globulins. At 10 ppb 1n the diet, ?,3,7,8-TCDD caused decreased Immune
response to tetanus toxold, sheep RBC, S. typhlmurlum and L. monocytogenes,,
and lowered contact sensitivity to DNFB. 7h1s study also sugggested that
although young animals are more susceptible to 2,3,7,8-TCDD, older animals
are still Immunosuppressed and exposure In utero 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-KDD 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 In 1-month-old mice than 1n
4-month-old mice.
The effect of single i.p. doses of 2,3,7,8-TCDD (1, 2, 6 and 30 pg/kg)
on peritoneal macrophage and splenic natural killer cell function 1n mice
(C57B1/6J) was studied by Mantovani et al. (1980) and Vecchi et al. (1980).
2,3,7,8-KDD treatment at all dose levels did not decrease the cytostatic
and cytoddal activity of macrophages or natural killer cells on a per cell
basis. Ihe total number of macrophages and splenic natural killer cells
recovered from 2,3,7,8-lCDD-treated animals, however, was reduced when com-
pared with untreated controls. Marked hypocellularity noted in the bone
marrow of 2,3,7,8-KDD-treated mice may account for the decrease in 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-lCDD-exposed animals (Mantovani 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).
1855A 8-36 03/29/84
-------
The actual mechanism of 2,3,7,8-TCDD 1mmunotox1cUy is 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 1-lymphocyte activity following 2,3,7,8-TCDD treatment.
Vos et al. (1973) measured serum cortlsol and corticosteron levels 1n guinea
pigs exposed to 2,3,7,8-TCDO 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 immunosuppresslon 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
in conjunction with 2,3,7,8-TCDD did not protect mice from the typical
2,3,7,8-lCDD-Induced Immunotoxlc alterations. Thymus weight was maintained
but not increased by thymosln, and thymus-derived cells continued to show
decreased responsiveness to mitogens (PHA, Con A). Thus, 1t 1s unlikely
that 2,3,7,8-TCDD affects the supply or synthesis of thymic hormones which
could lead to the observed Immunosuppresslon.
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 preventative
action on thymic involution. Hypophysectomized rats showed advanced thymlc
atrophy.
Sharma arid Gehring (1979) noted that 2,3,7,8-lCDD caused stimulation of
lymphocyte transformation to blast form cells (mitotically active precur-
sors) when no mitogens were present In the culture system. This represents
a phenomenon similar to actual antlgenlc challenge. At low doses (0.01 and
1855A 8-36 03/29/84
-------
0.1 yg 2,3,7,8-TCDD/kg/week for up to 8 weeks), serum ImmunoglobulIn
levels were elevated 1n male CD-I mice. Larger doses of 2,3,7,8-TCDD (1.0
and 10 pg/kg/week) resulted 1n a decrease \n the serum Immunoglobul1m
level. It was suggested that 2,3,7,8-KDO 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 Gehring (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-lCDD-exposure and Its possible antlgenlc complex.
Luster et al. (1979a,b) reported that 2,3,7,8-lCDD affects the Immune
system directly by altering lymphocyte function. The function of T-helper
cells was not altered, since no change In response to bovine gamma globulin
(requires T-helper cell cooperation) was noted 1n Wlstar/Flscher and Fischer
rats exposed to 2,3,7,8-lCDO. Jjn vitro, 2,3,7,8-lCDD (100 ng/mfc) sup-
pressed DNA, RNA and protein synthesis In 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 rnltogen 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-lCDD 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
1855A 8-37 03/29/84
-------
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 In thymlc transcription and
RNA synthesis that may lead to cell surface changes. Cell surface changes
could presumably result in altered antigen recognition and cell-to-cell
recognition, causing Immunosuppresslon and thymlc atrophy.
Clark et al. (1981) reported that 2,3,7,8-TCDD treatment (0.4, 4.0, 40
pg/kg weekly for 4 weeks by 1.p. Injection) caused functional Impairment
of cytotoxic T-cells in 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
et al. (1979a,b), 2,3,7,8-TCDD treatment Impaired the generation of cyto-
toxic T-cells by the spleen (at doses as low as 0.004 yg/kg when detected
In vitro) but did not appear directly toxic to the cytotoxic 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 cytotoxic T-cells in DBA/2 mice as
compared with C56B1/6 mice. This indicates that susceptibility to 2,3,7,8-
TCDD Immunotoxlclty segregates with the Ah locus which is 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 cytotoxic T-cells which Is again consistent
with the dominant Inheritance of Ah (Nagarkattl et al., 1984).
Few reports are available in which the Immunologlcal effects of 2,3,7,8-
TCDD exposure were studied 1n humans. Reggiani (1980) reported that the
1855A 8-38 03/29/84
-------
Immunocapabllity of M people, ranging In 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 Immunoglobin G, A, M, 0 and E,
as well as lymphocytes, T-cells, 8-cells, PHA response and blood cell
counts. These determinations were made 10 years after workers had developed
2,3,7,8-lCDD-1nduced chloracne. In this group of workers, there was a sig-
nificant Increase 1n the proportion of cases with reduced IgD and IgM. It
was suggested that the 2,3,7,8-TCDD-exposed group had a reduced Immune
capability and a deficiency 1n T-cell and B-cell cooperation. The Immuno-
toxldty of 2,3,7,8-TCDD In humans cannot be properly assessed because of
the paucity of data recorded soon after exposure. The most prominent
effects 1n animals (I.e., humoral responses) were not measured In humans.
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 In culture (Beatty et al., 1975; Bradlaw et al., 1976;
Knutson and Poland, 1980a; Yang et al., 1983), 1t 1s an extremely potent
enzyme Inducer 1n these systems (Kourl et al., 1974a; 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; Niwa et al., 1975a).
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, ERL-2, NRKE and Chang). ED values ranged
1855A 8-39 03/29/84
-------
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 In 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 C5/B1/6N 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-TCDO 1s thus
similar to the effects seen Iji vivo. The Inductive effect of 2,3,7,8-TCDD
was blocked by actinomydn D and cyclohexlmide, 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).
In all of these studies, there was no correlation between cytotoxlcity
and enzyme Induction. This Implies that, despite the correlation U[ vivo
(Section 8.3.5.), there may be no direct connection betweeen enzyme induc-
tion and the toxicity 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 In 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). Th)s 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 micro-
somal N-demethylation in 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 vg 2,3,7,8-TCDD/kg bw. The suppression of N-demethylase
activity was seen only in adult animals. In 10-day-old rats, 2,3,7,8-TCDD
had an inductive effect on this activity.
1855A 8-40 03/29/84
-------
Ihe Inductive effects of 2,3,7,8-lCDD have been demonstrated to be organ
specific. AHIo and Parkkl (1978) Investigated the effects of 2,3,7,8-TCDD
on the activities of AHH, ethoxycoumarin deethylase, cytochrome C reductase,
epoxide hydratase, UDP glucuronosyltransferase, and glutathlone S-trans-
ferase in the liver, kidney, lung, small Intestine and testes of male Wlstar
rats. Monooxygenase activity was stimulated in the liver, lung and kidney,
but not in any other tissue investigated. UDP glucuronosyltransferase
activity increased by a factor of 7 in the liver, by a factor of <2 in the
kidney, and not at all 1n any other tissue. Epoxide hydratase and gluta-
thione S-transferase activities were not affected in any of the tissues
studied, although stimulation of hepatic glutathlone S-transferase has been
reported by other investigators (Manis and Apap, 1979). Enzyme Induction
has also been reported in rat mammary gland (Rlkans et al., 1979), mouse
testes (Mattison and Thorgeirsson, 1978), and rat prostate gland (Lee and
Suzuki, 1980), but the rat adrenal gland is apparently Insensitive to induc-
tive effects of 2,3,7,8-lCDD (Guenthner et al., 1979b).
In the liver of rats and mice, 2,3,7,8-lCDD affects a wide range of
enzymatic activities, Including DT-d1aphorase (Beatty and Neal, 1976a,b),
bilirubin catabolism (Kapitulnik and Ostrow, 1978), ornlthlne decarboxylase
(Potter et al., 1982), 7-ethoxycoumarIn 0-demethylase (Greenlee and Poland,
1978), glutathione S-transferase (Baars et al., 1978; Manis and Apap, 1979),
aldehyde dehydrogenase (Llndahl et al., 1978; Deitrlch et al., 1977), uro-
porphyrinogen decarboxylase (Jones and Sweeney, 1977),
-------
2,3,7,8-lCDD 1s four orders of magnitude more potent than 3-MC 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 \n the absorption maximum
of the carbon monoxide difference spectrum from 450 to 448 nm. In a number
of studies, increased AHIi 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 (Kitchin 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.
8.1.1.5.3. In Rabbit — The response of the rabbit is quite different
from that observed in rats and mice (Hook et al., 1975a). The only changes
1n hepatic enzyme activities observed were suppression of benzpyrene
hydroxylase and benzphetamine N-demethylase. In the same study, biphenyl
4-hydroxylase was induced in the lung and benzpyrene hydroxylase was induced
1n the kidney. In a similar study, a hepatotoxic dose of 2,3,7,8-TCDD (30
yg/kg) failed to alter prostaglandin synthetase activity in hepatic or
renal tissue (Kohli and Goldstein, 1981).
In a series of studies, Johnson and Muller-Eberhard (1977a,b,c,d),
Johnson et al. (1979), Norman et al. (1978a,b), Hem et al. (1980) and Dees
et al. (1982) Isolated a series of cytochromes P-450 from rabbit liver
mlcrosomes. These cytochromes were Immunologlcally distinct, functioned In
different catalytic pathways, and responded differently to induction by
polycyclic aromatic hydrocarbons. 2,3,7,8-lCDD was found to induce two
1855A 8-42 03/29/84
-------
cytochromes, designated as form 4 and form 6. Form 4 Is the major cyto-
chrome Induced In adult rabbH liver by 2,3,7,8-KDD; however, form 6 Is the
major cyloihrome Induced In newborn rabbit liver (Norman et al., 19785),
adult rabbit lung, and adult rabbit kidney (L1em et al., 1980; Dees et al.,
198?).
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 rabbH 1n Us
response to 2,3,7,8-TCDD. Blphenyl 4-hydroxylase was Induced in the liver,
lung and kidney, blphenyl 2-hydroxylase was suppressed in the liver, and
benzpyrene hydroxylase was Induced 1n the kidney (Hook et al., 1975b).
Testicular microsomal 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 (Tofilon et al., 1980). Testicular microsomal
hcme levels and 6-aminolevulinic acid synthetase activity were unaffected
by this treatment. In contrast to the rat, 2,3,7,8-lCDD did not Induce
Dl-diaphorase in brain, spleen, kidney, lung, heart or liver of male guinea
pigs (Beatty and Neal, 1978).
Aryl hydrocarbon hydroxylase and
-------
an area containing soil contaminated with ?,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.
Kociba et al. (1976) exposed Sprague-Dawley rats to 2,3,7,8-TCDO for 13
weeks. The animals 1n groups of 12 males and 12 females received the com-
pound suspended In acetone-corn oil (1:9) by gavage 5 days/week at doses of
0.0, 0.001, 0.01, 0.1 or 1.0 yg/kg bw. At the end of the treatment period
5 rats of each sex were killed for h1stopatholog1c examination, and the
remaining animals were continued for postexposure observation. This report
on gross, hernatologlc, clinical chemistry and hlstopathologlc (on animals
terminated at the interim kill or killed when moribund) observations was
prepared on data available 13 weeks after termination of treatment. Signs
of toxicity were observed only at the two higher dose levels, and female
rats appeared more sensitive to the toxic effects of 2,3,7,8-lCOD. During
the study there were five treatment-related deaths in the high dose group
females, with throe occurring during treatment and two in the post-treatment
period. In male animals only two deaths occurred In the post-treatment
period in the high dose group. Both the male and female rats of the 0.1 and
1.0 ug/kg groups had depressed body weight; however, greater relative
depression of body weight was observed in the high dose females. Other
changes such as increases in blUrubin concentrations, urinary copropor-
phyrln excretion, and changes In relative thymus or liver weight to body
weight ratio occurred 1n the two high-dose female groups, but only 1n the
1.0 yg/kg male group. Although male rats had significantly decreased
hematologic values (packed cell volume, RBC count and hemoglobin) 1n the two
high-dose groups, and these values were normal in all female rats, the
1855A 8-44 03/29/84
-------
authors pointed out that these results may have been an artifact resulting
from dehydration-Induced hemoconcentration in the female rats. No specific
data were provided, however, to support this last conclusion.
After necropsy, gross examination revealed subcutaneous edema, a
decrease in the size of testes and uteri, and a decrease In the number of
corpora lutea. Histologic examination revealed Involution of the thymus,
decreased number of thymocytes, and focal necrosis and pigment accumulation
in the liver. 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 in
the group fed 0.1 yg/kg. Although hlstologlc evidence from animals killed
during the Interim sacrifice was consistent with the liver and thymus being
the primary target organs, in an animal that died during the study there
were signs of aortic thrombosis and adrenal hemorrhage, and in a second
animal there was severe anemia, suggesting possible involvement of the hema-
topoietic system near the time of death.
Liver toxicity was the only effect of treatment observed during hlsto-
logic examination of rats (Osborne-Mendel) and mice (B6C3F1) administered
2,3,7,8-lCDD for 13 weeks in a preliminary subchronlc toxicity study
designed to define an acceptable dose for a chronic toxldty study (NTP
1980a). Ihe animals in 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, ?, 4 and 8 yg/kg/week, and for mice at doses of 0.0, 1, 2, 5,
10 and 20 yg/kg/week. Deaths occurred at the two high-dose levels in
rats, with 4 females in the 8 yg/kg/week and 1 in 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
185SA 8-4'j 03/29/84
-------
other rats examined in groups administered 1-8 yg/kg/week; however, not
all animals in each group were submitted to necropsy. Normal liver his-
tology was observed 1n 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 1n mice, with a single death
occurring in 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 In all dose groups of male mice,
while the 1 and 2 yg/kg/week dose groups of female mice had normal
livers. Although the group sizes were small, making conclusions tenuous, 1t
appeared that sex differences in the sensitivity to the toxic effects of
2,3,7,8-KDD occurred, and that the more sensitive sex may vary with species
tested.
In a more extensive subchronic study 1n rats, King and Roesler (1974)
followed the development of toxldty by a series of Interim sacrifices
during 28 weeks of exposure to 2,3,7,8-KDD and a 12-week post-treatment
recovery period. Groups of 36 male and 3b female Sprague-Oawley rats were
intubated twice weekly with 2,3,7,8-KDD in 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.
1855A 8-46 03/29/84
-------
Besides a dose-related decrease in body weight gain in male rats and a
decrease in body weight gain 1n the high-dose female rats, the only effect
of exposure to 2,3,7,8-TCDD was hlstologlc changes In the liver. Liver
pathology was normal 1n all treated groups up through the Interim kill at 16
weeks. Fatty changes In the liver were considered the most Important obser-
vation. The fatty changes ranged from single large lipld droplets 1n a few
centrllobular hepatocytes to lipld droplets ^n all centrllobular hepatocytes
with extension into the mldzonal hepatocytes. No clear dose-response
pattern was observed 1n this study; however, H did appear that the severity
of fatty changes was greater In male rats. During the recovery period,
fatty changes progressively decreased 1n severity, but were still present in
some treated animals 12 weeks after cessation of exposure. Other hlstologlc
changes observed in the liver predominantly in the animals killed at 28
weeks included necrosis, Increased nuclear size, subtle distortion of liver
architecture, and hyperchromatlc nuclei. All of these lesions were consid-
ered to be slight or mild, and less tox1colog1cally relevant than the fatty
changes. Ihe 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 In a subchronic 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 In corn oil-acetone (7:1)
weekly by gavage for 16 weeks at doses of 0.0, 0.01, 0.1 or 10.0 yg/kg/
week and killed 1 week after the last treatment. Additional groups of rats
received doses of 0.0 or 1.0 ^g/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 toxldty was a decrease 1n body
1855A 8-47 03/29/84
-------
weight gain 1n the group receiving 1.0 pg/kg/week. After 16 weeks of
exposure to 2,3,7,8-TCOO, liver porphyrlns were elevated -1000-fold 1n 7 of
8 animals receiving 1.0 pg/kg/week, but only 1 of 8 animals in the 0.1
pg/kg/week group had elevated porphyrln levels. No effect was observed in
the low-dose animals. After a 6-month recovery period the porphyrln level
in animals exposed to 1 pg/kg/week was still 100-fold higher than values
in the control group. A similar pattern was observed for urinary excretion
of uroporphyrin. The rate limiting enzyme in heme synthesis,
-------
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-HxCOO. 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,
while mice received 0.0, 1.25, 2.5, 5, 10 and 50 pg/kg. At week 10 of the
study, the body weight 1n rats was decreased 1n a dose-related manner to a
maximum of -20% 1n the high-dose group. In mice, body weight 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 yg/kg/week for male and female rats, respectively,
and at 10 pg/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 patho-
logic findings or none were subjected to necropsy. In mice 1t was stated
that no changes were observed 1n males exposed to 2,3,7,8-TCDO at 1.25
vg/kg/week or In females exposed to 1.25 or 2.5 pg/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.
1855A 8-49 03/29/84
-------
In addition, a second subchronk range finding study conducted by NIP
(1980c) evaluated the dermal toxldty 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 in acetone. There was 100%
mortality in the 25 and 50 pg/application groups and 80% mortality 1n the
10 yg/application group. On histologic examination, there were signs of
liver damage at the lowest dose tested in 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 neoplasia, of long-term
exposure to 2,3,7,8-TCOD have been studied in rats and mice. The primary
purpose of many of the studies in rodents was to assess the carcinogenidty
of 2,3,7,8-lCDD. The observation of non-neoplastic systemic toxic effects
in these studies was often limited, and observations were made near the end
of the natural lifespan when conditions associated with aging may have
obscured some effects produced by 2,3,7,8-TCOD. tong-dura tion toxldty
assays were also conducted in monkeys. Many of the same organs in monkeys
as in rodents were adversely affected by long-term exposure to 2,3,7,8-TCOD;
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 which result in the observed
effects. There also are data on the chronic toxicity of a mixture of
1,2,3,6,7,8- and 1,2,3,7,8,9-HxCDD. No information was found in 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
Miller et al. (197/a,b) defined the dietary level of 2,3,7,8-TCDD which
adversely affected the longevity of rats following chronic exposure. Groups
1855A 8-50 03/29/84
-------
CO
Sir Effects of Chronic Exposure
Species/Strain Sex/No. Dose Treatment
Schedule
Rat/ M/10 0.0 ppt NA
Sprague-Dawley
M/10 1 ppt continuous 1n
diet for 78 weeks,
M/10 5 ppt continuous In
diet for 78 weeks
M/10 50 ppt continuous 1n
diet for 78 weeks
M/10 500 ppt continuous In
diet for 78 weeks
CD M/10 1000 and 5000 ppt continuous 1n
1 diet for 78 weeks
^
M/10 50,000, 500,000 and continuous 1n
1,000,000 ppt diet for 78 weeks
TABLE 8-5
to 2,3,7,8-TCDD
Duration
of Study
95 weeks
95 weeks
95 weeks
95 weeks
95 weeks
95 weeks
95 weeks
1n Laboratory
Parameters
Monitored
survival
survival
survival
survival
survival
survival
survival
Rodents
Effects of Treatment Reference
40% survived until 95 Van Miller
weeks, the first death et al., 1977a,b
occurred at week 68
SOX survived until 95
weeks, the first death
occurred at week 86
60% survived until 95
weeks, the first death
occurred at week 33
60% survived until 95
weeks, the first death
occurred at week 69
50% 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
Rats/ M&F/50&50 -2193 ppt
Sprague-Dawley (0.1 yg/kg/day)
continuous 1n 2 years
diet for 2 years
extensive hlsto-
pathology, hema-
tology, urine
analyses, and
clinical chemistry
o
'NJ
Cumulative mortality.
Increased (F);
bw gain,
decreased (M,F);
Red blood cell count,
decreased (M,F);
Packed cell volume,
decreased (M,E);
Hemoglobin,
decreased (M,F);
Retlculocytes,
Increased (M,F);
White blood cell count,
decreased (F);
Serum glutamlc pyruvlc
transamlnase, Increased
G-Glutamyl transferase,
'increased (F);
Alkaline phosphatase,
Increased (F);
Koclba et al.,
1978a, 1979
(F);
-------
OD
TABLE 8-5 (cont.
Species/Strain Sex/No. Dose Treatment Duration
Schedule of Study
Rats/
Sprague-Dawley
(cont.)
Rat/ M&F/50&50 -208 ppt continuous In 2 years
Sprague-Dawley (0.01 vg/kg/day) diet for 2 years
Parameters Effects of Treatment
Monitored
Urinary coproporphyr 1n,
Increased (F);
Urinary uroporphyr 1n ,
Increased (F);
Urinary del ta-amlno-
levullnlc add,
Increased hepatic
degeneration,
Increased (M.F)
extensive hlsto- Urinary coproporphyr 1n,
pathology, hema- Increased (F);
Reference
Koclba
1978a,
Koclba
1978a.
et al.,
1979
et al. ,
1979
M&F/50&50 -22 ppt continuous 1n
(0.001 pg/kg/day) diet for 2 years
Rat/ M&F/758J5 0.0 ng/kg/week
Osborne-Mendel
M&F/50&50 0.5 wg/kg/week
M&F/50&50 0.05 ug/kg/week
M&F/50&50 0.01 ng/kg/week
o M1ce/B6C3Fl MiF/758,75 0.0 pg/kg/week
NA
administered by
gavage biweekly
for 104 weeks
administered by
gavage biweekly
for 104 weeks
administered by
gavage biweekly
for 104 weeks
NA
2 years
106 weeks
107 weeks
107 weeks
107 weeks
tology, urine
analyses and
clinical chemistry
extensive hlsto-
pathology, urine
analyses and
clinical chemistry
extensive hlsto-
pathology
extensive hlsto-
pathology
extensive hlsto-
pathology
extensive hlsto-
pathology
Urinary uroporphyrIn,
Increased (F);
Hepatic degeneration,
Increased (M.F)
No differences from
values obtained from
control animals
Toxic hepatitis;
0/74 (M), 0/75 (F)
Toxic hepatitis;
14/50 (M), 32/50 (F)
Toxic hepatitis;
0/50 (M), 1/50 (F;
Toxic hepatitis;
1/50 (M), 0/50 (F)
NTP, 1980a
CO
M&F/50&50 0.5 pg/kg/week (M) administered by
2.0 pg/kg/week (F) gavage biweekly
for 104 weeks
105-106 weeks extensive hlsto- Toxic hepatitis;
pathology 1/73 (M), 0/73 (F)
107 weeks extensive hlsto- Toxic hepatitis;
pathology 44/50 (M), 34/47 (F)
NTP, 1980a
-------
TABLE 8-5 (cont. )
Species/Strain Sex/No. Dose
M1ce/B6C3Fl M&F/50&50 0.05 yg/kg/week (M)
(cont.) 0.2 yg/kg/week (F)
M8.F/50&50 0.01 yg/kg/week (M)
0.04 yg/kg/week (F)
oo
03 Mice/Swiss M/38 0.0 yg/kg/week
M/44 0.007 yg/kg/week
Treatment
Schedule
administered by
gavage biweekly
for 104 weeks
administered by
gavage biweekly
for 104 weeks
NA
administered by
gavage weekly
for 1 year
Duration
of Study
107 weeks
107 weeks
588 days
649 days
Parameters
Monitored
extensive hlsto-
pathology
extensive hlsto-
pathology
histology on all
organs
histology on all
organs
Effects of Treatment Reference
Toxic hepatitis; NTP, 1980a
3/49 (M), 2/48 (F)
Toxic hepatitis;
5/44 (M), 1/50 (F)
Dermatitis and Toth et a!.,
amyloldosls; 0/38 1978, 1979
Dermatitis and
amyloldosls; 5/44
M/44
M/43
0.7 yg/kg/week
7.0 yg/kg/week
administered by
gavage weekly
for 1 year
administered by
gavage weekly
for 1 year
633 days histology on all Dermatitis and
organs amyloldosls; 10/44
424 days histology on all Early mortality,
organs dermatitis and
amyloldosls; 17/43
NA = Not applicable
us
\
00
-------
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
?,3,7,8-lCDD. 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 In which the first death occurred In these high-dose
groups was 31, 31, 3, 2 and 2 weeks, with all animals In groups >50 ppb dead
by week 4. The mortality rate in 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 In
each group makes It Impossible to precisely define a dose-response relation-
ship, It was apparent that exposure to >1 ppb curtailed survival.
Increased mortality was also observed In female Sprague-Oawley rats
maintained for 2 years on a diet that provided a 2,3,7,8-TCOD dose of 0.1
yg/kg/day, while no Increased mortality was observed in male rats at this
dose or in animals receiving doses of 0.01 or 0.001 yg/kg/day (Kociba et
al., 1978a, 1979). The average dietary levels of 2,3,7,8-KDD associated
with these doses were 2193, 208 and 22 ppt. Interim hematologlc, clinical
chemistry and urine analyses revealed treatment-related changes in a number
of parameters in the high-dose group, along with some of the same changes
occurring in the mid-dose group, albeit to a lesser degree (see Table 8-6).
At termination of the study, gross arid histologic examination indicated that
the liver was the most severely affected organ, with degenerative, necrotic
and inflammatory changes observed. Increases in urinary excretion rates of
coproporphyHn and uroporphyrIn in 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 in the treated groups, and 85 males and 86
1855A 8-54 03/29/84
-------
females In the control groups) were reported, the description of the experi-
mental results did not enumerate the number of animals affected.
When ?,3,7,8-lCDD was administered by gavage 1n corn oil-acetone (9:1)
at dose levels of 0.0, 0.5, 0.05 or 0.01 pg/kg/wcek, "toxic hepatitis" was
observed respectively in male Osborne-Mendel rats at incidences of 0/74,
14/50, 0/50 and 1/50, and in female rats at incidences of 0/75, 32/49, 1/50
and 0/50 (NIP, 1980a). loxic hepatitis was defined as "lipldosls (lipoido-
sis) and hydropic degeneration of the cytoplasm of the hepatocytes" in the
central, midzonal and, at times, peripheral portions of the liver. No other
non-neoplastic lesions were observed even though extensive histologlc exami-
nations were performed. The two preceding studies support a NOEL for rats
of -0.001 pg/kg/day, with a tOAEL of 0.05 yg/kg/day, and a EEL for liver
Injury and possibly decreased survival of 0.5 yg/kg/day.
Non-neoplastic effects of chronic exposure to ?,3,7,8-lCDD in mice have
been briefly decribed in studies investigating the carcinogenic potential of
2,3,7,8 1COD. In an NTP (1980a) bioassay, extensive histologlc examinations
were performed on B6C3M mice treated biweekly with 2,3,7,8-lCDD by gavage
In 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
yg/kg/week, and for female animals, the doses were 0.0, 0.04, 0.2 and 2.0
yg/kg/week. The only non-neoplast1c lesion was toxic hepatitis, which
occurred in males at incidence of 0/73, 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 yg/kg/week for 1 year
resulted in amyloldosis of the kidney, spleen and liver, and dermatitis at
the time of death in male Swiss mice (loth et al., 1978, 1979). The inci-
dence of these lesions in the control, low-, medium- and high-dose groups,
1855A 8-55 03/29/84
-------
respectively, was 0/38, 5/44, 10/44 and 1//43. In the high-dose group, the
amyloldosis was extensive and considered to be the cause of early mortal
ity. Ihe amyloldosis may have resulted from the chronic dermal Inflammation
produced by the treatment. From the limited data presented 1n these
studies, It appears that mice and rats were approximately equally sensitive
to the toxic effects of ?,3,7,8-KDD following chronic exposure. Severe
toxic effects were observed at doses of 1 pg/kg/day (early mortality) and
0.28-0.07 pg/kg/day (toxic hepatitis), while a LOAEL for dermatitis and
amyloldosis of 0.001 pg/kg/day was reported. A NOAEl for mice was not
clearly defined by these studies.
Ihe only Information available on the effects of chronic exposure to
HxCDD was provided by an NIP (1980c) bloassay of a 1:2 mixture of
1,2,3,6,7,8- and 1,2,3,7,8,9-HxCOO. Male and female Sprague-Dawley rats and
B6C3H 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, 2.5 and 5.0 pg/kg/week,
while female mice received doses of 2.5, 5.0 and 10 pg/kg/week. The
treated male and female rats had a dose-related decrease in body weight gain
during the latter portion of the study, and the high dose females had re-
duced survival. No gross signs of toxlcity were observed in mice of either
sex. Although extensive hlstologic examinations were performed, the only
treatment-related effect was toxic hepatitis, which was defined as "degener-
ative hepatocytic changes and/or necrosis associated with mild flbrosls and
infiltration." Ihe incidence of this lesion 1n control-, low-, medium- and
high-dose groups, respectively, was, in male rats: 0/75, 28/48, 35/50 and
34/48; in female rats: 0/73, 33/50, 37/50 and 44/50; in male mice: 0/75,
28/50, 35/50 and 34/49; and 1n female mice: 0/75, 33/50, 37/50 and 44/50.
1855A 8-56 03/29/84
-------
The severity of the toxic hepatitis was dose-related; however, 1t Is 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
[ tl s for 1 iver toxIcHy.
8.1.3.2. STUDIES IN NONHUMAN PRIMATES — Initial studies Indicating
the effect of chronic exposure to PCODs Including 2,3,7,8-KDD 1n nonhuman
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, Macaca 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, with monkeys In the
high-dose group surviving only for an average of 91 days, while animals in
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 hematologic and gross clinical changes as well as hlstologlc
changes in the liver evaluated through needle biopsy samples. At death,
major organs were preserved for histologic evaluation. Since both clinical
and histologic 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
1855A 8-5/ 03/29/84
-------
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 retkulum and a loss of Mbosomes, which the authors suggested may
have resulted In the observed decrease 1n serum proteins. Skeletal muscle,
lungs, GI tract, skin and heart had signs of edema as observed under the
light microscope, while the electron micrographs of the heart revealed
vascular degeneration which, 1f 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 ICDDs represented 64% by mass (Norback and Allen,
1973).
Allen et al. (1977) have also assessed the toxlcity of 2,3,7,8-TCDD
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 toxicHy, monitored for hematologlc changes and, following
death or the termination of the study, were subjected to complete autop-
sies. Since no control animals were Included in this study, the data were
compared with pre-exposure values where possible.
As observed In monkeys fed "toxic fat," the monkeys fed 2,3,7,8-TCDD
lost hair and developed swollen eyelids and periorbHal 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
1855A 8-58 03/29/84
-------
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 in poor clotting and the widespread
hemorrhage observed in many organs, which was particularly severe In the
stomach. Also, the decreased RBC count and resultant loss of oxygen-carry-
ing capacity resulted 1n an Increase In 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, It was
concluded that the ultimate cause of death was related to the severe pan-
cytopenia.
The total dose of ?,3,7,8-TCDD used over 9 months in this study by Allen
et al. (1977) was estimated to be between ? and 3 jjg/kg/day, which is
approximately the same dose that resulted in severe toxic effects following
chronic exposure in rats and mice. Schantz et al. (1979) reported in an
abstract that similar, though less severe, effects were observed in female
monkeys following chronic Ingestion of diets containing 50 ppt of 2,3,7,8-
TCDO. It was also noted that this exposure resulted 1n a decreased ability
to successfully bear young (see Allen et al., 1977, in 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 ?,3,7,8-TCDD are nausea and vomiting, headache and signs of Irrita-
tion to the eyes, skin and respiratory tract. Initially a chemical burn-
1855A 8-59 03/29/84
-------
type cutaneous reaction will occur (possibly due to other chemicals),
usually followed by chloracne after several days to weeks (Taylor, 1979).
Chloracne is the most characteristic and frequently observed dermal lesion
produced by 2,3,7 ,8-KDD and other chlorinated aromatic hydrocarbons in
humans (Crow, 1981; Taylor, 1979). This lesion consists of hyperplasia and
hyperkeratosis of the interfollicular epidermis, hyperkeratosis of the hair
follicle, especially at the infundibulum, and squamous metaplasia of the
sebaceous glands which form keratlnaceous comedones and cysts (Kimbrough,
1974). These cutaneous eruptions of comedones, cysts and possibly pustules
In severe cases, usually occurs on the face and shoulders (Crow, 1978a;
Passi et al., 1981). The persistence of chloracne varies greatly, with
severe cases lasting for up to lb years, while mild cases may resolve in a
matter of months. Similar epidermal changes have been produced by 2,3,7,8-
1CDD in rhesus monkeys (McConnell et al., 1978a; Allen et al., 1977), the
ear of the rabbit (Poiger and Schlatter, 1980), and hairless mice (Knutson
and Poland, 1982a). These changes have not generally been observed in other
laboratory animals, such as guinea pigs, hamsters, rats and mice.
Chronic exposure to 2,3,7,8-TCDD most often occurs in chemical industry
workers exposed to low levels of this contaminant during the manufacture of
2,4,5-1 on a daily basis. Chloracne is normally the first symptom noted in
chronic exposure. Systemic symptoms, Including altered function of the
neuromuscular system, liver, kidneys, and pancreas, altered blood chemistry
(serum bilirubin, GOT, GP1, lipid and cholesterol levels), porphyria cutanea
tarda, hyperpigmentation and hyperkeratosis, have also been associated with
chronic 2,3,7,8-TCDD exposure (Crow, 1978b, 1981). A combination of acute,
high-level exposure to ?,3,7,8-lCDO followed by chronic exposure for many
years (or a lifetime) has been noted for residents of areas where PCDDs have
I8SSA 8-60 03/29/84
-------
boon accidentally released Into the environment (laylor, 1979). Residents
of Seveso, Italy, for example, where an explosion of a reactor vessel used
to manufacture 2,4,5-1 released PCDDs and other chemicals Into the atmo-
sphere, were exposed acutely for a few days and are now exposed dally to
diminishing levels of PCDOs In the soil.
Ihe first cases of chloracne associated with exposure to PCDDs occurred
after a 1949 explosion in a chemical factory producing 2,4,5-T In Nltro, WV
(Holmstedt, 1980). A total of 228 workers were exposed. Symptoms Included
nausea, headaches, fatigue, muscular aches and pains, and chloracne (Zack
and Suskind, 1980). Chemical tests revealed elevated I1p1d levels and
prolonged prothrombln time. Chronic symptoms, lasting up to 2 years, were
severe aches and pains, fatigue, peripheral neuropathy and some residual
chloracne. four additional Industrial explosions were reviewed by Holmstedt
(1980). In 1953, 75 workers were exposed during an accident at a factory
(BASF) in Ludwlgshafen, Germany. Most of the workers developed chloracne,
while 21 workers developed nervous system and Internal organ damage in
addition to severe chloracne. In 1963, an explosion at a 2,4,5-T producing
factory in Amsterdam resulted In the exposure of 106 men to chlorinated
dioxin 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 liver function tests, full blood counts and urlnalysls,
were conducted on 14 employees who were In the building at the time of the
explosion (May, 1973). Eleven of these 14 men showed abnormal liver func-
tion (?1nc turbidity, thymol turbidity and serum transamlnase) and altered
1855A 8-61 03/29/84
-------
hcrtuito loy ir.a I parameter s or qlutosuria. later, aftor normal plant opera-
tions were resumed, additional workers apparently were exposed to 2,3,7,8-
1CDO by contact and developed chloracne. Seventy-nine cases of chloracne
developed by the end of 1968. Ihe condition appeared on the face 1n all
cases, with other parts of the body being involved in more severe cases
(May, 1973).
The most recent and extensively studied chemical plant explosion
occurred on July 10, 1976, at the ICMESA (Industrie Ch1miche-Meda-Soc1eta
A/lonaria) plant at Seveso, Italy. This accident, caused by the release of
the reactor contents into the atmosphere, exposed workers and residents
(>865S people) of the area to 2,3,7,8-TCDD (Garattini, 1982). A total of
447 patients developed chloracne and some complained of nausea, vomiting,
headache, diarrhea, hyperhidrosis and irritation of the eyes (Taylor, 1979).
Serious cases of chloracne and dermal blistering occurred in children and
appeared within several weeks of their exposure (Glanotti, "1977; Crow, 1981;
Taylor, 1979). Pocchlari et al. (1979) cited unpublished data reported to
the Lombardy Regional Authority (Boeri, T978; Ch1app1no et al., 1978;
Sirchia, 1978) on the health effects of 2,3,7,8-TCDD to children and adults
at Seveso. Reduced peripheral nerve conduction velocities were noted in
adults and children, with abnormalities being more frequent in people resid-
ing nearer the chemical plant. The immunology of a group (n^-45) of exposed
children was compared with a similar unexposed group. No significant dif-
ferences were noted, but total serum complement activity, lymphocyte blasto-
genic response and peripheral blood lymphocytes were elevated to some degree
In the exposed children (Tognoni and Bonaccorsi, 1982). Exposure to
2,3,7,8-TCDD has been associated with Increased serum gTutamate-oxalacetate
transaminase (GOT), serum GPT and gamma-glutamyl transferase (g-GT) levels
1856A 8-62 03/29/84
-------
In exposed children (PocchlaM et al., 19/9). Compared with normal values
for "healthy" Individuals, lymphocyte aberrations appeared more frequently,
but 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 In the frequency of head-
aches and eye Irritation (p-0.01), GI tract symptoms (nausea, vomHIng, loss
of appetite, abdominal pain or gastritis) (p^l.6xlO~"), and abnormal g-61,
serum GP1 and amlnolevul 1n1c acid levels (p=-2.3xKT4, 0.035 and
1.2x10 b, respectively) was noted 1n those children who had chloracne
(Caramaschl et al., 1981). Ideo et al. (198?) measured urinary D-glucarlc
acid levels to assess liver mlcrosomal en/yme activity In 67 children
exposed to ?,3,7,8 -ICDl) at Seveso. A significant (p<0,0b) Increase In the
glucarlc acid levels, used to Indicate Increased mlcrosomal enzyme activity,
was noted In exposed children 3 years after the accident when compared with
unexposed children (n-86).
The decontamination and cleanup of the ICMESA plant at Seveso began In
May, 1980, and the possible contamination of clean-up workers was closely
monitored arid safely measures were Implemented (Ghez/1 et al., 1982).
laboratory tests on the blood (GOI, Gl'l, g-Gl, alkaline phosphatase, blU-
rubln, hemoglobin, toll counts, thrombopIdstIc partial time, albumin, gamma
globulin, cholesterol and tr Iglycer1dcs) 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 ?,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 lognonl and Bonaccorsl (198?).
1B!)')A 8 63 03/79/84
-------
Ihrce cases of accidental exposure to PCDDs (Isomer not specified) while
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, head-
aches, fatlgability and hypercholesterolemia (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.
Additional reports of toxic effects as a result of acute 2,3,7,8-lCDD
exposure in humans were noted by Kimbrough et al. (1977). Children were
exposed to soil in horse arenas (in Eastern Missouri) sprayed with oil con-
taminated with 2,4,5-trichlorophenol (5000 ppm In the soil) and 2,3,7,8-TCDD
(30 ppm in the soil). A 6-year-old girl developed headaches, diarrhea,
epistaxis and hemorrhagic cystitis, and became lethargic. Two 3-year-old
boys developed chloracne -1.5 months after playing 1n a contaminated horse
arena. Ihree additional individuals who had exposure to the arenas devel-
oped less severe symptoms of headache, skin lesions and polyarthralgia. The
girl was re-examined 5.3 years following exposure to the soil of the horse
arena and showed no residual signs of toxlcity (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 In the manufacture of 2,4,5-1. These workers, how-
ever, were also exposed to di- and trichlorophenols, PCOD contaminants and
2,4-0. Thirteen employees deveToped moderate to severe chloracne, while
another 35 had minimal "active acne" (cysts, comedones or pustules). Other
complaints noted by the workers were eye irritation, hyperplgmentatlon and
hirsutism. Gastrointestinal symptoms (nausea, vomiting, diarrhea, abdominal
pain or blood in the feces) were reported by 22 of the 73 workers. Findings
1855A 8-64 03/29/84
-------
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 assigning a causa-
tive agent(s) would be conjecture (Poland et al., 1971).
In a brief report, Walker and Martin (1979) reported on some of the
clinical findings of eight men who had chloracne as a result of occupational
exposure to ?,3,7,8-lCDD. Five men had elevated g-GT and trlglycerlde
levels. Average cholesterol levels were higher In exposed men than In a
control group of 100 men. Abnormal Upld levels, reported 1n 6 men, were
attributed to enzyme induction. May (1982), however, observed no differ-
ences in triglyceride, cholesterol, alkaline phosphatase D glucarlc acid or
g-Gl levels in 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.
In a survey of ?04 employees engaged 1n the manufacture of 2,4,5-1 for 1
month to 10 years, Ott et al. (1980) reported no cases of chloracne, por-
phyria cutanea tarda or other effects Indicative of dioxin exposure. Maxi-
mum allowable 2,3,7,8-TCDD levels In the final product were <1 mg/kg in 1966
and <0.1 mg/kg in 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, in 49 of 61
employees exposed to 2,3,7,8-TCDD during the manufacture of trIchlorophenol.
Changes in industrial and personal hygiene techniques decreased potential
exposure to 2,3,7,8-TCDD and subsequent chloracne. Additional toxic effects
were not reported. Pazderova-Vejlupkova et al. (1981) reported that 80
workers developed chloracne, nausea, fatigue and weakness in the lower
extremities while engaged In the production of 2,4,5-sodlum trlchloro-
phenoxyacetate and trichlorophenoxyacetate butylester. Prominent clinical
1855A 8-65 03/29/84
-------
symptoms among 55 of the 80 workers Included hypercholesterolemla, hyper-
lipemia and hyperphosphol ipemia, Increased plasma alpha and gamma globulins,
and decreased plasma albumin. Porphyria cutanea tarda was observed 1n 11 of
the 55 workers tested. In some cases Illness subsided, while other cases
became more severe during a 3-4 year follow-up period. Long-term pathologi-
cal symptoms (remaining evident 5 years after exposure) Include deviations
in lipid metabolism, abnormal glucose tolerance and high urinary excretion
of uroporphyrins (Pazderova-Vejlupkova et al., 1981). Polyneuropathy,
usually of the lower extremities, occurred during the period of Illness and
remained evident after 4 years. Singer et al. (1982) also indicated a
decrease in nerve conduction velocities of sural nerves 1n workers exposed
to phenoxy acid herbicides (average exposure, 7 years) when compared with a
similar group of nonexposed workers (40.3 m/sec In exposed vs. 42.8 m/sec in
nonexposed, p-0.02). Although the causative agent Is not known, PCDO
contaminants are suggested.
The toxic effects attributed to 2,3,7,8-TCDD exposure were studied over
a 10-month period in 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). It
is mentioned that these patients had previously been chronically ill and had
frequent infections and allergies (Bogen, 1979). This study was apparently
based on personal evaluations of health in a survey-type format. No control
group was used for comparison and no clinical or medical evaluations of
health were made. Most of these complaints are nonspecific, judgmental and
occur commonly in the general public.
1855A 8-66 03/29/84
-------
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-lCDD and determined the amount of this contaminant to which
veterans may have been exposed during Agent Orange spraying. Based on
studies in which rhesus monkeys were fed small amounts of dietary 2,3,7,8-
KOO and analogy with human data on the minimum toxic dose of 2,3,7,8-tetra-
chlorodlbenzo-p-furan (TCDF), the cumulative minimum toxic dose of 2,3,7,8-
TCDD In man was estimated to be 0.1 pg/kg (Stevens, 1981). Based on
application rates (4.1 g Agent Orange/m2) and 2,3,7,8-lCDD concentration
in the herbicide (2 ppm), the average concentration of 2,3,7,8-TCDD on
sprayed surfaces of Vietnam was estimated to be ~8 pg/m2. Based on
accidental exposures to 2,3,7,8-TCDD In humans (industrial accidents,
Eastern Missouri cases), Stevens (1981) estimated an average Intake transfer
factor (ratio of absorbed compound to environmentally available compound) of
1:2050 for 2,3,7,8-lCDD. Assuming this absorption-to-exposure ratio and
even assuming that a soldier was directly sprayed (exposed to 8 yg/m2)
for each day of his 1-year service 1n Vietnam, his cumulative intake would
be only 1.4 yg or 0.02 vg/kg of 2,3,7,8-lCDD (Stevens, 1981). Based on
these calculations and assumptions, Stevens (1981) reported that 5 years of
direct daily contact with Agent Orange would be necessary to reach a toxic
level of 2,3,7,8-lCDD and felt that claims of illness caused by 2,3,7,8-TCDD
in Agent Orange were without merit. Exception 1s made, however, for certain
workers (forest Industries) who could be exposed to 2,4,5-1 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-
ity of 2,3,7,8-TCDD. The ultimate purpose is to provide a better estimate
1855A 8-67 03/29/84
-------
of man's relative sensitivity to 2,3,7,8-lCDD and other compounds having a
similar mode of action. Specifically, these studies may be able to explain
the reason for the marked Interspecles differences in ?,3,7,8-KDD toxldty
and, thus, help determine if humans possess factors that are associated with
sensitivity to ?,3.7,8-lCDD toxldty.
8.3.1. Receptor-Mediated Toxldty. Pharmacogenetlc studies have played
an important role in understanding the biologic and toxic effects of drugs
and xenobiotics. Nebert and coworkers have shown that carcinogenic poly-
cyclic aromatic hydrocarbons (PAHs) Induce the cytochrome P-450-dependent
monooxygenase AHH in certain responsive strains of mice (e.g., C57B1/6J,
BAtBc, C3HF/He) whereas this PAH Induction activity 1s minimal or nonexis-
tent 1n nonresponsive strains (DBA/2J) (Nebert, 1979, 1982; Nebert and
Glelen, 1972; Nebert and Jensen, 1979; Nebert et al., 1972, 1981, 1983).
The gene complex responsible for the induction of AHH and several other
enzymes has been designated the Ah locus which comprises regulatory, struc-
tural and possible temporal genes. Extensive studies on genetically Inbred
responsive and nonresponsive mice (and their backcrosses) Indicate that
these differences are related to the Ah regulatory gene and Us gene
product, the Ah cytosollc receptor protein. This receptor protein interacts
with PAH ligands and the resultant PAH:Ah receptor complex translocates Into
the nucleus and presumably Initiates the induction of AHH via a process
comparable to that proposed for the steroid hormones.
Since the carcinogenic and toxic effects of PAHs are dependent on their
oxidative metabolism to reactive electrophlHe forms, 1t is not surprising
that the Ah receptor plays an important role 1n mediating their toxicity and
carcinogenicity (Kouri, 1976; KouM et al., 1974; Benedict et al., 1973;
1855A 8-68 03/29/84
-------
Shum et al., 1979; Thomas et al., 1973; Legraverend et al., 1980; Duran-
Reynolds et al., 1978; Robinson et al., 1975; Mattlson and Thorgelrsson,
1979). Responsive mice are more susceptible to the toxic (Inflammation,
fetotoxldty, primordial oocyte depletion) and carcinogenic effects of PAH
at organs/tissues 1n direct contact with the applied chemical; 1n contrast,
nonresponslve mice are more susceptible to the tumoMgenlc 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.
8.3.1.1. 2,3,7,8-TCDD: SEGREGATION 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
al., 1975) demonstrated that the mlcrosomal AHH-1nduc1ng activity of
2,3,7,8-lCDD and 3-MC In several genetically Inbred mice strains were
similar. Like MC and related PAHs, 2,3,7,8-lCDD Induced AHH In several
responsive mouse strains (I.e., C57B1/6J). In contrast to 3-MC, 2,3,7,8-
KDD Induced mlcrosomal AHH in the DBA/2J nonresponslve mice; however, the
ED 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 H was also shown for both 3-MC and
2,3,7,8-TCDD that the trait of responsiveness is Inherited In a simple
autosomal dominant mode (Poland and Knutson, 1982). It has been suggested
that the observed differences In 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 pharmacokinetic and metabolic factors which would more rapidly
diminish the "available" concentrations of 3-MC caused by metabolism and
excretion.
1855A 8-69 03/29/84
-------
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, i-am1nolevul1n1c add, glutath1one-S-transferase B,
T-aldehyde dehydrogenase and chollne klnase by 2,3,7,8-TCDD or 3-MC 1n
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.,
1978; 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 porphyMa, 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) have 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 keratlnlzatlon by 2,3,7,8-TCOO which resembles
some of the characteristics of chloracne (Knutson and Poland, 1980). This
cell line 1s 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 C57B1/6J mice
(Greenlee and Poland, 1979; Okey et al., 1979, 1980; Poland et al., 1976;
Mason and Okey, 1982; Gas1ew1cz 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 phenobarbltal which 1s the only known agent at present
that has been demonstrated to affect tissue concentrations of the receptor
1855A 8-70 03/29/84
-------
(Okey and 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 Is detected 1n the nuclei
of the nonresponslve 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). Several reports have also demonstrated that the cytosollc Ah
receptor protein migrates Into the nucleus of the cell only after binding
with 2,3,7,8-TCDD (Greenlee and Poland, 1979; Okey et al., 1979, 1980) and
this parallels the observations noted for the Interactions between steroids
and their receptor proteins.
8.3.1.2. 2,3,7,8-TCDO AND RELATED TOXIC HAL06ENATED ARYL HYDROCARBONS:
STRUCTURE-ACTIVITY CORRELATIONS — The evidence for a receptor mediated
mechanism of action for 2,3,7,8-TCDO 1s supported by data reported for the
effects of other halogenated aryl hydrocarbons 1n genetically Inbred mice
and other diverse animal species. A number of reviews and comparative
studies (Allen et al., 1979; Allen and Norback, 1977; Klmbrough, 1974;
Klmbrough et al., 1978; McConnell 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 which Include 1) a wasting syndrome which 1s manifested
by a progressive weight loss and decreased food consumption by the treated
animals; 2) skin disorders Including acneform eruptions or chloracne,
alopecia, edema, hyperkeratosls, and hypertrophy of the Me1bom1an glands;
3) lymphold Involution and atrophy; 4) porphyMa (resembling porphyrla
cutanea tarda); 5) endocrine and reproductive disorders; 6) modulation of
1855A 8-71 03/29/84
-------
chemical cardnogenesls; and 7) the Induction of numerous enzymes Including
the cytochrome P-448 (or P-450c) dependent monooxygenases. It 1s apparent
that the effects of these compounds are not manifested In all the animal
species tested. McConnell and Moore (1979) summarized the pathologic
findings observed 1n several animal species after pretreatment with PCDDs,
PCDFs, PCBs and PBBs and 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 which also contain the cytosollc receptor
protein (Carlstedt-Duke, 1979; Carlstedt-Ouke 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, 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 1s substituted In 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 toxldty 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 in culture and their binding affinities for the C57B1/6J mouse hepatic
cytosolic receptor protein (Poland et al., 1976, 1979; Bradlaw et al., 1980;
Bradlaw and Casterline, 1979). Comparable structure-activity correlations
have been reported for the PCDFs in which the most active compound, 2,3,7,8-
KDF, is 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
1855A 8-72 03/29/84
-------
correlation between the toxldty of several Individual PCOFs (Yoshlhara et
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., unpublished).
The most active PC8 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-MC 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, 1977; Goldstein et al., 1977).
Like Aroclor 1254, all the monoortho and at least eight dlortho-chloro
analogs of the coplanar PCBs exhibited a "mixed-type" Induction pattern and
Induced mlcrosomal AHH, OMAP 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 (ED5Q) 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
3,3',4,4',5,5'-hexachlorobiphenyls) > 3,4,4',5~tetrachlorob1phenyl > mono-
ortho coplanar PCBs > d1o£thj) coplanar PCBs. It was also apparent that the
relative toxlcities of this group of PCBs paralleled their biological
potencies (Blocca et al., 1981; Yoshlhara et al., 1979; Marks et al., 1981;
McKinney et al., 1976; Yamamoto et al., 1976; Ax and Hansen, 1975; Kuroki
and Masuda, 1977).
1855A 8-73 03/29/84
-------
The coplanar and monoortho coplanar PCBs also exhibit differential
effects 1n the Inbred C57B1/6J and OBA/2J mice. These compounds Induce AHH
and cause thymlc atrophy 1n the former "responsive" mice whereas at compar-
able or higher doses none of these effects are observed In the nonresponslve
DBA/2J mice (Parkinson et al., 1982; Robertson et al., 1984). The results
obtained for structurally diverse PCDDs, PCBs and PCDFs clearly support the
role of the receptor protein 1n Initiating the broad spectrum of biologic
and toxic effects elicited by these chemicals. Bandlera et al. (1983) have
demonstrated that the 2,3,7,8-KDD receptor protein Is not only susceptible
to halogen substitution patterns but also the structure of the substHuent.
Ihe 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 substHuent. The binding
data for 13 different substltuents was subjected to multlparameter regres-
sion analysis to correlate binding avidities with the physical chemical
characteristics of the critical lateral X substltuents. The equation
log (1) = 1.53o t 1.47 1 <- 1.09 HB + 4.08
ESo
showed that Ugand binding was dependent on substHuent electronegatlvHy
(o), I1poph1l1c1ty (1) and hydrogen binding (HB) with a correlation
coefficient (r) equal to 0.978 for 13 different substltuents.
The receptor mediated hypothesis for the mechanism of action of 2,3,7,8-
KDD still requires further confirmation and numerous problems must be
clarified. For example:
1. Several cell culture lines which appear to have the Ah
receptor are highly resistant to the toxldty of TCDD; the
nonresponslve HK and responsive H-4-II-E cell lines (I.e.,
for AHH 1nducib1lity by TCDD) do not possess cytosollc
receptor; however, the nonresponslve HTC cells possess more
nuclear receptor binding protein than the responsive H-4-II-E
cells (Okey, 1983; Okey et al., 1980).
1855A 8-74 03/29/84
-------
2. Hepatic cytosollc receptor levels In 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
1CDD (LD50 - 1-2 vg/kg) whereas the susceptibility of the
other species follows the order rat > C57B1/6J mice > DBA/2J
mice > hamster (Neal et al., 1982).
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-TCOD-
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 In feces suggests, however, that some routes of excretion may
not be dependent on prior metabolism of the toxin (Olson et al., 1983).
Thus, 1t 1s not possible to directly correlate the half-life for elimination
of 2,3,7,8-lCDD with Us jjn vivo rate of metabolism 1n a given species. The
relative persistence of ?,3,7,8-TCDD 1n a given species may be related to
the jn vivo rate of 2,3,7,8-lCDD metabolism, excretion of the toxin not
dependent upon metabolism (direct Intestinal elimination, lactation, sebum),
and the relative tissue distribution of 2,3,7,8-TCDD, particularly to
adipose stores. Qualitative and quantitative differences In 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 toxldty (Olson et al., 1983).
Polger et al. (1982a) suggest that 2,3,7,8-TCDD metabolism represents
detoxification, since they observed relatively little toxldty 1n 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-lnduced
1855A 8-75 02/29/84
-------
porphyrla (DeVerneull et al., 1983). Current data on the structural Identi-
fication of 2,3,7,8-TCDD metabolites suggest that reactive epoxlde Inter-
mediates may be formed during metabolism (Polger et al., 1982b; Sawahata et
al., 1982). Poland and Glover (1979) reported that the maximum possible in
vivo covalent binding of 1,6-3H-2,3,7,8-TCDD derived radioactivity to
hepatic DNA was 4 orders of magnitude less than the levels of binding
observed with other chemical carcinogens. The study did find 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 1s 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, polar
metabolites of 2,3,7,8-TCOO, H remains to be shown whether unexcreted
reactive metabolites Initiate some of the toxic responses associated with
exposure to this toxin.
8.3.3. VHamln 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 1mmunosuppress1on (see Section 8.1.1.; reviewed In Thunburg et
al., 1980). The administration of a single oral dose of 0.1, 1.0 or 10 yg
2,3,7,8-TCDD/kg bw produces a dose-related decrease In the hepatic storage
of retlnol In Sprague-Dawley rats (Thunburg et al., 1979, 1980). The
authors suggested, but did not demonstrate, that the low storage of retlnol
1n the 2,3,7,8-TCDD-treated animals Is the result of an Increased turnover
of retlnol. 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 supple-
ments could not fully compensate for the 2,3,7,8-TCDD-produced decrease 1n
hepatic retlnol content.
1855A 8-76 03/29/84
-------
8.3.4. Llpld Peroxldatlon. Increased I1p1d peroxldatlon has been sug-
gested as a possible mechanism of 2,3,7,8-7CDD-1nduced toxlclty (Sweeney and
Jones, 1983). This hypothesis Is based on the following limited pieces of
evidence. First, Iron deficiency Inhibits in vitro llpld peroxldatlon (Bus
and Gibson, 1979; Sweeney et al., 1979) and reduces the hepatotoxlc effects
of 2,3,7,8-lCDO (Sweeney et al., 1979). Secondly, llpofusdn pigments,
by-products of lipld 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 0.7b% 1n the diet provided some protection from
2,3,7,8-TCDD-lnduced prophyrla and neutral llpld 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 llpld peroxldatlon 1n Sprague-Dawley rats. Groups of 4-8 female
rats were treated for 3 days with 2,3,7,8-lCDD at doses of 0, 10, 20 or 40
vi'j/kg by gavage (In a corn oil vehicle). At days 1, 6 and 11 after the
last treatment the animals were sacrificed and llpld peroxldatlon was deter-
mined In isolated liver mlcrosomes by the reaction of formed malondlaldehyde
with thlobarbltur1c acid. At all sacrifice periods, Increased Upld 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 llpld peroxldatlon In
vivo by the determination of conjugated dlenes In rats receiving 2,3,7,8-
KOO at 40 yg/kg. Using this latter method, similar Increases In I1p1d
1855A 8-77 02/29/84
-------
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 J£ v1y o formation of reactive free radicals during llpld peroxlda-
tlon could account for the nonspecific nature of 2,3,7,8-TCDD toxldty.
8.3.5. Endocrine Imbalance. Some of the toxic response to 2,3,7,8-TCDD,
Including h1rsut1sm and diminishing Hbldo, Indicate that 2,3,7,8-TCOD may
produce some of Us toxldty through endocrine disturbances (Oliver, 1975).
Menstedt et al. (1979) reported that a single oral dose of 20 pg 2,3,7,8-
TCDD/kg bw significantly reduced testosterone catabollsm. Catabollsm of
exogenous estrogen In ovarlectomlzed rats 1s also decreased by 2,3,7,8-TCDD
pretreatment (Sh1ver1ck and Muther, 1982). In this study, there was a 57%
Increase 1n serum estrone concentrations following administration of 10 mg
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-lCDD-med1ated change 1n estrone disposition.
Shlverick and Muther (1983) also measured estradlcl metabolism 1n female
Holtzman rats given 2,3,7,8-TCDD at a dose of 1 pg/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 In the 7a-hydroxylat1on of testosterone, while there was no
change In the 16a- or 68-hydroxylase activity. Although steroid metabo-
lism was altered In mlcrosomes Isolated from 2,3,7,8-TCDD-treated pregnant
rats, similar exposure of pregnant rats on days 4-15 of gestation did not
result 1n any change 1n circulating levels of serum !7B-estrad1ol. The
1855A 8-78 03/29/84
-------
authors suggested 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 In steroid metabolism In female Sprague-Dawley rats
than In male rats of the same strain, resulting 1n a liver enzyme pattern
displaying less sex differentiation than In unlnduced rats. Based on this
result, they propose that some of the effects of 2,3,7,8-TCDD result 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
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 yg/kg (the ~LD5Q), there was a slight
depression In blood glucocortlcoids during post-treatment days 1-4, followed
by an ~2.5-fold Increase on post-treatment days 7 and 14. While In competi-
tive binding assays between 2,3,7,8-TCDD and a synthetic hormone, dexametha-
sone, 2,3,7,8-KDD had no affinity for the hormone receptor. Thus, 2,3,7,8-
1CDD may stimulate 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 glucocortlcoids was likely not to
participate in the toxIcHy of 2,3,7,8-TCDD through adrenal hyperfunction,
since prior adrenalectomy did not provide any protection from the lethal
effects of 2,3,7,8-TCDD in rats.
1855A 8-79 02/29/84
-------
8.4. SUMMARY
8.4.K 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 LD value of 0.6 yg/kg, while the
male hamster was the least sensitive, with an LDcn value of 5051 vg/kg
bu
(Schwetz et al., 1973; Henck et al., 1981). At least for acute exposure,
the toxlclty 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 In the guinea pig, the most sensitive species
to 2,3,7,8-TCDD, extensive liver damage was reported 1n rats and mice (Gupta
et al., 1973). In general, no specific cause of death could be Identified.
In a limited comparison of the LD for 9 congeners of PCDDs, It appeared
that biologic activity required chlorine In 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
toxlclty In rats and mice. Single high doses, 200 v»9/kg, of 2,3,7,8-TCDD
produced liver necrosis In rats (Jones and Butler, 1974), while 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 8.1.1.5.). 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,
1855A 8-80 02/29/84
-------
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 Immunotoxlc substance In
animal models, causing decreases In 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 polysacchaMde) 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
negated Indirect routes of 1mmunosuppress1on (hormonal controls). 2,3,7,8-
TCDD at Immunotoxlc levels that alter all function, however, 1s not directly
cytotoxic to lymphocytes (Koclba 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 In mice
at oral doses as low as 0.01 and 0.1 ^g/kg/week when administered for up
to 8 weeks (Sharma and Gehrlng, 1979). Thomas and Hlnsdlll (1979) reported
reduced hypersensltlvlty to DNFB, decreased Immune response to £. coll LPS
and decreased thymlc weight In young mice exposed to 2.5 and 5 ppb 2,3,7,8-
TCDD (0.33 and 0.65 pg/kg) through maternal dosing. Thlgpen et al. (1975)
postulated a NOEL of 0.5 Mg 2,3,7,8-TCDD/kg/week for 4 weeks, but more
precise tests of Immunotoxlclty suggest a lower NOEL would be appropriate,
especially for neonatal and young animals.
1855A 8-81 02/29/84
-------
The mechanism of 2,3,7,8-TCDD-induced immunotoxlclty 1s presently
unknown. 2,3,7,8-TCDD Is not likely to decrease Immune responsiveness
through an endocrine control. 2,3,7,8-TCDD may act as an antlgenic agent
causing Immunosuppresslon and thymlc atrophy (Sharma and Gehrlng, 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 In 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 In rats. Hlstologlc changes 1n the
liver of rats killed 2, 4, 8, 16 and 28 weeks after exposure to weekly doses
of 1 ug/kg bw did not reveal fatty changes until week 28, while 12 weeks
after termination of the 28-week exposure, there was still evidence of fatty
changes In the liver (King and Roesler, 1974). A similar long Induction
period was observed by Goldstein et al. (1982b) for porphyrln accumulation
In the liver of rats. Following 16 weeks of exposure to 2,3,7,8-TCDD and a
6-month postexposure period, porphyrln levels were still elevated. The only
study In mice (NTP, 1980a) described toxic hepatitis as the only effect of
subchronlc exposure to low levels of 2,3,7,8-lCDD. In these and other sub-
chronic studies, NOELs of 0.01 vg/kg/day (Koclba et al., 1976), 0.5 v>g/
kg/week (NTP, 1980a) and 0.01 yg/kg/week (Goldstein et al., 1982b) have
been reported for rats. In mice, a NOEL of 2 ng/kg/week was obtained 1n
females, while males exposed to 1 pg/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 pg/kg/week
reported for rats and mice, respectively (NTP, 1980b).
1855A 8-82 02/29/84
-------
In chronic toxldty studies 1n rats and mice, H was again the liver
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 pg/kg/day was a NOEL, while 0.05 and
0.1 pg/kg/day were the NOAEL and EEL 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 pg/kg/day, producing liver damage In
male and female B6C3F1 mice (NTP, 1980a), while the lowest dose tested 1n
Swiss mice, 0.001 pg/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-TCDO 1n the diet at 50 or 500 ppt resulted In 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 avail-
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 pg/kg/week for males and females, respectively, produced toxic
hepatitis and represented a EEL (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, hlrsutlsm and some peripheral neuropathy. Only one estimate
was available, which speculates a cumulative minimum toxic dose of 0.1
vg/kg for man (Stevens, 1981). The available follow-up reports and eplde-
m1olog1cal studies, primarily on populations exposed occupationally, acci-
dentally or in Vietnam, Indicate that toxic effects noted soon after expo-
sure to 2,3,7,8-TCDO may subside or may persist for many years.
1855A 8-83 03/29/84
-------
8.4.3. Mechanisms of ToxIcHy. 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
than the parent compound, although Intermediate or minor metabolites of
2,3,7,8-TCDD may be Involved In toxlclty. Vitamin A depletion, Increased
llpld peroxldatlon and effects on the hypothalamo-pHuHary axis have all
been Implicated as possible mechanisms for 2,3,7,8-TCDD-lnduced 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 which 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 In 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 In the toxic response to 2,3,7,8-TCDD do not correlate with
species differences In receptor concentration or affinity, or with the
degree of enzyme Induction. It thus appears that the toxlclty of 2,3,7,8-
1CDD may be mediated by binding to the cytosollc receptor responsible for
enzyme Induction; however, this theory does not apply In various species,
and cell culture studies Indicate that enzyme Induction Is not necessarily a
cytotoxlc process.
185SA 8-84 02/29/84
-------
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 in eliciting the teratogenlc response. These studies are
summarized 1n 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 2,4,5-1 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 Dlllmann (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 in 2,4,5-T-1nduced cleft palates. When the
1856A 9-1 03/28/84
-------
CO
TABLE 9-1
Studies on the Potential TeratogenU Effects of 2.3,7,8-TCDD Contaminated 2,4,5-T
Spedes/Straln Vehicle
M1ce/NMRI Rape-seed oil
M1ce/NMRI Rape-seed oil
M1ce/NMRI Rape-seed oil
Form of
2,4,5-T TCDD Level
add <0.02 ppm
(Sample A)
add 0.05^0.02 ppm
(Sample B)
add NR (Sample C)
Dally Dose
8, 15, 30,
45, 60. 90
and 120 mg/kg
30, 60 and
90 mg/kg
90 mg/kg
Treat- Obser-
ment vatlon Maternal Response
Days Day
6-15 18 No toxic effects;
decreased maternal
weight at doses of
90 mg/kg and
greater
6-15 18 No toxic effects;
decreased maternal
weight at 90 mg/kg
6-15 18 No toxic effects
but decreased
Fetal Response
Significant Increases 1n
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.
Increases 1n the Incidence
of cleft palate at 60 and
90 mg/kg; significant
(p<0.005) at all dose
levels
Increase 1n the Incidence
of cleft palate; s1gn1f1-
Reference
Neubert
DUlmann
Neubert
Dlllmann
Neubert
Dlllmann
and
, 1972
and
, 1972
and
, 1972
M1ce/NMRI
Rape-seed oil butyl NR
ester
12 and 17
mg/kg
6-15
18
maternal weight
No toxic effects
Mice/NMRI
NR
add
0.05±0.02 ppm 20. 35. 60.
90 and 130
mg/kg
6-15
NR
Toxic effects
observed at 90
and 130 mg/kg
Mice/CD-I
CO
•t*
Corn oil:acetone acid
(9:1)
<0.05 ppm
115 mg/kg
10-15
18
No significant
effect on weight
gain or I1ver-to-
bw ratios
cant (p<0.005) decrease
1n fetal weight
Significant decrease 1n Neubert and
fetal weight but no effect Dlllmann, 1972
on mortality; Increase 1n
the frequency of cleft
palate similar to that seen
with add {see text)
Increases 1n the percent- Roll, 1971
age of resorptlons and/or
dead fetuses at 90 and 130
mg/kg; Increases 1n the
Incidence of cleft palate
and retardation of skeletal
development at 35 mg/kg
and above
No effect on fetal mortal- Courtney. 1977
1ty or fetal weight but
an Increase In the Inci-
dence of cleft palate
-------
as
TABLE 9-1 (cont.)
Form of Treat- Obser-
Spedes/Straln Vehicle 2,4,5-T TCOD Level Dally Dose ment vatlon Maternal Response
Days Day
Fetal Response
Reference
M1ce/C57BL/6 Honey:water
(1:1)
add
30 ppm
46.4 and 113
mg/kg
6-14
18 NR
M1ce/AKR
Honey:water
(1:1)
add
30 ppm
113 mg/kg
6-15
Rats/Sprague- Gavage/hydroxy- add 0.5 ppm 1, 3, 6, 12 or 6-15
Dawley propyl-methyl- 24 mg/kg/day
(groups of cellulose
25 rats)
19
20
Rats/WUtar
Gavage/aqueous
gelatin or
corn oil
acid <0.5 mg/kg
25, 50, 100 or
150 mg/kg/day
6-15
22
ro
to
co
-f*
Rats/W1star
Gavage/aqueous
gelatin or
corn oil
butyl
ester
<0.5 mg/kg
50 or 150
mg/kg/day
6-15
22
Increase 1n Hver-
to-bw ratio
No effect on bw
and no observable
signs of toxldty
Some maternal
mortality and
decreased bw
gain at 150 mg/kg;
no signs of
toxldty at
100 mg/kg or
below
NR
Significant (p<0.01)
Increases 1n the Incidence
of cleft palate 1n the high
dose group and cystic
kidney 1n both dose groups;
Increased fetal mortality
also observed 1n the high
dose group
Significant (p<0.05)
Increases 1n the Incidence
of cleft palate and fetal
mortality
A slight but statistically
significant (p<0.05)
decrease In Implantations
and Utter size 1n lowest
dose group only; no frank
teratogenlc effects based
on a detailed examination
of the control and 24 mg/kg
dose group; the only effect
noted was an Increase 1n
the Incidence of 5th par-
tially ossified sternebrae
At 100 or 150 mg/kg.
decreased fetal weight,
Increased fetal mortality
and an Increase 1n the
Incidence of skeletal
anomalies; no significant
effect at the two lower
dose levels
No significant effect on
fetal mortality, fetal
weight or the Incidence
of anomalies
Courtney
et al., 1970a,b
Courtney
et al., 1970a,b
Emerson et al.
1970, 1971
Khera and
McKlnley, 1972;
Khera et al.,
1971
Khera and
McKlnley, 1972;
Khera et al.,
1971
-------
oo
<_n
o>
3>
TABLE 9-1 (cont.)
Spedes/Straln Vehicle
Form of Treat-
2,4,5-T TCOO Level Dally Dose ment
Days
Obser-
vation Maternal Response
Day
Fetal Response Reference
Rats/Holtzman Gavage/1:! add 30 ppm 4.6, 10.0 and 10-15
solution of 46.4 mg/kg/day
honey and water
20
Rats/CD Gavage/15X add
sucrose solution
0.5 ppm 10.0, 21.5, 6-15
46.4 and 80.0
mg/kg/day
20
Rats/strain Gavage/methocel add
not specified
0.5 ppm 50 mg/kg
6-15
NS
Kats/straln Gavage/methocel add 0.5 ppm 100 mg/kg 6-10
not specified
Syrian Gavage/acetone, add
hamsters/ corn on, and
MesocMcetus carboxymethyl
euratus cellulose In
ratio of 1:5.8:10
<0.1-4.5ppm 20, 40, 80 6-10
and 100 mg/kg
NS
14
NR
Reduced maternal
weight gain at the
2 higher dose
levels (p<0.05)
and Increased
I1ver-to-bw ratio
at the highest dose
level (p<0.05)
No effect on mor-
tality or bw gain
Increased mor-
tality and
decreased bw gain
NS
Significant (p<0.01) Courtney
Increases In fetal mor- et al., 1970a,b
talHy at the 2 higher
dose levels; dose-related
Increases 1n the percent
of abnormal fetuses per
Utter; a high Incidence
of cystic kidneys 1n
treated groups
Increase In the Incidence Courtney and
of kidney anomalies, but Moore, 1971
no Increase 1n cleft
palate
No significant effect on Sparschu
fetal mortality or fetal et al., 1971a
weight; a significant
(p<0.05) Increase 1n the
Incidence of delayed
ossification
Increase 1n the Incidence Sparschu
of delayed ossification et al., 1971a
and poorly ossified or
malallgned sternebrae
(p<0.05)
Dose-related Increases 1n Collins et al.
fetal mortality, gastro- 1971
Intestinal hemorrhages,
and fetal abnormalities;
see text for discussion
of effect TCDD level on
development
CD
NS = Not specified; NR = Not reported
-------
Utter is used as the basic experimental unH, the Incidences of cleft
palate (number of Utters with cleft palate/total numbers of Utters) versus
the dose can be plotted on log dose/probH response paper, correcting for
background response using Abbott's equation. According to this method, the
ED (by eye-fit) for cleft palate Induction are:
2,3,7,8-TCDD: 4.6 Pg/kg bw
?,4,5-l (Sample A): 115 mg/kg bw
2,4,5-1 (Sample B): 46 mg/kg bw
If the assumption were made that all teratogenic activity 1n the 2,4,5-T
samples were attributable to 2,3,7,8-KDD contamination, the expected ED
for samples A and 8 would be ?30,000 mg/kg (0.0046 mg/kg x 0.02 ppnT1) and
92,000 mg/kg (0.0046 mg/kg x 0.05 ppm '), respectively. Since the
observed f I) was lower by a factor of over 1000, 1t 1s suggestive that
2,3,7,8-lCDD Is not the sole factor In 2,4,5 l-1nduced cleft palate.
The nature of possible Interaction between 2,4,5-1 and 2,3,7,8-TCDD Is
more difficult to define. Based on assays of five mixtures of 2,3,7,8-TCDO
and the highly purified 2,4,5-1, Neubert and Dlllmann (1972) noted a greater
than additive effect on the Induction of cleft palates. A similar conclu-
sion can be reached 1f one assumes that Sample A was a "totally pure" sample
of 2,4,5-1. Using the assumptions of simple similar action (Flnney, 1971)
and treating Sample B as a mixture of 2,3,7,8-lCOO and 2,4,5-T, the expected
LD for Sample B would bo 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
in these analyses. Furthermore, the inability to define precisely the
1856A 9-5 03/28/84
-------
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 In 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 f_ 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 1n 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-KDD are presented 1n Table 9-2. CD-I, DBA/2J and C5781/6J mice
were given subcutaneous Injections of 2,3,7,8-TCDD at 1 or 3 yg/kg/day on
days 6-15 of gestation 1n the study by Courtney and Moore (1971). This dose
regime did not result In maternal toxldty, although an Increase 1n the
maternal llver/bw ratio was observed 1n DBA/2J and C57B1/6J mice. 2,3,7,8-
TCDD had no measurable effect on fetal mortality; however, anatomical
abnormalities were observed In 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 yg/kg/day on days 10-13 of gestation, or 1 ug/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
1856A 9-6 03/28/84
-------
oo
u-i
TABLE 9-2
"*" Studies on the Potential leratogenlc Effect of 2,3,7,8-TCDD
i
02/29/84
Species/Strain
House/C57Bl/6
House/AKR
Mouse/CD-I
Mouse/DBA/2J
Mouse/C57Bl/6J
Mouse/C57Bl/6
Mouse/CD-I
Mouse/CF-1
Mouse/NMRI
Rat/CD
Rat/Sprague-
Dawley
Rat/W1star
Vehicle
DMSO or
honey:water
(1:1)
DMSO
acetone:
corn oil
(1:9)
DMSO or
corn oil
corn oil/
acetone
(98:2)
rape-seed
oil
DMSO
corn oil/
acetone
corn oil/
anlsole
Dally Dose Treatment Observation
Days Day
21.5, 46.4, 6-14 or 9-17 19a
113.0 mg/kg
0.5, 1, 3 wg/kg 6-15 17a or 18
1, 3 pg/kg 10-13 or 10 18a
25, 50, 100, 7-16 18b
200. 400 pg/kg
0.001, 0.01, 6-15 18a
0.1, 1.0,
3.0 pg/kg
0.3, 3.0, 4.5, 6-15 18
9.0 w9/kg
0, 0.5, 6-15, 9 and 20a
2.0 w9/kg 10. or 13
and 14
0, 0.03, 0.125, 6-15 20a
0.5, 2.0 and
8.0 wg/kg
0.0, 0.125, 6-15 22
0.25. 1, 2, 4,
8, 16 w9/kg
Maternal 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 w9/kg
maternal toxlclty
observed at or
above 1 w9/kg
Fetal Response
fetoddal, cleft palate,
cystic kidney
cleft palate,
kidney anomalies
cleft palate, kidney
anomalies
cleft palate, hydronephrotlc
kidneys, hydrocephalus , open
eyes, edema, petechlae
cleft palate, dilated renal
pelvis
fetocldal at the high dose,
cleft palate at doses at or
above 5 w9/kg
kidney malformations at
both dose levels
Intestinal hemorrhage at
0.125 and 0.5 pg/kg.
fetal death at higher doses,
subcutaneous edema
Increased fetal death
observed at or above
1 wg/kg. subcutaneous
edema and hemorrhages 1n
the 0.25-2 wg/kg groups
Reference
Courtney
et al., 1970b
Courtney and
Moore, 1971
Moore et al . ,
1973
Courtney, 1976
Smith et al. ,
1976
Neubert and
Dlllmann. 1972
Courtney and
Moore, 1971
Sparschu
et al.. 1971b
Khera and
Ruddlck. 1973
-------
CD
U i
TABLE 9-2 (cont. )
LO
1
OD
Species/Strain
Rat/Sprague-
Dawley
Rat/Sprague-
Dawley
RabbH/
New Zealand
Vehicle
corn oil/
acetone
(9:1)
diet
corn oil/
acetone
(9:1)
Dally Dose Treatment
Days
0.1, 0.5, 1-3
2.0 ygAg
0.001, 0.01 throughout
and 0.1 vg/kgc gestation
0.0, 0.1, 6-15
0.25, 0.5
and 1 (ig/kg
Observation Maternal Response
Day
21 decrease In bw
gain In the high
dose group
post- low fertility at
parturition 0.01 and 0.1 wg/kg
decreased bw at
0.01 and 0.1 wg/kg
dilated renal pelvis
28 maternal toxldty
at doses of 0.25
wg/kg and above
Fetal Response
decreased fetal weight 1n
the 0.5 and 2 wg/kg group
low survival at 0.01 and
0.1 ug/kg, decreased bw at
0.01 wg/kg, slight dilated
renal pelvis at 0.001 wg/kg
In the FI but not succeeding
generations'1
Increases 1n extra Mbs and
total soft tissue anomalies
Reference
G1av1n1
et al..
Murray
et al.,
61av1n1
et al.,
1982a
1979
1982b
aF1rst day of gestation designated day zero
bF1rst day of gestation designated day one
cThe high dose level (0.1 wQ/kg/day) was discontinued due to very low fertility 1n adults
dN1sbet and Paxton (1982) re-evaluated the study by Murray et al. (1979) using different statistical methods and considered the effects ^n the 0.001
group to be statistically significant.
o
\
ru
-------
an average of 95.1% of the fetuses/Utter, with 83.1% having bilateral
kidney anomalies. When the dose was decreased to 1 ^g/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 58.9%. On
the average, bilateral kidney anomalies occurred In 36.3% of the fetuses/
Utter. A single dose of 1 yg/kg on day 10 of gestation produced kidney
anomalies in 34.3% of the fetuses; however, no cleft palates were observed.
Ir'hen C57B1/6 mice were treated with 1 yg/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-TCDO-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 in 4/14 litters. In the pups exposed
to 2,3,7,8-TCDO both 1_n utero and during the postnatal period, kidney
anomalies were observed in 5/7 Utters. 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 ?,3,7,8-TCDD 1n mammalian species. Also reported were their own studies
and previous work (Neubert and Olllmann, 1972) using NMRI mice, 1n 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-
TCOD 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 pg/kg. At day 18 of gestation, extensive
reabsorptlon was observed in the high dose group with 6/9 Utters totally
resorbed. In the few surviving fetuses, there was an 81% incidence of cleft
1856A 9-9 03/28/84
-------
palate. At lower doses, there were 9 and 3% Incidences at doses of 4.5 and
3.0 yg/kg, respectively, and no cleft palates were observed In 138 fetuses
examined In 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
vg/kg. A maximum number of Induced cleft palates occurred when animals
were treated on either day 8 or 11 of gestation, while 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-TCDO
administered orally with 2,3,7,8-TCDD administered subcutaneously. CO-1
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 pg/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 fetotoxic, resulting In decreased fetal weight and
survival. Anatomic abnormalities were observed at all dose levels, with
cleft palate and hydronephrotic kidneys being most common. Other abnormal-
ities observed included hydrocephalus, open eye, edema and petechiae.
Subcutaneous administration of 2,3,7,8-TCDD produced a greater teratogenic
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-KDD on the Incidence of fetal anomalies were
also studied by Smith et al. (1976) 1n CF-1 mice. The mice were given
1856A 9-10 03/28/84
-------
0.001-3.0 pg 2,3,7,8-lCDD/kg/day by gavagc from day 6 through 15 of gesta-
tion. The Incidence of cleft palate was found to be significantly Increased
In 1.0 and 3.0 pg/kg/day dose groups, and the Incidence of kidney
anomalies was significantly Increased at 3.0 pg/kg/day. There were no
observable teratogenlc effects 1n the study at 0.1 pg/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-TCOO
In the responsive C57B1/6J, the nonresponslve DBA/2J and the hybrid B602F1/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 ?,3,7,8-TCDO dissolved In
p-d1oxane or the solvent (control) alone (0.4 mil/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 C57B1/6J mice at
the higher dose level. At a dose of 30 pg/kg, the Incidence of cleft
palates among live fetuses for the C57B1/6J, B6D2F1/J 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-lCOO, the Incidence of cleft palates among live fetuses for the
responsive C57B1/6J, A/J, BALB/cByJ and SEC/1REJ mice was 54, 73, 65 and
95%, respectively. The only responsive mouse (CBA/J) 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/J, AKR/J, SWR/J and 129/J mice was
1856A 9-11 03/28/84
-------
between 0 and 3% at the 30 ng/kg dose level. Thus the responsive mice,
containing high levels of the Ah receptor, are highly susceptible to the
effects of 2,3,7,8-TCDD In producing cleft palate, whereas the non-respon-
sive 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, 198?) suggest that cleft palate formation
elicited by 2,3,7,8-TCDD segregates with the Ah locus.
9.1.3. 2,3,7,8-TCDO Studies 1n Rats. In an early study, Courtney and
Moore (1971) tested the teratogenlc potential of 2,3,7,8-TCDO 1n pregnant
rats (CD) Injected subcutaneously on a dally basis with 2,3,7,8-TCDD (0.5 or
2 vg/kg) in DMSO on days 6 through 15, 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-
centally 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 GI
tracts were observed in 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 pg/kg/day on days 6 through 15 of gestation (Sparschu et al.,
1971b). No adverse teratogenic effects were reported 1n fetuses exposed
transplacentally at the 0.03 pg/kg level. At the 0.125 yg/kg level,
three dead fetuses were reported, fetal weights were slightly depressed, and
Intestinal hemorrhage was noted in 18 of 127 examined fetuses. In the group
1856A 9-12 03/28/84
-------
given doses of 0.5 yg/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 yg/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 1n the group exposed to
8.0 yg 2,3,7,8-TCDD/kg/day. Subcutaneous edema appeared dose-related,
occurring 1n a considerable number of fetuses from the higher dose groups.
Male 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 Ruddkk (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-lCDD at doses of 0.125, 0.25, 1, 2, 4, 8 or 16
yg/kg on days 6 through 15 of gestation. At day 22 of gestation, there
were no live fetuses 1n groups exposed to >4 yg/kg, and reduced litter
size was observed In the 1 and 2 yg/kg group. Unspecified maternal toxlc-
Hy was reported 1n all groups where there was fetal mortality. In groups
exposed to 0.25-2 yg/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 yg/kg dose groups, respectively (the study was
conducted 1n two parts, and the 1 yg/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
1856A 9-13 03/28/84
-------
the control, 0.25, 0.5, 1, 1 and 2 yg/kg groups was 0/10, 1/33, 3/31,
3/10, 3/6 and 3/7, respectively. There were no effects of treatment
observed In the 0.125 yg/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 yg/kg on days 6 through 15 of gestation and allowed
the dams to Utter and wean the pups. In this experiment, maternal toxldty
was reported in the 0.5 and 1 yg/kg group. At birth, there were fewer
viable pups, and the pups had lower body weight In all but the 0.125 yg/kg
group. At weaning on day 21 after birth, there were no surviving pups 1n
the 1 yg/kg group, and 40% of the pups 1n the 0.5 yg/kg group did not
survive. Fostering pups from dams exposed to 2,3,7,8-TCDD at 1 yg/kg onto
control dams did not appreciably Increase survival, while fostering control
pups onto darns exposed to 2,3,7,8-TCDD did not Increase pup mortality.
These data suggest that poor pup survival was a result of delayed toxldty
from lii uterp exposure to 2,3,7,8-TCDD.
G1av1n1 et al. (1982a) assessed the effect of small doses of 2,3,7,8-
TCDD administered during the prelmplantatlon period 1n Sprague-Dawley 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 yg/kg on days 1-3 of gestation. (The legends
to the tables In this paper Indicated that the low dose was 0.125 yg/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 signif-
icantly reduced (P<0.05) 1n the 0.5 and 2 yg/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, In the control 0, 0.1, 0.5 and 2 yg/kg
groups; however, these Increases 1n the treated animals were not statlstlc-
1856A 9-14 03/28/84
-------
ally 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 In fetal weight was considered a result of
2,3,7,8-TCDD delayed Implantation.
In a second study, G1av1n1 et al. (1983) administered the same doses of
2,3,7,8-TCDD (0.0, 0.125, 0.5 or 2 pg/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
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 pg/kg), there were no overt
clinical signs of toxldty In the dams or adverse effects In any of the
fetal parameters examined. At the 0.5 and 2 pg/kg levels, average
maternal weight was decreased. Also, one animal In 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 In the animals of the high-dose group. The only signif-
icant (P<0.01) fetal effect observed 1n the 0.5 pg/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% 1n control and 14.9% 1n treated animals, and 15.5%
1n control and 12.0% 1n treated animals, respectively), and Increases In
both pre- and postlmplantatlon losses of 11.7% for controls and 19.5%
(P<0.05) In 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-
ity, 9/10 Utters 1n the high-dose group contained at least one malformed
1856A 9-15 03/28/84
-------
fetus as compared with 1/13, 2/13 and 2/13 1n the control, 0.125 and 0.5
yg/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-lCDD was administered during gestation.
The reproductive effects of 2,3,7,8-lCDD were also studied In a 3-gener-
atlon study using Sprague-Dawley rats (Murray et al., 1979). Throughout the
study, animals were continuously maintained on diets providing doses of 0,
0.001, 0.01 or 0.1 yg 2,3,7,8-TCDD/kg/day. The parental group (f ) was
maintained for 90 days on the test diets prior to mating. The f rats
were mated twice, producing the filial generations (f,. and f-io)-
Selected f,D and f_ rats were mated at -130 days of age to produce the
I D C
f? and f~ Utters, respectively. In later generations, the high dose
group (0.1 yg 2,3,7,8-lCDD/kg/day) was discontinued because few offspring
were produced 1n this group. At the Intermediate dose (0.01 yg/kg/day),
2,3,7,8-TCDD caused lower body weight 1n exposed rats of both sexes (f,
and fp). At the low dose, no toxic effects were discerned.
Fertility was greatly reduced In the f generation exposed to 0.1 yg
2,3,7,8-lCDD/kg/day. At 0.01 yg 2,3,7,8-TCOD/kg/day, fertility was
significantly (P<0.05) reduced in the f] and f2 rats. Fertility 1n rats
(of any generation) exposed to 0.001 yg 2,3,7,8-TCOD/kg/day was not
different from that of control rats. Decreases 1n litter size were noted In
the f group exposed to 0.1 yg/kg/day and the f and f Utters
IA c »J
exposed at 0.01 yg/kg/day. Statistically significant decreases 1n fetal
survival throughout gestation were noted 1n f and f Utters of the
c *J
0.01 yg 2,3,7,8-TCDD/kg/day exposed dams. At 0.001 yg 2,3,7,8-TCDD/kg/
day, a decreased gestational survival was reported for the f Utters, but
not for other generations. Decreased neonatal survival was noted among
1856A 9-16 03/28/84
-------
f,. and f~ pups exposed to 0.01 Pg 2,3,7,8-TCDO/kg/day, but not among
I n £
fin or f0 pups. Postnatal body weights of the f9 and f« Utters at
1 B O £ 0
0.01 yg 2,3,7,8-TCDD/kg/day were significantly depressed. At the low dose
(0.001 Pg 2,3,7,8-TCDO/kg/day), necropsy of 21-day-old pups revealed a
statistically significant (P<0.05) Increase 1n dilated renal pelvis 1n the
f generation. Subsequent generations at this dose level or any at the
Intermediate dose (0.01 vg 2,3,7,8-TCDD/kg/day) did not have a significant
Increase 1n this abnormality. Significantly decreased thymus weight and
Increased liver weight were reported In the f generation, but not 1n the
O
f, generation (f? generation data not obtained) of the Intermediate dose
group. Murray et al. (1979) concluded that 2,3,7,8-lCDD Ingested at 0.01 or
0.1 yg/kg/day Impaired reproduction among rats, and NOAELs were associated
with 0.001 Pg 2,3,7,8-TCDD/kg/day.
Nisbet and Paxton (1982) reevaluated the primary data of Murray et al.
(1979) using different statistical methods. From this reevaluatlon 1t was
concluded that 2,3,7,8-lCDD significantly reduced the gestational Index,
decreased fetal weight, and Increased liver to body weight ratios and the
incidence of dilated renal pelvis In both lower dose groups. Nisbet and
Paxton (1982) concluded that the dose of 0.001 pg/kg/day was not a NOAEL
1n this study. The FIFRA Scientific Advisory Panel has also reviewed the
data from this three generation study and concluded that the effects
observed at the 0.001 pg/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 pg/kg represented a NOEL.
Crampton and Rogers (1983) 2,4,5-trichlorophenoxyacetic acid (2,4,5-T)
contaminated with 30 ppb of 1CDD appears to have behaviorally teratogenlc
1856A 9-17 03/28/84
-------
effect 1n Long-Evans rats at doses as low as 6 mg 2,4,5-T/kg bw admin-
istered to mother rats on day 8 of gestation.
9.1.4. 2,3,7,8-TCDD Studies 1n Rabbits and Ferrets. A single report by
G1av1n1 et al. (1982b) describes the effects of exposure to 2,3,7,8-TCDD on
fetal development In rabbits. Groups of 10-15 New Zealand rabbits were
administered 2,3,7,8-TCDD by gavage at doses of 0.0, 0.1, 0.25, 0.5 and
1 yg/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 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-KDD 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 in abortions and resorptlons at a dose of >0.25 yg/kg, with
no live fetuses detected 1n the high dose group. In the fetuses, the most
common observation was a significant increase in extra ribs from 33.3% In
the controls to 82, 66.6 and 82% in the 0.1, 0.25 and 0.5 yg/kg dose
groups. Although there was no significant increase in 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) in total soft-tissue anomalies 1n the control, 0.1, 0.25 and 0.5
yg/kg groups. The most prevalent soft-tissue anomaly was hydronephrosis,
which the authors point out was a common finding in rat fetuses exposed to
2,3,7,8-lCDD 1_n_ utero. These effects were considered to be signs of embryo-
toxldty rather than a teratogenlc effect.
In addition to the fetotoxlc effects of prenatal exposure to 2,3,7,8-
lCDD, Norman et al. (1978b) demonstrated that 2,3,7,8-TCDD 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
1856A 9-18 03/28/84
-------
were examined for enzyme activity within 12 hours after birth. While this
treatment Increased the liver cytochrome P-450 levels 1n the adults ~2-fold,
from 1.8-3.7 nmol/mg protein, the Increase 1n 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 liver. The Identity of form 6 was confirmed by 1mmunolog1c
reaction and Us 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.
Muscarella et al. (1982) reported 1n an abstract the fetotoxlc and
teratogenic 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 in fetal deaths and resorbed fetuses, along with growth
retardation. Terata observed Included unilateral and bilateral patalos-
chisl, open eyelids, anasarca and brachygnatha. The author concluded that
2,3,7,8-TCDD was a teratogen in ferrets.
9.1.5. 2,3,7,8-TCOD Studies 1n Nonhuman Primates. Dougherty et al.
(1975) failed to find evidence of teratogenlcity or embryotoxldty 1n rhesus
monkeys that were given on days 22-38 of gestation daily 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
1856A 9-19 03/28/84
-------
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 Is 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
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 pg/kg, corresponding to a calculated dally dose of 0.0015 pg
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 pg 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.
1856A 9-20 03/28/84
-------
McNulty (1978) treated pregnant rhesus monkeys by gastric gavage to
2,3,7,8-TCDD 1n a vehicle of corn o1l:acetone solution. Group I animals
were administered total dosage of 5 yg/kg bw (two animals), 1 yg/kg bw
(four animals) and 0.2 yg/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 pg/kg bw of
2,3,7,8-lCDO 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
pg/kg) had fetal losses. In the next lower-dosed animals (1 yg/kg 1n
both groups), 12 of 16 females had fetal losses; and In the lowest-dosed
animals (0.2 yg/kg 1n Group I), one abortion occurred 1n four pregnancies.
Maternal toxldty was observed 1n many of these treated females. The
difference 1n frequency of fetal loss between all pregnant animals given 1
yg/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
yg/kg bw of 2,3,7,8-TCDD during early pregnancy results In fetal loss In
rhesus monkeys and the results appear to be related to the adverse effects
of 2,3,7,8-KDD on the fetus.
9.1.6. Studies 1n Chickens. The effects of 2,3,7,8-TCDD on the develop-
ment of the heart In 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 acetone:corn oil (0.5:9.5 v/v) on day zero of
embryo development. Administered doses ranged from 0.009-77.5 pmol/egg
1856A 9-21 03/28/84
-------
(0.00029-2.5x10 2 pg/egg) in 5 p8.. 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 1n all types of malformations (ventricular septal
defect, aortic arch anomaly, aortic arch anomaly and ventricular septal
defect, and conotruncal malformations) occurred. Hydroper1card1um was
observed 1n some embryos (not enumerated), but 1t could not be concluded
that this was the cause of the cardiovascular malformations. Malformed legs
and crossed beaks associated with mlcropthalmla was observed 1n treated
embryos, however, the Incidence, 7/284 and 2/284, respectively, was low.
9.1.7. Studies of the TeratogenU and Reproductive Effects of HxCOO. In
addition to 2,3,7,8-TCDD, the teratogenlc potential of a related chlorinated
d1benzo-g_-d1ox1n compound, HxCDD (congeners not specified), has been Invest-
igated In 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
al., 1973). Treatment with high levels of HxCDD (10 and 100 pg/kg) was
highly lethal to fetuses during late gestation. There was a significant
dose-related Increase 1n late resorptlons from 0% (at 0.1 pg/kg/day) to
79% (at 100 pg/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
1n fetuses of rats treated with 100 pg HxCDD/kg/day. No Increase 1n fetal
anomalies was noted 1n fetuses exposed to 0.1 pg HxCDD/kg, and only
subcutaneous edema was more prevalent 1n groups exposed at 1 or 10 pg
HxCDD/kg/day when compared with controls.
Pertinent Information regarding the teratogenldty or reproductive
effects of PeCDDs was not located 1n the available literature.
1856A 9-22 03/28/84
-------
9.2. STUDIES ON HUMAN POPULATIONS
A positive association between 2,4,5-T exposures and Increases in birth
defects or abortions has been reported 1n human populations 1n Oregon (U.S.
EPA, 1979c), New Zealand (Hanlfy et al., 1981), and Australia (Field and
Kerr, 1979). A lack of any such association has been reported In human
populations In 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
epidemiologlc investigation, as well as the difficulties in 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 abortifacients in humans.
Based on a report of a high incidence of abortions in 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
if spontaneous abortion rates differed between the exposed and unexposed
populations, if spontaneous abortion rates evidenced seasonal variation in
these two groups, and if such seasonal variations were associated with
2,4,5-T spray application.
The Spontaneous Abortion Rate Index, as defined by the U.S. EPA, is
"basically the ratio of the number of hospitalized spontaneous abortions to
the number of births corresponding to the spontaneous abortions, based on
1856A 9-23 04/05/84
-------
the residence zip code of the women contributing to each event." Upon com-
pletion of the study, the U.S. EPA concluded that (1) the 1972-77 Spontane-
ous Abortion Rate Index for the study area was significantly higher than 1n
the Rural Control Area or the Urban area; (2) there was a statistically sig-
nificant seasonal cycle 1n the abortion Index 1n each of the areas with a
period of ~4 months. In particular there was an outstanding peak In the
study area 1n June; and (3) there was a statistically significant correla-
tion between the Spontaneous Abortion Rate Index and spray patterns In the
study area when a lag-time of 2 or 3 months was Included. The U.S. EPA con-
cluded, however, that "This analysis 1s a correlational analysis, and corre-
lation does not necessarily mean causation."
M1lby 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. Ihe 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 in the underestimation of
abortions, particularly 1n the urban area (the Incidence of abortions 1n all
three groups was within the expected background rate of 8-1554); 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.
1856A 9-24 03/28/84
-------
EPA 1s attempting or has attempted to correlate 2,3,7,8- TCDD levels 1n the
affected areas with the observed rate of abortion. No published reports
have been located on the outcome of this effort.
In the only other report encountered on a population in the United
States, Nelson et al. (1979) noted a general Increase in the reported
Incidence of facial cleft In both high and low exposure groups 1n Arkansas
from 1948-1974. In this study, exposure estimates were based on average
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 1n 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 al.,
1982a), the report by the Department of Health is essentially anecdotal,
Involving two women who gave birth to malformed children (one with an atrial
septal defect and a malformation of the tricuspid valve of the heart, and
the other with biliary atresia). 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 if
the spraying had been implicated, a lack of Information on 2,3,7,8-TCOD
levels in the spray and the absence of any monitoring data on 2,4,5-T or
2,3,7,8-lCDD would limit the usefulness of this report.
1856A 9-25 03/28/84
-------
The study by McQueen el al. (1977) 1s not published 1n the open litera-
ture but 1s summarized by M1lby 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.1" No addi-
tional details are provided.
Hanlfy et al. (1981) performed an ep1dem1olog1c study 1n Northland, New
Zealand, 1n areas where spraying of 2,4,5-1 was carried out by various
companies for a number of years. The rate of birth defects was obtained
from an examination of hospital records 1n seven nonoverlapping 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, hypospadias and eplspadlas, and
talipes, had elevated rate ratios of >1 (p-0.05) 1n 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 statist-
ically significant association between the occurrence of talipes and expo-
sure when the fractional removal rate was assumed to be 0.25. There was,
however, no statistically significant association where 1.0 was used as the
fractional removal rate.
1856A 9-26 03/28/84
-------
Smith et al. (1982a) Investigated the outcome of pregnancy in 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 mail with 89% of
the chemical applicators responding and 83% of the agricultural contractors
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) is not
published in the open literature, but the following summary is taken from
Milby et al. (1980): "The report concluded that birth defects 1n a group of
babies born in the [Yarrarn] 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 In the
summary. The report by Field and Kerr (1979) plotted the Incidence of
neural-tube defects (anencephaly and meningomyelocele) in New South Wales,
Australia, over the years 1965-1975, and the previous years usage of 2,4,5-T
in all of Australia. The authors noted a decrease in the Incidence of
1856A 9-27 03/28/84
-------
neural-tube defects expected on the basis of the plotted line 1n 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 ep1dem1olog1cal data
on neural-tube defects to be "relatively complete," they do not comment on
the Increasing Incidence of neural-tube defects during the time period of
this study and whether or not an Increase 1n the thoroughness of reporting
neural-tube defects could have contributed to the apparent correlation of
2,4,5-T exposure with these defects. A replotUng 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 In 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 1n 1969 to 1,200,000 kg 1n 1975, Thomas (1980) considered Hungary
to be "...probably the best country 1n which to examine possible health
effects of this herbicide." In any event, all Indices of birth defect rates
decreased or remained stable over the period of study.
1856A 9-28 04/05/84
-------
In addition to contamination of 2,4,5-T being a potential source of
2,3,7,8-TCDD exposure, 2,3,7,8-TCDO 1s also an Inadvertant contaminant of
2,4,5-trlchlorophenol (TCP). Chronic exposure to 2,3,7,8-TCDD may occur
during the manufacture of 1CP and high level acute exposure to ?,3,7,8-TCDO
has occurred after an accident 1n July, 1976 at the ICMESA TCP chemical
factory 1n Seveso, Italy (Bonaccorsl et al., 1978). In this accident, the
reaction used to produce TCP became uncontrolled, producing conditions
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-TCDD, 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-
TCDD (Townsend et al., 1982).
Ep1dem1olog1c studies to determine the reproductive effects 1n 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), Regglani (1980) and B1sant1 et al. (1980). The spontaneous abortion
rate In 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 15-20%
frequency of spontaneous abortion. Subdividing the contaminated area Into
highly, moderately, and least contaminated, and examining the rates for each
1856A 9-29 03/28/84
-------
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 reasons why these studies would not Indicate that the effect of
2,3,7,8-TCDD exposure 1n this accident had no effect on human reproduction.
The authors note that there are many difficulties 1n Interpreting these
data. Adequate data on the Incidence rates of spontaneous abortions and
birth defects were not adequately available 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 preg-
nancies were surgically aborted for fear of 2,3,7,8-TCDD Induced birth
defects. In a recent review of the progress of ep1dem1olog1c Investigations
of the Seveso accident, Tognonl and Bonaccorsl (1982) Indicated that the
data on spontaneous abortions and malformation rates still needed verifica-
tion, and that these data were too preliminary to allow for conclusions.
lownsend et al. (1982) Investigated the reproductive history of wives of
employees potentially exposed to 2,3,7,8-TCDD during chlorophenol production
1n Midland, MI. A total of 930 potentially exposed males were Identified
who had worked for >1 month between January, 1939, and December, 1975, In 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 In a number of cases of chloracne. The con-
trol 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
1856A 9-30 03/28/84
-------
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 1n the control category (conceptions that occurred In the
exposed group before work records Indicating potential exposure to 2,3,7,8-
TCDO were placed 1n the control group), there was no statistically signif-
icant 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.
It was suggested that many confounding factors could account for these nega-
tive results, such as the Inappropriate selection of the populations, the
use of "exposed" persons 1n both exposed and control groups, unidentified
covaMables and low power; however, 1t was asserted that these results were
consistent with animal data, which report that paternal exposure to 2,3,7,8-
TCOD 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
lip 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
1856A 9-31 03/28/84
-------
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
different. The Michigan Department of Public Health (1983a) also reported
these results and, 1n addition, demonstrated that the same results occurred
If 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 In 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 mainten-
ance 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 1n
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.
1856A 9-32 03/28/84
-------
9.3. OTHER REPRODUCTIVE EFFECTS
The effects of a mixture of 2,4,5-T, 2,4-0 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 pg of
2,3,7,8-KDD (Group II); 40 mg each of 2,4,5-T and 2,4-D and 0.16 vq of
2,3,7,8-TCDD (Group III); or 20 mg each of 2,4,5-T and 2,4-D and 1.2 yg 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 motllHy and sperm abnormalities
were evaluated. In addition, the males were mated with virgin females (3
females/week for 8 posttreatment 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 In any groups of treated
mice when compared to controls. Lamb et al. (1981b), 1n 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 in offspring whose sires were exposed to the
phenoxy acids/2,3,7,8-TCDD mixture 1n this study. The only effects noted
were dose-related decreases 1n body weight in the treated males, and these
effects were reversed when treatment was terminated.
1856A 9-33 03/28/84
-------
9.4. SUMMARY
2,3,7,8-lCDD has been demonstrated to be teratogen\c 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 a MED of 1 pg/kg/day, 2,3,7,8-TCDD 1s the most potent terato-
gen known. At higher doses, 2,3,7,8-TCDD has a marked fetotoxlc effect, as
measured by decreased fetal weight and Increased fetal toxlclty. Hemor-
rhaglc GI tract has been associated with 2,3,7,8-TCDD fetal toxlclty.
In rats, 1t has also been consistently observed that 2,3,7,8-TCDD pro-
duced teratogenlc 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 yg/kg/
day. In addition, there 1s some evidence that 2,3,7,8-TCDD can Induce
mlcrosomal enzymes 1n the fetus exposed j£ utero, and this Induction 1s
accompanied by damage to the fine structure of the liver cell; however,
other reports indicate that enzyme Induction occurs only after birth follow-
ing exposure to 2,3,7,8-TCDD through the mother's milk. As 1n mice, hemor-
rhaglc GI tracts have been observed 1n rat fetuses exposed In utero to
2,3,7,8-TCOD.
Rabbits and monkeys are also susceptible to the fetotoxlc effects of
2,3,7,8-lCOD; however, the studies of these species have been too limited to
clearly demonstrate 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-TCDD 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,
1856A 9-34 03/28/84
-------
other studies have failed. 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 2,3,7,8-7CDD being only a
minor component. Hence, 1t 1s not possible to attribute with certainty any
positive finding to 2,3,7,8-TCDO. 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 elldt a detectable response. Although the evidence from
human studies 1s Insufficient to prove 2,3,7,8-TCDD 1s teratogenlc, the
animal data clearly Indicate teratogenlc or fetotoxlc effects 1n all animal
species tested.
1856A 9-35 03/28/84
-------
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 carclnogenldty) have not
been fully assessed (Bartsch et al., 1982).
Mutagenlcity 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).
Hussain et al. (1972) exposed S. typhlmurlum h1st1d1ne-dependent strains
1A1530 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 1A1530 at exposure levels of 1
and 10 yg/ms.. 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 pg/ma,, which resulted 1n
a 0-50% decrease in survival; however, at 2,3,7,8-TCDD levels that resulted
in a 99% decrease 1n survival, there was an Increased number of revertant
colonies/surviving cells. The dose levels were not specified. The source
of the 2,3,7,8-TCDD sample studied in this paper was the Food and Drug
1857A 10-1 02/29/84
-------
CO
— J
o
1
ro
O
ro
The Results of
Type of Assay
Spot test
Plate
Incorporation
Plate
Incorporation*
Fluctuation
test
Spot test
Plate
Incorporation
Plate
Incorporation
Suspension
assay
Suspension
assay
*The assay was
NT = Not tested
S-9 TA98
+/- NT
+/- NT
+/- 0
+/- 0
NT
+• 0
NT
NT
<-/- 0
TA1530
NT
NT
0
0
0
NT
NT
0
NT
performed under both
; QR = Questionable
TA1535
0
0
0
0
NT
0
NT
NT
0
aerobic
response
TABLE 10-1
Mutaqenlclty Assays for 2,3,7,8-TCDD 1n Salmonella typhlmurlum
Strains of Salmonella typhlmurlum
TA1537 TA1538 TA1532 TA1950 TA1975 TA1978 G46 TA100 TA1531 TA1534
0
0
0
0
NT
0
0
NT
0
and
; o =
0 0 NT NT NT NT NT NT NT
0 0 NT NT NT NT NT NT NT
0 0 0 0 000 NT NT
0 0 0 0 000 NT NT
NT +• NT NT NT 0 NT OR QR
0 NT NT NT NT NT 0 NT NT
NT NT NT NT NT NT NT NT NT
NT * NT NT NT NT NT NT NT
NT NT NT NT NT NT 0 frT NT
anaerobic conditions.
Negative response; + = Positive response
Reference
McCann, 1978
McCann, 1978
Gilbert et al., 1980
Gilbert et al., 1980
Seller, 1973
Gelger and Neal, 1981
Gelger and Neal, 1981
Hussaln et al., 1972
Zelger. 1983
-------
Administration, and Us reported purity was 99%. Also, Seller (1973)
observed a positive mutagenlc response 1n a spot test of 2,3,7,8-TCDO per-
formed 1n the absence of a metabolic activation system. However, the purity
of the sample studied was not provided. In tester strains G46 and TA1530,
the ratio of revertants/108 cells 1n the treated plates divided by spon-
taneous revertants/108 cells was <1. In strains 1A1531 and TA1534, the
ratio was between 1 and 2, which was considered a "doubtful" mutagenlc
response, while In 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-amlnopurlne 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 mutagenldty test of 2,3,7,8-TCDD
In two other mlcroblal test systems. A positive response was observed 1n
Escherlchia 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 v»g/mj,. 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/ms.. The absolute number of
colonies/plate was 7 for the control and 46 for the treated plate. The dose
of 2 vg/m8. caused an 89% decrease In cell survival. In the second test
system, the ability of 2,3,7,8-TCDD to Increase prophage Induction 1n £.
coll K-39 cells was examined. The vehicle control, DMSO, Inhibited prophage
induction as compared with the untreated controls, while the most effective
dose level of 2,3,7,8-TCDD (0.5 vg/mi) resulted 1n an Increased prophage
185/A 10-3 02/29/84
-------
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 In the reverse mutation rate 1n t^. col 1 Sd 4
and that 2,3,7,8-TCDD had a weak ability to Induce prophage )n £. 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
In S. typhlmurlum. Wassom et al. (1978) cited a personal communication from
McCann (1978), which 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, 1A1535, 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 pg/plate under both aerobic
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 mutagenlclty 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 In the standard plate
Incorporation assay using S-9 prepared from different sources. In order to
1857A 10-4 02/29/84
-------
maximize the amount of compound tested, dloxane, a better solvent for
2,3,7,8-lCDD than the commonly employed DMSO, was used. Even with the use
of dloxane, the limited solubility of 2,3,7,8-lCDD allowed only 20 vq/
plate to be tested, a dose that was shown to be non-toxic to the cells. The
S-9 used In these assays was prepared from the livers 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-TCOD
concentrations of 0.2, 2, 5 or 20 yg/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 ?,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
mutagenic In the Ames test, even though there 1s positive evidence of
carc1nogen1c1ty.
Mutagenlc effects of 2,3,7,8-TCDD In yeast were observed by Bronzettl et
al. (1983). Positive results for reversion and gene conversion were ob-
tained in vitro and 1n the host-mediated assay. The in vitro experiments
yielded small dose-related Increases 1n trp convertants and 1lv
revertants. An S10 metabolic activation system was required. Exposure of
the yeast to 2,3,7,8-TCDD at the highest level tested (10 yg/m9.)
resulted in 16% survival and yielded 4-fold Increases In reversion and gene
conversion.
1857A 10-5 02/29/84
-------
In the host-mediated assay, male mice were exposed to 25 ^g of
2,3,7,8-TCDD/kg (Bronzettl et al., 1983). After 5, 10, 20 or 30 days, 0.2
ml of a yeast culture (4 x 10B cells) was Instilled retroorbltally.
Four hours later, the liver and kidneys were removed and the yeast cells In
these organs were assayed for mutagenlc responses. Increases (4- to 6-fold)
In 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 1n 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 In DMSO transformed
baby hamster kidney cells (BHK) In. vitro. The dloxln Isomers 2,8-d1chloro-
and 1,3,7-tr1chlorod1benzo-£-d1ox1n also transformed BHK cells, but the
response was weak. The unchlorlnated d1benzo-£-d1ox1n and the fully chlori-
nated octachlorod1benzo-£-d1ox1n were both negative In the BHK assay (I.e.,
there was no cell transformation). More recently, Rogers et al. (1982)
reported that 2,3,7,8-TCDD Induced mutations 1n the excess thymldlne,
thloguanlne and methotrexate selective systems 1n L5178Y mouse lymphoma
cells In culture.
The National Toxicology Program (NTP) (Zelger, 1983) 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 reces-
sive lethal test In Drosophlla, and cytogenetic studies (sister chromatld
exchange and chromosome aberrations) 1n Chinese hamster ovary cells. Nega-
tive results were obtained in all of these assays. These studies cannot be
evaluated, however, because the procedures used to obtain the data were not
described.
1857A 10-6 02/29/84
-------
10.1.2. Interactions with Nucleic Acids, in vitro reactions of 2,3,7,8-
1CDO with bacterlophage QB RNA were evaluated by Kondorosi 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 Pg/mst of 2,3,7,8-TCDD. At all concentra-
tions tested, 2,3,7,8-TCDD had no effect on the transfectlvity of QB RNA.
Other compounds tested Included the alkylatlng agents methyl, ethyl and
Isopropyl methane-sulfonate, and dlethyl pyrocarbonate, all of which Inacti-
vated QB RNA under the same experimental conditions. The authors suggested
that 2,3,7,8-TCDD Inactivity 1n this assay Indicated that 2,3,7,8-TCDD was
an Intercalating agent, and hence would require double stranded DNA In order
to Interact. The data presented 1n this study, however, were Insufficient
to support this conjecture.
In v^vo 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 non-
extractable 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
1857A 10-7 02/29/84
-------
2,3,7,8-KDD binding. As a result of the extremely low levels of radio-
activity associated with RNA and DNA, H Is uncertain whether 2,3,7,8-TCDD
truly binds covalently to these macromolecules and, 1f so, whether there Is
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 In the bone marrow of male
rats were reported In 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 yg/kg. In the
second portion of this study, rats were exposed by a single Intraperltoneal
Injection of 2,3,7,8-TCDD at 5, 10 or 15 yg/kg or a single gavage treat-
ment at 20 yg/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 In chromosomal aberrations was
observed, except In the positive control group exposed to trlethy-
lenemelamlne.
In a later report, a small but significant Increase 1n chromosomal
aberrations was observed 1n the bone marrow cells of male and female
Osborne-Mendel rats (Green et al., 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 al., 1977). The animals
1n groups of 8 males and 8 females received twice weekly Intubations of
2,3,7,8-TCDD at respective doses of 0.25, 1.0, 2.0 and 4.0, or 0.25, 0.5,
2.0 and 4.0 yg/kg for 13 weeks. Because H was not required for the
range-finding study, a control group was not Included. Bone marrow cells
were analyzed for abnormalities and cells In mitosis 1n the animals that
survived to the end of the study (4-8 animals/group). The only significant
1857A 10-8 02/29/84
-------
Increases 1n chromosomal aberrations 1n comparison with the low dose group
were 1n males at 2 and 4 yg/kg and females at 4 yg/kg. The greatest
Incidence observed was 4.65% of the cells with chromosomal breaks 1n the
high-dose males, and 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 toxldty, makes the conclusion from this study that 2,3,7,8-
TCDD produced chromosomal breaks tenuous.
A similar weak response was observed by LopMeno et al. (1982) 1n male
and female CD-I mice which received an Intraperltoneal Injection of 2,3,7,8-
TCDD at a dose of 10 yg/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 any 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 1n the final product were
<0.1 mg/kg; however, the exposure levels for Individual workers were not
available.
Mulcahy (1980) reported no Increased Incidences of chromosomal aberra-
tions 1n the lymphocytes of 15 soldiers exposed to Agent Orange. The expo-
1857A 10-9 02/29/84
-------
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 note that the
long time between exposure and analysis may have accounted for the negative
results.
Also, both Regg1an1 (1980) and Mottura et al. (1981) have studied Inhab-
itants 1n Seveso, Italy, exposed to 2,3,7,8-TCDD from an accident 1n a tr1-
chlorophenol manufacturing plant. 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 In chromosomal aberra-
tions was detected. The methods of performing the analyses and the actual
number of aberrations detected were not described. Similar negative results
were reported 1n 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.
1857A 10-10 02/29/84
-------
D1Lern1a et al. (1982) conducted additional studies on lymphocytes pre-
pared In 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 1n 1976 and six In 1979). Cells were examined for average
number of SAs (evidence for functional rlbosomal genes), both on a cell
basis and for the large acrocentrlc 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 chromo-
somes of acutely exposed subjects examined 1n 1977 and 1n ICMESA workers at
both the 1976 and 1979 examinations. Although the biologic relevance of
these observations has not yet been confirmed, D1Lern1a et al. (1982)
observed a similar decrease 1n SAs after exposure of lymphocytes to
x-1rrad1at1on. It was concluded that the decrease 1n SAs may have resulted
from mutagenlc damage to functional nucleolar organizing regions.
10.2. SUMMARY. A limited number of Initial studies on the mutagenUHy of
2,3,7,8-TCDD 1n bacteria reported positive results 1n S. typh1mur1um strain
TA1532 1n the absence of a mammalian metabolic activation system (Hussaln et
al., 1972; Seller, 1973). More recent attempts to repeat these results with
strain TA1532 or related strains have failed (Gelger and Neal, 1981; Nebert
et al., 1976; Gilbert et al., 1980; McCann, 1978). These authors have also
reported no Increase 1n mutation rate when 2,3,7,8-TCDD was tested In the
presence of a mammalian metabolic activation system. In other jji vitro
assays, 2,3,7,8-TCDD has produced a positive response 1n reversion to
streptomycin Independence 1n £. coll Sd-4 cells and questionable positive
1857A 10-11 02/29/84
-------
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 1n both the _1jn vitro assay with S-10 and the host-mediated assay
(Bronzettl et al., 1983). Rogers et al. (1982) have also reported positive
mutagenldty results 1n 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 diffi-
cult. WHh the data available, 1t 1s not possible to resolve the conflict-
ing reports on the mutagenlc potential of 2,3,7,8-TCDD.
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-TCDD to produce
chromosomal aberrations. Kondorosl 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. Jji vivo studies using radlolabeled 2,3,7,8-TCDD Indicated some
association of non-extractable label with RNA and DNA (Poland and Glover,
1979); however, the level of bound label was very low. Similar marginal
data were available on the clastogenlc effect of 2,3,7,8-TCDD. Although two
in vivo studies 1n rats (Green and Moreland, 1975; Loprleno et al., 1982)
failed to demonstrate any 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
Intraperltoneal 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,
while no Increase was detected 1n Individuals exposed to 2,3,7,8-TCDD
following an Industrial accident 1n Seveso, Italy (Regg1an1, 1980; Mottura
1857A 10-12 02/29/84
-------
et al., 1981). 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 acids 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 toxldty of 2,3,7,8-TCDD. This chemical m?.y 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 1n mammalian cells In culture, additional yeast and
bacterial studies and the sex-linked recessive lethal test 1n DrosophHa.
Pertinent Information regarding the mutagenldty of PeCDDs and HxCDDs
were not located 1n the available literature.
1857A 10-13 02/29/84
-------
United States
Environmental Protection
Agency
Office of Health and
Environmental Assessment
Washington DC 20460
EPA-600/8-84-014A
May 1984
External Review Draft
Research and Development
Health Assessment
Document for
Polychlorinated
Dibenzo-p-Dioxins
Review
Draft
(Do Not
Cite or Quote)
Part 2 of 2
Notice
This document is a preliminary draft. It has not been formally
released by EPA and should not at this stage be construed to
represent Agency policy. It is being circulated for comment on its
technical accuracy and policy implications.
-------
EPA-600/8-84-01AA
May 1984
External Review Draft
DRAFT
Do not cite or quote
HEALTH ASSESSMENT DOCUMENT
FOR
POLYCHLORINATED DIBENZO-p_-DIOXINS
Part 2 of 2
Notice
This document 1s a preliminary draft. It has not been
formally released by EPA and should not at this stage be
construed to represent Agency policy. It 1s being circu-
lated for comment on Us technical accuracy and policy Im-
plications.
U.S. ENVIRONMENTAL PROTECTION AGENCY
Office of Research and Development
Office of Health and Environmental Assessment
Environmental Criteria and Assessment Office
Cincinnati, Ohio 45268
Project Manager: Dr. Debdas Mukerjee
U.5. *"•••• ' ' •"-. •••"• . Agenqfc
&••;. •
2&'j ^ .--'-•' ;-
ago, Illinois &wG04
-------
DISCLAIMER
This report 1s an external draft for review purposes only and does not
constitute Agency policy. Mention of trade names or commercial products does
not constitute endorsement or recommendation for use.
NOTE
For Information concerning this document, please contact the project
manager, Debdas Mukerjee (513/684-7531), of the Environmental Criteria and
Assessment Office, Cincinnati, OH 45268.
11
-------
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-£-
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 has been reviewed by a panel of expert scientists during the
peer review workshop held at the Cincinnati Convention/Exposition Center,
Cincinnati, OH, on July 27, 28 and 29, 1983.
111
-------
AUTHORS, CONTRIBUTORS, AND REN7IEWERS
The EPA Office of Health and Environmental Assessment (OHEA) was responsible
for the preparation of this draft health assessment document. The OHEA
Environmental Criteria and Assessment Office (ECAO-Cincinnati) had overall
responsibility for coordination and direction of the document preparation and
production effort (Debdas Mukerjee, Project Manager, Jerry F. Stara, Director,
ECAO-Cincinnati).
The participating members of the Environmental Criteria and Assessment
Office-Cincinnati, Ohio are:
D. Mukerjee, M.Sc., Ph.D.*
H. Ball, M.S.
C. DeRosa, Ph.D.
L. Erdreich, Ph.D.
R. Hertzberg, Ph.D.
R. Bruins, M.S.
M. Dourson, Ph.D.
B. Farren, B.S.
S. Lutkenhoff, B.S.
C. Mullin, M.S.
W.B. Peirano, M.S.
D.J. Reisman, M.S.
J.F. Stara, D.V.M.; D.S., Director*
M.W. Neal, Ph.D. (Syracuse Rsch. Corp.)*
S. Que Hee, Ph.D. (Univ. Cinc./Med.Ctr.)*
M.A. Schneiderman, Ph.D. (Env. Law Inst.)*
O.K. Basu, Ph.D. (Syracuse Rsch. Corp.)*
J.R. Olson, Ph.D. (State Univ./New York)*
S. Safe, Ph.D. (Texas A&M Univ.)*
W. Dorough, Ph.D. (Univ. Kentucky)*
F. Mink, Ph.D.
J. Orme, M.S.
W. Pepelko, Ph.D.
J. Risher, M.S.
The OHEA Carcinogen Assessment Group (CAG) was responsible for preparation
of the sections on carcinogenicity. Participating members of the CAG are listed
below:
Roy E. Albert, M.D. (Chairman)
Elizabeth L. Anderson, Ph.D.
Larry D. Anderson, Ph.D.
Steven Bayard, Ph.D.*
David L. Bayliss, M.S.*
Chao W. Chen, Ph.D.
Herman J. Gibb, B.S., M.P.H.
Bernard H. Haberman, D.V.M., M.S.
Charalingayya B. Hiremath, Ph.D.*
James W. Holder, Ph.D.
Robert E. McGaughy, Ph.D.
Jean C. Parker, Ph.D.
Dharm V. Singh, D.V.M., Ph.D.
Todd W. Thorslund, Sc.D.
The OHEA Reproductive Effects Assessment Group (REAG) was responsible for
the preparation of the sections on mutagenicity. Participating members of
the REAG are listed below:
John R. Fowle III, Ph.D.
Ernest R. Jackson, M.S.
David Jacobson-Kram, Ph.D.
Casey Jason, M.D.
K. S. Lavappa, Ph.D.
Sheila L. Rosenthal, Ph.D.*
Carol N. Sakai, Ph.D.
Vicki Vaughan-Dellarco, Ph.D.
Peter E. Voytek, Ph.D. (Director)
*Authors
i v
-------
The following individuals were asked to review this document and
earlier drafts of this document:
Bernard II. Haberman EPA Carcinogen Assessment Group
Franklin L. Mink ECAO-Cincinnati
Charles H. Nauman EPA Exposure Assessment Group
William E. Pepelko ECAO-Cincinnati
David J. Reisman ECAO-Cincinnati
John L. Schaum EPA Exposure Assessment Group
The following members of the ECAO-Cincinnati Technical Services Staff
were responsible for document production:
Cynthia Cooper Karen Mann
Patricia Daunt Judith Olsen, B.A.
Erma Durden, M.A. Bette Zwayer
Cindy Fessler
-------
POLYCHLORINATEO DIBENZO-p-DIOXINS PEER REVIEW PANEL MEMBERS
July 27, 28 and 29, 1983 Cincinnati, Ohio
Co-chairmen:
Oebdas Mukerjee. ECAO-CIN
Jerry f. Stara, ECAO-CIN
MEMBERS
Roy Albert
Institute of Environmental Medicine
New York University Medical Center
Donald G. Barnes
Office of Pesticides and Toxic
Substances
U.S. Environmental Protection Agency
K. Diane Courtney
Health Effects Research Laboratory
Research Triangle Park
U.S. Environmental Protection Agency
Frederick Coulston
White Sands Research Center
David Firestone
Food and Drug Administration
S. Garatt1n1
Institute dl
Farmacologlc
Milan, Italy
Recerche
"Mario NegM
Dolores Graham
Health Effects Research Laboratory
Research Triangle Park
U.S. Environmental Protection Agency
Richard Grelssmer
Oak Ridge National Laboratory
Lennart Hardell
University Hospital
Umea, Sweden
Robert Harless
Environmental Monitoring Systems
Laboratory, Research Triangle Park
U.S. Environmental Protection Agency
Rolf Hartung
University of Michigan
Al1sta1r W.M. Hay
University of Leeds
Leeds, United Kingdom
Otto Hutzlnger
University of Amsterdam
Amsterdam, The Netherlands
R.D. Klmbrough
Centers for Disease Control
Richard J. Kodba
Dow Chemical Company
Frederick Kopfler
Health Effects Research Laboratory
Cincinnati
U.S. Environmental Protection Agency
Marvin Legator
University of Texas Medical Branch
Ruth L1l1s
Mt. S1na1 School of Medicine
Prab D. Lotllkar
Temple University School of Medicine
Benjamin Lyklns, Jr.
Municipal Environmental Research
Laboratory, Cincinnati
U.S. Environmental Protection Agency
Fumlo Matsumura
Michigan State University
E. McConnell
National Institute of Environmental
Health Sciences
W.P. McNulty
Oregon Regional Primate Research
Center
V I
-------
Robert miler
National Cancer Institute
Ralph Nash
U.S. Department of Agriculture
James 01 sen
State University of New York
Francesco Pocch1ar1
Institute Superlore d1 Sanlta
Rome, Italy
Shane Que Hee
University of Cincinnati Medical
Center
Chrlstoffer Rappe
University of Umea, Sweden
Steven H. Safe
Texas A&M University
Marvin Schnelderman
Environmental Law Institute
Larry SUbart
National Wildlife Federation
Ellen SUbergeld
Environmental Defense Fund
David Stalling
Columbia National Fisheries Research
Laboratory
Lewis Thlbodeaux
University of Arkansas
Thomas Tlernan
Wright State University
V I I
-------
TABLE OF CONTENTS
Page
1. INTRODUCTION 1-1
2. SUMMARY AND CONCLUSIONS 2-1
2.1. SUMMARY 2-1
2.2. CONCLUSIONS 2-5
2.3. NEEDS FOR FUTURE RESEARCH 2-6
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 4-16
4.3.5. Photochemical Process 4-16
4.4. RELATIONSHIP BETWEEN SOURCES AND CONTAMINATION IN
ENVIRONMENTAL MATRICES 4-17
i x
-------
4.5. ENVIRONMENTAL LEVELS 4-17
4.5.1. Water 4-19
4.5.2. A1r 4-20
4.5.3. Soil 4-24
4.5.4. Foods and Biological Samples 4-27
4.6. EXPOSURE 4-30
4.7. SUMMARY 4-36
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 PHARMACOKINETICS 7-1
7.1. ABSORPTION 7-1
7.1.1. Absorption from the Gastrointestinal Tract 7-1
7.1.2. Absorption Through the Skin 74
7.2. DISTRIBUTION 7-5
7.3. METABOLISM 7-10
7.4. ELIMINATION 7-14
7.5. SUMMARY 7-18
-------
Page
8. TOXICOLOGY: ACUTE, SUBCHRONIC AND CHRONIC 8-1
8.1. EXPERIMENTAL ANIMALS 8-1
8.1.1. Acute 8-1
8.1.2. SubchronU 8-43
8.1.3. Chronic 8-50
8.2. HUMAN 8-59
8.2.1. Acute Exposure 8-59
8.2.2. Chronic Studies 8-64
8.3. MECHANISM OF TOXICITY 8-67
8.3.1. Receptor-Mediated Toxldty 8-68
8.3.2. Metabolism 8-75
8.3.3. Vitamin A Depletion 8-76
8.3.4. L1p1d Perox1dat1on 8-77
8.3.5. Endocrln Imbalance 8-78
8.4. SUMMARY 8-80
8.4.1. Experimental Animal Data 8-80
8.4.2. Human Data 8-83
8.4.3. Mechanisms of Toxldty 8-84
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 1n Mice 9-6
9.1.3. 2,3,7,8-TCDD Studies 1n Rats 9-12
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 9-19
9.1.6. Studies 1n Chickens 9-21
9.1.7. Studies of the Teratogenlc and Reproductive
Effects of HxCDD 9-22
9.2. STUDIES ON HUMAN POPULATIONS 9-23
9.3. OTHER REPRODUCTIVE EFFECTS 9-33
9.4. SUMMARY 9-34
10. MUTAGENICITY AND OTHER INDICATIONS OF GENOTOXICITY 10-1
10.1. RELEVANT STUDIES 10-1
10.1.1. Assays 1n 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 ..... 10-11
-------
Page
11. CARCINOGENICITY -\-\_-\
11.1. ANIMAL STUDIES H_1
11.1.2. Van Miller et al. 2,3,7,8-TCDO Oral Rat
Study (1977a,b) 11_2
11.1.3. Kodba et al. (Oral) Rat Study (1978a) 11-4
11.1.4. National Toxicology Program (Oral) Rat
Study (1980a,b) 11-12
11.1.5. Toth et al. (Oral) Mouse Study (1979) 11-15
11.1.6. National Toxicology Program (Oral) Mouse
Study (1980a,b) 11-20
11.2. SUMMARY OF ANIMAL CARCINOGENICITY 11-49
11.3. EPIDEMIOLOGICAL STUDIES 11-58
11.3.1. Case Reports 11-58
11.3.2. Soft-Tissue Sarcomas 11-62
11.3.3. Malignant Lymphoma 11-83
11.3.4. Stomach Cancer. .... 11-90
11.3.5. Summary of Ep1dem1olog1cal Studies 11-100
11.4. QUANTITATIVE ESTIMATION OF RISKS OF EXPOSURE 11-101
11.4.1. Introduction 11-101
11.4.2. Procedures for the Determination of Unit
Risk for Animals 11-102
11.4.3. Description of the Low-Dose Animal
Extrapolation Model ... 11-104
11.4.4. Selection of Data 11-106
11.5. ORAL 11-107
11.5.1. Calculation of the Unit Risk from Animal Studies. . 11-109
11.5.2. Interpretation of Quantitative Estimates. ..... 11-109
11.5.3. Alternative Methodological Approaches 11-111
11.5.4. Unit Risk Estimates for 2,3,7,8-TCDD via the
Oral and Inhalation Route 11-112
11.5.5. Unit Risk Estimate for HxCDOs (1,2,3,6,7,8 and
1,2,3,7,8,9) Via the Inhalation Route 11-115
11.5.6. Relative Potency 11-120
11.6. SUMMARY AND CONCLUSIONS 11-126
11.6.1. Qualitative Assessment-2,3,7,8-TCDD 11-126
11.6.2. Qualitative Assessment-HxCDO 11-129
11.6.3. Quantitative Assessment - 2,3,7,8-TCDD and HxCDD. . 11-129
11.6.4. Conclusion 11-130
X I I
-------
Page
12. SYNERGISM AND ANTAGONISM 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
13.1.1. Ambient Water 13-1
13.1.2. Drinking 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. Tox1c1ty 14-2
14.1.2. Mutagenldty 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 ToxUHy Data 14-10
14.4.2. Animal Carclnogenldty 14-11
15. REFERENCES 15-1
APPENDIX A A-l
APPENDIX 8 B-l
APPENDIX C C-1
XI
-------
LIST OF TABLES
No. Title Page
3-1 Physical Properties of a Few Selected PolychloMnated
01ox1ns 3-4
3-2 A Few Estimated Physical Parameters of Chlorinated
D1benzo-[)-d1ox1ns 3-6
3-3 Potential Interferences 1n the Determination of TCOOs
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 TCDDs . . 3-17
3-6 Some Published Method Validation Data for 2,3,7,8-TCDD
Recovered from Fortified Matrices and Determined by 6C/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 Major Producers and Formulators of
Chlorophenols and Their Derivatives 4-10
4-3 Levels of TCDD 1n Soils and Sediments from Different
Locations 4-25
4-4 Predicted BCFs from Calculated and Measured Values of Kow . . 4-34
4-5 Measured B1oaccumulat1on Factor for 2,3,7,8-TCDD 1n
Freshwater Aquatic Organisms. .... 4-35
5-1 B1oconcentrat1on 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
X I V
-------
No. Title Page
7-1 Gastrointestinal Absorption of 2,3,7,8-TCOD 7-2
7-2 Distribution of 2,3,7,8-TCDO 7-6
7-3 Elimination of 2,3,7,8-TCOD 7-15
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 8-51
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 Male 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, With
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, With
Dr. Squire's Review (8/15/80) Sprague-Dawley Male Rats -
Spartan Substraln (2 years) 11-14
XV
-------
No. TUIe Page
11-9 Incidence of Primary Tumors 1n Male Rats Administered
2,3,7,8-TCDD by Gavage 11-16
11-10 Incidence of Primary Tumors 1n Female Rats Administered
2,3,7,8-TCDD by Gavage 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 Gavage 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 Tumor1genes1s 1n
D2 Mice 11-32
11-18 Incidence of Tumors 1n Mice Treated With 3-MC and With
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 Carc1nogen1c1ty Bloassays of 2,3,7,8-TCDD and HxCDD
by Dermal Application to Mice 11-46
11-23 Cardnogenldty Bloassays of PCDD Administration by the
Oral and Dermal Route 11-50
11-24 Distribution of Tumor Types 1n Two Case-Controls Studies
of Soft-Tissue Sarcoma 11-65
XV I
-------
No. Title Page
11-25 Exposure Frequencies 1n Two Case-Control Studies of
Soft-Tissue Sarcoma 11-66
11-26 Relative Risks of Soft-Tissue Sarcoma 1n Relation to
Exposure to Phenoxyacetlc Adds and Chlorophenols 1n
Two Case-Control Studies 11-68
11-27 Distribution of H1stolog1cal Types of Soft-Tissue
Sarcomas 11-72
11-28 Midland County Soft and Connective Tissue Cancer
Deaths 1960-1981 11-81
11-29 Other Occupations (Minus Forestry/Agriculture) 11-87
11-30 Other Occupations (Minus Forestry/AgMculture/Woodworkers . . 11-88
11-31 Analysis of Stomach Cancer Mortality In a Group of West
German Factory Workers Exposed to 2,3,7,8-TCDD 11-93
11-32 Reanalysls of Stomach Cancer Mortality 1n a Group of
West German Factory Workers Exposed to 2,3,7,8-TCDD 11-95
11-33 Stomach Cancer Mortality 1n Three Studies of Workers
Exposed to Phenoxyacetlc Add Herbicides and/or
2,3,7,8-TCDD 11-97
11-34 NTP HxCDD (Gavage) Bloassay. Osborne-Mendel Rats
(2 years) Incidences of Neoplastlc Nodules and Hepato-
cellular Carcinomas 11-116
11-35 NTP HxCDD (Gavage) Bloassay. 86C3F1 Mice (104 weeks)
Incidences of Neoplastlc Nodules and Hepatocellular
Carcinomas 11-118
11-36 Relative Carcinogenic Potencies Among 54 Chemicals
Evaluated by the Carcinogen Assessment Group as Suspect
Human Carcinogens 11-122
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 14-3
14-2 No-Observed-Effect Levels and Low-Observed-Effect Levels
Obtained from Subchronlc and Chronic Oral Toxlclty
Studies of HxCDD 14-5
14-3 Cardnogenldty Bloassays of 2,3,7,8-TCDD 14-12
14-4 Cardnogenldty Bloassays of a 1:2 Mixture of 1,2,3,6,7,8-
and 1,2,3,7,8,9-HxCDD 14-16
XV I I
-------
LIST OF FIGURES
No. Title
4-1 Ullmann Condensation Reactions 4-3
4-2 Possible potential relationship between various sources
of PCDDs and the environmental matrices where PCDOs have
been detected 4-18
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 54 suspect carcinogens evaluated by
the Carcinogen Assessment Group 11-121
XVI I I
-------
LIST OF ABBREVIATIONS
ADI
AHH
bw
BCF
BromoPeCDD
DCDO
DMSO
DNA
EC/GC
"50
PEL
GC/MS
GC/SIM/MS
HPLC
HRGC
HRMS
HxCDDs
L050
LOAEL
LRMS
MFO
NICI
NOAEL
NOEL
Acceptable dally Intake
Aryl hydroxycarbon hydroxylase
Body weight
Bloconcentratlon factor
Bromopentachlorod1benzo-p_-d1ox1n
D1chlorod1benzo-p_-d1ox1n
Dlmethylsulfoxlde
Deoxyr1bonucle1c acid
Electron capture/gas chromatography
Median effective dose
Frank effect level
Gas chromatography/mass spectrometry
Gas chromatography/spedflc 1on monitoring/mass spectrom-
etry
High performance liquid chromatography
High resolution gas chromatography
High resolution mass spectrometry
Hexachloro derivatives of d1benzo-p_-d1ox1ns
Concentration lethal to SOX of recipients
Dose lethal to 50% of recipients
Lowest-observed-adverse-effect level
Low resolution mass spectrometry
Mixed function oxldase
Negative 1on chemical 1on1zat1on
No-observed-adverse-effect level
No-observed-effect level
X I X
-------
OCDD
PCDDs
PCP
PeCDDs
ppb
ppm
ppt
RBC
RNA
SA
TCODs
TrICDD
2.4.5-T
TWA
UV
WCOT
Octachlorlnated 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
R1bonucle1c add
Satellite association
Tetrachloro derivatives of d1benzo-p_-d1ox1ns
Tr1chlorod1benzo-£-d1ox1n
2,4,5-Tr1chlorophenoxyacet1c add
Time-weighted average
Ultraviolet
Wall-coated open tubular
XX
-------
11. CARCINOGENICITY
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 1,2,3,6,7,8-hexachlorod1benzo-p_-d1ox1n (HxCDO), 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
carc1nogen1c1ty depends heavily on animal bloassays and epidemlologic
evidence. However, Information on mutagenldty and metabolism, particularly
1n relation to Interaction with ONA, as well as to pharmacoklnetic behavior,
has an Important bearing on both the qualitative and quantitative assessment
of carc1nogen1c1ty. The available Information on these subjects 1s reviewed
1n other sections of this document. This chapter presents an evaluation of
the animal bloassays, the human epidemlologic evidence, the quantitative
aspects of assessment, and finally, a summary and conclusions dealing with
all of the relevant aspects of cardnogenldty.
11.1. ANIMAL STUDIES
The polychlorlnated d1benzo-p_-d1ox1ns (PCDOS), 2,3,7,8-TCDO and a
mixture of 1,2,3,7,8,9- and 1,2,3,6,7,8-HxCDD, have been tested for cardno-
genldty 1n rats and mice by administering the compound 1n the diet and by
gavage. Also, the tumor Incidence 1n 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-TCDO and a mixture of 1,2,3,7,8,9- and 1,2,3,6,7,8-HxCDD have
1858A 11-1 03/28/84
-------
been tested for tumor 1 gen 1dty by dermal application. Using the skin
two-stage tumorigenicity model, 2,3,7,8-TCDD has been tested for promoting
and Initiating activity as well as antlcarclnogenlc 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 potential of chemical carcinogens.
11.1.2. Van Miller et al. 2,3,7,8-TCDD 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 consumption 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 yg/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 in
Table 11-1.
All animals in groups maintained on diets containing 1-1000 ppb of
2,3,7,8-TCDD were dead by week 90 of treatment with the first deaths in
groups at the 1000 and 1 ppb levels observed at 2 weeks and 31 weeks of
treatment, respectively. Animals exposed to 0.001-0.5 ppb of 2,3,7,8-TCOO
had similar food consumption and survival as control animals; however, all
treated animals had hlstopathologic degenerative changes 1n the kidneys.
1858A 11-2 03/28/84
-------
TABLE 11-1
2,3,7,8-TCDD Intake and Mortality 1n Male Sprague-Dawley Rats^
Doseb
(ppb)
0.0
0.001
0.005
0.05
0.5
1
5
Weekly Dose/Rat
(yg/kg bw)
0
0
0
0
0
2
.0003
.001
.01
.1
.4
.0
Week of
First Death
68
86
33
69
17
31
31
Number
Dead at
6/10
2/10
4/10
4/10
5/10
10/10
10/10
of Rats
95th Week
(6054)
(20%)
(4054)
(4054)
(5054)
(10054)
(10054)
^Source: Van Miller et al., 1977a,b
bRats at 50, 500 and 1000 ppb dose levels were all dead within 4 weeks.
1858A
11-3
03/28/84
-------
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 In the diagnosis of neo-
plasms. Various benign and malignant tumors were found in each treatment
group. No tumors were observed in the controls (Table 11-2).
Statistically significant increases of squamous cell tumors of the lungs
and neoplastlc nodules of the liver were observed in rats ingesting 5 ppb
TCDO (Table 11-3). In addition, two animals in the 5 ppb dose group and one
animal in the 1 ppb dose group had liver cholangiocarcinomas, which are rare
in Sprague-Dawley rats. These results provide evidence of a carcinogenic
effect.
The observation of no tumors of any kind in the controls is unusual for
Sprague-Oawley rats. In addition, the reporting of the study was not exten-
sive. These factors may tend to lessen the reliance which can be placed on
the positive results of this study. However, this study is suggestive of a
carcinogenic response upon exposure to TCOD in rats.
11.1.3. Kodba et al. (Oral) Rat Study (1978a). Although this study was
published as Kociba et al., 1978a, a fuller version was submitted in an
unpublished report (Kociba et al., 1977).
In this study, groups of 50 Sprague-Dawley rats (Spartan substrain) of
each sex were maintained for up to 2 years on diets providing 0.1, 0.01 or
0.001 yg/kg/day 2,3,7,8-TCDD. Vehicle control groups comprised 86 animals
of each sex. The test was appropriately conducted with the high-dose group
*The audit of this study brought out the fact that it 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.
1858A 11-4 03/28/84
-------
TABLE 11-2
Benign and Malignant Tumors 1n Rats Ingesting 2,3,7,8-TCDDa
Ooseb
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
51
10J
Number of Rats
With Tumors
0/10
0/10
5/10
3/10
4/10
4/10
7/10
(0%)c
(0%)
(50%)e
(30%)
(40%)n
(40%)
(70%)
aSource: Van Miller et al., 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.
dl rat had ear duct carcinoma and lymphocytlc leukemia
1 adenocarclnoma (kidney)
1 malignant hlstlocytoma (retroperltoneal)
1 anglosarcoma (skin)
1 Leydlg cell adenoma (testls)
3 rats died with aplastlc anemia
^1 flbrosarcoma (muscle)
1 squamous cell tumor (skin)
1 astrocytoma (brain)
9l fibroma (striated muscle)
1 carcinoma (skin)
1 adenocarclnoma (kidney)
1 scleroslng semlnoma (testls)
1 rat had a severe liver Infarction
^1 rat cholang1ocarc1noma and malignant hlstlocytomas (retroperltoneal)
1 anglosarcoma (skin)
1 glloblastoma (brain)
1 malignant hlstlocytoma (retroperltoneal)
^1 rat had squamous cell tumor (lung) and neoplastlc nodule (liver)
2 cholanglocarclnomas and neoplastlc nodules (liver)
3 squamous cell tumors (lung)
1 neoplastlc nodule (liver)
1858A 11-5 04/12/84
-------
TABLE 11-3
Liver Tumors 1n Rats Ingesting 2,3,7,8-TCDDa
Dose (ppb)
0
1
5
Neoplastlc
Nodules
0/10
0/10
4/10
p=0.
(0%)
(0%)
(40%)
043
Cholanglocardnomas
0/10 (0%)
1/10 (10%)
2/10 (20%)b
Squamous Cell
Tumors of the Lungs
0/10
0/10
4/10
p=0.
(40%)
043
aSource: Van Miller et al., 1977a,b
''The two animals had both neoplastlc nodules of the liver and Cholanglo-
cardnomas.
1858A 11-6 04/12/84
-------
given a dose which Induced signs of tissue toxlclty, reduced weight Incre-
ments In both sexes, and shortened llfespans 1n female rats. Clinical tests
performed at Intervals during the study monitored organ specific toxldty,
particularly of the liver. Pathologic examinations Included 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 1n lower-dose groups. This approach 1s suitable for the
Identification of a carcinogenic effect, but does not determine actual tumor
Incidences 1n all groups except 1n those organs Identified as target organs.
It, therefore, 1s adequate to define dose-response relationships only 1n
these target organs. Tissues examined from most animals 1n all dose groups
Included liver, lungs, kidneys, urinary bladder, tongue, brain, testes/ovar-
1es and prostate/uterus. For these tissues, a quantitative analysis can be
performed using the actual number of tissues examined h1stopatholog1cally
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 1s the appropriate denominator to
determine Incidence, because detection of these tumors 1s based on observa-
tion 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. Reviewers concluded that the study was pro-
perly 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
1858A 11-7 04/12/84
-------
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
shdw progressive mortality beginning as early as the 12th month and leading
to 50% mortality by 21 months.* The effects of this early mortality are a
reduction In 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-TCDO
1s carcinogenic 1n 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 yg/kg/day (2200
and 210 ppt 1n the diet, respectively). The Increase of hepatocellular
carcinomas alone, In 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-
mous cell carcinomas of the lungs 1n 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 Wh1tney-W1lcoxon test and the
Kolmogorov-Slmonov test. These tests show that mortality was significantly
higher 1n the high-dose females than 1n controls, and while Indications of
Increased mortality were found 1n other groups, they were not part of a
consistent pattern.
1858A 11-8 03/28/84
-------
TABLE 11-4
Hepatocellular Carcinomas and Hepatocellular Hyperplastlc Nodules
1n Female Sprague-Dawley Rats Maintained on Diets Containing 2,3,7,8-TCDOa
Dose Level
(yg/kg/day)
0
0.001
(22 ppt)
0.01
(210 ppt)
0.1
(2200 ppt)
Hepatocellular
Hyperplastlc Hepatocellular
Nodules Carc1nomasb
8/86 (9%) 1/86
3/50 (6%) 0/50
18/50 (36%) 2/50
23/49 (48%) 11/49
(p=5.6
(1*)
(0%)
(4%)
(22%)
x 10~5)
Total Number
With Both
Types of Tumorsb
9/86 (10%)
3/50 (6%)
18/50 (36%)c
(p=4.36 x 10~")
34/50 (71%)
(p=4.56 x 10 13)
^Source: Koclba et al., 1977
bP values calculated using the Fisher Exact Test (one-tailed).
cTwo rats had both hepatocellular carcinomas and hyperplastlc nodules.
1858A 11-9 03/28/84
-------
TABLE 11-5
Tumor Incidence 1n Female Rats Fed Diets Containing 2,3,7,8-TCDDa
Dose Level Stratified Squamous Cell Kerat1n1z1ng Squamous
(yg/kg/day) Carcinomas of Hard Palate Cell Carcinomas of
or Nasal Turblnates Lungs
0 1/54 (2%) 0/86 (OX)
0.001 0/30 (0%) 0/50 (OX)
(22 ppt)
0.01 1/27 (4X) 0/50 (OX)
(210 ppt)
0.1 5/24 (21X) 7/49 (14X)
(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).
1858A 11-10 03/28/84
-------
TABLE 11-6
Tumor Incidence 1n Male Rats Fed Diets Containing 2,3,7,8-TCDOa
Dose Level Stratified Squamous Cell Hard Palate/Nasal Turblnates
(pg/kg/day) Carcinomas of the Tongue Stratified Squamous Cell Carc1nomab
0 0/76 (0%) 0/51 (OX)
0.001 1/49 (2%) NS 1/34 (3%) NS
(22 ppt)
0.01 1/50 (2%) NS 0/27 (0%) NS
(210 ppt)
0.1 3/42 (7%) (p=4.3 x 10~2) 4/30 (13%) (p=0.016)
(2200 ppt)
aSource: Kodba et al., 1977
blncludes examinations from both original and updated report (5/20/79).
NS = Not significant at p=0.05.
1858A 11-11 04/12/84
-------
Or. Robert Squire, pathologist at the Johns Hopkins University Medical
School and consultant to the CAG, evaluated the h1stopatholog1c slides from
Dow Chemical Company's 2-year rat feeding studies on 2,3,7,8-TCDD by Koclba
et al. (1978a). Dr. Squire and his associates examined all liver, lungs,
tongues, hard palates and nasal turblnates available from the 2,3,7,8-TCDD
study. Their hlstopathologlcal findings, as well as Dr. Kodba's hlsto-
pathologlcal evaluations, are summarized 1n Tables 11-7 and 11-8 and
Appendix B. Although there are some differences between the diagnoses of
Drs. Kodba and Squire, the conclusions about the target organ for cancer
Induction and the dose levels at which Induction occurred are the same.
11.1.4. National Toxicology Program (Oral) Rat Study (1980a,b). A cancer
bloassay for the possible carc1nogen1c1ty of 2,3,7,8-TCDD was tested by the
Illinois Institute of Technology 1n rats and mice under a contract sponsored
by the National Cancer Institute (NCI).
In the rat study, 50 Osborne-Mendel rats of each sex were administered
2,3,7,8-TCDD* suspended 1n a vehicle of 9:1 corn oil-acetone by gavage 2
days/week for 104 weeks at doses of 0.01, 0.05 or 0.5 pg/kg/week.
Seventy-five rats of each sex served as vehicle controls. One untreated
control group containing 25 rats of each sex was present 1n the 2,3,7,8-TCDD
treatment room and one untreated control group containing 25 rats of each
sex was present 1n the vehicle control room. All surviving rats were killed
at 105-107 weeks.
*Pur1ty of 2,3,7,8-TCDD was found to be 99.4%; two Impurities tentatively
Identified as a tr1chlorod1benzo-p_-d1ox1n and a pentachlorod1benzo-p_-
dloxln. The presence of 0.1-0.2% hexachlorod1benzo-p_-d1ox1n was also
detected by gas chromatography and mass spectrometry.
1858A 11-12 03/28/84
-------
CO
-------
oo
CD
3>
TABLF 11-8
Dow 2,3,7,8-TCOD Oral Rat Study by Dr. Koclba, WHh Dr. Squire's Review (8/15/80)
Sprague-Dawley Male Rats - Spartan Substraln (2 years)
Tissues and Diagnoses
^ Nasal turblnate/hard palate
-«» squamous cell carcinomas
Tongue
squamous cell carcinomas
Total - 1 or 2 above
(each rat had at least
one tumor above)
Dose Levels (ug/kg/day)
0 (control) 0.001 0.01 0.1
,S K S K S K S K
0/55 0/51 1/34 1/34 0/26 0/27 6/30 (20%) 4/30 (13%)
(p=l .36 x 10~3) (p=l .6 x 10~?)
0/77 0/76 2/44 1/49 1/49 1/49 3/44 (7%) 3/42 (7%)
(p-4.60 x 10"*) (p=4.34 x 10~2)
0/77 2/44 1/49 9/44
5% 2% 20%
(p=6.28 x 10"5)
S =
S K
Dr. Squire's hlstopathologlc analysis
Dr. Koclba's hlstopathologlc analysis
CD
-------
In rats, a dose-related depression 1n mean body weight gain became
evident 1n the males after week 55 of the bloassay and In the females after
week 45.
The results of hlstopathologlc diagnosis of primary tumors caused by the
oral administration of 2,3,7,8-TCOO are presented in Table 11-9. In male
rats an Increased incidence of folUcular-cell adenomas or carcinomas of the
thyroid was dose-related and was statistically significantly higher 1n the
low-, mid- and high-dose groups than in the vehicle controls. In addition,
a statistically significant Increase in subcutaneous tissue fibromas was
found in males of the high-dose group.
In female rats, a statistically significant Increase of each of the
following tumors was found in the high dose group: hepatocellular carci-
nomas and neoplastic nodules (p=0.001), subcutaneous tissue fibrosarcomas
(p=0.023) and adrenal cortical adenomas (p=-0.039), as shown in Table 11-10.
These results confirm the carcinogenic effect observed in the Kociba et
al. (1978a) study using Sprague-Dawley (Spartan substrain) rats.
11.1.5. Toth et al. (Oral) Mouse Study (1979). This study investigated
the carcinogenlcity of 2,3,7,8-TCDD 1n Swiss mice. Ten-week-old outbred
Swiss/H/R1op mice were used. 2,3,7,8-TCDD was administered in 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 pg/kg bw for a year (groups 9, 10, 11, respectively, In
Table 11-11). Matched male vehicle controls were administered sunflower oil
once a week. Matched controls to a companion study investigating the
cardnogenicity of (2,3,5-trichlorophenoxy)ethano1 (TCPE) contaminated with
low levels of 2,3,7,8-TCDD, were administered carboxymethyl cellulose (the
vehicle used in that study) once a week. Two untreated controls were also
maintained.
1858A 11-15 03/28/84
-------
| TABLE 11-9
Incidence of Primary Tumors 1n Male
Type of Tumor Vehicle Control
Subcutaneous tissue 3/75 (4%)
Fibroma
Liver
__ Neoplastlc nodule
^ or hepatocellular
^ carcinoma 0/74 (0%)
Adrenal
Cortical adenoma 6/72 (8%)
Thyroid
Folllcular cell
adenoma 1/69 (1%)
Thyroid
Folllcular cell
adenoma or
carcinoma 1/69 (1%)
o
-p»
Rats Administered
Low Doseb
0.01
1/50 (2%)
0/50 (0%)
9/50 (18%)
5/48 (10%)
p=0.042
5/48 (10%)
p=0.042
2,3,7,8-TCDD by Gavage3
yg/kq/week
M1d Doseb
0.05
3/50 (6%)
0/50 (0%)
12/49 (24%)
6/50 (16%)
p=0.021
8/50 (16%)
p=0.004
High Doseb
0.5
7/50 (14%)
p-0.048
3/50 (6%)
9/49 (18%)
10/50 (20%)
p=0.001
11/50 (22%)
p<0.001
CO
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 Gavage*
ug/kg/week
Type of Tumor Vehicle Control
Low Ooseb
0.01
M1d Dose
0.05
High Doseb
0.5
Subcutaneous tissue
Fibrosarcoma
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 (2%)
11/73 (15%)
2/50 (4%) 3/50 (6%) 4/49 (8X)
p=0.023
1/49 (2X) 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 (7X)
p=0.044
8/49 (16%) 4/49 (8X) 14/46 (30X)
p=0.039
^Source: NTP, 1980a
bP values calculated using the Fisher Exact Test.
1858A
11-17
03/28/84
-------
00
en
CD
3>
TABLE ,,_,,
Cumulative Data on Tumor Incidence3
i
oo
CO
Treatment
Group
1
2
3
4
5
6
7
8
9
10
11
12
TCPE°
(mg/kg) TCDO
(vgAg)
67.0 0.112
(1.6 ppm)
70.0 0.007
{0.1 ppm)
control
7.0 0.07
(10 ppm)
7.0 0.0007
(0.1 ppm)
0.7 0.00007
(0.1 ppm)
-_
control
7.0
0.7
0.007
__
Veh1cleb
(mg/kg)
50
50
50
50
50
50
10
10
10
10
Sex
M
F
H
F
H
F
M
F
H
F
H
F
M
F
H
F
H
M
M
M
Effective
Number of
MUe
88
83
98
96
93
84
93
96
94
93
97
94
96
84
96
91
43
44
44
38
Number
of Tumor
Bearing
HUe
69
61
78
59
63
57
79
60
77
71
78
64
74
55
78
57
27
36
39
27
Liver
(X)
42<:
7
571
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:
Lymphomas
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: Toth et al., 1979
TCPE = TrUhlorophenoxy ethanol
CCarboxymethyl cellulose 1n groups 1-8, sunflower oil 1n groups 9-12.
(t
P<1*
Vo.ix
CO
-------
This study appears to have been generally well conducted. However, the
administration of 2,3,7,8-lCDD over a period of only one 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-TCOO
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 In the matched controls (group 12) and 1n the males In the three
other control groups. Males In 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 In all four
control groups were compared to 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%).
1858A 11-19 03/28/84
-------
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 to the matched sunflower oil controls (13/43 = 30%), but
this Increase was not statistically significant (p=0.11). The Incidence of
liver tumors In the high-dose group was comparable to that of the pooled
controls. The highest-dose group, however, had a much reduced average
survival In comparison to any of the control groups (only 424 days compared
to 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 In liver tumors In the high-dose group. Furthermore, 1f 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 liver tumors that was observed 1n the
high-dose group 1n comparison to 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.
11.1.6. National Toxicology Program (Oral) Mouse Study (1980a,b). A
cancer bloassay for the possible carc1nogen1c1ty of 2,3,7,8-TCDD 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 yg/kg/week for male mice
and 0.04, 0.2 and 2.0 yg/kg/week for female mice. Seventy-five mice of
1858A 11-20 03/28/84
-------
each sex were used as vehicle controls. One untreated control group of 25
mice of each sex was present 1n the 2,3,7,8-TCDD treatment room. One
untreated control group of 25 mice of each sex was present 1n the vehicle
control room. In mice, the mean body weight gain 1n 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-
TCDD 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) 1n the high-dose group. In addition, a statistically
significant Increase 1n tumor Incidences of fIbrosarcoma, h1st1ocyt1c
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 1n liver tumors 1n the male mouse study
performed by Toth et al. (1979).
11.1.6.1. OTHER RELATED STUDIES PITOT ET AL. PROMOTION STUDY IN RATS
(1980) — PHot et al. (1980) Investigated a two-stage model of hepato-
cardnogenesls. Twenty-four hours after a partial hepatectomy (to enhance
cell proliferation), female Sprague-Dawley rats were divided Into seven
groups (Table 11-14). The animals 1n groups 1, 5, 6 and 7 received dlethyl-
nltrosamine (DEN). The rats 1n group 1 were then maintained on a standard
1858A 11-21 03/28/84
-------
TABLE 11-12
Incidence of Primary Tumors 1n Male Mice Administered
2,3,7,8-TCOO by Gavage3
Type of Tumor Vehicle Control
pg/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%)
15/73 (21%)
9/49 (18%) 8/49 (16%) 17/50 (34%)
p=0.002
12/49 (24%) 13/49 (27%) 27/50 (54%)
p=<0.001
aSource: NTP, 1980a
bP values calculated using the Fisher Exact Test.
1858A
11-22
03/28/84
-------
TABLE 11-13
Incidence of Primary Tumors 1n Female Mice Administered
2,3,7,8-TCDD by Gavage3
yg/kg/week
Type of Tumor Vehicle Control
Low Dose
0.04
M1d Dose
0.2
High Doseb
2.0
Subcutaneous tissue
Flbrosarcoma
Hematopo1et1c
system
H1st1ocyt1c
lymphoma
Hematopoletlc
system
All lymphoma
Hematopoletlc
system
Lymphoma or
leukemia
Liver
Hepatocellular
carcinoma
L 1ver
Hepatocellular
adenoma or
carcinoma
Thyroid
FolUcular-cell
adenoma
1/74
9/74 (12%)
18/74 (24%)
1/73 (1%)
1/50 (2%) 1/48 (2%) 5/47 (11%)
p=0.032
4/50 (8%) 4/48 (17%) 14/47 (30%)
p=0.016
18/74 (24%) 11/50 (22%) 13/48 (27%) 20/47 (43%)
12/50 (24%) 13/48 (27%) 20/47 (43%)
p=0.029
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
aSource: NTP, 1980a
bp values calculated using the Fisher Exact Test.
1858A
11-23
03/28/84
-------
oo
-------
laboratory diet for 32 weeks. The rats 1n groups 2 and 3 received no DEN,
but starting 1 week after hepatectomy received biweekly subcutaneous Injec-
tions of 0.14 or 1.4 yg/kg of 2,3,7,8-TCDO 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 pg/kg of 2,3,7,8-TCDO. 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 liver were removed and frozen on solid
CO . Serial sections of the frozen blocks of liver were cut and stained
consecutively for glucose-6-phosphatase (G6Pase), canallcular ATPase,
glutamyl transpeptldase (GGTase) with hematoxylln and eosln. The number of
enzyme-altered fod were determined from photographs of hlstochemlcally
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 fod 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
yg/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 yg/kg 1n addition to DEN had hepatocellular
1858A 11-25 03/28/84
-------
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 hepatocardnogenesls, causing
Increased neoplasla and Increases 1n enzyme-altered fod at exceedingly low
levels.
11.1.6.2. NATIONAL TOXICOLOGY PROGRAM SKIN PAINTING STUDY IN MICE
(19805) — This cancer bloassay of 2,3,7,8-TCDD for possible carclnogenlc-
1ty 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 yg 2,3,7,8-TCDO per application, while the female mice received
0.005 vg 2,3,7,8-TCOO per application.
In another experiment, the same number of animals were pretreated with
one application of 50 vq 7,l2-d1methylbenz{l)anthracene (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 ma 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-TCDD 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%.
1858A 11-26 03/28/84
-------
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 fIbrosarcoma 1n the Integumentary systems of female mice
treated with 2,3,7,8-TCDD alone and 2,3,7,8-TCDD following a single Initial
application of DMBA.
11.1.6.3. 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 yg/
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 1n acetone. After 30 weeks of promotion with 2,3,7,8-TCDO,
no papillomas were observed on the DMBA-1n1t1ated mice. In the positive
controls, DMBA-1n1t1ated mice were treated with 12-0-tetradecanoylphorbol-
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-dimethylbenz(a)anthracene (DMBA) 1n a two-stage tumorlgenesis
bloassay In 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 vg of 2,3,7,8-TCDD/mouse was applied, but was only
slightly less effective (89%) when the dose was Increased to 10 yg/mouse.
1858A 11-27 03/28/84
-------
TABLE 11-15
Incidence of Primary Tumors 1n Mice Administered 2,3,7,8-TCDO
or 2,3,7,8-TCDO Following DMBA by Dermal Application3
Type of Tumors Vehicle Control
Dose Levels
TCDD
DMBA (50 Pg)
plus TCDD6
Integumentary
system
Flbrosarcoma
3/42 (7%)
MALE
0.001 v«g x 3/weeks 0.001 pg x 3/weeks
6/28 (21%)
p-0.08
6/30 (20X)
p=0.10
Flbrosarcoma
2/41 (5%)
FEMALE
0.005 yg x 3/weeks
8/27 (30%)
p=0.007
0.005 pg x 3/weeks
8/29 (28%)
p-0.010
aSource: NTP, 1980b
bP value calculated using the Fisher Exact Test.
1858A
11-28
03/28/84
-------
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 large (maximum 65%) with BaP as with DMBA.
11.1.6.4. COHEN ET AL. SKIN PAINTING STUDY IN MICE (1979) — Cohen et
al. (1979) showed that pretreatment of mice with dermally applied 2,3,7,8-
KDD resulted In the Inhibition of skin tumor Induction by subsequent treat-
ment with DMBA and BaP. The Inhibition of skin cardnogenesls by BaP 1n
mice after pretreatment with 2,3,7,8-TCDD was associated with an Increase In
covalent binding of BaP metabolites to DNA, RNA and protein (1n contrast to
the results with DMBA, which showed a reduction 1n binding to DNA and RNA).
However, the BaP metabolites that were bound to DNA and RNA In mice pre-
treated with 2,3,7,8-TCOD differed from those 1n untreated mice. In partic-
ular, 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 Us covalent binding with guanoslne 1n DNA.
11.1.6.5. KOURI 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
86) and DBA/2Cum (abbreviated 02), were used. These strains are responsive
and nonresponslve, respectively, to the Induction of aryl hydrocarbon
hydroxylase (AHH) by 3-MC.
1858A 11-29 03/28/84
-------
Groups of mice of both sexes were Injected subcutaneously at 4-6 weeks
of age with either 150 yg of 3-MC dissolved 1n trloctanoln or with trioc-
tanoln alone. Some groups were also Injected with 2,3,7,8-TCDD dissolved 1n
p_-d1oxane, either simultaneously with the administration of 3-MC or 2 days
earlier. Two doses of 2,3,7,8-TCDD (1 Pg/kg and 100 pg/kg) were used,
and the effects of both 1ntraper1toneal and subcutaneous Injections were
Investigated. Two sets of experiments Involving 29 groups of mice were
conducted ~1 year apart (Table 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 h1stolog1cally as fIbrosarcomas 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 11-17.
No subcutaneous tumors were observed 1n controls or in 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-MC.
However, in both experiments involving D2 (nonresponslve) mice, the adminis-
tration of 2,3,7,8-TCDD simultaneously with 3-MC appeared to enhance the
carcinogenic response. The "carcinogenic index" increased from 1-6 in
groups treated with 3-MC alone to 14 1n the group treated subcutaneously
1858A 11-30 03/28/84
-------
CD
1ABL1 11 16
CD
3>
Effects of
Intraperl toneal Administration of 2,3,7,8
Treatment
-.
i
CO
— '
o
CO
o
03
Inbred
Strain
B6 1.p.
1.p.
None
None
None
None
None
l.p.
l.p.
D2 l.p.
1.p.
None
None
None
None
None
l.p.
l.p.
Source: Kourl
-2
Days
p-dloxln
TCDO
TCDD
TCDD
(100 yg/kg)
(100 yg/kg)
(1 yg/kg)
p-d1oxane
TCDD
TCDD
TCDD
et
During the first
(100 yg/kg)
(100 yg/kg)
(1 yg/kg)
al., 1978
s .
s .
s .
1.
1.
s .
1.
1 .
s .
s.
s.
s .
s.
s .
1 .
1 .
5.
1.
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
28 days following
Defined as the number of mice
H
0 Days
. trloctanoln
. trloctanoln
. 3-MC
. TCDD (100 yg/kg)
. TCDD (100 yg/kg) i
. 3-MC
. TCDD (1 yg/kg)
. TCDD (1 yg/kg) *•
. 3-MC
. 3-HC
. 3-MC
. trloctanoln
. trloctanoln
. 3-HC
. TCDD (100 yg/kg)
. TCDD (100 yg/kg) «•
. 3-MC
. TCDD (1 yg/kg)
. TCDD (1 yg/kg) f
. 3-MC
. 3-MC
. 3-MC
treatment.
No. of Mice
Dying Because
of Treatment'3
1
20
1
20
30
4
6
20
6
6
24
3
30
43
5
5
20
6
-icnn on 3 -MC -
No. of Mice
at Klsk for
1 umor sc
39
27
36
30
43
46
27
25
23
22
25
34
38
43
48
34
28
31
Initiated Suh( utanpous Tumors3
No. of
Mice with
lumor s1^
0
0
29
0
33
0
27
21
16
0
0
1
0
10
0
5
0
0
Avpraqp
X of Mice 1 ,iteni y
wl th Tumors (days )
0
0
81 125
0
71 123
0
100 132
84 129
70 140
0
0
3 217
0
23 178
0
15 199
0
0
Care inogenlc
index*
65
63
76
65
50
1
13*
7
surviving the 36-week observation period.
CD
At the end of the 36-week experiment.
Percentage of Incidence of tumors, divided by the average latency 1n days, multiplied by 100 (8).
fTh!s carcinogenic Index value lies outside (greater than) the 99% confidence Interval (l.e , n
-------
00
en
CO
TABIF 11-17
Effect of IntraperHoneal or Subcutaneous Administration of 2,3,7,8-TCDO Given 2 Days Before or Simultaneous
With Subcutaneous Administration of 3-MC on TumorIgenesIs In 02 Mice3
Treatment
i
CO
O
CO
-v.
O
CO
-2 Days
None
l.p. p-dloxane
1.p. TCOD (100 Mg/kg)
None
None
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-MC
3-MC
3-MC
p-d1oxane * s.c. 3-HC
TCOD (100 pg/kg) t
3-MC
TCOD (1 pg/kg) +
3-MC
p-dloxane * s.c. 3-MC
TCOD (100 pg/kg)
TCOD (100 pg/kg) ^
3-MC
TCDO (1 yg/kg)
TCDD (1 pg/kg) *
3-MC
aSource: Kour 1 et al. , 1978
''These carcinogenic Index values He outside the 99X
No. of Mice
Dying Because
of Treatment
0
10
35
5
38
22
2
8
18
2
2
confidence Interval.
No. of Mice
at Risk for
Tumors
30
40
65
45
62
78
68
42
82
48
98
No. of
Mice with
Tumors
3
3
9
5
1 7
8
8
0
46
0
21
X of Mice
with Tumors
10
10
14
11
27
10
12
0
55
0
21
Average
Latency
(days)
177
194
145
176
183
162
180
145
154
Carcinogenic
Index
6
5
10
6
15°
6
6
38°
14"
-------
with 2,3,7,8-TCDD at 1 pg/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-MC-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-MC at the higher dose
(100 ug/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).
11.1.6.5.1. Poland et al. Study (1982) — Poland et al. (1982) have
described studies which Indicate that genetic differences 1n mice affect the
tumor-promoting capacity of 2,3,7,8-TCDD In the mouse skin two-stage tumoM-
genesls model. Both 2,3,7,8-TCDD and TPA were compared for tumor-promoting
activity in DMBA-1n1t1ated HRS/J mice which were either heterozygous
(hourA) or homozygous (hour/hour) for the recessive "hairless" trait.
1858A 11-33 03/28/84
-------
TABLE 11-18
Incidence of Tumors 1n Mice Treated With 3-MC
and With 3-MC and 2,3,7,8-TCDDa
Experiment
1
2
2
2
Dose of
TCOD
(yg/kg)
100
100
100
1
Tumor Incidence
Route of
Administration
1ntraper1toneal
1ntraper1toneal
subcutaneous
subcutaneous
TCDO and 3-MC
10/43
17/62
46/82
21/98
3-MC
l/34b
5/45
5/42
5/45
p Valueb
p=0.01
p=0.03
p=3.0 x 10~7
p=0.1
aSource: Kourl et al., 1978
bp value calculated using the Fisher Exact Test (one-tailed).
cVeh1cle (p-d1oxane) not administered
1858A
11-34
03/28/84
-------
Promotion with biweekly applications of 2 yg of 1PA for 25 weeks resulted
1n papllloma Incidences of 100 and 70% 1n (hour/O and (hour/hour) mice,
respectively. Promotion of DMBA-1n1t1ated (hourA) mice with 2,3,7,8-TCDD
(50 ng/app!1cat1on for 8 weeks followed by 20 ng/appl1cat1on) did not result
1n the formation of tumors, while promotion of (hour/hour) mice resulted 1n
both the same Incidence and multiplicity of tumors as observed 1n TPA-pro-
moted mice. With either DMBA or methyl-N-n1trosoguan1d1ne (MNNG)-1n1t1ated
(hour/hour) mice, the effective dose of 2,3,7,8-TCDD was -100-fold less than
1PA on a molar basis. H1stolog1c examination of the skin showed that TPA
produced both acute Inflammation and hyperplasla 1n (hourA) and (hour/hour)
mice, while 2,3,7,8-TCDD produced hyperplasla and hyperkeratosls only 1n
(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 paplllomas 1n (hour/hour) mice by a mechanism different from
TPA.
11.1.6.5.2. Di61ovann1 et al. Study (1977) — 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 papilloma 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
in the Incidence of DMBA-1n1t1ated tumors when CD-I mice were pretreated 3
days before DMBA initiation with 1 Mg/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
1858A 11-35 04/12/84
-------
critical in the antHumorlgenic effects of 2,3,7,8-TCDD (Berry et al., 1979;
DiGiovannl 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 DMBA
Initiation, the tumor yield was decreased by 78%, while 2,3,7,8-TCDD treat-
ment 5 minutes before or 1 day after DMBA 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 DMBA Initiation) and the
Incidence of tumors. 2,3,7,8-TCDD doses of 0.0, 0.01, 0.1 and 2 yg/mouse
resulted in decreased tumor yields, respectively, of 0, 83, 92 and 96%
(D1G1ovann1 et al., 1979a). Also under similar experimental conditions
Cohen et al. (1979) observed a 75% decrease 1n the Incidence of skin tumors
1n Sencar mice pretreated with 1 yg of 2,3,7,8-TCDD 3 days prior to
Initiation by DMBA.
11.1.6.5.3. D1G1ovann1 et al. Study (1980) — 01G1ovann1 et al. (1980)
Investigated the antUumorigenlc effect of 2,3,7,8-TCDD In CD-I mice with
chemical carcinogens other than DMBA (see Figure 11-1). As observed with
DMBA, 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, while pretreatment with 2,3,7,8-TCDD 5
minutes before or 1 day after Initiations was Ineffective 1n changing the
tumor yield. The maximum decrease In 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-diol-epox1de as compared with the previously studied poly-
aromatic hydrocarbons (PAH). When 2,3,7,8-TCDD was applied 3 days or 5
minutes before, or 1 day after Initiation with BaP-d1ol epoxlde, there was
1858A 11-36 03/28/84
-------
CD
U-l
03
o r\i
eu O
3 3
O 3
O
O _>
-tl
CO
B) -» CO
—J O> C
«• o o>
m 3 -I
A 30862 -U
TUMOR RESPONSE (PERCENT OF CONTROL
I
CO
O
CO
00
•x.
CO
CD
3
Q.
§
~H?
-" * re
5 TO
CO 3
_, BJ 5.
o -o«
-
^^
00 '
re
7T
§5.
3 Q,
O
re
o
CO
cu
TJ
XI
&> rr^
•m ^^ «h *
^o cu *^»
T3 co
—i a. •
o -* -*1
00*
-, T" i.
a! o
—» c
OJ O 3
3-^0
a. • -i
ra
•a
-------
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 a 21-fold Increase 1n aryl
hydrocarbon hydroxylase (AHH) activity 72 hours after treatment (D1G1ovann1
et al., 1980). The In vitro metabolism of DMBA by dermal homogenates from
2,3,7,8- TCDD-treated mice Indicated both qualitative and quantitative
changes 1n metabolism (Cohen et al., 1979; 0161ovann1 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
antHumor1gen1c properties of 2,3,7,8-TCDD resulted from 2,3,7,8-TCDD
Induced alteration 1n the metabolism of the Initiating chemical. Although
metabolic change was a possible mechanism for the Inhibition of DMBA, 3-MC
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
antlcardnogenk effect of 2,3,7,8-TCDD.
11.1.6.6. COCKERHAM ET AL. 1980 FIELD STUDY ON BEACH MICE — Cockerham
et al. (1980) performed a field study on beach mice, Peramyscus pollenotus,
that Inhabited an area which was heavily treated with the herbicide 2,4,5-T,
of which 2,3,7,8-TCOD 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
1858A 11-38 04/12/84
-------
neoplastlc lesions, observed 1n the liver as compared to 9 male and 6 female
mice captured 1n a noncontamlnated area. The only observed difference 1n
the two groups of mice was a statistically significant (95% confidence)
Increase 1n liver to body weight ratios. The authors back-calculated from
the 2,3,7,8-TCOD levels of the liver and estimated a dally 2,3,7,8-TCDD dose
of 0.0012 yg/kg bw. It was noted that this exposure was much lower than
the exposures used 1n laboratory studies to produce tumors.
11.1.6.7. NATIONAL TOXICOLOGY PROGRAM BIOASSAY OF HxCDD 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 cardnogenlcHy, the NTP has performed a
chronic bloassay 1n both Osborne-Mendel rats and B6C3F1 mice to determine
the cardnogenlcHy 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-HxCDD congener and
67% of the 1,2,3,7,8,9-HxCOD congener, with a total HxCDO purity of 98%.
The following Impurities were detected 1n HxCDD used for this bloassay:
PeCDD, 0.04%; TCDO, 0.09%i0.03%; 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 pg/kg/week. Treatment was con-
tinued for 104 weeks followed by a 3-4 week observation period. Complete
necropsies, Including extensive hlstologlc examinations, were performed on
1858A 11-39 04/12/84
-------
animals at the time of natural death, when moribund or at the termination of
the study.
A decrease 1n body weight gain was seen at the two higher exposure
levels. A dose-related "toxic hepatitis" which was noninflammatory and
consisted of degenerative changes 1n the liver, eos1nophH1c 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 which 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 In male rats showed a positive dose-related trend
by the Cochran-Arm1tage 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 cardnogenlclty 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 (p<0.001)
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 Dr.
Robert A. 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
1858A 11 40 03/28/84
-------
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
Untreated
Control
2/75b
0/75
2/75
Vehicle
Control
0/74
0/74
0/74
Treatment Group
Low Dose M1d Dose
MALE
0/49 1/50
0/49 0/50
0/49 1/50
High Dose
3/48
1/48
4/48
p=0.002C
Neoplastlc
nodule (NN)
Combined NN f HC
1/73
Hepatocellular
carcinoma (HC) 0/74
1/73
FEMALE
5/75 10/50 12/50 30/50
p=0.026 p=0.006 p=6.94xlO"11
0/75
5/75
0/50
0/50
4/50
p=0.024
10/50 12/50 30/50
p=0.026 p^O.006 P-6.94X10"11
^Source: Adapted from NTP, 1980c
Incidence =
No. of rats with lesion
No. of rats examined microscopically
Cstat1st1cally significant by Fisher's Exact Test compared with vehicle
control
1858A
11-41
03/28/84
-------
hepatocardnogenlc effect 1n rats and mice. Drs. R. Schueler and B. Haber-
man 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. Bellln (U.S. EPA, 1983b) with an attached report
prepared by Or. R. Schueler, Research Pathology Associates, Inc. (Schueler,
1983). Finally, Dr. E. McConnel of NTP requested that Or. P. Hlldebrandt of
Tracor-JHco, Inc., review the microscopic slides of the HxCDD bloassay
(gavage) 1n the female rat; his findings (Hlldebrandt, 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 find-
Ings, still confirmed that the HxCDO mixture administered by gavage produced
an Increased Incidence of liver tumors 1n treated female rats as compared
with control animals, as well as an Increase 1n "toxic hepatitis."
In mice there were no gross signs of HxCDO 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 which 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 In control and dosed
groups metastaslzed to the lungs. The Incidence of hepatocellular adenomas
or carcinomas were 15/73, 14/50, 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
1858A 11-42 03/28/84
-------
TABLE 11-20
Liver Tumor Incidences 1n Female Osborne-Mendel Rats Administered
HxCDD by Gavage for 104 Weeks3
Diagnoses
Neoplastlc
nodule (NN)
Hepatocellular
carcinoma (HC)
Combined NN f HC
Untreated
Control
1/73&
0/73
1/73
Vehicle
Control
2/75
0/75
2/75
Low Dose
1.25
5/50
0/50
5/50
yg/kg/week
Mid Dose
2.5
7/50
0/50
7/50
p=0.02
High Dose
5
16/50
p=6.0xlO~6
2/50
18/50
aSource: Adapted from Hlldebrandt, 1983
Incidence =
No. of rats with lesion
No. of rats examined microscopically
C8tat1st1cally significant by Fisher's Exact Test compared with vehicle
control.
1858A
11 43
03/28/84
-------
TABLE 11-21
Liver Tumor Incidences 1n Male and Female B6C3F1 Mice Administered
HxCDO by Gavage for 104 Weeks3
Diagnoses
Hepatocellular
adenoma (HA)
Hepatocellular
carcinoma (HC)
Combined HA f HC
Hepatocel lular
adenoma (HA)
Hepatocellular
carcinoma (HC)
Combined HA + HC
aSource: Adapted
b
TnrlHonro
Untreated
Control
15/75b
12/75
27/75
2/74
0/74
2/74
Treatment Group
Vehicle Low Dose M1d Dose
Control
MALE
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=7.33xlO~«
9/47
p=0.003
2/47
10/47
p=0.004
from NTP, 1980c
No. of rats
with lesion
No. of rats examined microscopically
GStat1st1cally significant by Fisher's Exact Test compared with vehicle
control.
1858A
11-44
03/28/84
-------
Incidence showed a significant dose-related trend by the Cochran-Armltage
test, and the Incidence of tumors In the high-dose group was significantly
higher than the Incidence 1n the control group by the Fisher exact test.
Under the test conditions of this bloassay, the 1:2 mixture of
1,2,3,7,8- and 1,2,3,7,8,9-HxCDO was carcinogenic, as Indicated by a statis-
tically significant Increased Incidence 1n tumors of the liver 1n female
rats and in both male and female mice, and by a borderline liver tumor
response In male rats.
11.1.6.8. NTP SKIN-PAINTING STUDY OF HxCDD ON MICE (NTP, 1980b,c) —
Both ?,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 pre-
sented 1n the discussion of NTP (1980a,d). There was no Information found
in the literature searched on the tumorlgenlc effect of 1,2,3,7,8-PeCOD
following dermal exposure. The tumorlgenlc response after chronic dermal
exposure to HxCDD was presented 1n Table 11-22.
In both NTP bloassays (1980b,c), groups of 30 male and 30 female Swiss-
Webster mice were treated with 100 pS, 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
ug/appl1cat1on 1n male mice and 0.005 v>g/appl1cat1on 1n female mice,
while the concentration of HxCDD used resulted 1n a dose of 0.005 pg/
application for the Initial 16 weeks of the study, followed by a subsequent
Increase to 0.01 pg/app!1cat1on for the remainder of the study. Sub-
chronic toxldty studies used to define the dose levels for the chronic
1858A 11 45 03/28/84
-------
CD
U1
CO
01
03/09/8
Cardnogenldty Bloassays of 2,3,7
Compound Sex Doseb
2,3,7,8-TCDD M 0.01 Vg/appl1cat1on
M 0.0 ^/application
(vehicle control }
M 0.0 yg/appHcatlon
(untreated control )
2,3,7,8-TCDD F 0.005 yg/appl 1cat1on
F 0.0 pg/appl1cat1on
(vehicle control )
F 0.0 yg/app!1cat1on
(untreated control )
HxCDD M 0.01 yg/appT1cat1onc
M 0.0 yg/appHcatlon
(vehicle control )
TABLE 11 -22
,8-TCDD and
Duration
of
Exposure
104 weeks
104 weeks
NA
104 weeks
104 weeks
NA
104 weeks
104 weeks
HxCDD by Dermal Appl
Target Organ
Integumentary
system
Integumentary
system
Integumentary
system
Integumentary
system
Integumentary
system
Integumentary
system
Tung
lung
IcaUon to M1cea
Tumor Type
fibrosarcoma
fibrosarcoma
fibrosarcoma
fibrosarcoma
fibrosarcoma
fibrosarcoma
alveolar/
bronchlolar
carcinoma
alveolar/
bronchlolar
carcinoma
Tumor
Incidence
6/28
3/42
0/28
7/28
2/41
1/27
5/30
1/41
-------
oo
01
oo
TABLE 11-22 (cont.)
o
CO
o
US
CD
Compound Sex
HxCDD (cont.) M
HxCDD F
F
F
Doseb
0.0 yg/appl1cat1on
(untreated control )
0.01 yg/appl1cat1onc
0.0 vg/appl1cat1on
(vehicle control )
0.0 ^/application
(untreated control )
Duration
of
Exposure
NA
104 weeks
104 weeks
NA
Target Organ
lung
skin
skin
skin
Tumor Type
alveolar/
bronchlolar
carcinoma
f Ibrosarcoma
f Ibrosarcoma
f Ibrosarcoma
Tumor
Incidence
4/28
4/27
2/41
0/30
^Source: NTP, 1980b,c
''The compound was applied 3 times/week In 100 yi of acetone.
cFor the Initial 16 weeks of the study, the dose was 0.005 yg/appl1cat1on,
NA = Not applicable
-------
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 examinations were also made of all major organs.
In mice exposed to 2,3,7,8-TCOD (NTP, 1980b), there was no treatment-
related difference In body weight of either sex between exposed animals and
control groups; however, male mice treated with 2,3,7,8-TCDD had a signif-
icant shortening of Hfespan. Nontumor1gen1c hepatic lesions were observed
In treated female mice, while no mention was made of these lesions occurring
1n male mice. The only tumors that were treatment-related were Integument-
ary system fIbrosarcomas, with tumors developing on or near the site of
application. The Incidence of these tumors In 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 1n
female mice was statistically (p--0.007) greater than control values;
however, life table analyses Indicated that the time to tumor was shorter In
both male and female treated mice. The Incidence of tumors in untreated and
vehicle control groups was similar.
In the bloassay of HxCDD (NTP, 1980c), no gross or nonneoplastlc histo-
loglc effects associated with treatment were observed. Although there was a
slight increase 1n the Incidence of skin f Ibrosarcomas 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/bronchiolar 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 in a carcinogenic response in both male
1858A 11-48 03/28/84
-------
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 1n a carcinogenic response under
the conditions of this bloassay. A summary of the carclnogenlcHy bloassays
1s given 1n Table 11-23.
11.2. SUMMARY OF ANIMAL CARCINOGENICITY
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 In site-
specific 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 Intake of 2,3,7,8-TCDD
from the diet was 0.0, 0.001, 0.01 and 0.1 pg/kg/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, while 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-TCDD at levels that produce a dally dose of 0.1
results 1n Increased tumor Incidences 1n both male and female rats.
1858A 11-49 04/12/84
-------
CD
co
Exposure
Route/ Species/Strain
Compound
Gavage/ rats/
2.3,7,8-TCDD Osborne-Mendel
i
0 Gavage/ rats/
2,3,7,8-TCOD Osborne-Mendel
Gavage/ m1ce/B6C3H
2,3,7,8-TCDD
O
GJ
o
Carclnogenldty Bloassays of PCDD
Duration
Sex Dose or Exposure of
Treatment
M 0.0 pg/kg/week 104 weeks
0.1 pg/kg/week 104 weeks
0.05 pg/kg/week 104 weeks
0.5 pg/kg/week 104 weeks
F 0.0 pg/kg/week 104 weeks
0.1 pg/kg/week 104 weeks
0.05 pg/kg/week 104 weeks
0.5 pg/kg/week 104 weeks
M 0.0 pg/kg/week 104 weeks
0.1 pg/kg/week 104 weeks
TABLE 11-23
Administration by the Oral
Duration Vehicle
of Study
105 weeks corn oil-
acetone
(9:1)
107 weeks corn oil-
acetone
(9:1) ,
107 weeks corn oil-
acetone
(9:1)
105 weeks corn oil-
acetone
(9:1)
105 weeks corn oil-
acetone
(9:1)
107 weeks corn oil-
acetone
(9:1)
107 weeks corn oil-
acetone
(9:1)
107 weeks corn oil-
acetone
(9:1)
105 weeks corn oil-
acetone
(9:1)
107 weeks corn oil-
acetone
(9:1)
and Dermal Route
Tumor Type
folllcular-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 liver
neoplastlc nodule or
hepatocellular carcinoma
of the liver
hepatocellular carcinoma
hepatocellular carcinoma
Tumor
Incidence
1/69
5/48
8/50
11/50
5/75
1/49
3/50
14/49
8/73
9/49
Reference
NTP, 1980a
NTP. 1980a
CO
-------
CD
on
CD
I
tn
O
O
vD
X.
Exposure
Route/ Species/Strain Sex Dose or Exposure
Compound
Gavage/ m1ce/B6C3Fl 0
2,3,7,8-TCDD
(cont.)
0
Gavage/ mice/B6C3Fl F 0
2,3,7,8-TCDD
0
0
2
Oral/ rat/ M 0
2,3,7,8-TCDD Sprague-Dawley
0
0
0
0
1
5
05 ug/kg/week
5 ug/kg/week
0 ug/kg/week
04 pg/kg/week
2 ug/kg/week
0 ug/kg/week
0 ppb
001 ppb
005 ppb
05 ppb
5 ppb
0 ppb
0 ppb
TABLE 11-23 (cont
Duration
of Duration
Treatment of Study
104 weeks 107 weeks
104 weeks 107 weeks
104 weeks 105 weeks
104 weeks 107 weeks
104 weeks 107 weeks
104 weeks 107 weeks
78 weeks 95 weeks
78 weeks 95 weeks
78 weeks 95 weeks
78 weeks 95 weeks
78 weeks 95 weeks
78 weeks 95 weeks
78 weeks 95 weeks
.)
Vehicle
corn oil-
acetone
(9:1)
corn oil-
acetone
(9:1)
corn oil-
acetone
(9:1)
corn oil-
acetone
(9:1)
corn oil-
acetone
(9:1)
corn oil-
acetone
(9:1)
In diet
1n diet
In diet
1n diet
1n diet
In diet
1n diet
Tumor Type
hepatocellular
hepatocellular
hepatocellular
folllcular-cell
of the thyroid
hepatocellular
folllcular-cell
of the thyroid
hepatocellular
folllcular-cell
of the thyroid
hepatocellular
folllcular-cell
of the thyroid
all tumors
all tumors
all tumors
all tumors
all tumors
all tumors
all tumors
carcinoma
carcinoma
carcinoma,
adenomas
carcinoma,
adenomas
carcinoma,
adenomas
carcinoma,
adenomas
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
NTP, 1980a
NTP, 1980a
Van MUler
et al.. 1977a
oo
-------
CD
in
co
TABLE 11-23 (cont. )
Exposure Duration
Route/ Species/Strain Sex Dose or Exposure of Duration Vehicle
Compound Treatment of Study
Tumor Type
Tumor
Incidence
Reference
i
en
o
to
CO
Oral/ rat/ M 0.0 Pg/kg/day
2,3,7,8-TCDD Sprague-Dawley
Oral/
2,3,7.8-TCDD
rat/
Sprague-Dawley
Oral/ rat/
2,3,7,8-TCDD Sprague-
Dawley
105 weeks 105 weeks 1n diet
0.001 vg/kg/day 105 weeks 105 weeks 1n diet
0.01 yg/kg/day
105 weeks
105 weeks
0.1 pg/kg/day
105 weeks
105 weeks
1n diet
1n diet
F 0.0 pg/kg/day 105 weeks 105 weeks 1n diet
0.001 vg/kg/day 105 weeks 105 weeks 1n diet
0.01 pg/kg/day 105 weeks 105 weeks 1n diet
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 liing
0/85 Kodba
et al., 1978a
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
Kodba
et al.. 1978a
Kodba
et al., 1978a
-------
oo
in
CO
i
en
CO
03/28/84
Exposure
Route/ Species/Strain Sex Dose or Exposure
Compound
Oral/ rat/ F 0.1 yg/kg/day
2,3,7,8-TCDD Sprague-Dawley
Gavage/ mice/Swiss/ M 0.0 yg/kg/week
2,3,7,8-TCDD H/R1op
0.007 yg/kg/week
0.7 yg/kg/week
7.0 vg/kg/week
Oral/ mice/ MiF 0.0012 yg/kg/day
2,3,7,8-TCDD Peramyscus
pollenotus
0.0 yg/kg/day
Gavage/HxCDD rats/ M 0.0 yg/kg/week
Osborne-Mendel (vehicle control )
Gavage/HxCDD rats/ M 1 .25 yg/kg/week
Osborne-Mendel
2.5 yg/kg/week
5.0 yg/kg/week
TABLE 11-23 (cont
Duration
of Duration
Treatment of Study
105 weeks 105 weeks
365 days 588 days
365 days 649 days
365 days 633 days
365 days 424 days
NA NA
NA NA
104 weeks 105 weeks
104 weeks 106 weeks
104 weeks 107 weeks
104 weeks 107 weeks
"'
Vehicle
In diet
sunflower
oil
sunflower
oil
sunflower
oil
sunflower
oil
contami-
nated soil
contami-
nated soil
corn oil-
acetone
(9:1)
corn oil-
acetone
(9:1)
corn oil-
acetone
corn o-1l-
acetone
Tumor Type Tumor Reference
Incidence
hepatocellular carcinoma, 11/49 Kodba
squamous cell carcinoma et al., 1978a
of the tongue, 4/49
squamous cell carcinoma
of the lung 7/49
liver tumors 7/38 Toth et al.,
1979
liver tumors ' 13/44
liver tumors 21/44
liver tumors 13/43
liver 0/15 Cockerham
et al., 1980
liver 0/15
liver neoplastlc nodules 0/74 NTP, 1980d
or hepatocellular
carcinoma
liver neoplastlc nodules 0/49 NTP, 1980d
or hepatocellular
carcinoma
liver neoplastlc nodules 1/50
or hepatocellular
carcinoma
liver neoplastlc nodules 4/48
or hepatocellular
carcinoma
-------
TABLE 11-23 (cont.)
03
(Jl
03
Exposure
Route/ Species/Strain Sex Dose or Exposure
Compound
Gavage/HxCDD rats/ F 0.0 yg/kg/week
Osborne-Mendel
1 .25 yg/kg/week
2.5 yg/kg/week
i
tn
4k
5.0 yg/kg/week
Gavage/HxCDD m1ce/B6C3Fl M 0.0 yg/kg/week
1 .25 yg/kg/week
2.5 yg/kg/week
5.0 yg/kg/week
Gavage/HxCDD m1ce/B6C3Fl F 0.0 yg/kg/week
2.5 yg/kg/week
o 5.0 yg/kg/week
CO
o
vD
c» 10.0 yg/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 oil-
acetone
(9:1)
corn oil-
acetone
(9:1)
corn oil-
acetone
(9:1)
corn oil-
acetone
(9:1)
corn oil-
acetone
(9:1)
corn oil-
acetone
(9:1)
corn oil-
acetone
(9:1)
corn oil-
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
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 adenomas
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
hepatocellular adenomas
or carcinomas
Tumor Reference
Incidence
5/75 NTP, 1980d
10/50
12/50
30/50
15/73 NTP, 1980d
14/50
14/49
24/48
3/73 NTP, 1980d
4/48
6/47
10/47
NA = Not available
-------
Under the National lexicology Program, 2,3,7,8-TCDD was tested for
cardnogenldty 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 folHcular cell adenomas or carcinomas of the thyroid 1n male animals,
and neoplastlc nodules or hepatocellular carcinomas of the liver In 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-
1CDD was concluded to be carcinogenic 1n both male and female rats.
Further studies In mice exposed by gavage have provided support for the
cardnogenldty 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, Us contaminant 2,3,7,8-TCDD or both were
carcinogens. At the 0.7 yg/kg/week level there was a significant
Increased incidence of liver tumors. Liver tumors were not significantly
Increased in the high-dose group; however, early mortality 1n this group may
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 In the high-dose female
mice exposed to 2 yg/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 yg/kg/day, depending on sex and the Individual
study.
The mouse skin two-stage tumor1gen1dty model has also been used to test
the carcinogenic potential of 2,3,7,8-TCDD. Following long-term dermal
1858A 11-55 03/28/84
-------
application 3 times/week of 2,3,7,8-TCDD at levels of 0.01 and 0.005
lag/application 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. PHot et al. (1980) also
reported that 2,3,7,8-TCDD was a promoter for DEN-1n1 tlated hepatocarcino-
genesis in 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-
1CDD in the assays. In rat liver Initiated with DEN, 2,3,7,8-TCDD was a
tumor promoter.
In studies of the interaction of 2,3,7,8-TCDD with other chemical
carcinogens, Kouri et al. (1978) reported that 2,3,7,8-TCOD was a cocar-
cinogen with 3-MC when administered by subcutaneous Injection. In the mouse
skin bloassay, Initiation with simultaneous administration of 2,3,7,8-TCDD
and DMBA, however, did not affect tumor yield (D161ovann1 et al., 1977).
Similarly, no effect was observed when 2,3,7,8-TCDD was administered either
immediately prior to (5 minutes) or 1 day after DMBA Initiation (Berry et
al., 1979; D1G1ovann1 et al., 1977, 1979b; Cohen et al., 1979). When treat-
ment with ?,3,7,8-7CDD occurred 1-10 days before DMBA initiation, 2,3,7,8-
1858A 11-56 03/28/84
-------
1CDD demonstrated a potent antlcardnogenlc action. Although 1-5 days prior
exposure to 2,3,7,8-TCDO Inhibited tumor Initiation by BaP, 3-MC and BaP-
dlol-epoxide, the tumor Initiating ability of the latter compound was also
Inhibited when 2,3,7,8-TCDD exposure occurred either 5 minutes prior to or 1
day after Initiation (D1G1ovann1 et al., 1980). The Increased AHH activity
resulting from 2,3,7,8-TCDO exposure may account for the antlcardnogenic
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 In the antlcardnogenic activity of 2,3,7,8-TCDD.
HxCDD has also been tested for carc1nogen1c1ty 1n 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 HxCOD 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 either sex only tumors of the liver
occurred at a significantly greater Incidence than 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 Incidence was significantly greater at both the medium- and
high-dose levels. In the study of HxCDD cardnogenlclty In mouse skin con-
ducted by NTP (1980c), there were no treatment-related tumors In either the
cardnogenlclty bloassay or the tumor promotion assay using DMBA as an
Initiator. It was concluded that this mixture of HxCDD was carcinogenic to
rats and mice following administration by gavage; however, there was no
tumoMgenlc activity when HxCDD was applied to mouse skin.
1858A 11-57 03/28/84
-------
No chronic animal bloassays were found In the literature searched on the
cardnogenlcHy of 1,2,3,7,8-PeCOD.
11.3. EPIDEMIOLOGICAL STUDIES*
11.3.1. Case Reports. Observations of an unusual occurrence of relatively
rare soft-tissue sarcomas were first made by Hardell (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 adds 10-20
years earlier. The tumors were 2 leiomyosarcomas, 1 Uposarcoma, 1 rhabdo-
myosarcoma, 1 myxofIbrosarcoma and 2 additional sarcomas of which the hlsto-
pathology was uncertain but one was probably a neurofIbrosarcoma 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 if exposure to phenoxy acids and the Impurities they
contain are related to the occurrence of soft-tissue sarcomas.
Zack and Suskind (1980) reported a soft-tissue sarcoma death 1n a cohort
study of workers exposed to 2,3,7,8-TCDO 1n a trlchlorophenol process
accident in N1tro, West 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) In a cohort mortality study of 61 male employees of a
trlchlorophenol 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 fibrosarcoma. The authors did not seem to attribute any
special significance to this finding at the time.
*Port1ons of this section were taken from U.S. EPA (1980c).
1873A 11-58 03/12/84
-------
Ott et al. (1980) 1n a cohort mortality study of 204 employees exposed
to 2,4,5-T during Us 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, Cook, an unpublished
study by Zack (1n which a Uposarcoma was found), and a study by Ott et al.
(1980) and Honchar and Halperln (1981) noted 3 (2.9%) soft-tissue sarcomas
1n a total of 105 deaths. Among United States 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 somewhat of
an underestimate because of the posslbHty that some soft-tissue sarcomas
may be coded to categories other than ICD 171. Individually, none of the
reported case studies reported a significant excess of soft-tissue sarcomas.
Cook (1981a) found an additional malignant fibrous hlstlocytoma 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 fibrosarcoma
worked in a trichlorophenol (TCP) process area contaminated with 2,3,7,8-
TCDD. This individual exhibited facial dermatitis but no diagnosis of
chloracne. The fourth case (diagnosed as a Uposarcoma) had been employed
earlier in a plant producing 2,4,5-T. Cook noted that although chloracne
was not reported, it could not be discounted. He also noted that all four
*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.
1873A 11-59 03/29/84
-------
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 Hardell's seven cases exhibited chloracne before the appearance
of the soft-tissue saromcas, and that 1n their subsequent later case control
study, they found no difference 1n smoking habits between his cases and
controls.
Moses and Sellkoff (1981) reported a fifth soft-tissue sarcoma In a
worker employed at the Monsanto Chemical Company at a time when tMchloro-
phenol and 2,4,5-T were being produced. He died of a retroperltoneal neuro-
genlc sarcoma (malignant schwanoma) In 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 did not have a history of chloracne.
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 llposarcoma). Both were exposed
to halogenated phenol derivatives. The author noted that 2,4-d1chlorophenol
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 1s the follow-up time given. The
exposure of the son 1s not likely related to that of the father.
1873A 11-60 03/29/84
-------
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 h1st1ocytoma, medlastlnal
fIbrosarcoma, 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. M1ld, moderate and severe cases of chloracne were
seen 1n 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
NHL. Four of the five were reported to have repeatedly sprayed large areas
with phenoxy add herbicides. In the remaining 118 NHL patients, only seven
had a similar occupational exposure to phenoxy adds. 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 believe their observations were similar to
those of Bishop and Jones (1981).
1873A 11-61 03/29/84
-------
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 1s unusual that so many cases of soft-tissue sarcomas have
occurred. A Lancet editorial (Anonymous, 1982) calls this phenomenon
"disturbing."
11.3.2. Soft-Tissue Sarcomas. Soft-tissue sarcomas (STS) constitute a
collection of heterologous lesions that Include both malignant and non-
malignant tumors. Not all of them have their origin 1n primordial mesen-
chymal cells. Some exceptions are tumors of peripheral nerves, and neuro-
ectodermal tumors which are classified as STS, but are derived from non-
mesenchymal cells. Classification, grading and staging of STSs 1s 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 Intracellular 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,
STSs can be induced in any of these tissue types (Hajdu, 1983). The classl-
flcation of STSs for cause of death coding 1n the ninth and latest revision
of the International Classification of Diseases (ICO, 1975) places STSs into
one of several categories. But chiefly, they fall into "malignant neoplasms
of connective and other soft-tissue" (ICD 171). Lymphosarcomas, retroperi-
toneal sarcomas and extra skeletal SISs of the bone are coded elsewhere. In
some Instances, 1f site 1s mentioned, 1t Is coded to the site, I.e., Ie1o-
myosarcoma of the stomach (ICD 151.9), neuroflbroma of the chest wall
(215.4).
1873A 11-62 03/29/84
-------
Questions have been raised concerning the appropriateness of lumping
together malignant tumors of different sites and tumor types In 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 \s 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, and therefore, H 1s Impossible to derive
risk estimates for particular types at given sites. Altogether, -2000
deaths/year can be attributed to STSs In the United States, most of which
are coded to ICD category 171 for purposes of developing Incidence and mor-
tality 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
underestimated. 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 are lumped together while expected STSs are based upon ICD 171 only.
Thus, action of this sort, especially with respect to cohort studies of
Individuals exposed to d1ox1n-conta1n1ng herbicides 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 sar-
comas in seven men with previous occupational exposure to phenoxyacetlc acid
1873A 11-63 03/29/84
-------
herbicides (Harden, 1977), researchers at the Department of Oncology, Uni-
versity Hospital, Umea, Sweden, Initiated case-control ep1dem1olog1c studies
(Cole, 1979) to test the hypothesis of an etlologlc association (Hardell and
Sandstrom, 1979). Cases were defined as male patients with sarcomas of soft
connective tissue, such as smooth muscle (lelomyosarcoma) and fat (lipo-
sarcoma). The distribution of tumor types in the two studies 1s shown in
lable 11-24. 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 in patients with
these tumors as compared with that of STS (Hardell, 1983).
Two case-control studies were conducted: the first in 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 in Table 11-25. In the north (Study A), occupational exposure to
phenoxyacetic acids took place in both forestry and agricultural work. In
the south (Study B), these exposures were predominantly agricultural. The
phenoxyacetic acids to which exposure occurred consisted predominantly of
2,4,5-1 and 2,4-D in both studies. Exposure to 2,4,5-T in the absence of
2,4-0 was rarely reported in either study. Exposure to chlorophenols, which
contain chlorinated dibenzodioxin Impurities (Levin et al., 1976) occurred
mostly in sawmill work and paper pulp production. Very few persons reported
exposure both to phenoxyacetic add and chlorophenols in these studies. Of
the two predominant phenoxyacetic acids, only 2,4,5-T Is known to be con-
taminated with 2,3,7,8-TCDD. In Study B, a relative risk of 4.9 (90% confi-
dence intervals 1.6-11.1) was found In relation to exposure to phenoxyacetic
acid herbicide other than 2,4,5-T (2,4-0, MCPA, mecoprop, dichloroprop).
1873A 11-64 03/29/84
-------
TABLE 11-24
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=110)
Lelomyosarcoma
Fibrous h1st1ocytoma
Uposarcoma
Neurogenlc sarcoma
Anglosarcoma
Myxosarcoma
Fibrosarcoma
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 Hardell to EPA (Hardell and Sand-
strom, 1979)
bEr1ksson et al., 1979, 1981
1873A
11-65
03/09/84
-------
TABLE 11-25
Exposure Frequencies In 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, Hardell and Sandstrom, 1979; Study B, Eriksson et al.,
1979, 1981
1873A 11-66 03/09/84
-------
Relative risks 1n relation to the three major categories of exposure are
shown 1n Table 11-26.* Studies A and B Indicate a risk of developing STSs
among workers exposed to phenoxyacetlc acids only, chlorophenols only, or
phenoxyacetlc adds and/or chlorophenols several times higher than among
persons not exposed to these chemicals. In each comparison, a high relative
risk 1s estimated and was thus unlikely to have resulted by chance alone.
Since Uttle 1s known of the etiology of STSs, the consideration of
confounding 1n these studies was largely a hypothetical matter. The authors
prevented the effects of age, sex, and place of residence as possible
confounding factors In the selection of controls.t Because of the high
correlation between exposure to the substances of Interest and employment 1n
agriculture and forestry, a possible alternative hypothesis could be that
some other unknown factor present In these occupations was responsible for
the elevated relative risks.
To test this hypothesis, it is possible to calculate the relative risk
in relation to the phenoxyacetlc add exposure In Study B, restricting the
analysis to workers within agriculture and forestry. The result is a rela-
tive risk of 6.1 (90% confidence interval 2.4-15.4). This finding suggests
that a confounding risk factor for SIS distributed throughout agriculture
and forestry work was not responsible for the overall increase in risk found
in relation to phenoxyacetlc acid exposure.
*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 adds 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 acids and/or
chlorophenols were 6.2 (p<0.001) in Study A and 5.1 (p<0.001) 1n Study B.
1873A 11-67 04/09/84
-------
CD
~J
00
01
03
o
10
co
TABLE 11-26
Relative Risks of Soft-Tissue Sarcoma in Relation to Exposure to
Phenoxyacetlc Adds and Chlorophenols 1n Two Case-Control Studies3
Phenoxyacetlc Adds
Onl^_
Relative riskb
90% Confidence interval0
Significance level1*
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
Phenoxyacetic Acids
and/or
Chlorophenols
Study A
5.7
3.2-10.2
<0.001
Study
4.7
2.7-8.
<0.001
B
3
aSource: Study A, Hardell and Sandstrom, 1979; Study B, Eriksson et al., 1979, 1981
^Unmatched odds ratio
cTest-based method of Miettinen, 1976
square statistic, no continuity correction, one-tailed test
-------
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 1t 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 to unexposed persons 1n other occupations
was 0.9 (90% confidence Interval 0.3-2.4) In Study B. This suggests that
Uttle 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 1mpur1-
t1es--polychlor1nated dlbenzodloxlns and dlbenzofurans. The risk ratio
given exposure to phenoxy adds free of polychlorlnated dlbenzodloxlns and
dlbenzofurans equaled 4.2 based upon 7 out of 14 respondents who Indicated
exposure to phenoxy add 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 non-exposure to dloxlns 1s not well presented 1n this study.
The author concluded that exposure to phenoxy adds and chlorophenols
"might constitute a risk factor In the development of soft-tissue sarcomas."
This risk relates not only to 2,4,5-trlchlorophenoxy adds containing dloxln
Impurities, but to other phenoxy adds as well. Some doubt was raised con-
cerning the possible m1sclass1f1cat1on of Individuals who were exposed to
phenoxy adds free of polychlorlnated dlbenzodloxlns (I.e., 1n particular,
"dlchoroprop" 1n the Eriksson study). In a recent communication from
1873A 11-69 04/09/84
-------
Hardell (1983), Eriksson recalculated his risk estimates after reclasslfylng
his dlchoroprop-exposed cases and controls Into the category of probable
exposure to phenoxy adds contaminated with polychlorlnated dibenzodloxins
and removing them from the nonexposed category. His new estimates were 4.0
based upon 5 out of 8 respondents who were exposed to phenoxy adds free of
contamination and 10.9 for those exposed to contaminated phenoxy add. The
first estimate was of only borderline significance utilizing the M1et1nen
test based statistic, thus, weakening any finding that the risk of STS
extends to phenoxy acids free of dloxln.
In a cohort mortality Investigation Cook et al. (1980a) studied 61 males
Involved in 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 1n the air were provided by
the author. On the other hand, the author did divide the cohort of 61 males
Into potentially "high" vs. "low" exposure by place of work based upon
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) fIbrosarcoma, 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
1873A 11-70 04/09/84
-------
any of these four deaths were smoking related. SHe 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 liver 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 in the Hardell 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.
Smith et al. (19825) 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 hlstological types 1n the
cases is given in Table 11-27. An interview to elicit occupational history
Information was accomplished via the telephone either with the next of kin
to the patient or the patient himself 1f he was well enough, although the
information was not used in this preliminary analysis.
1873A 11-71 03/29/84
-------
TABLE 11-27
Distribution of H1stolog1cal Types of Soft-Tissue Sarcomas*
Cell Type
Flbrosarcoma
Llposarcoma
Rhabdomyosarcoma
Lelomyosarcoma
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
1873A
11-72
04/09/84
-------
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. Ihe 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
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, e.g., separation of the earlier work history
from the latter; purging of earlier employment records, etc. Besides the
"first 20 Interviews" where a change could be noted 1s not necessarily
representative of the entire cohort 1n any case.
1873A 11-73 03/29/84
-------
Furthermore, the authors do not know absolutely that any of their cases
and controls were exposed to phenoxy adds and/or chlorophenols since
apparently no effort was made to confirm "potential" exposures. Only dif-
ferences in occupational classification were noted where "potentially" cases
or controls could have had exposure to the dloxln-contalnlng herbicides. It
was pointed out that the risk estimates noted do not "preclude" the possi-
bility that an association may be found In this study when the cases and
controls (or surviving kin) are Interviewed for chemical spraying at a later
time. The authors themselves conclude that the preliminary study results
"should not be taken as substantial evidence against the hypothesis that
phenoxy herbicides and chlorophenols may cause human cancer."
Ihe 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 fIbrosarcomas and llposarcomas appear
prominently 1n the Smith study. More attention 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 New 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
1873A 11-74 03/29/84
-------
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 If
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 In 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 acids 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 in all remaining
situations that 1f the member sprayed "gorse" and/or "blackberries" this was
tantamount to potential exposure to phenoxyacetlc add. Smith calculated
elevated but nonsignificant relative risks of exposure to phenoxyacetlc 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 riot 1n the
previous 5 years before cancer registration. When risk ratios were calcu-
lated 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 non-
significant. If the numbers would allow, 1t would be of Interest to repeat
1873A 11-75 03/29/84
-------
the above calculations excluding only those with potential exposure occur-
ring 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 1n contact
with enough phenoxyacetlc add to justify such a designation. It could be
that, 1n 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 state that If exposures of >5 days pr 1 or
to 10 years before cancer registration are not Included they would expect an
Increase, and since they do not see an Increase, there Is 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
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 In workers
exposed In phenoxy adds, the author alludes to the fact that there are
currently 500 full-time workers registered 1n New Zealand who do full time
ground spraying 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 1n
this study. This kind of argument has appeal 1f these workers could be
shown to have had their exposure sufficiently far 1n the past that latency
considerations could be adequately addressed. However, the real question
1873A 11-76 03/29/84
-------
again remains how much real exposure did those patients 1n the study* really
have 10-15 years earlier, and 1n what numbers. The author remarks that H
1s surprising that he found no STS victims who had ever worked full-time 1n
phenoxyacetlc add herbicide spraying. Perhaps they have not yet been ob-
served for a long enough period. However, as was pointed out by the author,
the findings do not support the hypothesis that exposure to phenoxyacetlc
add herbicides causes STS. But neither do they support a negative finding
without better documentation regarding actual exposure and time of actual
exposure. The author does note (Smith, 1983), however, that his documenta-
tion of exposure to 2,4,5-T (and 2,4-D) 1s at least as good as that 1n the
Hardell study, and that although Hardell noted higher relative risks of <30
days exposure, Smith did not. Hence the paradox. Smith does admit the
possibility that 2,3,7,8-TCDO contaminations might be lower In New Zealand
as opposed to 2,3,7,8-TCOD contamination 1n the Swedish studies, although
there 1s no evidence for H. He still maintains that his study shows that
exposure to phenoxyacetlc adds may not be associated with STS.
Pazderova-Vejlupkova et al. (1981) studied 80 workers Involved 1n the
production of 2,4,5-sodlum trlchlorophenoxyacetate and butylester of tr1-
chlorophenoxyacetlc add who subsequently became 111 from exposure to
2,3,7,8-lCDD 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. Most patients developed chloracne
while 11 developed porphyda cutanea tarda. Chief chemical signs were
metabolic disturbances, pathologically elevated llplds 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, pedportal flbrosls or activation of Kupffer
1873A 11-77 03/09/84
-------
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 predpue cerebl and 2 1n auto accidents. No SISs 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 six deaths that occurred during the
10-year observation period )n the 55 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-Hodgkin'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
(e.g., the forestry Authority, Highway Authority, State Railways and a
state-owned electric power company). Chlorinated phenoxyadds had been used
since the 1950's 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 1960's revealed that these
mixtures contained 0.1-0.9 mg/kg of 2,3,7,8-TCDO).
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.
1873A 11-78 03/29/84
-------
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 SIS and 0.5 lymphomas were expected.) In no Instance was
cancer of any site significantly elevated.
The authors note that this unusual deficit of mortality and morbidity of
between 70 and 82% (even after 15 years from Initial exposure) 1s probably a
consequence of the "healthy worker effect" 1n 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 vital statistics.
1873A 11-79 03/29/84
-------
The authors correctly note 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 is 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.
Recently, the Michigan Department of Public Health (1983b), produced an
ecological study of soft and connective tissue cancer mortality rates in
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
estimates are based upon 5 deaths and 7 deaths, respectively, and are listed
in Table 11-28. 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 is 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 1n the period
1970-1979. Midland County 1s the home of a major chemical company that pro-
duced phenoxyacetic acid herbicides until recently. The authors state that
a detailed review of death certificates, hospital records, residency and
occupational histories of the 20 male and female cases revealed no "common-
alities" 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 instituted to evaluate possible influences, such as
lifestyle, occupation or location of residence on the risk of STS.
1873A 11-80 03/09/84
-------
oo
-J
CO
TABLE 11 -28
Midland County Soft and Connective Tissue Cancer Deaths 1960-1981*
oo
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
Hemangiosarcoma
Liposarcoma
Leiomyosarcoma
Liposarcoma
Flbrosarcoma
Leiomyosarcoma
Kaposi sarcoma
Fibrosarcoma
Leiomyosarcoma
Rhabdomyosarcoma
Liposarcoma
Type of
Primary Site
Face
Right gluteal
Uterus
Spine
Right thigh
Uterus
Right leg
Right thigh
Abdominal wall
Inguinal area
Right thigh
Malignancy
Metastases
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
o
CO
o
10
1978
lymph nodes
64
Leiomyosarcoma
Left knee
Liver, lymph nodes,
lunq. bone
7-70
CD
-------
33 IHDLC i i -^o iiuiu.;
00
Identification
Year of
Death
1978
1978
1979
1962
CO
1967
1967
1969
1971
1972
O
o 1976
LO
\
CO
Sex
F
F
F
M
M
M
M
M
M
M
Age
26
88
27
63
77
20
32
76
89
53
Type
Rhabdomyosarcoma
Flbrosarcoma
Lelomyosarcoma
Rhabdomyosarcoma
Mesothelloma
Rhabdomyosarcoma
Llposarcoma
Lelomyosarcoma
Lelomyosarcoma
Flbrosarcoma
Type of Malignancy
Primary Site
Rectum
Right cheek
Left thigh
Left lower leg
Lung
Pharynx
Left arm
Small
Intestine
Retro-
perHonal
region
Per1t1oneum
Metastases
Lung, neck, Inguinal
region
Facial area
Lung
Lung and right outer
chest wall
Lung, peritoneum and
diaphragm
Per1orb1tal area and
liver
Perineum and buttock
Liver
Hepatic system
Lung, liver
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: Michigan Department of Public Health, 1983b
-------
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
positive studies of Harden and Eriksson as being biased, and 1t makes no
reference to the later validity study by Hardell (1981) of his own work
utilizing colon cancer controls (see Section on Malignant Lymphoma). In
this report Hardell answered these early criticisms that were reiterated by
the British In 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 method). In short, the British
review appears to be overly optimistic about the safety of 2,4,5-T
herbicides.
In summary, the associations reported 1n the two Swedish soft-tissue
sarcoma studies are strong enough to make H 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 add herbicides, chlorophenols or their Impurities are
carcinogenic 1n humans.
11.3.3. Malignant Lymphoma. A separate series of clinical observations at
the Department of Oncology 1n Umea, Sweden (Hardell, 1979), led the re-
searchers to conduct a case-control study of malignant lymphoma In relation
to phenoxyacetlc add, chlorophenols, and other organic compounds (Hardell
1873A 11-83 04/09/84
-------
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-Hodgk1n's lymphomas.
This study employed essentially the same methods and produced results
comparable to those of the SIS studies: statistically significant 5-fold to
6-fold relative risks In relation to phenoxyacetlc adds and chlorophenols.
In addition, an elevated relative risk was found 1n connection with exposure
to organic solvents, such as ben/ene, trichloroethylene, 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 who 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 adds are excluded and consideration
1s 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
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 Is examined, given
1873A 11-84 03/09/84
-------
that cases and controls exposed to only phenoxy acids 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 notes 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 adds
constitutes a risk factor for malignant lymphoma."
This latter study 1s still subject to the same methodological criticisms
to which the earlier study was subjected. Chief among those Is the possi-
bility 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 potentially carcinogenic
organic solvents and other agents could have had an effect, although the
author assures the reader that they did not.
Other research has tentatively suggested that lumberjacks may be at
Increased risk of lymphoma (Edllng and Granstam, 1979). The NHro study
found three deaths from cancers of the lymphatic and hematopoletic system,
against only 0.88 expected (p-0.06, one-tailed Polsson test).
The lymphoma case-control study (Hardell 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.
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
1873A 11-85 04/09/84
-------
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 In exclusively agriculture/forestry workers who were exposed to
phenoxy adds compared with workers found 1n other occupations having no
apparent exposure to phenoxy adds 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
acid 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 11-29).
When woodworkers are separated out (possible exposure to chlorophenols
1n treatment of wood) the risk ratio becomes 9.7 (Table 11-30). These data
suggest the presence of some recall bias.
Another focus of this 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 compared the exposure data
1873A 11-86 03/29/84
-------
TABLE 11-29
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 EMkkson, 1981
1873A 11-87 03/09/84
-------
TABLE 11-30
Other Occupations (Minus Forestry/AgMculture/Woodworkers)*
Group Phenoxy Adds/Chlorophenols Non-exposed
Cases 4 66
Referents 1 160
RR =, 9.7 X2 = 5.98 (P<0.05)
*Source: Hardell and EMkkson, 1981
1873A 11-88 03/09/84
-------
derived from the Interviewee's returned questionnaires only with the com-
bined Information from both the phone Interviews and questionnaires. The
study found no substantial differences 1n the frequency of reporting
exposure.
Still a third consideration of possible bias Involves recall of exposure
to phenoxy adds 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 (Hardell and
Sandstrom, 1979) and the 169 malignant lymphomas (Hardell et al., 1981) a
group of 164 colon cancer cases from the same population source and compared
their exposure to phenoxy acids 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 acids remaining significantly high at 5.5 and to chloro-
phenols 5.4 1n the SIS 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 is assumed 1n this study that
recall bias was and is 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 it does not rule out the possi-
bility that recall bias can still be present 1n their data for other
reasons. Hardell refers to an Intense "debate about phenoxy adds and their
presumptive risk" in Sweden at the time the colon cancer study was conduct-
ed. But, there 1s no reason to think that colon cancer victims would assume
1873A 11-89 03/29/84
-------
their disease was brought about from exposure to dloxln containing chemicals
1f no connection was suggested.
It seems plausible that STS and/or non-Hodgkln's lymphoma patients would
either learn at the time of their diagnosis that exposure to dloxln contain-
ing 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 their 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 dloxln containing 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 this study may explain any biases Introduced from
secondary recall of occupation, observational bias Introduced from the tele-
phone 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-Hodgkln's lym-
phoma that his condition may have been caused by exposure to dloxln contain-
ing herbicides.
11.3.4. Stomach Cancer. Studies of two of the oldest cohorts of workers
known to have been exposed to phenoxyacetlc add herbicides and/or 2,3,7,8-
1CDD report stomach cancer mortality rates significantly higher than expect-
ed. The results 1n each study were based on small numbers of deaths. In
one study (Axelson et al., 1980), 348 Swedish railroad workers with at least
46 days of herbicide exposure between 1955 and 1972 were followed through
1873A 11-90 03/29/84
-------
October 1978. The workers were grouped on the basis of their primary herbi-
cide 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 herbi-
cides. After a 10-year latency was achieved, 3 stomach cancer deaths were
observed vs. 0.71 expected (p<0.05). None were attributable to amltrol
alone, but two were assigned to phenoxy adds alone while the remaining
stomach cancer death occurred 1n a worker exposed to both amltrol and
phenoxy adds. The excess was more pronounced (3 observed vs. 0.57 expect-
ed, 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 acid alone and amltrol
In combination, the excess Is enhanced further (3 observed vs. 0.41
expected, p<0.01).
Axelson et al. (1980) also notes an excess 1n total "tumors" after 10
years latency as well (15 observed vs. 6.87 expected, p<0.005). This 1s
pronounced In those exposed early to phenoxy adds alone (6 observed vs.
2.60 expected, p<0.01) and phenoxy adds 1n combination with amltrol (5
observed vs. 1.34 expected, p<0.05). Presumably, "tumors" 1n Sweden are
analogous to malignant neoplasms 1n 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 tdchlorophenol 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
1873A 11-91 03/29/84
-------
period 1971-1974, and 1972-1975 rates for Rh1nehessen-Palat1nate, the region
1n which Ludwlgshafen 1s located.*
The results, shown 1n Table 11-31, 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 1n 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 1970's were
used to generate expected deaths to compare with observed mortality over a
much longer period (1953-1977). The substantial decline 1n stomach cancer
mortality In West Germany during the late 1950's and 1960's would likely
make these expected figures too large.
The researchers also used an internal control group which 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
Ludwigshafen, which were later shown to be Inaccurate.
1873A 11-92 03/29/84
-------
TABLE 11-31
Analysis of Stomach Cancer Mortality 1n a Group of
West German Factory Workers Exposed to 2,3,7,8-TCDO*
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
1873A 11-93 03/29/84
-------
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 11-32).
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 is 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
1873A 11-94 03/29/84
-------
TABLE 11-32
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 Cancer Deaths
Observed Expected
Relative
Risk
Significance
Level
Federal Republic of
Germany 1971-1974
Rhlnehesstn-
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 al., 1982
1873A
11-95
03/29/84
-------
since one stomach cancer death has been noted in an Internal control group
1n the updated version, 1t appears that this finding has been weakened some-
what. Furthermore, as was pointed out earlier, trends 1n stomach cancer
mortality during the 1950's, 1960's and 1970's 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 is
warranted, but firm conclusions cannot yet be made.
Four additional cohort studies have reported results that do not show
Increased stomach cancer mortality rates In 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
trichlorophenol 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 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 11-33. Results of neither the Midland study nor the
Nitro study contradict the findings of the Swedish and West German investi-
gations previously discussed. This can be shown 1n two ways. First, the
1873A 11-96 03/29/84
-------
TABLE 11-33
Stomach Cancer Mortality 1n Three Studies of Workers Exposed
to Phenoxyacetlc Add Herbicides and/or 2,3,7,8-TCDD
Stomach Cancer Deaths Relative
Risk
Observed Expected
0 0.1 4a 0
5 6.9a-° 0.7
0 0.5b 0
95% Confidence
Interval Reference
0-26.3 Ott et al., 1980
0.2-1.7 R11h1mak1 et al.,
1978
0-7.4 Zack and Susklnd,
1980
aEst1mated from total cancer expected deaths (see footnote 1n text).
DEnt1re follow-up period without regard for minimum time for cancer Induc
tion (Ott et al., 1980 used a 10-year minimum Induction period).
1873A
11-97
03/09/84
-------
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 Tables 14 and 19).
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 1n the Midland study
(a = 0.05,
-------
Statistically, the study of Finnish herbicide applicators 1s 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 which 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 In the source records from which vital status was
determined.
In summary, four cohort studies of workers exposed to phenoxyacetlc acid
herbicides and/or 2,3,7,8-TCOD 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.
1873A 11-99 03/09/84
-------
11.3.5. Summary of Ep1dem1olog1cal 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 1s shown, considering the rarity
of the disease. This excess 1s suggestive of an association of cancer with
exposure to phenoxy adds and/or chlorophenols, and consequently, with the
Impurities found 1n these herbicides, Including 2,3,7,8-TCDO.
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, 1n one study,
the risk was found to extend to phenoxy adds free of dloxln Impurities. In
that study, the risk Increases to 17 when phenoxy adds known to contain
dloxln Impurities (polychlodnated d1benzod1ox1ns 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 1n
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 adds and/or chloro-
phenols and, consequently, 2,3,7,8-TCDD.
The Swedish case-control studies provide limited evidence for the
cardnogenldty of phenoxy adds and/or chlorophenols 1n humans. However,
with respect to the dloxln Impurities contained therein, the evidence for
the human cardnogenld ty for 2,3,7,8-TCDO based on the ep1dem1olog1c
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.
1873A 11-100 03/29/84
-------
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.
11.4. QUANTITATIVE ESTIMATION OF RISKS OF EXPOSURE
11.4.1. Introduction. This quantitative section deals with the unit risk
for 2,3,7,8-TCDD via Inhalation and oral routes, and the potency of 2,3,7,8-
TCDD relative to other carcinogens that the CAG has evaluated. The unit
risk estimate for an air pollutant 1s defined as the lifetime cancer risk
occurring 1n a hypothetical population 1n which all Individuals are exposed
continuously from birth throughout their lifetimes to a concentration of
1 yg/m3 of the agent 1n the air they breathe. The unit risk from oral
exposure 1s similarly defined 1n terms of either mg/kg bw/day or 1n terms of
yg/Sl 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 carcinogenic potency of several agents
with each other, and 2) to give a crude Indication of the population risk
which might be associated with known (or anticipated) air or water exposure
to these agents.
The unit risks for both the Inhalation and oral routes will be estimated
from animal oral bloassays, since there are no animal Inhalation studies.
The animal to man extrapolations for the oral route will assume 100% absorp-
tion 1n both species. However, the unit risk for the ambient air concentra-
tion 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 1n
small amounts from sources Including the production of 2,4,5-T, trlchloro-
phenol, sllvex and hexachlorophene; the application of 2,3,7,8-TCDD-contaml-
nated herbicides or wood preservatives; the burning of municipal waste, wood
and PCBs; and, possibly, dust from 2,3,7,8-TCDD-contam1nated soil.
1873A 11-101 04/23/84
-------
Physically, 2,3,7,8-TCDD has a very low vapor pressure and 1s not
normally airborne. At room temperature 1t 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 1n the general air environment.
11.4.2. Procedures for the Determination of Unit Risk for Animals. In the
development of quantitative estimates of carcinogenic risk, one or both of
the following two types of data are utilized: 1) lifetime animal studies,
and 2) human studies where excess cancer risk has been associated with
exposure to the agent. In animal studies 1t 1s assumed, unless evidence
exists to the contrary, that 1f 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.
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 epidemiologic studies. We must, therefore, depend on our
current understanding of the mechanisms of cardnogenesls for guidance as to
which risk model to use. At the present time, the dominant view of the
carcinogenic process Involves the concept that most cancer-causing agents
also cause irreversible damage to DNA. This position 1s reflected by the
fact that a large proportion of agents that cause cancer are also mutagenlc.
1873A 11-102 04/17/84
-------
There 1s reason to expect that the quantal type of biological response,
which 1s characteristic of mutagenesls, 1s associated with a linear non-
threshold dose-response relationship. Indeed, there 1s substantial evidence
from mutagenlclty studies with both Ionizing radiation and a wide variety of
chemicals that this type of dose-response model 1s the appropriate one to
use. This 1s particularly true at the lower end of the dose-response curve;
at higher doses, there can be an upward curvature, and eventual flattening
out as saturation 1s reached. The linear nonthreshold dose-response
relationship 1s also consistent with those ep1dem1olog1c studies of cancer
responses to specific agents that contain enough Information to make the
evaluation possible (e.g., radiation-Induced leukemia, breast and thyroid
cancer, skin cancer Induced by arsenic 1n drinking water, liver cancer
Induced by aflatoxlns 1n the diet). There 1s also some evidence from animal
experiments that 1s consistent with the linear nonthreshold model (e.g.,
liver tumors Induced 1n mice by 2-acetylam1nofluorene 1n the large-scale
EOQ, study at the National Center for Tox1colog1cal Research, and the
Initiation stage of the two-stage cardnogenesls model In rat liver and
mouse skin). The multistage model of cancer (Arm1tage-Doll), derived from
human data 1s also consistent with a linear, nonthreshold dose-response
curve.
Because Us scientific basis, although limited, 1s the best of any of
the current mathematical extrapolation models, 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 Is not likely to be higher
than the estimate, but 1t could be lower.
1873A 11-103 04/16/84
-------
The mathematical formulation chosen to describe the linear nonthreshold
dose-response relationship at low doses 1s the linearized multistage model.
The multistage model employs enough arbitrary constants to be able to fit
almost any monotonlcally Increasing dose-response data, and 1t Incorporates
a procedure for estimating the largest possible linear slope (1n the 95%
upper confidence limit sense) at low extrapolated doses that Is consistent
with the data at all dose levels of the experiment.
11.4.3. Description of the Low-Dose Animal Extrapolation Model. Let P(d)
represent the lifetime risk (probability) of cancer at dose d. The multi-
stage model has the form
P(d) = 1 - exp [-(qQ + q^ + q?d2 + ... + qRd )]
where
q > 0, 1 = 0, 1, 2, ..., k
Equlvalently,
k
Pt(d) = 1 - exp [(q^ + q2d2 + ... + qRd )]
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.
Ihe 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 95% upper confidence limit,
q * on parameter q . Whenever q * > 0, at low doses the extra risk
1873A 11-104 04/17/84
-------
P.(d) has approximately the form Pt(d) = q^* x d. Therefore,
q * x d is a 95% upper confidence limit on the extra risk and R/q,* 1s a
95% lower confidence limit on the dose producing an extra risk of R. Let
L be the maximum value of the log-likelihood function. The upper limit,
q,*, 1s calculated by Increasing q, to a value q,* such that when the
log-likelihood 1s remax1m1zed 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^ = 2.70554
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, P (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 non-threshold
concept discussed earlier. The slope, q * Is 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, P,(d) will be 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 fH the data sufficiently well,
data at the highest dose are deleted and the model 1s 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 Is acceptable, the ch1-square
X2 * l
1873A 11-105 04/16/84
-------
statistic 1s calculated where N1 1s the number of animals 1n the 1th
dose group, R, 1s the number of animals In 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 1s the
number of remaining groups. The fH 1s 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.
11.4.4. Selection of Data. For some chemicals, several studies 1n differ-
ent animal species, strains and sexes, each run at several doses and differ-
ent 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 procedures
used In 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 1s 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-)*, estimated from each of these data
sets, 1s used for risk assessment. The geometric mean of
numbers A], &£• ••• Am 1s defined as
(A] x A2 x ... x Am)
1/m
In some cases one or more of these studies may be negative, but
the 95% upper limit q]* will still be greater than zero.
1873A 11-106 04/17/84
-------
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 1n the model. Alterna-
tively, the total number of significant tumors may also be used
1n some cases.
11.4.1.5. CALCULATION OF HUMAN EQUIVALENT DOSAGES — 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 Is
assumed that mg/surface area/day provides an equivalent dose between
species. To a close approximation, since the surface area Is proportional
to the 2/3 power of the weight, as would be the case for a perfect sphere,
the exposure In 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 In 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 Is
. lp x m
d = e
Le x W2/3
11.5. ORAL
Exposures are often not given 1n units of mg/day, and 1t becomes neces-
sary to convert the given exposures Into mg/day. For example, 1n drinking
1873A 11-107 04/16/84
-------
water studies, exposure Is in ppm 1n the water. Similarly, 1n most feeding
studies exposure 1s 1n terms of ppm 1n the diet. In these cases the
exposure 1n mg/day 1s
m = ppm x F x r
where ppm 1s parts per million of the carcinogenic agent 1n the diet or
water, F Is the weight of the food or water consumed/day 1n kg, and r 1s the
absorption fraction. In the absence of any data to the contrary, r Is
assumed to be equal to one. For a uniform diet, the weight of the food
consumed 1s proportional to the calories required, which 1n turn 1s roughly
proportional to the surface area, or 2/3 power of the weight 1n kilograms.
Water demands are also assumed to be proportional to the surface area, so
that
2/3
m a ppm x W x r or
m
a ppm
rW2/3
as a result, ppm 1n the diet or water 1s often assumed to be an equivalent
exposure between species. However, this 1s not fully appropriate since the
yield of calories/kg of food 1s very different 1n the diet of man in compar-
ison to that of laboratory animals, largely becuase of moisture content
differences. Consequently, the amount of drinking water required by each
species also differs because of the amount of moisture 1n the food. There-
fore, we use an empirically derived factor, f = F/W, which is the fraction
of a species' body weight that is consumed/day as food. The following rates
are used:
Fraction of body weight
Consumed as
Species W fFood fWater
Man
Rats
Mice
70
0.35
0.03
0.028
0.05
0.13
0.029
0.078
0.17
1873A 11-108 04/16/84
-------
Thus, when exposure 1s given as a certain dietary or water concentration 1n
2/3
ppm, the exposure 1n mg/W 1s
m
= ppmx F = ppm x f x H ^ ppm x f x wi/3
rW2/3 w2/3 w2/3
When exposure Is given 1n terms of mg/kg/day = m/Wr = s, the conversion Is
m = s x W1/3
rW2/3
11.5.1. Calculation of the Unit Risk from Animal Studies. The excess risk
associated with d mg/kg /day 1s obtained from GLOBAL 79, and for most
cases of Interest to risk assessment can be adequately approximated by
P (d) =- 1 - exp (-q *d). A "unit risk" 1n units X 1s the risk corre-
sponding to an exposure of X = 1. To estimate this value, we find the
number of mg/kg" /day corresponding to one unit of X and substitute this
value Into the above relationship. For example, 1f X 1s 1n units of
ug/m3 1n the air, then for partlculates such as 2,3,7,8-TCDD, d - 0.29 x
1/3 _ 2/3
70 x 10 3 mg/kg /day, when pg/m3 Is the unit used to compute
parameters 1n animal experiments.
If exposures are given 1n terms of ppm 1n air, we may use the fact that
1 ppm -- 1.2 x molecular weight (gas) mg/m3
molecular weight (air)
Note that an equivalent method of calculating unit risk would be to use
mg/kg/day for the animal exposures and then to Increase the j polynomial
coefficient by an amount
(Wh/Wa)j/3 J = 1, 2, .... k
and use the mg/kg/day equivalents for the unit risk values. In the section
calculating the unit risks for animal data, the final q * will always be
the upper-limit potency estimate for humans.
1873A 11-109 04/23/84
-------
11.5.2. Interpretation of Quantitative Estimates. The unit-risk estimate
based on animal bloassays 1s an approximation to the absolute risk 1n
populations exposed to known carcinogen concentrations. This 1s because
1) there may be Important species differences 1n uptake, metabolism and
organ distribution of carcinogens, as well as species differences 1n
2) target site susceptibility, 3) 1mmunolog1cal responses, 4) hormone
function, 5) dietary factors and other diseases. The concept of equivalent
doses for humans compared with animals on a mg/surface area basis has little
experimental verification regarding carcinogenic response. Human popula-
tions are more variable than laboratory animals with respect to genetic
constitution and diet, living environment, activity patterns and other
cultural factors.
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 In the regulatory decision-making process,
e.g., setting regulatory priorities, evaluating the adequacy of technology-
based controls, etc. However, the estimation of cancer risks to humans at
low levels of exposure Is uncertain. At best, the linear extrapolation
model used here provides a rough but plausible estimate of the upper limit
of risk; I.e., 1t 1s not likely that the true risk would be much more than
the estimated risk, but 1t could very well be considerably lower. The risk
estimates presented In subsequent sections should not be regarded as an
accurate representation of the true cancer risks even when the exposures are
1873A 11-110 04/16/84
-------
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.
11.5.3. 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 in the appendix. The models presented
there are the one-hit, problt and Weibull models. The CAG feels that with
the limited data available from these animal bioassays, 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
is taken by the CAG that the risk estimates obtained by use of the linear
nonthreshold model are upper limits, and the true risk could be lower.
Another modification of the method described here Involves the choice of
the specific animal bioassay as the basis for extrapolation. The present
approach is to use the most sensitive responder. Alternatively, the average
responses of all of the adequately tested bioassay 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 in human pharmacological responses; it is not clear which of
the two approaches is more appropriate for carcinogens. In the absence of
information on this point, it seems appropriate to use the most generally
1873A 11-111 04/16/84
-------
accepted method, which also Is 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 unit risk estimates by a factor of
5.8 for rats and about 13 for mice.
11.5.4. Unit Risk Estimates for 2,3,7,8-TCDD via the Oral and Inhalation
Routes. The positive animal cancer data available for calculating a unit-
risk estimate for 2,3,7,8-TCDD are presented 1n Appendix B 1n Tables 8-1
through B-5. These are as follows:
1. The Dow (1978) diet study on Sprague-Dawley rats, Spartan
substraln. Significantly Increased cancers 1n 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 (Kodba) and that of an
Independent reviewer (Squire) are presented (Table 8-1).
Significant cancers 1n the females Included lung, nasal turbi-
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.
2. The NCI gavage study 1n Osborne-Mendel rats and B6C3F1 mice.
a. 2,3,7,8-TCDD 1n male rats caused an Increase 1n 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 1n 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 flbrosarcomas, 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 fitted to the linearized multistage model
described 1n the methodology section. These results are presented 1n
Appendix B 1n some detail 1n Tables B-6 through B-12, and summarized in
Table B-13. The data from which the steepest slope factor (q *) (I.e.,
1873A 11-112 04/23/84
-------
greatest potency) was calculated were from an Independent pathologist's (Dr.
R. Squire) review of the Dow Chemical Company lifetime rat feeding study.
This factor 1s
q * - 4.25 x 10s (mg/kg/dayr1
based on the tumors 1n female Sprague-Dawley rats. For the purpose of these
calculations, the largest dose group In the study was eliminated because
Inclusion of all of the dose groups resulted In a poor fit of the model
(p<0.01). Early Increased mortality 1n the high-dose group was also adjust-
ed for by eliminating animals that died during the first year, so that the
first tumors considered were those detected during the 13th month of the
study. Tables B-8A and B-9A present the results of data analyses made
following the adjustments described above. The results yield acceptable fits
of the data without dropping the responses at the highest dose levels. The
slope estimates for the Kodba and Squire analyses, 1.51xl05 and
1.61xlOs (mg/kg/day)"1 were averaged by taking the geometric mean, and
the final estimate thus becomes
I/?
q^ = [(1.51 x 105) x (1.61 x 105)]"^ = 1.56 x 10s (mg/kg/ day)'1.
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 in this bioassay is 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 parti-
ally support either hypothesis. An adjustment of the multistage model
needed to incorporate such an effect or effects, however, is felt to be
1873A 11-113 04/16/84
-------
unwarranted by the sparslty of the supporting evidence. As an alternative,
to Incorporate this uncertainty, a range of 95% upper-limit estimates of
q-j* = 9.0xl04 to 4.25xl05 (mg/kg/day )" * has been chosen to accommo-
date this unusual data set.
In order to estimate a unit risk for a 1 yg/8. concentration 1n
drinking water, the following conversion 1s used:
1 yg/kg/day x 70 kg x 103 ng/pg x 1 day/2 8. = 3.5 x 104 ng/S.
based on human consumption of 2 a water/day for a lifetime. Therefore,
unit risk corresponding to 1 ng 2,3,7,8-TCDD/S. water 1s
qi* = 1.56xl02 (vg/kg/dayT1 x -- = 4.5xlO~3
3.5x10" ng/S.
Similarly, the lower and upper limits of the range vary from q * = 2.6xlO~3 to
1.2xlO~2 (rig/l)'1.
This unit-risk estimate from an oral study must be transformed before an
estimate can be made of the effect due to 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 partlculates. The amount of exposure depends on
the partlculate size distribution. Based on the report of the Task Group on
Lung Dynamics (1966), It 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 deposition of 5-m1cron particles 1n
the lower respiratory tract 1s not more than 30%. Even those larger parti-
cles retained by the upper respiratory tract, however, may be swallowed and
eventually absorbed by 1ngest1on. In the absence of any specific data on
the size distribution and eventual fate of the particles, the Information
developed by the International Commission on Radiological Protection,
Committee 2, will be used. The Committee developed the following estimates
1873A 11-114 04/16/84
-------
for retention of participate matter 1n the lungs. For compounds not readily
soluble, 25% will be exhaled, 50% will be deposited 1n the upper respira-
tory passages and subsequently swallowed, and the final 25% will be deposit-
ed in 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 1n the lung alveoli
for long periods of time, with some eventually transferred to pulmonary
lymph nodes.
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 m3/day
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-TCDD 1s 3.3 x 10~5, as calculated below:
q-|*(resp.) = 1.56 x 105 (mg/kg/day)'1 x 1 x 10~9 mg/pg x .75 x 20 mV70 kg
or
q-|*(resp.) = 3.3 x 10"5 (pg/m3)"1.
Slmllary, the range of estimates 1s 1.9 x 10~5 to 9.1 x 10~5 (pg/m3)"1.
11.5.5. Unit Risk Estimate for HxCDDs (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 (combined liver neoplastlc nodules
or hepatocellular carcinomas) with the greater response 1n 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
1873A 11-115 04/16/84
-------
presented In Table 11-34, which Includes the recent NTP reevaluatlon of the
female rat liver slides. The review shows responses 1n the range of 50%
less than that of the original analysis. The responses for neoplastlc
nodules and combined nodules and carcinomas are still statistically signifi-
cant. These results have been detailed 1n the qualitative section of this
document.
In female mice, there was a dose-related trend 1n 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 11-35).
Although no statistically significant Increase 1n carcinomas occurred 1n
mice or rats of either sex, when neoplastlc nodules 1n the rats and hepato-
cellular adenomas 1n the 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 11-34 and 11-35,
the 95% upper-limit unit risk estimates are:
Rat - male q * = 0.59 (pg/kg/dayT1
female q^ = 3.5 (
House - male q * =11.0 (pg/kg/day)~a
female q * = 2.9 (pg/kg/day)"1
The usual CAG procedure 1s to use the most sensitive sex-species for
estimating the 95% upper-limit unit risk. Under that procedure, which 1s
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 (mg/kg/day)"1 would be selected to provide the upper limit estimate
of potency. However, as examination of Tables 11-34 and 11-35 show, there
are several reasons to give weight to the female rat data base also. These
1873A 11-116 04/16/84
-------
CD
-J
CO
TABLE 11-34
NTP HxCDD (Gavage) Bloassay (NTP, 1980d)
Osborne-Mendel Rats (2 years)
Incidences of NeoplasUc Nodules and Hepatocellular Carcinomas
Tumor
Number of animals examined
Hepatocellular carcinoma (HC)
NeoplasUc nodule (NN)
HC + NN combined
Human equivalent dose
Vehicle
Control
74
0
0
0
0
Untreated
Control
HALE
75
0
2(3%)
2(3%)
0
Low-Dose
1.25
(700 g)b
49
0
0
0
0.04
yg/kg/week
Mid-Dose
2.5
50
0
1(2%)
1(2%)
0.08
High-Dose
5
48
1(2%)
3(6%)
4(8%)C
0.15
Estimates*
of q-j*
(vg/kg/dayr*
—
—
S.&xlO'1
5.9X10"1
—
ng/kg/day
(N3
CO
00
-------
CD
TABLE 11-34 (cont.)
CD
ro
CO
co
Tumor
Number of animals examined
Hepatocellular carcinoma (HC)
Neoplastlc nodule (NN)
HC + NN combined
Human equivalent dose
vg/kg/day
Vehicle
Control
75
0
2(354)
2(3%)
0
Untreated
Control
FEMALE
73
0
1(1%)
1(1%)
0
Low-Dose
1.25
(450 g)d
50
0
5(10%)
5(10%)
0.03
yg/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 qi*
(yg/kg/dayT1
--
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
-------
oo
CO
TABLE 11-35
NTP HxCDD (Gavage) Bloassay
(NTP, 1980d)
B6C3F1 Mice (104 weeks)
Incidences of Neoplastlc Nodules and Hepatocellular Carcinomas
uq/kg/week
Tumor Vehicle Untreated Low-Dose
Control Control 1.25
i
5 MALES
Number of animals examined 73 75 50
Hepatocellular carcinoma 8(11%) 12(16%) 9(18%)
(HC)
Hepatocellular adenoma 7(10%) 15(20%) 5(10%)
(HA)
Combined HA and HC 15(21%) 27(36%) 14(29%)
Human equivalent dally 0 0 0.014
Mid-Dose High-Dose Estimates of q-|*a
2.5 5 (vg/kg/day) 1
49 48
5(10%) 9(19%) 3.71
9(18%) 15(31%)b 6.99
14(29%) 24(50%)c 11.00
0.027 0.054
dose (pg/kg/day)
co
-------
CD
— J
00
TABLE 11-35 (cont.)
Tumor Vehicle Untreated Low-Dose
Control Control 1.25
FEMALES
Number of animals examined 73 74 48
JL Hepatocellular carcinoma 1(1%) 0 0
£ (HC)
Hepatocellular adenoma 2(3%) 2(3%) 4(8%)
(HA)
Combined HA and HC 3(4%) 2(3%) 4(8%)
Human equivalent dally 0 0 0.027
dose (yg/kg/day)
yg/kg/week
Mid-Dose High-Dose Estimates of q-|*a
2.5 5 (yg/kg/day)"1
47 47
2(4%) 2(4%) 9.5X10'1
4(9%) 9(19%)b 2.61
6(13%) 10(23%)b 2.94
0.054 0.107
a95% upper-limit estimate of linear term 1n the multistage model based on human equivalent dosages using
surface area correction.
bp<0.01 versus vehicle-control
cp<0.001
CD
-p.
-------
are: 1) low spontaneous (control) rates 1n the rat vs. the male mouse
liver; 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 1n the high dose group. There were none 1n 148
control animals. By comparison, the male mouse showed no clear trend 1n
carcinomas.
In addition to the above reasoning, we point to the uncertainty of the
surface area correction. Nearly all the quantitative Increase 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 factor 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 95% upper-limit
estimates from the male mouse and the female rat. The final estimate 1s
q^ = (3.5xll.0)1/2 = 6.2 (Pg/kg/day)~*
In terms of exposure to 1 yg/8, of HxCC contaminate and 2 a/day for a
lifetime, we use the same assumptions as with 2,3,7,8-TCDD:
1 yg/kg/day = 3.5xl04 ng/a.
Thus, for 1 ng/fc 1n the drinking water the unit risk 1s
-6.2/3.5,10-.
1873A 11-121 04/23/84
-------
In terms of continuous lifetime exposure to ambient air containing 1 pg/m3
HxCOD, the transformation as was done before with 2,3,7,8-TCDD, 1s
q^HxCOD) (resp.) = 6.2xl03 (mg/kg/dayJ"1 x lxlO~» mg/pg x 0.75x20 m3/70 kg
q1*(HxCDO) (resp.) = 1.3 x 10"6 (pg/m3)"1.
11.5.6. 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
basis, the unit risk slope factor 1s multiplied by the molecular weight, and
the resulting number Is expressed 1n terms of (mMol/kg/day)'1. This 1s
called the "relative potency Index."
Figure 11-2 1s a histogram representing the frequency distribution of
potency Indices of 54 chemicals evaluated by the CAG as suspect carcinogens.
The actual data summarized by the histogram are presented 1n Table 11-36.
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 1n 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 liver, lung and nasal turbl-
nate and hard palate tumors 1n the female rat 1n the Dow 2,3,7,8-TCDD feed-
Ing study (Kodba et al. (1978a) 1s 5xl07 (mMol/kg/day)'1. This number
1s derived by multiplying as follows: the 95% 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.
1873A 11-122 04/16/84
-------
CO
—J
LO
FREQUENCY
I 1 1 1 1 1 1 1 1 1 1 1 1 1 1
VI >/l
C i-f
in o
T3 (Q
(0 -1
o a>
(D
3 S
O (V
3
<£)
co
CO
ro
<
(u
tr>
zt '
o rr r\j
O
Q
m
O
O
m
X
GO
!-»• O
Q.
n
o>
ut
CO
^
x
_A
o
+
to
llllillillll
1st
QUARTILE
I I I I I
D
c
m
D
C
> CO
m
2nd
QUARTILE
-------
TABLE 11-36
Relative Carcinogenic Potencies Among 54 Chemicals Evaluated by the
Carcinogen Assessment Group as Suspect Human Carc1nogensa»b«c
Compounds
Acrylon1tr1le
Aflatoxln B]
Aldrln
Allyl Chloride
Arsenic
B[a]P
Benzene
Benzldlne
Beryllium
Cadmium
Carbon letrachlorlde
Chlordane
Chlorinated Ethanes
1 ,2-d1chloroethane
hexachloroethane
1,1,2,2-tetrachloro-
ethane
1 ,1 ,1-tr khloroethane
1 ,1 ,2-trlchloroethane
Chloroform
Chromium
DDT
Dlchlorobenzldlne
l,l-D1chloroethylene
Slope
(mg/kg/day)'1
0.24(W)
2924
11.4
1.19x10-2
15(H)
11.5
5.2xlO-2(W)
234(W)
1.40(W)
7.8(W)
1.30xlO-]
1.61
6.90x10-2
1.42x10-2
0.20
1.6xlO~3
5.73x10-2
7x10-2
41(W)
8.42
1.69
1.47,10-1(1)
Molecular
Weight
53.1
312.3
369.4
76.5
149.8
252.3
78
184.2
9
112.4
153.8
409.8
98.9
236.7
167.9
133.4
133.4
119.4
100
354.5
253.1
97
Order of
Potency Magnitude
Index (Iog10 Index)
IxlO*1
9xlO*5
4xlOt3
9X10-1
2xlOt3
3xlO+3
4x10°
4xlO*4
IxlO*1
9x10*2
2X10*1
7x10*2
7x10°
3x10°
SxlO*1
2X10-"1
8x10°
8x10°
4xlO*3
3xlO*3
4x10*2
1x10*1
,1
+6
+4
0
+3
+3
+ 1
+ 5
tl
+3
+ 1
+3
tl
0
-1
+ 1
+4
+ 3
+ 3
+ 1
1873A
11-124
04/16/84
-------
TABLE 11-36 (cont.)
Compounds
D1e1dr1n
Dlnltrotoluene
D1phenylhydraz1ne
Ep1chlorohydr1n
B1s(2~chloroethyl)ether
B1s(chloromethyl ) ether
Ethylene D1brom1de (EDB)
Ethylene Oxide
Heptachlor
Hexachlorobenzene
Hexachlorobutadlene
Hexachlorocyclohexane
technical grade
alpha Isomer
r
beta Isomer
gamma Isomer
Hexachlorod1benzod1ox1n
Methylene chloride
Nickel
N1 trosamlnes
D1methyln1trosam1ne
D1ethyln1trosam1ne
D1butyln1 trosamlne
N-n1 trosopyrrolldlne
N-n1troso-N-e thy 1 urea
N-n1troso-N-methylurea
N-n1 troso-d1phenylam1ne
Slope
(mg/kg/day)"1
30.4
0.31
0.77
9.9xlO-3
1.14
9300(1)
8.51
1.26(1)
3.37
1.67
7.75xlO-2
4.75
11.12
1.84
1.33
6.2xlO+3
6.3xlO-4
1.15(W)
25.9
(not by q-j*)
43.5
(not by q]*)
5.43
2.13
32.9
302.6
4.92xlO-3
Molecular
Weight
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
84.9
58.7
74.1
102.1
158.2
100.2
117.1
103.1
198
Order of
Potency Magnitude
Index (log-|0 Index)
lxlO+4
6xlO+1
lxlO+2
9X10-"1
2x10+2
lxlO+6
2xlO+3
6X10+1
1x10+3
5x10+2
2X10+1
1x10+3
n
3xlO+3
5x10+2
4x10+2
2xlO+6
5x10-2
7X10+1
2xlO+3
4x10+3
9x10+2
2x10+2
4x10+3
3xlO+4
1x10°
+4
+ 2
+ 2
0
+ 2
+6
+3
+2
+3
+3
+ 1
+3
+3
+3
+3
+6
-1
+ 2
+3
+4
+3
+ 2
+4
+4
0
1873A
11-125
04/16/84
-------
TABLE 11-36 (cont.)
Compounds
PCBs
Phenols
2,4,6-tMchlorophenol
Tetrachlorodibenzo-p-
dioxln
Tetrachloroethylene
Toxaphene
Trichloroethylene
Vinyl Chloride
Slope
(mg/kg/day)"1
4.34
1.99x10-2
1.56xlO+5
3.5x10-2
1.13
1.9x10-2
1.75x10-2(1)
Molecular
Weight
324
197.4
322
165.8
414
131.4
62.5
Potency
Index
lxlO+3
4x10°
5xlO+7
6x10°
5x10+2
2.5x10°
1x10°
Order of
Magnitude
(log^Q Index)
+3
+ 1
+8
,1
+3
0
0
aAn1mal slopes are 95% upper-limit slopes based on the linearized multi-
stage model. They are calculated based on animal oral studies, except for
those Indicated by I (animal Inhalation), W (human occupational exposure),
and H (human drinking water exposure). Human slopes are point estimates
based on the linear non-threshold model.
bThe potency index 1s a rounded-off slope in (mMol/kg/day)"1 and 1s
calculated by multiplying the slopes in (mg/kg/day)"1 by the molecular
weight of the compound.
cNot 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.
1873A
11-126
04/16/84
-------
Rounding off to the nearest order of magnitude gives a log 10 value of 8,
which 1s the scale presented on the horizontal axis of Figure 11-2. The
Index of 5xl07 Is the most potent of 54 chemicals which the CAG has evalu-
ated as suspect carcinogens. It 1s 50 times more potent than the third most
potent chemical, b1s(chloromethyl) ether, and 50,000,000 times as potent as
vinyl chloride. The potency Index of HxCOD, based on combined hepatocellu-
lar adenomas and carcinomas 1n male mice 1n the NTP gavage study (NTP,
1980d), and combined nodules and hepatocellular carcinomas 1n female rats by
gavage (NTP, 1980d) Is 2.4xlO+6 (mMol/kg/day)'1. This 1s derived by
multiplying the mean 95% upper-limit slope factor q * = 6.2xl03 (mg/kg/
day)"1 by the molecular weight, 391. This potency 1s about one-twentieth
that of ?,3,7,8-TCDO, making 1t the second most potent of 54 chemicals which
the CAG has evaluated as suspect carcinogens.
The ranking of relative potency Indices Is subject to the uncertainties
Involved 1n comparing a number of potency estimates for different chemicals
based on varying routes of exposure 1n 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 1f linearity does not
exist there. Nevertheless, the potency rankings of one and two for these
dloxlns cannot be easily dismissed.
11.6 SUMMARY AND CONCLUSIONS
11.6.1. Qualitative Assessment-2,3,7,8-TCDD. 2,3,7,8-Tetrachlorodlbenzo-
p_-d1ox1n (2,3,7,8-TCDD) 1s one of the most toxic chemicals known to man.
1873A 11-127 04/16/84
-------
In small amounts, 2,3,7,8-TCDD 1s a potent Inducer of arylhydrocarbon
hydroxylase in mammals. The enzyme epoxldase 1s known to mediate the forma-
tion of epoxldes, which are potentially active carcinogenic metabolites.
2,3,7,8-lCDD may be metabolized 1n mammalian species via the epoxlde to
dlhydrodlol and further conjugated. Persistent residues of 2,3,7,8-TCDD
were found 1n liver and fat in a 2-year feeding study 1n rats. Significant
covalent binding of 2,3,7,8-TCDD to protein has been demonstrated by two
Investigators. Covalent binding of 2,3,7,8-TCDD with DNA Is not significant
1n liver cells.
Currently available studies on the mutagenldty of 2,3,7,8-TCDD are
Inconclusive. Two bacterial systems, EscheMchia coll and S. typhlmurlum
(without metabolic activation), exhibited positive mutagenlc activity.
However, In another study of Salmonella typhlmurlum {with and without
metabolic activation), the results were negative.
There have been several cancer bloassay studies of 2,3,7,8-TCDD: 1) a
Dow Chemical Company (Kodba et al., 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 PHot et al. (1980) promotion study 1n rats; and 6) the Kourl et al.
(1978) cocardnogenlclty study 1n mice.
The 1978 study by the Dow Chemical Company of male and female Sprague-
Dawley rats fed 2,3,7,8-TCDD 1n doses of 22, 210 and 2200 ppt revealed a
highly statistically significant excess Incidence of hepatocel lular carci-
nomas 1n female rats at the highest dose level and hepatocellular carcinomas
and hepatocellular hyperplastlc nodules 1n female rats at the middle dose
level, as compared to the controls. In addition, at the high dose there was
1873A 11-128 04/16/84
-------
a significant Increase 1n 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 carci-
nogenic 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 In male mice (females were not tested) receiving 0.7 ^g/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-TCOD by gavage at three dose
levels: 0.01, 0.05 and 0.5 yg/kg/week. 2.3,7,8-TCDD Induced statistic-
ally significant Increases of hepatocellular carcinomas, subcutaneous flbro-
sarcomas and adrenal cortical adenomas 1n high-dose female rats. 2,3,7,8-
TCDD also Induced significant Increases of thyroid tumors 1n low-, mlddle-
and high-dose male rats.
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 jig/kg/week for males and 0.04, 0.2 and
2.0 pg/kg/week for females. 2,3,7,8-TCDD Induced statistically signifi-
cant Increased Incidences of hepatocellular carcinomas 1n the high-dose
males and females, and thyroid tumors, subcutaneous flbrosarcomas and
hlstiocytlc lymphomas 1n females.
In the study by PHot et al. (1980), 2,3,7,8-TCDD has been shown to be a
potent liver cancer promoter after Initiation with d1ethyln1trosam1ne.
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 1n one mouse
1873A 11-129 04/16/84
-------
strain. In the study by Kourl et al. (1978), 2,3,7,8-TCDO has been shown to
be a potent cocardnogen with 3-methyl chloranthrene.
Several ep1dem1olog1cal studies have been conducted which are relevant
to the assessment of the cardnogenldty of 2,4,5-T, sllvex and 2,3,7,8-
TCDD. Two Swedish ep1dem1olog1c case-control studies (Hardell and Sand-
strom, 1979; Eriksson et al., 1979, 1981) reported a significant association
between soft-tissue sarcomas and occupational exposure to phenoxyacetlc add
herbicides and/or chlorophenols. These studies Indicated ~5- to 7-fold
Increases 1n the risk of developing soft-tissue sarcomas among people
exposed only to phenoxyacetlc adds and/or chlorophenols 1n comparison with
people not exposed to these chemicals. When an attempt was made to separate
exposures Into two categories based on expected presence or absence of poly-
chlorinated dlbenzodioxln and dlbenzofuran Impurities, the relative risks
were 17 and 4.2, respectively. This Indicates that agents themselves
without the dloxln Impurities may be contributing to the risk of soft-tissue
sarcomas as well. Another Swedish case-control study (Hardell 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 1n humans.
Four other cohort studies by Ott et al. (1980), R11h1mak1 et al. (1978),
Cook et al. (1980a) and Zack and Susklnd (1980), did not Indicate a signifi-
cantly Increased risk of stomach cancer In people exposed to phenoxyacetlc
adds and/or chlorophenols, but two of these studies were of relatively low
statistical power, and another study has certain Inconsistencies requiring
clarification.
1873A 11-130 04/23/84
-------
11.6.2. Qualitative Assessment-HxCDD. Hexachlorod1benzo-p_-d1oxin has also
been tested for cardnogenldty 1n rats and mice treated 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-HxCOO was tested. In the oral
study, animals received HxCDO at doses of 0.0, 1.25, 2.5 or 5.0 vg/kg/
week, except for female mice, which received 0.0, 2.5, 5.0 and 10.0 pg/kg/
week. In both species and both sexes, only tumors of the liver occurred at
a significantly greater Incidence than In 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 in female rats the inci-
dence 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 in either the carcinogenlclty bio-
assay or the tumor promotion assay using DHBA as an initiator.
11.6.3. Quantitative Assessment - 2,3,7,8-TCDD and HxCDD. Because they
lack suitable exposure estimates, existing epidemlologic studies are not
appropriate for use in estimating 2,3,7,8-TCDD Inhalation risks to humans.
Several animal data sets are available for estimating an Inhalation unit
risk for 2,3,7,8-TCDD, but they are all based on either gavage or feeding
studies. The quantitative cancer unit risk estimate is q * = 1.56xlOs
(mg/kg/day)"1 derived from the Kodba et al. (1978a) 2,3,7,8-TCOD feeding
study in female rats that Induced a statistically significant Increased
incidence of tumors in the liver, lungs, hard palate and nasal turbinates.
Based on continuous lifetime exposure to 1 ng/a. 2,3,7,8-TCDD in drinking
water, the 95% upper limit estimate of Individual cancer risk is 4.5xlO~3
with a range of upper limit values of 2.6xlO~3 to 1.2xlO~2, depending
upon pathological Interpretation and mortality correction. Based on contin-
1873A 11-131 04/23/84
-------
uous lifetime exposure to 1 pg/m3 2,3,7,8-TCDD 1n ambient air, the 95%
upper-limit estimate of Individual cancer risk 1s 3.3xlO~s, with a range
of upper-limit estimates of 1.9xl(Ts to 9.1xlO~5 depending upon patho-
logic Interpretation and mortality correction.
An upper-limit unit risk estimate for a mixture of HxCOOs has been
calculated from the NCI gavage study (NTP, 1980d). Based on combined liver
heptacel lular carcinomas and nodules 1n female rats, and hepatocellular
adenomas and carcinomas 1n male mice, q * = 6.2xl03 (mg/kg/day)"1. A
continuous lifetime exposure to 1 ng/8. of HxCDD 1n drinking water 1s
estimated to result In a 95% upper limit risk of l.BxlO"4. Similarly, for
ambient air, a continuous lifetime exposure to 1 pg/m3 of HxCDD Is esti-
mated to yield an upper-limit unit risk of 2.4xlO~6.
The potency of 2,3,7,8-TCDD using the linearized multistage model Is
also estimated relative to 53 other chemicals which the CAG has evaluated as
suspect carcinogens. This relative potency Index 1s 5xl07 (mMol/kg/
day)'1, making 2,3,7,8-TCDD the most potent animal carcinogen that the CAG
has evaluated. It 1s about 50 times more potent than the third most potent
chemical, b1s(chloromethyl)ether and 50,000,000 times more potent than vinyl
chloride. The relative potency Index for HxCDD 1s 2x10* (mMol/kg/
day)"1, making 1t the second most potent carcinogen, about one-twentieth
the low dose potency of 2,3,7,8-TCDD.
11.6.4. Conclusion. Because of the Induction of hepatocellular carcinoma
1n two strains of female rats and both sexes of one mouse strain, along with
the induction of thyroid tumors, subcutaneous fibrosarcomas and lung and
tongue tumors 1n both rats and mice, the evidence of cardnogenlcity for
2,3,7,8-TCDD in animals is regarded as "sufficient" using the classifica-
tion system of the International Agency for Research on Cancer (IARC).
1873A 11-132 04/23/84
-------
These effects notably occur at extremely low doses. There 1s evidence that
2,3,7,8-lCDD 1s a promoter and a cocardnogen.
The human evidence for the cardnogenlcHy of 2,3,7,8-TCDD alone Is
regarded as "Inadequate" using the IARC classification, because of the
difficulty of attributing the effects to 2,3,7,8-TCDD, which occurred as an
Impurity 1n the phenoxyacetlc adds and chlorophenols to which the people
were exposed. However, the human evidence for the cardnogenlcHy of
chlorinated phenoxy acetic herbicides and/or chlorophenols with chlorinated
d1benzod1ox1n and dlbenzofuran Impurities 1s "limited" according to the IARC
criteria. Therefore, the overall evidence of cardnogenlcHy, considering
both animal and human studies, would place 2,3,7,8-TCDD alone 1n the 28
category of IARC, and 2,3,7,8-TCDD 1n association with the phenoxy herbi-
cides and/or chlorophenols 1n the 2A category. Chemicals 1n categories 2A
and 2B are regarded as being 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 In animals would be regarded as
"sufficient" according to the IARC classification scheme. Therefore, based
on animal evidence, HxCDD would be placed 1n Group 28, which IARC charac-
terizes as probably carcinogenic 1n humans.
Quantitatively, 1n 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 the U.S. EPA has evaluated.
1873A 11-133 04/16/84
-------
12. SYNERGISM 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 In either potentlatlon or Inhibition of their biological effects.
12.1. CHEMICAL CARCINOGENS
Synerglstlc and antagonistic activities of 2,3,7,8-TCDD with chemical
carcinogens have been discussed In depth In Chapter 11 of this document.
12.2. NON-CARCINOGENIC 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 Pg 2,3,7,8-TCDD/kg bw 1-3 days preceding treatment with 100
mg/kg zoxazolamlne hydrochlorIde 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 1n the sleeping time
produced by hexabarbltone. A recent report compares the ImmunotoxIcHy of
2,3,7,8-lCDD, 2,3,7,8-TCDF and 2,3,7,8-TCDF plus 2,3,7,8-TCDD (coadmln-
Istered) (Rlzzardlnl 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 by Intraperltoneal administration and plaque-forming cells (RFC) In
the spleen were counted 5 days later. 2,3,7,8-TCDD Inhibited antibody pro-
duction by 80%. In a parallel study, a dose of 2,3,7,8-TCDF was admin-
istered (10 vg/kg) and no significant Immunotoxlc effects were observed.
Coadmlnlstration of 2,3,7,8-lCDD (1.2 vg/kg) plus 2,3,7,8-TCDF (10
vg/kg) resulted In 50% reduction In antibody production and demonstrates a
significant antagonistic effect by 2,3,7,8-TCDF. Coadmlnlstration of these
1859A 12-1 03/02/84
-------
two Isostereomers resulted In antagonistic effects with respect to the
Induction of hepatic mlcrosomal cytochrome P-450 and 7-ethoxycoumarIn 0-de-
ethylase. Sweeney et al. (1979) found that Iron deficiency protected mice
against the development of hepatocellular damage (Including porphyMa) nor-
mally caused by 2,3,7,8-TCDD exposure.
12.3. SUMMARY
Exposure to 2,3,7,8-KDD has been observed to alter the biological
response of many species to some compounds. This altered response Is pre-
sumed to be the result of altered enzyme activities 1n tissue in 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 jji vitro test systems;
however, exposure to 2,3,7,8-TCDD often has an antlcardnogenic effect jjn
vivo (see Section 11.1.2). This antlcardnogenic effect may be the result
of Increased detoxification or an Increased cytotoxicity 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 car-
cinogens. This modification may reduce the effectiveness, as in the case of
zoxazolamine, or increase the effectiveness, as 1n the case of hexabarbitone
(Greig, 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.
1859A 12-2 02/08/84
-------
13. REGULATIONS AND STANDARDS
13.1. WATER
13.1.1. Ambient Water. Previous release of PCDD-conta1n1ng herbicides has
been one mechanism by which these agents enter the environment. Their high
environmental stability 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-TCOD has bio-
accumulated. The U.S. EPA has set criteria of 1.3xlO~7, 1.3xlO~8 or
1.3xlO~» pg 2,3,7,8-TCDD/J, based on estimated human lifetime cancer
risks of 10~5, 10~6 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 9. 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-
HxCOD 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 Regu- latlons (41 CFR 321), fish with a 2,3,7,8-TCOD 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).
1860A 13-1 03/29/84
-------
A tolerance for hexachlorophene methylenebis (2,3,6-tMchlorophenol) In
or on feedstock cottonseeds has been established at 0.05 ppm, with the con-
dition that 1t not contain >0.1 ppm of 2,3,7,8-TCDD (U.S. EPA, 1982c).
No Information regarding concentration limits of other dloxln Isomers Is
available.
13.4. SUMMARY
The regulation of dloxln by-products In substances such as chlorophenols
and 2,4,5-tr1chlorophenoxyacet1c add 1s apparently expected to eliminate
dloxln releases to the environment. The Canadian concentration limit for
2,3,7,8-TCDD 1n fish Is 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 is available regarding concentration limits for other
PCDDs.
1860A 13-2 03/26/84
-------
14. EFFECTS OF MAJOR CONCERN AND HEALTH HAZARD ASSESSMENT
Of the four congeners of PCDOs discussed 1n this report (I.e., 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
toxlcologlc data are on 2,3,7,8-TCDD. The limited data on the other con-
geners Indicate that they are qualitatively similar In 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 In mice,
1n which 2,3,7,8-TCDD has an LDcn value of 0.88 ptnol/kg and 1,2,3,7,8-
DU
PeCDD; 1,2,3,6,7,8- and 1,2,3,7,8,9-HxCDD have LD values of 0.94, 3.19
and 3.67 Mmol/kg, respectively (McConnell et al., 1978b). This suggests
that either the position or the number of chlorine effects the toxlcity of
the PCDOs.
In more recent studies using biochemical endpolnts, Poland et al.
(1979), Bradlaw and Casterline (1979) and Bradlaw et al. (1980) have sup-
ported 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 EDrn for the Induction of AHH activity in hepatoma cells
bu
in culture was used to establish a range of potency for congeners of PCDOs.
Although acute toxidty and induction of AHH activity have been used to
quantify the difference in the biologic activity of the congeners 2,3,7,8-
TCDD, 1,2,3,7,8-PeCDD and 1,2,3,7,8,9-HxCDO, the extrapolation of this data
to estimate quantitative dose-response relationships for the chronic tox-
icity of individual congeners is not sufficiently supported at the present
time. From the following data described, 1t is clear that sufficient Infor-
mation for quantitative hazard assessment is available only for 2,3,7,8-TCDD
and a mixture of the two HxCDD congeners.
1861A 14-1 03/27/84
-------
14.1. PRINCIPAL EFFECTS
14.1.1. ToxIcHy. 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
In the guinea pig, the most sensitive species to 2,3,7,8-TCDD toxlclty.
Necropsy revealed no remarkable alteration in any Internal organ except for
thymlc atrophy (Gupta et al., 1973). Although liver damage was observed 1n
rats, rabbits and mice (Schwetz et al., 1973), there are insufficient data
to indicate that this effect is the underlying cause of mortality after
acute exposure to 2,3,7,8-TCDD. Also, In the guinea pig and monkey, which
have the same general progression of gross signs of toxlcity as do rats,
rabbits and mice, there is only mild liver damage (see Section 8.1.). In
addition, 2,3,7,8-TCDD is 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, subchronic and chronic studies are better able to
define dose and effect relationships than are acute studies. Subchronic and
chronic animal studies that define NOELs and LOELs are summarized in Table
14-1 for orally administered 2,3,7,8-TCDD. The NOEL for subchronic exposure
is -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
toxicity. There are only limited data on the NOEL and LOEL for HxCDD
1861A 14-2 03/27/84
-------
TABLE 14-1
CO
j^ No-Observed-Effect Levels and Low-Observed-Effect Levels Obtained from Subchronic and
Chronic Oral Toxlcity Studies of 2,3,7,8-TCDD
yg/kg/day
Species/Strain
NOEL LOEL
Rat/Sprague-Dawley 0.01 0.1
Rat/Osborne-Mendel 0.07 0.14
Rat/Sprague-Dawley 0.0014 0.014
" Rat/Sprague-Dawley ND 0.014
Mice/B6C3F1 ND 0.014
Monkey/Rhesus ND <0.02
Rat/Sprague-Dawley 0.001 0.01
Rat/Osborne-Mendel 0.0014 0.007
Mice/Swiss ND 0.001
0
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 porphyrin
levels
fatty changes in
the liver,
decreased bw
toxic hepatitis
pancytopenia
degenerative and
necrotic changes
in the liver
toxic hepatitis
dermatitis and
amyloidosis
Reference
Kociba et al .
NTP, 1980a
Goldstein et
1982b
King and
Roesler, 1974
NTP, 1980a
Allen et al . ,
Kodba et al.
1978a, 1979
NTP, 1980a
Toth et al.,
, 1976
al.,
1977
>
1979
o
2 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-KDD, there 1s
a suggestion that the cumulative dose of this mixture Is an Important
consideration 1n defining a NOEL. For both 2,3,7,8-TCOD and the mixture of
HxCOD, 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 Giavini et al. (1982b) 1n which increases 1n extra ribs and
total soft-tissue anomalies were observed. In mice, 1 pg/kg/day given for
9-10 days during the middle of gestation was the minimum dose necessary to
elicit a teratogenic response (Smith et al., 1976; Moore et al., 1973),
while dilated renal pelvis and decreased fetal weight were observed in the
rat fetuses of dams receiving doses of 2,3,7,8-TCDD as low as 0.001
pg/kg/day throughout gestation. The statistical and biological signifi-
cance of effects at this later dose, however, Is argued (Murray et al.,
1979; Nisbet 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 in
chronic studies (see Table 14-1). Also, Schwetz et al. (1973) demonstrated
that HxCDD (Isomers not specified) was both fetotoxlc and teratogenic when
administered to pregnant rats at 100 pg/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 is a known contaminant, and birth
1861A 14-4 03/27/84
-------
TABLE 14-2
No-Observed-Effect Levels and Low-Observed-Effect Levels
Obtained from Subchronlc and Chronic Oral loxlclty Studies of HxCODa«b
yg/kg/day Duration
Species/Strain of
NOEL LOEL Exposure
Rat/Osborne-Mendel 0.35 0.7 13 weeks
M1ce/B6C3Fl 0.7 1.4 13 weeks
Rat/Osborne-Mendel ND 0.18 104 weeks
Mice/B6C3M ND 0.18 104 weeks
Duration Reported Effects
of Study
13 weeks hepatotoxldty
13 weeks hepatotoxldty
107 weeks toxic hepatitis
107 weeks toxic hepatitis
aSource: NTP, 1980b
blhe HxCDD was a 1:2 mixture of 1,2,3,6,7,8- and 1,2,3,7,8,9-HxCDD.
ND =. Not determined
1861A
14-5
02/29/84
-------
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 in
rats and mice, and the more intensive studies by Kociba et al. (1978a) and
NTP (1980a) support these early findings. Also, papillomas have been re-
ported in female mice after dermal application of 2,3,7,8-TCDD (NTP, 1980b),
and using the skin tumorigenesis model, it has been shown that 2,3,7,8-TCDD
may affect the carcinogenic potential of other chemical carcinogens (see
Section 11.1.3.). Human exposure to 2,3,7,8-TCDD has resulted from contami-
nation of other polychlorinated compounds with 2,3,7,8-TCDD (see Section
11.2.).
A 1:2 mixture of 1,2,3,6,7,8- and 1,2,3,7,8,9-HxCDD also has been tested
for careinogenicity In rats and mice treated by gavage and by dermal appli-
cation in mice (NTP, 1980c,d). In both species, this mixture produced liver
tumors when administered by gavage, while in the dermal study there was no
increase in the incidence of skin tumors.
Epidemiological studies of workers exposed to chemicals contaminated
with 2,3,7,8-TCDD such as 2,4,5-trichlorophenoxyacetic add and 2,4,5-tri-
chlorophenol are consistent with the position that 2,3,7,8-TCDD is probably
carcinogenic for humans; the available evidence Indicates an excess inci-
dence of soft tissue sarcoma. Because 2,3,7,8-TCDD Is almost always found
1861A 14-6 03/27/84
-------
1n association with the materials (e.g., chlorophenols, combustion products,
etc.) 1t may never be possible to evaluate the carc1nogen1c1ty of 2,3,7,8-
TCOO by Itself 1n humans.
14.1.2. Mutagenldty. 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 1s not a mutagen.
These negative results were obtained both 1n 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, H 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-TCOD 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 1n animal studies
were small, 1t 1s still not substantiated that 2,3,7,8-TCDD produces clasto-
genlc changes.
Pertinent data regarding the mutagenlc potential of 1,2,3,7,8-PeCDO,
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,
1861A 14-7 03/27/84
-------
rabbits, ferrets and monkeys have shown that in utero exposure to 2,3,7,8-
TCDD can result In malformations, fetal toxlclty and abortions (see Table
9-2). The lowest dose reported to adversely affect the fetus Ijn utero was
0.001 yg/kg/day administered to the dams throughout gestation (from Murray
et al., 1979, according to Nisbet and Paxton, 1982); this dose 1s similar to
the NOEl reported for chronic exposure of adult rats (see Table 14-1).
Moore 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). Scarpelll et al. (1980), for example, demon-
strated that pretreatment of hamsters with 2,3,7,8-TCDD resulted In greater
activation of mutagenlc nltrosamlnes when assayed in 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
ingestion of contaminated food and water, by Inhalation of the compound
absorbed to resplrable particulates, or through dermal contact. Although
potential exposure may occur by all routes, most of the toxlcologic informa-
tion is from studies of oral exposure. The limited observation of toxic
1861A 14-8 03/27/84
-------
effects In humans and animals after dermal contact with 2,3,7,8-TCDD 1n
organic solvents Indicates that dermal absorption occurs. Polger and
Schlatter (1980) have shown 1n rats that both dermal and GI absorption 1s
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 If the 2,3,7,8-lCDD was applied In a soil and water paste. Inhalation
exposure is likely to occur through airborne partlculate matter containing
absorbed 2,3,7,8-lCDD; 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 ASSESSMENT
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 LD value and studies of Induc-
tion 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,
teratogenlcity or carc1nogen1city.
1861A 14-9 03/27/84
-------
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 in 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, it is 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 In human population.
Without adequate exposure data, health assessments cannot be made.
14.4.1. Animal Toxldty 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 1s 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 toxicity studies would be more appropriately used for hazard
assessment. The NOEL from the two studies in rats (Kodba et al., 1978a,
1979; NTP, 1980a) are 0.001 and 0.0014 Pg/kg/day; however, In the mouse
(NTP, 1980a), the dose of 0.07 yg/kg/day was a PEL, as Indicated by fatty
changes in the liver, and 0.007 was a NOEL.
In addition, it may be inappropriate to derive a toxldty-based hazard
assessment for 2,3,7,8-TCDD from these chronic studies, since a 3-generatlon
study by Murray et al. (1979) Indicates that exposure of pregnant rats to
this dose of 2,3,7,8-TCDD (0.001 yg/kg/day) throughout gestation resulted
in the observation of dilated renal pelvis 1n the fetuses. Murray et al.
(1979) and U.S. EPA (1979c) consider this effect not to be treatment-related
because it occurred 1n only one generation at this dose and not at higher
1861A 14-10 03/27/84
-------
doses. Hence, 0.001 yg/kg/day represented a NOAEL. However, a reevalua-
tion 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 In 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 toxicity-based hazard assessment Is 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 Carc1nogen1dty. In addition to the Inadequate data base
for a tox1dty-based hazard assessment, the strong evidence of cardnogenl-
city 1n animals for 2,3,7,8-TCDD would justify a cardnogenldty-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; Kodba et
al., 1978a) (Table 14-3). In the NTP (1980a) study, male rats developed
follicular-cell adenomas or carcinomas of the thyroid. Female rats and mice
of both sexes had Increased incidences of folHcular-cel 1 adenomas of the
thyroid. In the study by Kociba et al. (1978a), rats maintained on diets
that provided doses of 0.0, 0.001, 0.01 and 0.1 yg/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.
1861A 14-11 03/27/84
-------
CD
3>
-p.
1
f\5
o
r\j
CO
Exposure Species/Strain Sex Dose or
Route Exposure
Gavage rats/ M 0.0 yg/kg/week
Osborne-Mendel
0.01 yg/kg/week
0.05 yg/kg/week
0.5 yg/kg/week
Gavage rats/ F 0.0 yg/kg/week
Osborne-Mendel
0.1 yg/kg/week
0.05 yg/kg/week
0.5 yg/kg/week
TABLE 14-3
Cardnogenld ty Bloassays
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 107 weeks
104 weeks 107 weeks
104 weeks 107 weeks
of 2,3,7,8-TCDD
Vehicle
corn oil-
acetone
(9:1)
corn oil-
acetone
(9:1)
corn oil-
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)
Tumor Type
folllcular-cell adenomas
or carcinoma of the
thyroid
folllcular-cell adenomas
or carcinoma of the
thyroid
folUcular-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
Tumor Reference
Incidence
1/69 NTP, 1980a
5/48
8/50
11/50
5/75 NTP. 1980a
1/49
3/50
14/49
-------
CD
cr>
3>
Exposure Species/Strain
Route
Gavage m1ce/B6C3Fl
_i
j^
i
«-J
00
Gavage m1ce/B6C3Fl
Oral rat/
Sprague-Dawley
o
PO
\
o
—
00
_^ —
Sex Dose or
Exposure
M 0.0 yg/kg/week
0.01 yg/kg/week
0.05 pg/kg/week
0.5 vg/kg/week
F 0.0 pg/kg/week
0.04 vg/kg/week
0.2 pg/kg/week
2.0 pg/kg/week
M 0.0 pg/kg/day
TABLE 14-3 (cont
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 107 weeks
104 weeks 107 weeks
104 weeks 107 weeks
105 weeks 105 weeks
-)
Vehicle
corn oil-
acetone
(9:1)
corn oil-
acetone
(9:1)
corn oil-
acetone
(9:1)
corn oil-
acetone
(9:1)
corn oil-
acetone
(9:1)
corn oil-
acetone
(9:1)
corn oil-
acetone
(9:1)
corn oil-
acetone
(9:1)
1n diet
Tumor Type
hepatocellular carcinoma
hepatocel lular carcinoma
hepatocellular carcinoma
hepatocellular carcinoma
hepatocellular carcinoma,
folUcular-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
cortex
Tumor
Incidence
8/73
9/49
8/49
17/50
1/73
0/69
2/50
3/50
2/48
1/47
6/47
5/46
0/85
0/85
0/85
Reference
NTP, 1980a
NTP, 1980a
Koclba et
al.. 1978a
-------
TABLE 14-3 (cont.)
oo
Exposure Species/Strain Sex Dose or
Route Exposure
Duration
of Duration
Treatment of Study
Vehicle
Tumor Type
Tumor
Incidence
Reference
Oral rat/
(cont.) Sprague-Dawley
I
4*
Oral rat/ F
Sprague-Dawley
0.001 pg/kg/day 105 weeks 105 weeks In diet
0.01 ^g/kg/day 105 weeks 105 weeks 1n diet
0.1 pg/kg/day
105 weeks
105 weeks
0.0 pg/kg/day
105 weeks
105 weeks
1n diet
1n diet
0.001 pg/kg/day 105 weeks 105 weeks 1n diet
0.01 wg/kg/day 105 weeks 105 weeks 1n diet
0.1 yg/kg/day 105 weeks 105 weeks 1n diet
CO
squamous cell carcinoma 0/50 Kodba et
of the hard palate, al., 1978a
squamous cell carcinoma 1/50
of the tongue,
adenoma of the adrenal 0/50
cortex
squamous cell carcinoma 0/50
of the hard palate,
squamous cell carcinoma 1/50
of the tongue,
adenoma of the adrenal - 2/50
cortex
squamous cell carcinoma 4/50
of the hard palate,
squamous cell carcinoma 3/50
of the tongue,
adenoma of the adrenal 5/50
cortex
hepatocellular carcinoma, 1/86 Kodba et
squamous cell carcinoma al., 1978a
of the hard palate, 0/86
squamous cell carcinoma
of the lung 0/86
hepatocellular carcinoma, 0/50
squamous cell carcinoma
of the hard palate, 0/50
squamous cell carcinoma
of the lung 0/50
hepatocellular carcinoma, 2/50
squamous cell carcinoma
of the hard palate, 1/50
squamous cell carcinoma
of the lung 0/50
hepatocellular carcinoma, 11/49
squamous cell carcinoma
of the hard palate, 4/49
squamous cell carcinoma
of the lung 7/49
-------
A single bloassay tested a mixture of the two congeners of HxCOD for
cardnogenicity (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
HxCDO and the HxCDO mixture used for this bloassay was found to be contami-
nated with other PCODs Including 0.09% (+0.03%) of KDD. The specific
Isomer of PCDOs 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 HxCDO, Information obtained from
this combined exposure may not be applicable to the Individual congeners.
1861A 14-15 03/27/84
-------
oo
en
TABLE 14-4
Cardnogenldty Bloassays of a 1:2 Mixture of 1.2,3,6.7.8- and 1,2,3,7,8,9-HxCDO
oo
-P.
Duration
Exposure Species/Strain Sex Dose or Exposure of Duration
Route Treatment of Study
Vehicle
Tumor Type
Tumor Reference
Incidence
Gavage rats/
Osborne-Mendel
M 0.0 ug/kg/week
104 weeks 105 weeks corn oil-
acetone (9:1)
liver neoplastlc nodules
or hepatocellular
carcinoma
0/74 NTP, 1980b
Gavage rats/
Osborne-Mendel
H 1.25
104 weeks 107 weeks corn oil-
acetone (9:1)
liver neoplastlc nodules
or hepatocellular
carcinoma
0/49 NTP, 1980d
01
2.5 yg/kg/week 104 weeks 107 weeks corn oil-
acetone (9:1)
5.0 pg/kg/week 104 weeks 107 weeks corn oil-
acetone (9:1)
liver neoplastlc nodules 1/50
or hepatocellular
carcinoma
liver neoplastlc nodules 4/48
or hepatocellular
carcinoma
Gavage rats/
Osborne-Mendel
0.0 ^g/kg/week 104 weeks 105 weeks corn oil-
acetone (9:1)
liver neoplastlc nodules
or hepatocellular
carcinoma
5/75 NTP, 1980d
1.25 yg/kg/week 104 weeks 107 weeks corn oil-
acetone (9:1)
liver neoplastlc nodules
or hepatocellular
carcinoma
10/50
2.5 yg/kg/week 104 weeks 107 weeks corn oil-
acetone (9:1)
liver neoplastlc nodules
or hepatocellular
carcinoma
12/50
o
CO
rv>
5.0 yg/kg/week 104 weeks 107 weeks corn oil-
acetone (9:1)
liver neoplastlc nodules
or hepatocellular
carcinoma
30/50
-------
CD
3>
TABLE 14-4 (cont.)
Exposure Species/Strain
Route
Duration
Sex Dose or Exposure of Duration
Treatment of Study
Vehicle
Tumor Type
Tumor Reference
Incidence
Gavage rats/
Osborne-Mendel
F 0.0 yg/kg/week
104 weeks 105 weeks corn oil- hepatocellular adenomas
acetone (9:1) or carcinomas
15/73 NTP, 1980d
1.25 yg/kg/week 104 weeks 108 weeks corn oil- hepatocellular adenomas
acetone (9:1) or carcinomas
14/50
-e-
i
2.5 yg/kg/week 104 weeks 107 weeks corn oil- hepatocellular adenomas
acetone (9:1) or carcinomas
14/49
5.0 yg/kg/week
Gavage m1ce/B6C3Fl F 0.0 pg/kg/week
2.5 yg/kg/week
5.0 yg/kg/week
10.0 yg/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
24/48
3/75 NTP, 1980d
4/48
6/47
10/47
O
CO
CD
-------
15. REFERENCES
ACP (Advisory Committee on Pesticides). 1980. Further Review of the Safety
for Use 1n the U.K. of the Herbicide 2,4,5-T, Pesticides Branch, Ministry of
Agriculture, Fisheries and Food, London, England.
Adamoll, P., E. Angell, G. Bandl, et al. 1978. Analysis of 2,3,7,8-tetra-
chlorod1benzo-p-d1ox1n 1n the Seveso area, in: Chlorinated Phenoxy Acids
and Their D1ox1ns, C. Ralnel, Ed. Ecol. Bull. 27: 31-38.
AH1o, A. and M.G. Parkkl. 1978. Organ specific Induction of drug metabo-
lizing enzymes by 2,3,7,8-tetrachlorod1benzo-p-d1ox1n 1n the rat. Toxlcol.
Appl. Pharmacol. 44(1): 107-114.
Albro, P.M. 1979. Problems 1n analytical methodology: Sample handling,
extraction, and clean up. Ann. NY Acad. Scl. 320: 19-27.
Albro, P.M. and B.J. Corbett. 1977. Extraction and clean up of animal
tissues for subsequent determination of mixtures of chlorinated d1benzo-p-
dloxln and dlbenzofurans. Chemosphere. 6: 381-385.
Albro, P.M., J.T. Corbett, M. Harrlss and L.D. Lawson. 1978. Effects of
2,3,7,8-tetrachlorod1benzo-p-d1ox1n on I1p1d profiles in tissue of the
Fischer rat. Chem. B1ol. Interact. 23(3): 315-330.
Albro, P.W., M.I. Luster, K. Chae, et al. 1979. A radlolmmunoassay for
chlorinated d1benzo-p-d1ox1ns. Environ. Blol. Chem. Branch, Natl. Inst.,
Environ. Health Sc1., Research Triangle Park, NC 27709.
1862A 15-1 03/12/84
-------
Aldred, J.E. 1978. Report of the Consultative Council on Congenital Abnor-
malities 1n the Yarrom District. Minister of Health, Melbourne, Victoria,
Australia. (Cited 1n M1lby et al., 1980)
Allen, J.R. and L.A. Carstens. 1967. Light and electron microscopic
observations 1n Macaca mulatta monkeys fed toxic fat. Am. J. Vet. Res. 28:
1613-1629.
Allen, J.R. and J.J. Lallch. 1962. Response of chickens to prolonged
feeding of crude "toxic fat." Proc. Sox. Exp. B1ol. Med. 109: 48-51.
Allen, J.R., J.P. Van Miller and D.H. Norback. 1975. Tissue distribution,
excretion, and biological effects of (14-c) tetrachlorod1benzo-p-d1ox1n 1n
rats. Food Cosmet. Toxlcol. 13(5): 501-505.
Allen, J.R., D.A. Barsottl, J.P. Van Miller, L.J. Abrahamson and J.J.
Lallch. 1977. Morphological changes 1n monkeys consuming a diet containing
low levels of TCDD. Food Cosmet. Toxlcol. 15: 401.
Allen, J.R., D.A. Barsottl, L.K. Lambrecht and J.P. Van Miller. 1979.
Reproductive effects of halogenated aromatic hydrocarbons on nonhuman
primates. Ann. NY Acad. Sc1. 320: 419-425.
Anonymous. 1979. D1ox1n 1s found 1n cleanup worker's blood. Chemical
Week. 124: 22.
Anonymous. 1982. Phenoxyherblcldes, trichlorophenols, and soft-tissue
sarcomas. May 8. Lancet, p. 1051-1052.
1862A 15-2 03/12/84
-------
Appelgren, L.-E., I. Brandt, E.B. BrHtebo, M. Glllner and J.-A. Gustafsson.
1983. Autoradlography of 2,3,7,8-tetrachloro[l«C]-d1benzo~p-d1ox1n
(TCDD): Accumulation 1n the nasal mucosa. Chemosphere. 12(4/5): 545-548.
AWPI (American Wood Preservers Institute). 1977. Memo to the Office of
Pesticide Program, U.S. EPA, Research Triangle Park, NC.
Ax, R.L. and L.G. Hansen. 1975. Effects of purified PCB analogs on chicken
reproduction. Poult. Sc1. 54: 895-900.
Axelson, 0. 1980. A note on observational bias case-referent studies.
Scand. J. Work Environ. Health. 6(1): 80-82.
Axelson, 0., L. Sundell, K. Anderson, C. Edllng, C. Hogstedt and H. Kllng.
1980. Herbicide exposure and tumor mortality: An updated ep1dem1olog1c
Investigation on Swedish railroad workers. Scand. J. Work Environ. Health.
6: 73-79.
Baars, A.J., M. Jansen and D.O. Brelmer. 1978. The Influence of pheno-
barbital, 3-methylcholanthrene, and 2,3,7,8-tetrachlorod1benzo-p-d1ox1n on
glutathione S-transferase activity of rat liver cytosol. Blochem.
Pharmacol. 27(21): 2487-2494.
Ball, L.M. and R.S. Chhabra. 1981. Intestinal absorption of nutrients In
rats treated with 2,3,7,8-tetrachlorod1benzo-p-d1oxin (TCDD). J. Toxlcol.
Environ. Health. 8/4: 629-638.
1862A 15-3 03/29/84
-------
Bandiera, S., T.W. Sawyer, M.A. Campbell, P. FujHa and S. Safe. 1983.
Comparative binding to the cytosollc 2,3,7,8-tetrachlorod1benzo-p-diox1n
receptor: Effects of structure on the affinities of substituted halogenated
blphenyls -- a QSAR analysis. Blochem. Pharmacol. 32: 3803-3813.
Barnes, D.G. 1983. "D1ox1n" Production from Combustion of Blomass and
Waste. Presented at the Symposium on Energy from Blomass and Wastes. VII.
Lake Buena Vista, FL. Jan. 24-28.
Barsotti, D.A., L.J. Abrahamson and J.R. Allen. 1979. Hormonal alterations
1n female rhesus monkeys fed a diet containing 2,3,7,8-tetrachlorod1benzo-p~
dloxln. Bull. Environ. Contam. Toxlcol. 21: 463-469.
Bartsch, H., L. Tomatis and C. Malavellle. 1982. Qualitative and quantita-
tive comparisons between mutagenlc activities of chemicals. In: Mutagenlc-
1ty: New Horizons in Genetic Toxicology, J.A. Heddle, Ed. Academic Press,
NY. p. 36-72.
Baughman, R. and M. Meselson. 1973. An analytical method for detecting
TCDD (dloxln): Levels of 1CDD 1n samples from Vietnam. Environ. Health
Perspect. 5: 27-35.
Beale, M.G., W.T. Shearer, M.M. Karl and A.M. Robson. 1977. Long-term
effects of dloxln exposure. Lancet. April 2. p. 748.
Beatty, P.W. and R.A. Neal. 1975. Effect of alteration of hepatic mixed
function oxldase activity on the toxlclty of 2,3,7,8-tetrachlorodlbenzo-
dloxln (TCDD) 1n rats. Toxlcol. Appl. Pharmacol. 33: 151.
1862A 15-4 03/29/84
-------
Beatty, P. and R.A. Neal. 1976a. Induction of DT-d1aphorase by 2,3,7,8-
tetrachlorod1benzo-p-dioxin (TCDD). Blochem. Biophys. Res. Commun. 12.
68(1): 197-204.
Beatty, P.M. and R.A. Neal. 1976b. Induction of DT-diaphorase activity of
rat liver by 2,3,7,8-tetrachlorod1benzo-p-d1ox1n. Toxlcol. Appl. Pharmacol.
37: 189.
Beatty, P. and R.A. Neal. 1978. Factors affecting the induction of
DT-diaphorase by 2,3,7,8-tetrachlorodibenzo-p-d1oxin. Blochem. Pharmacol.
27(4): 505-510.
Beatty, P.M., K.J. Lembach, M.A. Holscher and R.A. Neal. 1975. Effects of
2,3,7,8-tetrachlorodibenzo-p-d1oxin (TCDD) on mammalian cells 1n tissue
cultures, loxicol. Appl. Pharmacol. 31(2): 309-312.
Beatty, P.M., M.A. Holscher and R.A. Neal. 1976. Toxicity of 2,3,7,8-
tetrachlorod1benzo-p-d1oxin in larval and adult forms of Rana catesbeiana.
Bull. Environ. Contam. Toxlcol. 16(5): 578-581.
Beatty, P.W., W.K. Vaughn and R.A. Neal. 1978. Effect of alteration of rat
hepatic mixed-function oxidase (MFO) activity on the toxiclty of 2,3,7,8-
tetrachlorodibenzo-p-d1oxin (TCDD). Toxlcol. Appl. Pharmacol. 45(2):
513-519.
Benedict, W.F., N. Considine and D.W. Nebert. 1973. Genetic differences 1n
aryl hydrocarbon hydroxylase induction and benzo[a]pyrene-produced tumorl-
genesls in the mouse. Mol. Pharmacol. 9: 266-277.
1862A 15-5 03/29/84
-------
Benfenatl, E., F. G1zz1, R. Reglnate, R. Fanelll, M. Lod1 and R. Taghaferrl.
1983. Polychlorlnated d1benzo-p-d1ox1ns (TCDO) and polychlorlnated dlbenzo-
furans (PCDF) 1n emissions from an urban Incinerator. II. Correlation
between concentration of mlcropollutants and combustion conditions. Chemo-
sphere. (In press)
Berry, D.L., J. D1G1ovann1, M.R. Juchau, W.M. Bracken, G.L. Gleason and T.J.
Slaga. 1978. Lack of tumor-promoting ability of certain environmental
chemicals 1n a two-stage mouse skin tumor 1 genes 1s assay. 20(1): 101-108.
Berry, O.L., l.J. Slaga, J. D1G1ovann1 and M.R. Juchau. 1979. Studies with
chlorinated d1benzo-p-d1ox1ns, polybromlnated blphenyls, and polychlorlnated
blphenyls 1n a two-stage system of mouse skin tumorlgenesls: Potent anti-
carcinogenic effects. Ann. NY Acad. Sc1. 320: 405-414.
Blocca, M., B.W. Gupta, K. Chae, J.D. McKlnney and J.A. Moore. 1981.
loxIcHy of selected symmetrical hexachloroblphenyl Isomers 1n the mouse.
loxlcol. Appl. Pharmacol. 58: 461-474.
B1sant1, L., F. Bonettl, F. Caramaschl, et al. 1980. Experiences from the
accident of Seveso. Acta. Morphol. Acad. Sc1. Hung. 28(1-2): 139-157.
Bishop, C.M. and A.M. Jones. 1981. Non-Hodgk1n's lymphoma of the scalp In
workers exposed to dloxlns. Lancet. 2(8242): 369.
Boerl, E. 1978. Report to Lombardy Regional Authority (unpublished).
(Cited In Pocch1ar1 et al., 1979)
1862A 15-6 03/29/84
-------
Bogen, G. 1979. Symptoms in Vietnam veterans exposed to Agent Orange. J.
Am. Med. Assoc. 242(22): ?391.
Bollen, W.B. and L.A. Norris. 1979. Influence of 2,3,7,8-tetrachlorodl-
benzo-p-d1ox1n on respiration 1n a forest floor and soil. Bull. Environ.
Contam. Toxicol. 22(4-5): 648-652.
Bonaccorsi, A., R. Panel 11 and G. Tognoni. 1978. In the wake of Seveso.
Amblo. 7(5-6): 234-239.
Bonaccorsi, A., A. diDomenico, R. FanelH, et al. 1983. The Influence of
soil particle adsorption on TCDO biological uptake 1n the rabbit. Arch.
Toxicol. (In press)
Botre, C., A. Memoli and F. Alhaique. 1978. TCDD solubillzation and photo-
decomposition in aqueous solutions. Environ. Sd. Technol. 12(3): 335-336.
Bradlaw, J.A. and J.L.. Casterllne, Jr. 1979. Induction of enzyme activity
in cell culture: A rapid screen for detection of planar polychlorlnated
organic compounds. J. Assoc. Off. Anal. Chem. 62: 904-906.
Bradlaw, J.A., L.H. Garthoff, 0. Graff and N.E. Hurley. 1975. Detection of
chlorinated dloxins induction of aryl hydrocarbon hydroxylase activity 1n
rat hepatoma cell culture. Toxicol. Appl. Pharmacol. 33(1): 166.
Bradlaw, J.A., L.H. Garthoff, N.E. Hurley and D. Firestone. 1976. Aryl
hydrocarbon hydroxylase activity of twenty-three halogenated d1benzo-p-
dioxins (Meeting Abstract). Toxicol. Appl. Pharmacol. 37(1): 119.
1862A 15-7 03/29/84
-------
Bradlaw, J.A., L.H. Garthoff, N.E. Hurley and D. Firestone. 1980. Com-
parative Induction of aryl hydrocarbon hydroxylase activity Iji vitro by
analogues of d1benzo-p-d1ox1n. Food Cosmet. Toxlcol. 18: 627-635.
Brewster, D.W., B.V. Madhukar and F. Matsumura. 1982. Influence of
2,3,7,8-TCDD on the protein composition of the plasma membrane of hepatic
cells from the rat. Blochem. Blophys. Res. Commun. 107(1): 68-74.
Bronzettl, G., I. Lee, E. Zelger, et al. 1980. Genetic effects of TCOD in
yitr_o and in vWo using 07 strain of S. cerevlslae. Mutat. Res. 74/3 (75).
Bronzettl, G., C. Bauer, C. Corsl, R. Del Carratare, R. Neerl and M.
Paollne. 1983. Mutagenldty study of TCOD and ashes from urban Incinerator
"In. vitro" and "In vivo" using yeast 07 strain. Chemosphere. 12: 549-553.
Brumley, W.C., J.A.G. Roach, J.A. Sphon, et al. 1981. Low-resolution
multiple 1on detection gas chromatographlc-mass spectrometrlc comparison of
six extraction-cleanup methods for determining 2,3,7,8-tetrachlorod1benzo-p-
dloxln 1n fish. J. Agrlc. Food Chem. 29(5): 1040-1046.
Bumb, R.R., W.B. Crummet, S.S. Cutle, et al. 1980. Trace chemistries of
fire: A source of chlorinated dloxlns. Science. 210: 385-390.
Burns, L.H., D.M. CUne and R.R. Lasslter. 1981. Exposure analysis model-
Ing system (EXAMS). Prepared by the Environmental Research Laboratory, ORO,
U.S. EPA, Athens, GA.
1862A 15-8 03/29/84
-------
Bus, J.S. and J.E. Gibson. 1979. L1p1d peroxldatlon and Us role 1n
toxicology. _ln_: Reviews 1n Biochemical lexicology, E. Hodgson, J.R. Bend
and R.M. Phllpot, Ed. Elsevier/North Holland, NY. 1: 125-149.
Buser, H.R. 1975. Polychlorlnated d1benzo-p-d1ox1n: Separation and Identi-
fication of Isomers by gas chromatography-mass spectrometry. J. Chromatogr.
114: 95-108.
Buser, H.R. 1976. High-resolution gas chromatography of polychlorlnated
d1benzo-p-d1ox1ns and dlbenzofurans. Anal. Chem. 48: 1553-1557.
Buser, H.R. 1978. Analysis of TCDD by gas chromatography-mass spectrometry
using glass capillary columns. In: D1ox1n: lexicological and Chemical
Aspects, E. Cattabenl, A. Cavallaro and G. Gain, Ed. SP Medical and Scien-
tific Books, NY. p. 27-41.
Buser, H.R. 1979. Formation of polychlorlnated dlbenzofurans (PCDEs) and
d1benzo-p-d1ox1ns (PCODs) from pyrolysls of chlorobenzenes. Chemosphere.
6: 415-424.
Buser, H.R. and H.P. Bosshardt. 1976. Determination of polychlorlnated
d1benzo-p-d1ox1ns and dlbenzofurans 1n commercial pentachlorophenols by
combined GC-MS. J. Assoc. Off. Anal. Chem. 59: 562-569.
Buser, H.R. and H.P. Bosshardt. 1978. Polychlorlnated d1benzo-p-d1ox1ns,
dlbenzofurans, and benzenes 1n the fly ash of municipal and Industrial
Incinerators. M1tt. Geb. Lebens. Hyg. 69: 191-199.
1862A 15-9 03/29/84
-------
Buser, H.R. and C. Rappe. 1978. Identification of substitution patterns 1n
polychlorinated d1benzo-p-d1oxins (PCDOs) by mass spectrometry. Chemo-
sphere. 7: 199-211.
Buser, H.R. and C. Rappe. 1980. High resolution gas chromatography of the
22 tetrachlorod1benzo-p-d1ox1n Isomers. Anal. Chem. 52: 2257-2262.
Buser, H.R. and C. Rappe. 1983. Isomer-spedf1c separation and analysis of
2,3,7,8-substHuted polychlorlnated d1benzo-p-d1oxins (PCDOs) using high-
resolution gas chromatography and mass spectrometry. Anal. Chem. (In review)
Buser, H.R., H.P. Bosshardt and C. Rappe. 1978. Identification of poly-
chlorinated d1benzo-p-d1oxin Isomers formed 1n fly ash. Chemosphere. 7:
165-172.
Buu-Ho1, N.P., Pham-Huu-Chanh, G. Sesque, M.C. Azum-Gelade and G. Saint-Ruf.
1972. Organs as targets of dloxln (2,3,7,8-tetrachlorod1benzo-p-d1ox1n)
Intoxication. Naturwissenschaften. 59(4): 174-175.
Callahan, M.A., M.W. Slirnak, N.W. Gabel, et al. 1979. Water-Related
Environmental Fate of 129 Priority Pollutants. Vol. I. EPA 440/4-79-029a,
Office of Water Planning and Standards, Office of Water and Wastewater
Management, U.S. EPA, Washington, DC.
Camonl, I., A. DIMucdo, D. Pontecorvo, et al. 1983. Lack of in vitro
oxidation of 2,3,7,8-tetrachlorod1benzo-p-diox1n (TCDD) 1n the presence of
laccase from Polypoj-us versicolor fungus. Chemosphere. (In press)
1862A 15-10 03/29/84
-------
Cantoni, L., M. Rizzardini, G. Belvedere, R. Cantonl, R. FanelH and M.
Salmona. 1981. Induction of mixed-function oxldase by chronic treatment
with 2,3,7,8-tetrachlorod1benzo-p-d1oxin 1n female rats. Toxicology.
21(2): 159-167.
Caramaschl, F., G. del Crono, C. Favarett, S.E. Glambelluca, E. Montesarchlo
and G.M. Fara. 1981. Chloracne following environmental contamination by
TCDO 1n Seveso, Italy. Int. J. Ep1dem1ol. 10(2): P135-143.
Carlstedt-Duke, J.M. 1979. Tissue distribution of the receptor for
2,3,7,8-tetrachlorodibenzo~p-d1oxin 1n the rat. Cancer Res. 39(8):
3172-3176.
Carlstedt-Duke, J.M., G. Elfstrom, B. Hogberg and J.A. Gustafsson. 1979.
Ontogeny of the rat hepatic receptor for 2,3,7,8-tetrachlorod1benzo-p-d1ox1n
and Us endodne Independence. Cancer Res. 39(11): 4653-4656.
Carlstedt-Duke, J.M.B., V.8. Harnemo, B. Hogberg and J.A. Gustafsson. 1981.
Interaction of the hepatic receptor protein for 2,3,7,8-tetrachlorodibenzo-
p-d1ox1n with DNA. J. Blochim. Blophys. Acta. 672(2): 131-141.
Carter, C.D., R.D. Kimbrough, J.A. Uddle, et al. 1975. Tetrachlorodi-
benzo-p-dioxin: An accidental poisoning episode in horse arenas. Science.
188: 738-740.
1862A 15-11 03/29/84
-------
Cavallaro, A., G. Bartolozzl, D. CarreM, G. Bandl, L. Luclanl and G. Villa.
1980a. Method for the determination of 2,3,7,8-tetrachlorod1benzo-p-d1ox1n
at ppt levels 1n vegetables by high resolution gas chromatography and
low-resolution mass spectrometry. Chemosphere. 9(10): 623-628.
Cavallaro, A., G. Bandl, G. Invern1zz1, L. Luclanl, E. Mong1n1 and A. Cornl.
1980b. Sampling, occurrence and evaluation of PCDOs and PCDFs from Inciner-
ated solid urban waste. Chemosphere. 9(10): 611-621.
Cavallaro, A., G. Tebaldi and R. Gauldl. 1982. Analysis of transport and
ground deposition of the TCDD emitted on 10 July 1976 from the ICMESA
factory (Seveso, Italy). Atmos. Environ. 16: 731-740.
CEQ (Council on Environmental Quality). 1981. 12th Annual Report of the
Council on Environmental Quality. Washington, DC. p. 129.
Chen, J-Y.T. 1973. Infrared studies of chlorinated d1benzo-p-d1ox1ns and
structurally related compound. J. Assoc. Off. Anal. Chem. 56: 962-975.
Chess, E.K. and M.L. Gross. 1980. Determination of tetrachlorod1benzo-p-
dloxlns by mass spectrometr1c metastable decomposition monitoring. Anal.
Chem. 52: 2057-2061.
Cheung, M.O., E.F. Gilbert and R.E. Petersen. 1981. Cardiovascular
teratogenldty of 2,3,7,8-tetrachlorod1benzo-p-d1oxin 1n the chick embryo.
loxlcol. Appl. Pharmacol. 61: 197-204.
1862A 15-12 03/29/84
-------
Chhabra, R.S., T.J. Michael, R.M. Phllopot and J.R. Fouts. 1976. Relation
between Induction of aryl hydrocarbon hydroxylase and de novo synthesis of
cytochrome P 448 (Pl-450) 1n mice. Environ. Health Sc1., Research Triangle
Park, NC.
Ch1app1no, G., R. G1l1oli and V. Foa. 1978. Report of Lombardy Regional
Authority (unpublished). (Cited 1n Pocch1ar1 et al., 1979)
Clark, D.A., J. Gauldle, M.R. Szewczuk and G. Sweeney. 1981. Enhanced
suppressor cell activity as a mechanism of 1mmunosuppress1on by 2,3,7,8-
tetrachlorod1benzo-p-d1ox1n (41275). Proc. Soc. Exp. Blol. Med. 168(2):
290-299.
Clark, D.A., G. Sweeney, S. Safe, E. Hancock, D.G. Kllburn and J. Gauldle.
1983. Cellular and genetic basis for suppression of cytotoxlc T cell
generation by haloaromatlc hydrocarbons. Immunopharmacology. 6: 143-153.
Clement Associates. 1979. Exposure, toxldty and risk assessment of
2,4,5~T/TCDD. Prepared for U.S. EPA under contract no. 68-01-5095.
Clement, R.E. and F.W. Karasek. 1982. Distribution of organic compounds
adsorbed on size-fractionated municipal Incinerator fly ash particles. J.
Chromatogr. 234: 395-405.
Cochrane, W.P., J. Singh, W. Miles and B. Wakeford. 1981. Determination of
chlorinated d1benzo-p-d1ox1n contaminants 1n 2,4-0 products by gas chroma-
tography-mass spectrometrlc techniques. J. Chromatogr. 217: 289-299.
1862A 15-13 03/29/84
-------
Cockerham, L.G., A.L. Young and C.E. Thalken. 1980. H1stopatholog1cal and
ultrastructural studies of liver tissue from ICDD-exposed beach mice
(Peromyscus pollonotus). J. AD-A083 323/6 PC A04/MF A01. 61 p.
Cocucd, S., F. 01 Gerolamo, A. Verderlo, et al. 1979. Absorption and
translocation of tetrachlorod1benzo-p-d1ox1n by plants from polluted soil.
Experlentia. 35(4): 482-484.
Cohen, G.M., W.M. Bracken, R.P. Iyer, D.L. Berry, J.K. Selkirk and T.J.
Slaga. 1979. Antlcarclnogenlc effects of 2,3,7,8-tetrachlorod1benzo-p-
dloxln on benzo(a)pyrene and 7,l2-d1methylbenz(a)anthracene tumor Initiation
and Us relationship to DMA binding. Cancer Res. 39(10): 4027-4033.
Cole, P. 1979. The evolving case-control study. J. Chron. DIs. 32: 15-27.
Collins, I.F.S., C.H. Williams and G.C. Gray. 1971. Teratogenlc studies
with 2,4,5-1 and 2,4-D 1n the hamster. Bull. Environ. Contam. Toxlcol.
6(6): 559-567.
Conway, C.C. and F. Matsumura. 1975. Alteration of cellular utilization of
thymldine by KDD (?,3,7,8-tetrachlorod1benzo-p-d1ox1n). Bull. Environ.
Contam. Toxlcol. 13(1): 52-56.
Cook R.R. 1981a. D1ox1n, chloracne and soft-tissue sarcoma. Lancet. 1:
618-619.
1862A 15-14 03/29/84
-------
Cook, R.R. 1981b. Mortality experience of employees to 2,3,7,8-tetra-
chlorod1benzo-p-d1oxin (TCDD). Reply to comments. J. Occup. Med. 23(1): 8.
Cook, R.R., J.C. Townsend, M.G. Ott and L.G. S1lverste1n. 1980. Mortality
experience of employees exposed to 2,3,7,8-tetrachlorod1benzo-p-d1ox1n. J.
Occup. Med. 22{8): 530-532.
Cordle, F. 1981. The use of epidemiology In the regulation of dloxlns 1n
the food supply. Regulatory Toxlcol. Pharmacol. 1: 379-387.
Cordle, F. 1983. Use of epidemiology 1n the regulation of dloxlns In the
food supply. In: Accidental Exposure to Dloxlns: Human Health Aspects, F.
Coulston and F. Pocch1ar1, Ed. Academic Press, NY. p. 245-256.
Coulston, F. and E.J. Olajos. 1980. Panel Report: Panel to Discuss the
Epidemiology of 2,4,5-T. New York City, July 10-11, 1979. Ecotoxlcol.
Environ. Safety. 4: 96-102.
Courtney, K.D. 1976. Mouse teratology studies with chlorod1benzo-p-d1ox~
1ns. Bull. Environ. Contam. Toxlcol. 16(6): P674-681.
Courtney, K.D. 1977. Prenatal effects of herbicides evaluation by the
prenatal development Index. Arch. Environ. Contam. Toxlcol. 6(1): 33.
Courtney, K.D. and J.A. Moore. 1971. leratology studies with 2,4,5-T and
2,3,7,8-lCDD. Toxlcol. Appl. Pharmacol. 20: 396-403.
1862A 15-15 03/29/84
-------
Courtney, K.D., D.W. Gaylor, M.O. Hogan and H.L. Falk. 1970a. Teratogenlc
evaluation of pesticides: Large-scale screening study. Teratology. 3: 199.
Courtney, K.D., D.W. Gaylor, M.D. Hogan, M.L. Falk, R.R. Bates and I.
Mitchell. 1970b. Teratogenlc evaluation of 2,4,5-T. Science. 168:
864-866.
Courtney, K.D., J.P. Putnam and J.E. Andrews. 1978. Metabolic studies with
TCDD (dloxln) treated rats. Arch. Environ. Contam. Toxlcol. 7(4): 385-396.
Crampton, M.A. and L.J. Rogers. 1983. Low doses of 2,4,5-tMchlorophenoxy-
acetic acid are behavlorally teratogenlc to rats. Exper1ent1a. 39: 891-892.
Crosby, D.G. and A.S. Wong. 1976. Photochemical generation of chlorinated
dioxins. Chemosphere. 5: 327-332.
Crosby, D.G. and A.S. Wong. 1977. Environmental degradation of 2,3,7,8-
tetrachlorod1benzo-p-d1ox1n (TCDO). Science. 195: 1337-1338.
Crosby, D.G., A.S. Wong, J.R. PUmmer and E.A. Woolson. 1971. Photodecom-
posltlon of chlorinated d1benzo-p-d1ox1ns. Science. 173(3998): 748-749.
Crow, K. 1978a. Chloracne: The chemical disease. New Sci. 78(1098):
78-80.
Crow, K.D. 1978b. Chloracne -- An up to date assessment. J. Ann Occup.
Hyg. 21(3): 297-298.
1862A 15-16 03/29/84
-------
Crow, K.D. 1981. Chloracne and Us potential clinical Implications. Cl1n.
Exp. Dermatol. 6/3: 243-257.
Crummett, W.B. 1980. D1ox1n detection. Nature. 283: 330.
Crummett, W.B. 1983. Status of analytical systems for the determination of
PCDDs and PCDFs. Chemosphere. 12: 429-446.
Crummett, W.B. and R.H. Stehl. 1973. Determination of chlorinated
d1benzo-p-d1ox1ns and dlbenzofurans 1n various materials. Environ. Health
Perspect. 5: 15-25.
Crummett, W.B., R.R. Bumb, L.L. Lamparski, N.H. Mahle, 7.J. NestMck and
L.W. Whiting. 1981. Environmental chlorinated dloxlns from combustion: The
trace chemistries of fire hypothesis. In.: Impact of Chlorinated D1ox1ns and
Related Compounds 1n the Environment, 0. Hutzlnger et al., Ed. Pergamon
Press, Oxford, p. 253-263.
Crump, K.S. and W.W. Watson. 1979. GLOBAL 79: A Fortran program to
extrapolate dlchotomous animal carclnogenldty data to low dose. NIEHS,
Contract No. I-ES-2123.
Crump, K.S., H.A. Glvess and L.L. Deal. 1977. Confidence Intervals and
test of hypothesis concerning dose-response relations Inferred from animal
carclnogenlcity data. Biometrics. 33: 437-451.
1862A 15-17 03/29/84
-------
Cunningham, H.M. and D.T. Williams. 1972. Effect of 2,3,7,8-tetrachloro-
dibenzo-p-dioxin on growth rate and the synthesis of I1p1ds and proteins in
rats. Bull. Environ. Contam. Toxlcol. 7(1): 45-51.
CupHt, L.T. 1980. Fate of Toxic and Hazardous Materials 1n the A1r Envi-
ronment. EPA EPA 600/3-80-084, Environmental Sciences Research Laboratory,
Research Triangle Park, NC.
Cutle, S.S. 1981. Recovery efficiency of 2,3,7,8-tetrachlorod1benzo-p-
dioxln from active carbon and other partlculates. Anal. Chlm. Acta. 123:
25-31.
Czelzel, E. and J. Klraly. 1976. Chromosome examinations 1n workers
producing Klorlnol and Bumlnol. In: The Development of a Pesticide as a
Complex Scientific Task, L. Bankl, Ed. Medldna: Budapest. p. 239-256.
(Cited in NRCC, 1981a)
Dees, J.H., B.S. Masters, U. Muller-Eberhard and E.F. Johnson. 1982.
Effect of 2,3,7,8-tetrachlorod1benzo-p-d1oxin and phenobarbltal on the
occurrence and distribution of four cytochrome P-450 Isozymes 1n rabbit
kidney, lung, and liver. Cancer Res. 42(4): 1423-1432.
Deitrich, R.A., P. Bludeau, T. Stock and M. Roper. 1977. Induction of
different rat liver supernatant aldehyde dehydrogenases by phenobarbltal and
tetrachlorodibenzo-p-dioxin. J. B1ol. Chem. 252(17): 6169-6176.
1862A 15-18 03/29/84
-------
Delgado, S. 1983. Division of Regulatory Guidance, Case and Advisory
Branch, U.S. FDA. Personal communication.
Department of Health, New Zealand. 1980. Report to the Minister of Health
of an Investigation Into allegations of an association between human
congenital defects and 2,4,5-T spraying 1n and around Te Ku1t1. New Zealand
Med. J. p. 314-315.
De Verneull, H., S. Sassa and A. Kappas. 1983. Effects of polychloMnated
blphenyl compounds, 2,3,7,8-tetrachlorod1benzo-p-d1ox1n, phenobarbltal and
Iron on hepatic uroporphyrlnogen decarboxylase. Blochem. J. 214: 145-151.
Dickens, M., M.D. Seefeld and R.E. Peterson. 1981. Enhanced liver DNA
synthesis 1n partially hepateotomlzed rats pretreated with 2,3,7,8-tetra-
chlorod1benzo-p-d1ox1n. Toxlcol. Appl. Pharmacol. 58(3): 389-398.
D1Domen1co, A., V. Sllano, G. V1v1ano and G. Zapponl. 1980a. Accidental
release of 2,3,7,8-tetrachlorod1benzo-p-d1ox1n (TCDD) at Seveso, Italy.
I. Sensitivity and specificity of analytical procedures adopted for TCDD
assay. Ecotoxlcol. Environ. Safety. 4(3): 283-297.
D1Domen1co, A., V. Sllano, G. V1v1ano and G. Zapponl. 1980b. Accidental
release of 2,3,7,8-tetrachlorod1benzo-p-d1ox1n (TCDD) as Seveso, Italy.
VI. TCDD levels 1n atmospheric particles. Ecotoxlcol. Environ. Safety.
4(3): 346-356.
1862A 15-19 03/29/84
-------
DIDomenico, A., V. Sllano, 6. Vivlano and G. Zapponi. 1980c. Accidental
release of ?,3,7,8-tetrachlorod1benzo-p-d1oxin (TCDD) at Seveso, Italy.
II. TCDD distribution 1n the soil surface layer. Ecotoxlcol. Environ.
Safety. 4(3): 298-320.
DIDomenico, A., V. Sllano, G. Vlvlano and G. Zapponi. 1980d. Accidental
release of 2,3,7,8-tetrachlorod1benzo-p-d1oxin (TCDD) 1n Seveso, Italy.
V. Environmental persistence of TCDO In soil. Ecotoxlcol. Environ. Safety.
4(3): 339-345.
DIDomenico, A., V. Sllano, G. Vlvlano and G. Zapponi. 1980e. Accidental
release of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) at Seveso, Italy.
IV. Vertical Distribution of ICDD in soil. Ecotoxlcol. Environ. Safety.
4(3): 327-338.
DiDomenico, A., G. Viviano and G. Zapponi. 1982. Environmental persistence
of 2,3,7,8-TCDD at Seveso. Ijn: Chlorinated Dioxins and Related Compounds:
Impact on the Environment, 0. Hutzlnger et al., Ed. Pergamon Press, NY.
p. 105-114.
Dietrich, R.A., R. Bludeau, M. Roger and J. Schmuck. 1978. Induction of
aldehyde dehydrogenases. Biochem. Pharmacol. 27: 2343-2347.
DiGiovanni, J., A. Viaje, D.L. Berry, T.J. Slaga and M.R. Juchau. 1977.
Tumor-Initiating ability of 2,3,7,8-tetrachlorod1benzo-p-dioxin (TCDD) and
arochlor 1254 in the two-stage system of mouse skin carclnogenesls. Bull.
Environ. Contam. Toxlcol. 18(5): 552-557.
1862A 15-20 03/29/84
-------
D1G1ovanni, J., O.L. Berry, M.R. Juchau and T.J. Slaga. 1979a. 2,3,7,8-
Tetrachlorod1benzo~p-d1ox1n: Potent antlcardnogenlc activity 1n CD-I mice.
Blochem. Blophys. Res. Commun. 86(3): 577-584.
DlGlovann1, J., D.L. Berry, T.J. Slaga, A.M. Jones and M.R. Juchau. 1979b.
Effects of pretreatment with 2,3,7,8-tetrachlorod1benzo-p-d1ox1n on the
capacity of hepatic and extrahepatlc mouse tissues to convert procardnogens
to mutagens for Salmonella typh1mur1um auxotrophs. Toxlcol. Appl.
Pharmacol. 50(2): 229-239.
D1G1ovann1, J., O.L. Berry, G.L. Gleason, G.S. Klshore and T.J. Slaga.
1980. Time-dependent Inhibition by 2,3,7,8-tetrachlorod1benzo-p-d1ox1n of
skin tumorigenesis with polycycllc hydrocarbons. Cancer Res. 40(5):
1580-1587.
DUernla, R., C. Crimaudo and G. Pacchettl. 1982. The study of X-rays and
TCDD effects on satellite associations may suggest a simple model for
application In environmental mutagenesls. Hum. Genet. 61(1): 42-47.
Oobbs, A.J. and C. Grant. 1979. Photolysis of highly chlorinated
d1benzo-p-d1ox1ns by sunlight. Nature. 278: 163-165.
Dougherty, W.J., M. Herbst and F. Coulston. 1975. The non-teratogenldty
of 2,4,5-trlchlorophenoxyacetlc add In the rhesus monkey, Macaca mulatta.
Bull. Environ. Contam. Toxlcol. 13: 477-482.
1862A 15-21 03/29/84
-------
Dow Chemical Co. 19/8. The trace chemistries of fire -- A source of and
routes for the entry of chlorinated dloxlns Into the environment. The
Chlorinated D1ox1n Task Force, the Michigan Division, Dow Chemical, U.S.A.
Duran-Reynolds, M., F. Llllie, H. Bartsch and K.J. Blank. 1978. The
genetic basis of susceptibility to leukemia Induction 1n mice by 3-methyl-
cholanthrene applied subcutaneously. J. Exp. Med. 147: 459-469.
Edllng, C. and S. Granstam. 1979. XXXVI. National Conference of the
Medical Society sponsored by the Swedish Medical Society at Stockholmsmasan,
Alvifi, Stockholm, December 5-8, 1979. Summaries transaction of the Swedish
Medical Society. 88(3): 73-74. (translation)
Elceman, G.A., R.E. Clement and F.W. Karasek. 1979. Analysis of fly ash
from municipal Incinerators for trace organic compounds. Anal. Chem.
5.1(14): 2343-2350.
Elceman, G.A., A.C. V1au and F.W. Karasek. 1980. Ultrasonic extraction of
polychlorInated d1benzo-p-d1ox1ns and other organic compounds from fly ash
from municipal Incinerators. Anal. Chem. 52: 1492-1496.
Elceman, G.A., R.E. Clement and F.W. Karasek. 1981. Variations 1n concen-
trations of organic compounds Including polychlorlnated d1benzo-p-d1ox1ns
and polynuclear aromatic hydrocarbons 1n fly ash from a municipal Inciner-
ator. Anal. Chem. 53(7): 955-959.
1862A 15-22 03/29/84
-------
Elovaara, E., H. Savolainen, M.G. Parkkl, A. AH1o and H. Vainio. 1977.
Neurochemical effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin in Wistar and
Gunn rats. Research Commun. Chem. Pathol. Pharmacol. 18: 487-494.
Emerson, J.L., O.J. Thompson, C.G. Gerbig and V.B. Robinson. 1970. Terato-
genic study of 2,4,5-tMchlorophenoxyacetlc add 1n the rat. Toxlcol. Appl.
Pharmacol. 17: 317.
Emerson, J.L., O.J. Thompson, R.J. Streblng, C.G. Gerbig and B. Robinson.
1971. Teratogenlc studies of 2,4,5-T 1n the rat and rabbit. Food Cosmet.
Toxlcol. 9: 395.
Eriksson, M., L. Hardell, N.O. Berg, T. Moller and 0. Axelson. 1979.
Case-control study on malignant mesenchymal tumors of the soft-tissue and
exposure to chemical substances. Lakartldnlngen. 76: 3872-3875. (trans-
lation)
Eriksson, M., L. Hardell, N. O'Berg, T. Moller and 0. Axelson. 1981.
Soft-tissue sarcomas and exposure to chemical substances: A case-referent
study. Br. J. Ind. Med. 38: 27-33.
Facchetti, S., A. Fornari and M. Montagna. 1980. Distribution of
2,3,7,8-tetrachlorod1benzo-p-diox1n in the tissues of a person exposed to
the toxic cloud at Seveso (Italy). Adv. Mass Spectrom. 8B: 1405-1414.
Faith, R.E. and M.I. Luster. 1979. Investigations of the effects of
2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on parameters of various immune
functions. Ann. NY Acad. Sci. 320: 564-571.
1862A 15-23 03/29/84
-------
Fanelli, R., C. Chlabrando, M. Salmona, S. Garatt1n1 and P.6. Caldera.
1978. Degradation of 2,3,7,8-tetrachlorod1benzo-p-d1ox1n 1n organic
solvents by gamma ray Irradiation. Exper1ent1a. 34: 1126-1127.
Fanelli, R., M.P. Bertonl, M. Bonfantl, et al. 1980a. Routine analysis of
2,3,7,8-tetrachlorod1benzo-p-d1ox1n 1n biological samples from the contami-
nated area of Seveso, Italy. Bull. Environ. Contam. Toxlcol. 24(6):
818-823.
Fanelli, R., M.P. Bertonl, M.G. Castelll, et al. 1980b. 2,3,7,8-Tetra-
chlorod1benzo-p-d1ox1n toxic effects and tissue levels 1n animals from the
contaminated area of Seveso, Italy. Arch. Environ. Contam. Toxlcol. 9(5):
569-577.
Fanelli, R., M.G. Castelll, G.P. Martelll, A. Noseda and S. Garatt1n1.
1980c. Presence of 2,3,7,8-tetrachlorod1benzo-p-d1ox1n 1n wildlife living
near Seveso, Italy: A preliminary study. Bull. Environ. Contam. Toxlcol.
24(3): 460-462.
Fanelli, R., C. Chlabrando and A. Bonaccorsl. 1982. TCDO contamination 1n
the Seveso Incident. Drug Metab. Rev. 13: 407-422.
FDA (Food and Drug Administration). 1981. FDA advises Great Lakes states
to monitor d1ox1n-contam1nated fish. FDA talk paper dated August 28. In:
Food Drug Cosmetic Law Reports, Paragraph 41, 321. Commerce Clearing House,
Inc. September 8.
1862A 15-24 03/29/84
-------
FDA (Food and Drug Administration). 1983. Statement by S.A. Miller,
Director, Bureau of Foods, FDA, before The Subcommittee on Natural
Resources, Agriculture Research and Environment, U.S. House of
Representatives, June 30.
Federal Register. 1971. EPA Method 5. Determination of Participate
Emissions from Stationary Sources. 36: 24876-24895. December 23.
Federal Register. 1980a. Storage and Disposal of Waste Material;
Prohibition of Disposal of Tetrachlorod1benzo-p-D1ox1n. 45(98): 32676.
Federal Register. 1980b. Water Quality Criteria Documents; Availability.
45(231): 79318-79379.
Field, B. and C. Kerr. 1979. Herbicide use and Incidence of neural-tube
defects. Lancet. 1(8130): 1341-1342.
Flnney, D.J. 1971. Problt Analysis. Cambridge University Press, London.
333 p.
Firestone, D. 1973. Etiology of chick edema disease. Environ. Health
Perspect. 5: 59-66.
Firestone, D. 1977a. Chemistry and analysis of pentachlorophenol and Us
contaminants. FDA By-I1nes. 2: 57-89.
1862A 15-25 03/29/84
-------
Firestone, 0. 197/b. Determination of polychlorod1benzo-p-d1ox1ns and
polychlorodlbenzofurans 1n commercial gelatins by gas-liquid chromatography.
J. Agrlc. Food Chem. 25: 1274-1280.
Firestone, D., J. Ress, N.L. Brown, R.P. Barron and J.N. Damlco. 1972.
Determination of polychlorod1benzo-p-d1ox1ns and related compounds In
commercial chlorophenols. J. Assoc. Off. Anal. Chem. 55(1): 85-92.
Firestone, D., M. Clower, Jr., A.P. Borsettl, R.H. Teske and P.E. Long.
1979. Polychlorod1benzo-p-d1ox1n and pentachlorophenol residues 1n milk and
blood of cows fed technical pentachlorophenol. J. Agrlc. Food Chem. 27(6):
1171-1177.
Fowler, B.A., G.W. Lucier, H.W. Brown and O.S. McDanlel. 1973. Ultrastruc-
tural changes 1n rat liver cells following a single oral dose of TCDD.
Environ. Health Perspect. 5: 141-148.
Fries, G.F. and G.S. Marrow. 1975. Retention and excretion of 2,3,7,8-
tetrachlorod1benzo-p-diox1n by rats. J. Agrlc. Food Chem. 23(2): 265-269.
Garattini, S. 1982. TCDD toxicology with particular reference to Seveso:
Introductory remarks. Drug Metab. Rev. 13(3): 345-353.
Gaslewicz, l.A. and R.A. Neal. 1979. 2,3,7,8-Tetrachlorod1benzo-p-d1ox1n
tissue distribution, excretion, and effects on clinical chemical parameters
in guinea pigs. Toxlcol. Appl. Pharmacol. 51(2): 329-340.
1862A 15-26 03/29/84
-------
Gas1ew1cz, l.A. and R.A. Neal. 1982. The examination and quantHatlon of
tissue cytosollc receptors for 2,3,7,8-tetrachlorod1benzo-p-d1ox1n using
hydroxyapatlte. Anal. Blochem. 124: 1-11.
Gaslewkz, l.A., M.A. Holscher and R.A. Neal. 1980. The effect of total
parenteral nutrition on the toxldty of 2,3,7,8-tetrachlorod1benzo-p-d1ox1n
In the rat. Toxlcol. Appl. Pharmacol. 54: 469-488.
Gaslewlcz, T.A., J.R. Olson, I.E. Gelger and R.A. Neal. 1983a. Absorption,
distribution and metabolism of 2,3,7,8-tetrachlorod1benzo-p-d1ox1n (TCDD) 1n
experimental animals. in: Human and Environmental Risks of Chlorinated
D1ox1ns and Related Compounds, R.E. Tucker, A.L. Young and A.P. Gray, Ed.
Plenum Press, NY. p. 495-525.
Gaslewlcz, l.A., L.E. Gelger, G. Rucci and R.A. Neal. 1983b. Distribution,
excretion, and metabolism of 2,3,7,8-tetrachlorod1benzo-p-d1ox1ns 1n
C57B1/6J, DBA/2J and B6D2F /J mice. Drug Metab. Dlspos. (In press)
Gebefuegl, 1., R. Baumann and F. Korte. 1977. Photochemical degradation of
2,3,7,8-tetrachlorod1benzo-p-d1ox1n (TCDD) under simulated environmental
conditions. Naturwlssenschaften. 64(9): 486-487.
Gelger, L.E. and R.A. Neal. 1981. Mutagen1c1ty testing of 2,3,7,8-tetra-
chlorod1benzo-p-d1ox1n 1n h1st1d1ne auxotrophs of Salmonella typh1mur1um.
loxlcol. Appl. Pharmacol. 59(1): 125-129.
1862A 15-27 03/29/84
-------
Ghezzi, I., P. Cannatelll, G. Assennato, et al. 1982. Potential 2,3,7,8-
tetrachlorod1benzo-p-d1ox1n exposure of Seveso decontamination workers: A
controlled prospective study. Scand. J. Work Environ. Health. 8(Suppl. 1):
176-179.
Glanotti, F. 1977. Chloracne due to tetrachloro-2,3,7,8-d1benzo-p-d1ox1n
In children. Ann. Oermatol. Venereol. 104(12): 825-829.
G1av1n1, E., M. Pratl and C. Vlsmara. 1982a. Effects of 2,3,7,8-tetra-
chlorod1benzo-p-d1ox1n administered to pregnant rats during the prelmplanta-
tlon period. Environ. Res. 29(1): 185-189.
G1av1ni, E., M. Prati and C. Vlsmara. 1982b. Rabbit teratology study with
2,3,7,8-tetrachlorod1benzo-p-d1ox1n. Environ. Res. 27(1): 74-78.
G1av1ni, E., M. Pratl and C. Vlsmara. 1983. Embryotoxlc effects of
2,3,7,8-tetrachlorod1benzo~p-d1ox1n administered to female rats before
mating. Environ. Res. 31: 105-110.
Gilbert, P., G. Salnt-Ruf, F. Poncelet and M. Herder. 1980. Genetic
effects of chlorinated anilines and azobenzenes on Salmonella typhlmurlum.
Arch. Environ. Contam. Toxlcol. 5(5): 533-541.
G1zz1, F., R. Reglnato, E. Benfenatl and R. Fanelll. 1982. Polychlorlnated
d1benzo~p-d1ox1ns (PCOO) and polychloMnated dlbenzofurans (PCDF) 1n emis-
sions from an urban Incinerator. I. Average and peak values. Chemosphere.
II: 577-583.
1862A 15-28 03/29/84
-------
Glaser, J.A., O.L. Foerst, G.O. McKee, S.A. Quave and W.L. Budde. 1981.
Trace analyses for wastewaters. Environ. Sc1. Technol. 15(12): 1426.
Goldstein, J.A., P. Hickman, H. Bergman, J.D. McKlnney and M.P. Walker.
1977. Separation of pure polychloMnated blphenyl Isomers Into two types of
Inducers on the basis of Induction of cytochrome P-450 or P-448. Chem.-
B1o1. Interact. 17: 69-87.
Goldstein, J.A., M. Frlesen, T.M. Scottl, P, Hickman, J.R. Hass and H. Berg-
man. 1978. Assessment of the contribution of chlorinated d1benzo-p-d1oxlns
and dlbenzofurans to hexachlorobenzene-lnduced toxldty, porphyrla, changes
In mixed function oxygenases, and histopathologlcal changes. Toxicol. Appl.
Pharmacol. 46(3): 633-649.
Goldstein, J.A., P. Linko, J.N. Hucklns and D.L. Stalling. 1982a.
Structure-activity relationships of chlorinated benzenes as Inducers of
different forms of cytochrome P-450 1n rat liver. Chem. Biol. Interact.
41(2): 131-139.
Goldstein, J.A., P. Linko and H. Bergman. 1982b. Induction of porphyrla 1n
the rat by chronic versus acute exposure to 2,3,7,8-tetrachlorodlbenzo-p-
dloxln. Blochem. Pharmacol. 31(8): 1607-1613.
Gorski, T. 1981. Presence of polychlorlnated d1benzo-p-d1oxins 1n latex
nipples. Bull. Environ. Contam. Toxicol. 27: 68-71.
1862A 15-29 03/29/84
-------
Gray, A.P., S.P. Cepa and J.S. Cantrell. 1975. Intervention of the Smiles
rearrangement 1n synthesis of d1benzo-p-d1ox1ns: 1,2,3,6,7,8- and
l,2,3,7,8,9-hexachlorod1benzo-p-d1ox1n (HCOD). Tetrahedron Lett. 33:
2873-2876.
Gray, A.P., S.P. Cepa, I.J. Solomon and 0. Aniline. 1976. Synthesis of
specific polychlorlnated d1benzo-p-d1ox1ns. J. Org. Chem. 41: 2435-2437.
Green, S. and F.S. Moreland. 1975. Cytogenetlc evaluation of several
dloxlns 1n the rat. Toxlcol. Appl. Pharmacol. 33: 161.
Green, S., F. Moreland and C. Sheu. 1977. Cytogenlc effect of 2,3,7,8-
tetrachlorod1benzo-p-d1ox1n on rat bone marrow cells. U.S. FDA, Washington,
DC. FDA By-Lines. 6: 292.
Greenlee, W.F. and A. Poland. 1978. An Improved assay of 7-ethoxycoumar1n
0-deethylase activity: Induction of hepatic enzyme activity In C57B1/6J and
DBA/2J mice by phenobarbital, 3-methylcholanthrene and 2,3,7,8-tetrachloro-
d1benzo-p-d1ox1n. J. Pharmacol. Exp. Ther. 205(3): 596-605.
Greenlee, W.F. and A. Poland. 1979. Nuclear uptake of 2,3,7,8-tetrachloro-
dlbenzo-p-dloxln in C57B1/6J and DBA/2J mice. Role of the hepatic cytosol
receptor protein. J. B1ol. Chem. 254(19): 9814-9821.
Grelg, J.B. 1972. Effect of 2,3,7,8-tetrachlorod1benzo-l,4-d1ox1n on drug
metabolism In the rat. Blochem. Pharmacol. 21(23): 3196-3198.
1862A 15-30 03/29/84
-------
Greig, J.B., G. Jones, W.H. Butler and J.M. Barnes. 1973. Toxic effects of
2,3,7,8-tetrachlorod1benzo-p-d1ox1n. Food Cosmet. Toxlcol. 11: 585-595.
Greig, J.B., D.M. Taylor and J.D. Jones. 1974. Effects of 2,3,7,8-tetra-
chloro-d1benzo-p-d1ox1n on stimulated DNA synthesis In the liver and kidney
of the rat. Chem. B1ol. Interact. 8(1): 31-39.
Gross, M.L., T. Sun, P.A. Lyon, et al. 1981. Method validation for the
determination of tetrachlorod1benzod1ox1n at the low parts-per-trllllon
level. Anal. Chem. 53(12): 1902-1906.
Guenthner, T.M., J.M. Fysh and O.W. Nebert. 1979a. 2,3,7,8-Tetrachloro-
dibenzo-p-d1ox1n: Covalent binding of reactive metabolic Intermediates
principally to protein jm vitro. Pharmacology. 19: 12-22.
Guenthner, T.M., D.W. Nebert and R.H. Menard. 1979b. Mlcrosomal aryl
hydrocarbon hydroxylase in rat adrenal: Regulation by ACTH but not by
polycycllc hydrocarbons. Mol. Pharmacol. 15(3): 719-728.
Gupta, B.N., J.G. Vos, J.A. Moore, J.G. Z1nkl and B.C. Bullock. 1973.
Pathologic effects of 2,3,7,8-tetrachlorodibenzo-p-d1ox1n in laboratory
animals. Environ. Health Perspect. 5: 125-140.
Gustafsson, J.A. and M. Ingelman-Sundberg. 1979. Changes 1n steroid
hormone metabolism in rat liver mlcrosomes following administration of
2,3,7,8-tetrachlorod1benzo-p-dioxin (TCDD). Biochem. Pharmacol. 28(4):
497-499.
1862A 15-31 03/29/84
-------
Halley, C.L., J.S. Stanley, A.M. Magln, R.V. Northcutt and D.P. Redford.
1983. Emissions of Organic Pollutants from Coal-Fired Utility Boiler
Plants. Presented at the ACS Annual Meeting, Seattle, WA. March 20-25.
Hajdu, S.I. 1983. Classification of soft tissue tumors and tumor-like
lesions. Presented at Symposium on Public Health Risks of the D1ox1ns,
Rockefeller Univ., October 19-20.
Hallett, D. 1984. Environment Canada, Toronto, Ontario. Private communi-
cation with Or. S.H. Safe, Texasd A&M Univ., College Station, TX.
Hanlfy, J.A., P. Metcalf, C.L. Nobbs and R.J. Worsley. 1981. Aerial spray-
ing of 2,4,5-1 and human birth malformations: An ep1dem1olog1cal Investiga-
tion. Science. 212: 349-351.
Hardell, L. 1977. Malignant mesenchymal tumors and exposure to phenoxy-
acids: A clinical observation. Lakart1dn1ngen. 74: 2753-2754. (transla-
tion)
Hardell, L. 1979. Malignant lymphoma of h1st1ocyt1c type and exposure to
phenoxyacetic adds or chlorophenols. Lancet. 1: 55-56.
Hardell, L. 1981. Relation of soft-tissue sarcoma, malignant lymphoma and
colon cancer to phenoxy acids, chlorophenols and other agents. Scand. J.
Work Environ. Health. 7: 119-130.
1862A 15-32 03/29/84
-------
Hardell, L. 1983. letter to D. Bayllss, Carcinogen Assessment Group, U.S.
EPA, Washington, DC, August 5.
Hardell, L. and M. Eriksson. 1981. Soft-tissue sarcomas, phenoxy herbi-
cides, and chlorinated phenols. Lancet. 2(8240): 8/1/81.
Hardell, L. and A. Sandstrom. 1979. Case-control study: Soft-tissue sar-
comas and exposure to phenoxyacetlc adds or chlorophenols. Br. J. Cancer.
39: 711-717.
Hardell, L., M. Eriksson and P. Lenner. 1980. Malignant lymphoma and
exposure to chemical substances, especially organic solvents, chlorophenols,
and phenoxy adds. Lakart1dn1ngen. 77: 208-210. (translation)
Hardell, L., M. Eriksson, P. Lenner and E. Lundgren. 1981. Malignant
lymphoma and exposure to chemicals, especially organic solvents, chloro-
phenols and phenoxy acids: A case-control study. Br. J. Cancer. 43:
169-176.
Harless, R. 1981. Internal memo to M. Oellarco. Office of Toxic Sub-
stances, U.S. EPA, Washington, DC.
Harless, R.L. and R.G. Lewis. 1980a. Quantitative capillary column gas
chromatography-mass spectrometry methods of analysis for toxic organic
compounds. Sym.: Practical Solutions to Quantitative Capillary Column Gas
Chromatography. Cong.: Anal. Chem. Appl. Spectro., Atlantic City, NJ.
March, 1980.
1862A 15-33 03/29/84
-------
Harless, R.L. and R.G. Lewis. 1980b. Quantitative determination of
?,3,7,8-tetrachlorod1benzo-p-d1ox1n residues by gas chromatography/mass
spectrometry. Presented at Workshop on Impact of Chlorinated D1ox1ns and
Related Compounds on the Environment. Institute Superlore d1 Sanlta, Rome,
Italy. October 22-24.
Harless, R.L. and R.G. Lewis. 1982. Quantitative determination of 2,3,7,8-
tetrachlorod1benzo-p-d1ox1n (TCDO) Isomers. In: Chlorinated D1ox1ns and
Related Compounds. Impact on the Environment, 0. Hutzlnger, R.W. Fre1, E.
Merlan and F. Pocch1ar1, Ed. Pergamon Press, NY. p. 25-35.
Harless, R.L. and E.O. Oswald. 1978. Gas chromatograpy/mass spectrometry
Interface for glass capillary columns. Paper presented at the 26th Annual
Conference Mass Spectrometry. May 28-June 2, 1978.
Harless, R.L., E.O. Oswald, M.K. Wilkinson, et al. 1980. Sample prepara-
tion and gas chromatography-mass spectrometry determination of 2,3,7,8-
tetrachlorodibenzo-p-d1ox1n. Anal. Chem. 52(8): 1239-1245.
Harris, H.W., J.A. Moore, J.G. Vos and B.N. Gupta. 1973. General biologi-
cal effects of TCDD 1n laboratory animals. Environ. Health. Perspect. 5:
101-109.
Harvan, O.J., J.R. Mass, J.L. Schroeder and B.J. Corbett. 1981. Detection
of tetrachlorod1benzod1ox1ns 1n air filter samples. Anal. Chem. 53(12):
1/55-1759.
1862A 15-34 03/29/84
-------
Mass, J.R. and M.D. Frlesen. 1979. Qualitative and quantitative methods
for dloxln analysis. Ann. NY Acad. Sc1. 320: 28-42.
Mass, J.R., M.D. Frlesen, O.J. Harvan and C.E. Parker. 1978. Determination
of polychlorlnated d1benzo-p-d1ox1ns In biological samples by negative
chemical 1on1zat1on mass spectrometry. Anal. Chem. 50(11): 1474-1479.
Hassoun, E.M. and L. Oencker. 1982. TCOO embryotoxldty 1n the mouse may
be enhanced by B-naphthflovone, another Ugand of the Ah-receptor. Toxic.
Lett. 12: 191-198.
Hawkes, C.L. and L.A. Morris. 1977. Chronic oral toxldty of 2,3,7,8-
tetrachlorod1benzo-p-d1ox1n (TCDD) to rainbow trout. Trans. Am. F1sh Soc.
106(6): 641-645.
Hay, A. 1979. Dispute over Dow Chemical's theory of dloxln traces.
Nature. 281: 619-620.
Hay, A. 1982. Toxicology of dloxlns. In: The Chemical Scythe: Lessons of
2,4,5-T and D1ox1n. Plenum Press, NY and London, p. 41-47.
Helda, H. 1983. TCDD In bottom sediments and eel around a refuse dump near
Amsterdam, Holland. Chemosphere. 12: 503-509.
Helder, T. 1980. Effects of 2,3,7,8-tetrachlorod1benzo-p-d1ox1n (TCDD) on
early life stages of the pike (Esox luclus L.). Sc1. Total Environ. 14(3):
255-264.
1862A 15-35 04/05/84
-------
Helder, 1. 1981. Effects of 2,3,7,8-tetrachlorod1benzo-p-dioxin (TCDO) on
early life stages of rainbow trout (Salmo qairdneri. Richardson). Toxlcol.
19(2): 101-112.
Helling, C.S., A.R. Isensee, E.A. Woolson, et al. 1973. Chlorod1ox1ns 1n
pesticides, soils and plants. J. Environ. Quality. 2(2): 171-178.
Henck, J.W., M.A. New, R.J. Kodba and K.S. Rao. 1981. 2,3,7,8-Tetra-
chlorod1benzo-p-d1ox1n: Acute oral toxldty In hamsters. Toxlcol. Appl.
Pharmacol. 59: 405-407.
Hildebrandt, P.K. 1983. Letter to E.E. McConnell, NTP, October 31, 1983,
with attached tables.
Miles, R.A. and R.D. Bruce. 1976. 2,3,7,8-Tetrachlorod1benzo-p-d1ox1n
elimination in the rat: First order or zero order? Food Cosmet. Toxicol.
14(6): 599-600.
Hlnsdill, R.D., D.L. Couch and R.S. Spelrs. 1980. Immunosuppression in
mice induced by dloxln (1COD) 1n feed. J. Environ. Pathol. Toxlcol.
4(2-3): 401-425.
Holmstedt, B. 1980. Prolegomena to Seveso Ecclesiastes I 18. Arch.
Toxlcol. 44(4): 211-230.
Honchar, P.A. and W.E. Halperin. 1981. 2,4,5-Trichlorophenol and
soft-tissue sarcoma. Lancet. 1(8214): 268-269.
1862A 15-36 03/29/84
-------
Hook, G.t.R., J.K. Haseman and G.W. Luder. 1975a. Induction and suppres-
sion of hepatic and extrahepatlc mlcrosomal fore1gn-compound-metabol1z1ng
systems by 2,3,7,8-tetrachlorod1benzo-p-d1oxin. Chem.-B1ol. Interact.
10(3): 199-214.
Hook, G.E.R., l.C. Orton, J.A. Moore and G.W. Lucier. 1975b. Tetrachloro-
d1benzo~p-diox1n-1nduced changes 1n the hydroxylatlon of blphenyl by rat
liver mlcrosomes. Blochem. Pharmacol. 24(3): 335-340.
Hook, J.B., K.M. McCormack and W.M. Kluwe. 1977. Renal effects of
2,3,7,8--tetrachlorodibenzo-p-d1oxin. in: Pentachlorophenol, K.R. Rao, Ed.
Plenum Press, NY. p. 381-387.
Houk, V.N. 1983. Testimony by Dr. Vernon W. Houk, Director, Center for
Environmental Health, Center for Disease Control, Public Health Service,
Department of Health and Human Services before the United States House of
Representatives Science and Technology Committee, Subcommittee on Natural
Resources, Agricultural Research and Environment. March 23.
Hryhorczuk, D.O., W.A. WUhrow, C.S. Hesse and V.R. Beasley. 1981. Wire
reclamation incinerator as a source of environmental contamination with
tetrachlorodibcnzo-p-d1oxins and tetrachlorodlbenzofurans. Arch. Environ.
Health. 36(5): 228-234.
Huckins, J.N., D.L. Stalling and W.A. Smith. 1978. Foam-charcoal chroma-
tography for analysis of polychlorlnated d1benzod1ox1ns in Herbicide Orange.
Assoc. Off. Anal. Chem. 61(1): 32-38.
1862A 15-37 03/29/84
-------
Hueper, W.C. and W.D. Conway. 1964. Chemical Carclnogenesls and Cancers.
Springfield, Thomas.
Huetter, R. and M. Phillppi. 1982. Studies on mlcroblal metabolism of TCOO
under laboratory conditions. Pergamon Ser. Environ. Sc1. 5: 87-93.
Hummel, R.A. 1977. Clean-up techniques for the determination of parts per
trillion residue levels of 2,3,7,8-tetrachlorod1benzo-p-d1ox1n (TCDO). J.
Agrlc. Food Chem. 25: 1049-1053.
Hummel, R.A. and L.A. Shadoff. 1980. Specificity of low resolution gas
chromatography-low resolution mass spectrometry for the detection of tetra-
chlorod1benzo-p-d1ox1n 1n environmental samples. Anal. Chem. 52: 191-192.
Hussaln, S., L. Ehrenberg, G. Lofroth and 1. Gejvall. 1972. Mutagenlc
effects of 1CDD on bacterial systems. Amblo. 1: 32-33.
Hutzlnger, 0., K. Olie, J.W. Lustenhouwer, A.B. Okey, S. Bandlera and S.
Safe. 1981. Polychlorinated d1benzo-p-d1ox1ns and dlbenzofurans: A bio-
analytical approach. Chemosphere. 10(1): 19-25.
Hwang, S.W. 1973. Effect of 2,3,7,8-tetrachlorod1benzo-p-d1ox1n on the
biliary excretion of Indocyanlne green 1n rat. Environ. Health Perspect.
5: 227-231.
1862A 15-38 03/29/84
-------
IARC (International Agency for Research on Cancer). 1977. IARC Monographs
on the Evaluation of the Carcinogenic Risk of Chemicals to Man. Some Fum1-
gants, the Herbicides 2,4-D and 2,4,5-T, Chlorinated D1benzod1ox1ns and
Miscellaneous Industrial Chemicals. Vol. 15, IARC, Lyon, France, p. 41-102.
Ideo, G., G. Bellatl, A. Bellobuono, P. Mocarelll, A. Marocchl and P.
Brambllla. 1982. Increased urinary D-glucar1c add excretion by children
living 1n an area polluted with tetrachlorod1benzoparad1oxin (TCDD). Cl1n.
Chlm. Acta. 120(3): 273-283.
ICO (International Classification of Diseases). 1975. 9th revision. WHO,
Geneva.
Isensee, A.R. 1978. B1oaccumulat1on of 2,3,7,8-tetrachlorod1benzo-para-
dioxln. Ecol. Bull. C. Ramel, Ed. 27: 255-262.
Isensee, A.R. and G.E. Jones. 1971. Absorption and translocatlon of root
and foliage applied 2,4-d1chlorophenol, 2,7-d1chlorod1benzo-p-d1ox1n, and
2,3,7,8-tetrachlorod1benzo-p-d1ox1n. J. Agrlc. Food Chem. 19: 1210-1214.
Isensee, A.R. and G.E. Jones. 1975. Distribution of 2,3,7,8-tetrachlorodl-
benzo-p-d1ox1n (TCDD) 1n aquatic model ecosystem. Environ. Sc1. Technol.
9: 668-672.
Ishldate, K., M. Isuruoka and Y. Nakazawa. 1980. Induction of chollne
klnase by polycycllc aromatic hydrocarbon carcinogens 1n rat Hver. B1o-
chem. Blophys. Res. Commun. 96: 946-952.
1862A 15-39 03/29/84
-------
Jackson, W.T. 1972. Regulation of mitosis. III. Cytologlcal effects of
2,4,5-tr1chlorophenoxyacet1c add and of dloxln contaminants In 2,4,5-T
formulations. J. Cell Scl. 10: 15-25.
Jensen, D.J. and R.A. Hummel. 1982. Secretion of TCOD 1n milk and cream
following the feeding of TCDD to lactatlng dairy cows. Bull. Environ.
Contam. Toxlcol. 29: 440-446.
Jensen, D.J., R.A. Hummel, N.H. Mahle, C.W. Kocher and H.S. H1gg1ns. 1961.
Residue study on beef cattle consuming 2,3,7,8-tetrachlorod1benzo-p-d1ox1n.
J. Agrlc. Food Chem. 29(2): 265-268.
Jensen, D.J., M.E. Getzendaner, R.A. Hummel and J. Turley. 1983. Residue
studies for (2,4,5-trIchlorophenoxy) acetic add and 2,3,7,8-tetrachlorodl-
benzo-p-d1ox1n In grass and rice. J. Agrlc. Food Chem. 31: 118-122.
Jett, G. 1982. Memorandum to J.S. Bellin (Office of Solid Waste, U.S. EPA,
Washington, DC). Re: Hazardous waste estimates for phenoxyacetlc compounds
and priority pollutant chlorophenols 1n the pesticide Industry. June 1.
Johnson, E.F. and U. Muller-Eberhard. 1977a. Resolution of two forms of
cytochrome P-450 from liver mlcrosomes of rabbits treated with 2,3,7,8-
tetrachlorod1benzo-p~d1ox1n. J. B1ol. Chem. 252(9): 2839-2845.
Johnson, E.F. and U. Muller-Eberhard. 1977b. Multiple forms of cytochrome
P-450 from liver mlcrosomes of rabbits treated with 2,3,7,8-tetrachlorodl-
benzo-p-d1oxin (Meeting Abstract). Fed. Proc. 36(3): 833.
1862A 15-40 03/29/84
-------
Johnson, E.F. and U. Muller-Eberhard. 1977c. Purification of the major
cytochrome P-450 of liver mlcrosomes from rabbits treated with 2,3,7,8-
tetrachlorodibenzo-p-d1oxin (TCDD). Blophys. Res. Commun. 76(3): 652-659.
Johnson, E.F. and U. Muller-Eberhard. 1977d. Multiple forms of cytochrome
P-450 resolution and purification of rabbit liver aryl hydrocarbon hydroxyl-
ase. Blochem. Blophys. Res. Commun. 76(3): 644-651.
Johnson, E.F., G.E. Schwab and U. Muller-Eberhard. 1979. Multiple forms of
cytochrome P-450: Catalytic differences exhibited by two homogeneous forms
of rabbit cytochrome P-450. Mol. Pharmacol. 15(3): 708-718.
Johnson, F.E., M.A. Kugler and S.M. Brown. 1981. Soft-tissue sarcomas and
chlorinated phenols. Lancet. 2(8236): 40.
Jones, G. 1975. A histochemlcal study of the liver lesion Induced by
2,3,7,8-tetrachlorodibenzo-p-dioxln (dioxln) In rats. J. Pathol. 116(2):
101-105.
Jones, G. and W.H. Butler. 1974. A morphological study of the liver lesion
induced by 2s3,7,8-tetrachlorod1benzo-p-d1ox1n in rats. J. Pathol. 112:
93-97.
Jones, G. and J.B. Grelg. 1975. Pathological changes 1n the liver of mice
given 2,3,7,8-tetrachlorod1benzo-p-d1oxin. Exper1ent1a. 31(11): 1315-1317.
1862A 15-41 03/29/84
-------
Jones, K.G. and G.D. Sweeney. 1977. Association between Induction of aryl
hydrocarbon hydroxylase and depression of uroporphyrlnogen decarboxylase
activity. Res. Commun. Chem. Pathol. Pharmacol. 17(4): 631-638.
Jones, K.G. and G.D. Sweeney. 1980. Dependence of the porphyrogenlc effect
of 2,3,7,8-tetrachlorod1benzo-p-d1ox1n upon Inheritance of aryl hydrocarbon
hydroxylase responsiveness. Toxlcol. Appl. Pharmacol. 53(1): 42-49.
Josephson, J. 1983. Chlorinated dloxlns and furans 1n the environment.
Environ. Sd. Technol. 17: 124A-128A.
Junk, G.A. and J.J. Richard. 1981. Dloxlns not detected In effluents from
coal/refuse combustion. Chemosphere. 10: 237-241.
KapUulnlk, J. and J.D. Ostrow. 1978. Stimulation of b1!1rub1n catabollsm
1n jaundiced Gunn rats by an Inducer of mlcrosmal mixed-function mono-
oxygenases. Proc. Natl. Acad. Sd. 75(2): 682-685.
Karasek, F.W., R.E. Clement and A.C. V1an. 1982. Distribution of PCDDs and
other toxic compounds generated on fly ash partlculates 1n municipal Incin-
erators. J. Chromatogr. 239: 173-180.
Karlckhoff, S.W., D.S. Brown and T.A. Scott. 1979. Sorptlon of hydrophollc
pollutants on natural sediments. Water Res. 13: 241-248.
Kearney, P.C., E.A. Woolson and C.P. Ellington, Jr. 1972. Persistence and
metabolism of chlorodloxins 1n soils. Environ. Sc1. Technol. 6(12):
1017-1019.
186PA 15-42 03/29/84
-------
Kenaga, E.E. 1980. Correlation of bloconcentratlon factors of chemicals In
aquatic and terrestrial organisms with their physical and chemical proper-
ties. Environ. Sci. Technol. 14: 553-556.
Kenaga, E.E. and C.A.I. Goring. 1980. Relationship between water solubil-
ity, soil sorption, octanol-water partitioning, and concentration of chemi-
cals in biota. In: J.G. Eaton et al., Ed., Aquatic Toxicology ASTM STP 707,
American Society for Testing and Materials, Philadelphia, PA. p. 78-115.
Kende, A.S. and J.J. Wade. 1973. Synthesis of new stemic and electronic
analogs of 2,3,7,8-tetrachlorodibenzo-p-dioxin. Environ. Health Perspect.
5: 49-57.
Kende, A.S., J.J. Wade, D. Ridge and A. Poland. 1974. Synthesis and
Fourier transform carbon-13 nuclear magnetic resonance spectroscopy of new
toxic polyhalodibenzo-p-dioxins. J. Org. Chem. 39: 931-937.
Khora, K.S. and W.P. McKinley. 1972. Pre- and postnatal studies on 2,4,5-T
and 2,4-D and their derivatives in rats. Toxicol. Appl. Pharmacol. 22: 14.
Khera, K.S. and J.A. Ruddick. 1973. Polychlorodibenzo-p-dioxins: Perinatal
effects and the dominant lethal test in Wistar rats. In: Chlorodioxins -
Origins and Fate, E.H. Blair, Ed. Adv. Chem. Ser. No. 120. Am. Chem. Soc.,
Washington, DC. p. 70-84.
Khera, K.S., B.L. Huston and W.P. McKinley. 1971. Pre- and postnatal
studies on 2,4,5-1, 2,4-D, and derivatives in Wistar rats. Toxicol. Appl.
Pharmacol. 19: 369-370.
1862A 15-43 03/29/84
-------
Kim, P., G.C. Yang, D. Firestone and A.E. Pohland. 1975. Photochemical
DechloMnatlon of Chlorinated D1benzo-p-d1ox1ns. Presented at the 1st
Chemical Congress of the North American Continent, Mexico City, November.
Klmble, B.J. and M.L. Gross. 1980. Tetrachlorod1benzo-p-d1ox1n quantHa-
tlon 1n stack-collected coal fly ash. Science. 207: 59-61.
Klmbrough, R.O. 1974. The toxlclty of polychlorlnated polycycllc compounds
and related chemicals. CRC CrH. Rev. Toxlcol. 2: 445-489.
Klmbrough, R.D., C.O. Carter, J.A. Mddle and R.E. Cllne. 1977. Epidemiol-
ogy and pathology of a tetrachlorod1benzod1ox1n poisoning episode. Arch.
Environ. Health. 32(2): 77-86.
Klmbrough, R.D., J. Buckley, L. F1shbe1n, et al. 1978. Animal toxicology.
Environ. Health Perspect. 24: 173-184.
K1mm1g, J. and K.H. Schulz. 1957. Berufllche akna (sog. chlorakne) durch
chloMette aromatlsche zykllsche ather. Dermatologla. 115: 540-546. (Ger.)
King, M.E. and A.R. Roesler. 1974. Subacute Intubation study on rats with
the compound 2,3,7,8-tetrachlorod1ox1n. U.S. EPA. NTIS PB-257 677. p. 27.
Kirsch, et al. 1975. Structural and functional studies of UganCMn, a
major renal organic an1on-b1nd1ng protein. J. Cl1n. Invest. 55: 1009.
1862A 15-44 03/29/84
-------
KHchln, K.I. and J.S. Woods. 1977. 2,3,7,8-Tetrachlorod1benzo-p-d1ox1n
(TCDD) Induced synthesis of cytochrome P-448 and aryl hydrocarbons hydroxyl-
ase (AHH) activity In female rat liver (Meeting Abstract). Pharmacol.
19(2): 232.
KHchln, K.T. and J.S. Woods. 1978a. 2,3,7,8-Tetrachlorod1benzo-p-d1oxin
Induction of aryl hydrocarbon hydroxylase (AHH) In hepatic mlcrosomes from
female rats. Toxlcol. Appl. Pharmacol. 45(1): 297.
KHchln, K.I. and J.S. Woods. 1978b. 2,3,7,8-Tetrachlorod1benzo-p-d1ox1n
Induction of aryl hydrocarbon hydroxylase 1n female rat liver, evidence for
de novo synthesis of cytochrome P-448. Mol. Pharmacol. 14: 890-899.
Knutson, J.C. and A. Poland. 1980. 2,3,7,8-7etrachlorod1benzo-p-d1ox1n:
Failure to demonstrate toxldty In twenty-three cultured cell types.
Toxlcol. Appl. Pharmacol. 54(3): 377-383.
Knutson, J.C. and A. Poland. 1982. Response of murlne epidermis to the
2,3,7,8-tetrach!orod1benzo-p-d1ox1n: Interaction of the Ah and hr lod.
Cell. 30(1): 225-234.
Kocher, C.W., N.H. Mahle, R.A. Hummel, L.A. Shadoff and M.E. Getzendaner.
1978. A search for the presence of 2,3,7,8-tetrachlorod1benzo-p-d1ox1n 1n
beef fat. Bull. Environ. Contam. Toxlcol. 19: 229-236.
Koclba, R.J. and B.A. Schwetz. 1982. Toxldty of 2,3,7,8-tetrachlorodl-
benzo-p-d1oxin (TCDD). Drug Metab. Rev. 13: 387-406.
1862A 15-45 03/29/84
-------
Kociba, R.J., P.A. Keeler, C.N. Park and P.J. GehMng. 1976. 2,3,7,8-
Ietrachlorod1benzo-p-d1ox1n results of a 13-week oral toxlclty study 1n
rats, loxicol. Appl. Pharmacol. 35: 553-573.
Kociba, R.J., D.G. Keyes, J.E. Beyer, et al. 1977. Results of a two-year
chronic toxlclty and oncogenldty study of 2,3,7,8-tetrachlorod1benzo-p-
dloxln 1n rats. Toxlcol. Appl. Pharmacol. 46: 279-303.
Kociba, R.J., D.G. Keyes, J.E. Beyer, et al. 1978a. Results of a two-year
chronic toxlclty and oncogenldty study of 2,3,7,8-tetrachlorod1benzo-p-
dloxln In rats. Toxlcol. Appl. Pharmacol. 46(2): 279-303.
Kociba, R.J., D.G. Keyes, J.E. Beyer and R.M. Carreon. 1978b. Tox1colog1c
studies of 2,3,7,8-tetrachlorod1benzo-p-dioxin (TCDD) In rats. Toxlcol.
Occup. Med. (Dev. Toxlcol. Environ. Sd.) 4: 281-287.
Kociba, R.J., D.G. Keyes, J.E. Beyer, R.M. Carreon and P.J. Gehrlng. 1979.
Long-term toxlcologlc studies of 2,3,7,8-tetrachlorod1benzo-p-d1oxin (TCDD)
In laboratory animals. Ann. NY Acad. Sc1. 320: 397-404.
Kohll, K.K. and J.A. Goldstein. 1981. Effects of 2,3,7,8-tetrachlorodl-
benzo-p-d1oxin on hepatic and renal prostaglandln synthetase. Life Sc1.
29(3): 299-305.
Kondorosl, A., I. Fedorcsak, F. Solymosy, L. Ehrenberg and S. Osterman-
Golkar. 1973. Inactlvation of QBRNA by electrophlles. Mutat. Res. 17:
149-161.
1862A 15-46 03/29/84
-------
Kooke, R., J.W.A. Lustenhouwer, K. Ol1e and 0. Hutzinger. 1981. Extraction
efficiencies of polychlorlnated d1benzo-p-d1ox1ns and polychlorlnated
dlbenzofurans from fly ash. Anal. Chem. 53: 461-463.
Kourl, R. 1976. Relationship between levels of aryl hydrocarbon hydroxy-
lase activity and susceptibility to 3-methylcholanthrene and benzo(a)pyrene-
Induced cancers 'In Inbred strains of mice. In: Polynuclear Aromatic Hydro-
carbons: Chemistry, Metabolism and Cardnogenesls, Vol. 1, R.J. Freudenthal
and P.M. Jones, Ed. Raven Press, NY. p. 139.
Kourl, R.E., H. Ratrie, III, S.A. Atlas, A. Nlwa and O.W. Nebert. 1974.
Aryl hydrocarbon hydroxylase Induction 1n human lymphocyte cultures by
2,3,7,8-tetrachlorod1benzo-p-d1ox1n. Life Sd. 15(9): 1585-1595.
Kourl, R.E., l.H. Rude, R. Joglekar, et al. 1978. 2,3,7,8-Tetrachlorodl-
benzo-p-d1oxln as cocarclnogen causing 3-methylcholanthrene-1n1t1ated subcu-
taneous tumors In mice genetically "nonresponsive" at Ah locus. Cancer Res.
38(9): 277/-27S3.
Kurl, R.N., J. Lund, L. Poelllnger and J.A. Gustafsson. 1982. Differential
effect of 2,3,7,8-tetrachlorod1benzo-p~d1ox1n on nuclear RNA polymerase
activity in the rat liver and thymus. Biochem. Pharmacol. 31(15):
2459-2462.
Kurokl, H. and Y. Masuda. 1977. Structures and concentrations of the main
components of polychlorlnated blphenyls retained 1n patients with Yusho.
Chemosphere. 6: 465-469.
1862A 15-47 03/29/84
-------
Lamb, J.C., J.A. Moore and T.A. Markes. 1980. Evaluation of 2,4-0,
2,4,5-T, and 1CDO toxldty In C57BL/6 mice: Reproduction and fertility 1n
treated male mice and evaluation of congenital malformation 1n their
offspring. National Toxicology Program, Research Triangle Park, NC.
Lamb, J.C., l.A. Markes, B.C. Gladen, J.W. Allen and J.A. Moore. 1981a.
Male fertility, sister chromatld exchange, and germ cell toxldty following
exposure to mixtures of chlorinated phenoxy adds containing 2,3,7,8-tetra-
chlorod1benzo-p-d1ox1n. J. Toxlcol. Environ. Health. 8: 825-834.
Lamb, J.C., J.A. Moore, T.A. Marks and J.K. Haseman. 1981b. Development
and viability of offspring of male mice treated with chlorinated phenoxy
acids and 2,3,7,8-tetrachlorod1benzo-p-d1ox1n. J. Toxlcol. Environ. Health.
8(5-6): 835-844.
Lamparskl, L.L. and T.J. Nestrlck. 1980. Determination of tetra-, hexa-,
hepta-, and octachlorod1benzo-p-d1ox1n Isomers 1n partlculate samples at
parts per trillion levels. Anal. Chem. 52: 2045-2054.
Lamparskl, L.L., N.H. Mahle and L.A. Shadoff. 1978. Determination of
pentachlorophenol, hexachlorod1benzo-p-d1ox1n, and octachlorod1benzo-p-
dloxln 1n bovine milk. J. Agrlc. Food Chem. 26(5): 1113-1116.
Lamparskl, L.L., l.J, Nestrlck and R.H. Stehl. 1979. Determination of
part-per-tr1H1on concentrations of 2,3,7,8-tetrachlorod1benzo-p-d1ox1n In
fish. Anal. Chem. 51(9): 1453-1458.
1862A 15-48 03/29/84
-------
Lamparski, L.L., R.I. Stehl and R.L Johnson. 1980. Photolysis of penta-
cblorophenol-treated wood; chlorinated d1benzo-p-d1ox1n formation. J.
Environ. Sc1. Tech. 14: 196-200.
Langhorst, M.L. and L.A. Shadoff. 1980. Determination of part-per-tMlllon
concentrations of tetra-, hexa-, hepta-, and octa-chlorod1benzo-p-d1ox1ns In
human milk samples. Anal. Chem. 52: 2037-2044.
Lavy, T.L., J.S. Shepard and J.D. Mattlce. 1980. Exposure measurements of
applicators spraying (2,4,5-trlchlorophenoxy) acetic add 1n the forest. J.
Agrlc. Food Chem. 28: 626-630.
Lee, I.P and K. Suzuki. 1980. Induction of aryl hydrocarbon hydroxylase
activity In the rat prostate glands by 2,3,7,8-tetrchlorod1benzo-p-dioxins.
J. Pharmacol. Exp. Ther. 215(3): 601-605.
Legraverend, C., B. Mansour, O.W. Nebert and J.M. Holland. 1980. Genetic
differences in benzo[a]pyrene-1n1tiated tumorlgenesls 1n mouse skin. Phar-
macology. 20: 242-255.
Leo, A.J. 1979. Letter to C. Stephan. Pamona College, Claremont, CA.
May 11.
Levin, J.O., C.F. Rappe and C.A. Nllssen. 1976. Use of chlorophenols or
fungicides in sawmills. Scand. J. Work Environ. Health. 2: 71.
1862A 15-49 03/29/84
-------
L1bert1, A. and D. Brocco. 1981. Formation of polychlorod1benzod1ox1ns 1n
urban Incinerator emissions. Iji: Impact of Chlorinated Dloxlns and Related
Compounds on the Environment, 0. Hutzlnger et al., Ed. Pergamon Press,
Oxford, p. 245-251.
Liem, H.H., U. Muller-Eberhard and E.F. Johnson. 1980. Differential
Induction by 2,3,7,8-tetrachlorod1benzo-p-d1ox1n of multiple forms of rabbit
microsomal cytochrome P-450: Evidence for tissue specificity. J. Mol.
Pharmacol. 18(3): 565-570.
Llndahl, R., M. Roper and R.A. Dietrich. 1978. Rat liver aldehyde dehydro-
genase -- Immunochemlcal Identity of 2,3,7,8-tetrachlorod1benzo-p-d1ox1n
Induclble normal liver and 2-acetylam1nof luorene Indudble hepatoma
Isozymes. Blochem. Pharmacol. 27(20): 2463-2465.
Liss, Q.S. and P.G. Slater. 1974. Flux of gases across the air-sea Inter-
face. Nature. 247: 181.
Loprieno, N., I. Sbrana, D. Rusclano, D. Lasc1alfar1 and T. Larl. 1982. Jhi
vivo cytogenetlc studies on mice and rats exposed to 2,3,7,8-tetrachlorodl-
benzo-p-dloxln. In: Chlorinated Dloxlns and Related Compounds. Impact on
the Environment, 0. Hutzlnger, R.W. Fre1, E. MeMan and P. Pocch1ar1, Ed.
Pergamon Press, NY. p. 419-428.
Lustenhouwer, J.W.A., K. Ol1e and 0. Hutzlnger. 1980. Chlorinated dlbenzo-
p-d1ox1ns and related compounds 1n Incinerator effluents: A review of
measurements and mechanisms of formation. Chemosphere. 9: 501-522.
1862A 15-50 03/29/84
-------
Luster, M.I., R.E. Faith and G. Clark. 1979a. Laboratory studies on the
Immune effects of halogenated aromatlcs. Ann. NY Acad. Sci. 320: 473-484.
Luster, M.I., G. Clark, L.O. Lawson and R.E. Faith. 1979b. Effects of a
brief jm vitro exposure to 2,3,7,8-tetrachlorod1benzo-p-d1ox1n (TCDD) on
mouse lymphocytes. J. Environ. Pathol. Toxlcol. 2(4): 965-977.
Luster, M.I., G.A. Boorman, J.H. Dean, et al. 1980. Examination of bone
marrow, 1mmunolog1c parameters and host susceptibility following pre- and
postnatal exposure to 2,3,7,8-tetrachlorod1benzo-p-d1ox1n (TCDD). Int. J.
Immunopharmacol. 2(4): 301-310.
Mabey, W.R., J.H. Smith, R.T. Podoll, et al. 1981. Aquatic Fate Processes
Data for Organic Priority Pollutants. EPA 440/4-81-014, Monitoring and Data
Support Div., Office of Water and Regulations and Standards, Washington, DC.
p. 107-108.
Madge, D.S. 1977. Effects of trlchlorophenoxyacetlc add and chlorodloxlns
on small Intestinal function. Gen. Pharmacol. 8: 319-324.
Mahle, N.H. and L.A. Shadoff. 1982. The mass spectrometry of chlorinated
d1benzo-p -dloxins. Blomed. Mass Spectrom. 9: 45-60.
Mahle, N.H., M.S. H1gg1ns and M.E. Getzendaner. 1977. Search for the pres-
ence of 2,3,7,8-tetrachlorod1benzo-p-d1ox1n 1n bovine milk. Bull. Environ.
Contam. Toxlcol. 18(2): 123-130.
1862A 15-51 03/29/84
-------
Malik, N. and I.S. Owens. 1977. Studies on the requirements for Induction
of UDP glucuronosyl-transferase (T'ASE) activity 1n the Reuber hepatoma H-4-
II-E established cell line (Meeting Abstract). Pharmacology. 19(2): 150.
Malik, N., G.M. Koteen and I.S. Owens. 1979. Induction of UDP-glucuro-
nosyl-transferase activity In the Reuber H-4-II-E hepatoma cell culture.
Mol. Pharmacol. 16(3): 950-960.
Manara, L., P. Cocda and T. Crod. 1982. Persistent tissue levels of TCDO
In the mouse and their reduction as related to prevention of toxldty. Drug
Metab. Rev. 13(3): 423-446.
Manls, J. and R. Apap. 1979. Intestinal organic anlon transport, gluta-
thlone transferase and aryl hydrocarbon hydroxylase activity: Effect of
dloxln. Life Sci. 24(15): 1373-1380.
Manis, J. and G. K1m. 1979a. Stimulation of Iron absorption by polychlorl-
nated aromatic hydrocarbons. Am. J. Physlol. 236(6): E763-E768.
Manis, J. and G. Kim. 1979b. Introduction of Iron transport by a potent
Inducer of aryl hydrocarbon hydroxylase, 2,3,7,8-tetrachlorod1benzo-p-d1ox-
in. Arch. Environ. Health. 34(3): 141-145.
Mantel, N. and M.A. Schnelderman. 1977. Estimation of "safe" levels, a
hazardous undertaking? Cancer Res. 35: 1379-1386.
1862A 15-52 03/29/84
-------
Mantovanl, A., A. Vecchl, W. Lu1n1, et al. 1980. Effect of 2,3,7,8-tetra-
chlorod1benzo-p-d1ox1n on macrophase and natural killer cell-mediated cyto-
toxlclty In mice. B1omed1c1ne. 32(4): 200-204.
Marks, T.A., G.A. Klmmel and R.E. Staples. 1981. Influence of symmetrical
polychlorlnated blphenyl Isomers on embryo and fetal development 1n mice.
loxlcol. Appl. Pharmacol. 61: 269-276.
Marselos, H., R. Torronen and A. A1t1o. 1978. Responses of the D-glucuron-
1c acid pathway In rat tissues to treatment with tetrachlorodlbenzo dloxln.
Xenobiotlcs. 8(7): 397-402.
Mason, M.E. and A.B. Okey. 1982. Cytosollc and nuclear binding of 2,3,7,8-
tetrachlorod1benzo-p-d1ox1n to the AH receptor 1n extra-hepatic tissues of
rats and mice. tur. J. Blochem. 123(1): 209-215.
Matsumura, F. and H.J. Benezet. 1973. Studies on the bloaccumulatlon and
mlcroblal degradation of 2,3,7,8-tetrachlorod1benzo-p-d1ox1n. Environ.
Health Perspect. 5: 253-258.
Matsumura, F., J. Quensen and G. Isushlmoto. 1983. Mlcroblal degradation
of TCDO in a model ecosystem. Iji: Human and Environmental Risks of Chlori-
nated Dloxins and Related Compounds, R.E. Tucker, A.L. Young and A.P. Gray,
Ed. Plenum Press, NY. p. 191-219.
Mattison, D.R. and S.S. Thorgelrsson. 1978. Gonadal aryl hydrocarbon
hydroxylase In rats and mice. Cancer Res. 38(5): 1368-1373.
1862A 15-53 03/29/84
-------
Mattison, D.R. and S.S. Thorgelrsson. 1979. Ovarian aryl hydrocarbon
hydroxylase activity and primordial oocyte toxldty of polycycllc aromatic
hydrocarbons 1n mice. Cancer Res. 39: 3471-3475.
May, G. 1973. Chloracne from the accidental production of tetrachlorodl-
benzodioxln. Br. J. Ind. Med. 30(3): 276-283.
May, G. 1982. Tetrachlorod1benzod1ox1n: A survey of subjects ten years
after exposure. Br. J. Ind. Med. 39(2): 128-135.
McCann. 1978. Unpublished study. (Cited In Wassom et al., 1978)
McConnell, E.E. 1980. Acute and chronic toxldty, carclnogenesls, repro-
duction, teratogenesls and mutagenesls 1n animals. In: Halogenated Bi-
phenyls, Terphenyls, Naphthalenes, D1benzod1ox1ns and Related Products, R.O.
Klmbrough, Ed. Elsevler/North-Holland B1omed1cal Press, NY. p. 109-150.
McConnell, E.E. and J.A. Moore. 1979. Toxlcopathology characteristics of
the halogenated aromatlcs. Ann. NY Acad. Sc1. 320: 138-150.
McConnell, E.E., J.A. Moore and O.W. Dalgard. 1978a. Toxldty of 2,3,7,8-
tetrachlorod1benzo-p-d1ox1n In rhesus monkeys (Macacas mulatta) following a
single oral dose. Toxlcol. Appl. Pharmacol. 43: 175.
McConnell, E.E., J.A. Moore, J.K. Haseman and M.W. Harris. 1978b. The com-
parative toxldty of chlorinated d1benzo-p-d1ox1ns 1n mice and guinea pigs.
Toxlcol. Appl. Pharmacol. 44(2): 335-356.
1862A 15-54 03/29/84
-------
McKlnney, J.D., K. Chae, B.N. Gupta, J.A. Moore and J.A. Goldstein. 1976.
lexicological assessment of hexachloroblphenyl Isomers and 2,3,7,8-tetra-
chlorodlbenzofuran 1n chicks. I. Relationship of chemical parameters.
loxlcol. Appl. Pharmacol. 36: 65-80.
McKlnney, J., P. Albro, M. Luster, 8. Corbett, J. Schroeder and L. Lawson.
1981. Development and reliability of a radio Immunoassay for 2,3,7,8-tetra-
chlorod1benzo-p-d1ox1n. In: Impact of Chlorinated 01ox1ns and Related Com-
pounds on the Environment, 0. Hutzlnger et al., Ed. Pergamon Press, Oxford.
p. 67-75.
McNulty, W.P. 1978. Direct testimony before the administrator, U.S.
Environmental Protection Agency, FIFRA Docket No. 415, EPA Exhibit No. 106.
McNulty, W.P., K.A. Nielsen-Smith, J.O. Lay, Jr., et al. 1982. Persistence
of ICDD 1n monkey adipose tissue. Food Cosmet. Toxlcol. 20: 985-987.
McQueen, E.G., A.M.O. Veale, W.S. Alexander and M.N. Bates. 1977. 2,4,5-T
and human birth defects. Report of the Division of Public Health, New
Zealand Dept. of Health. (Cited 1n M1lby et al., 1980)
Meselson, M,, P.W. O'Keefe and R. Baughman. 1978. The evaluation of possi-
ble health hazards from TCDD 1n the environment. Presented at the Symposium
on the Use of Herbicides In Forestry, Arlington, VA. February 21-22.
1862A 15-55 04/05/84
-------
Michigan Department of Public Health. 1983a. Evaluation of Congenital
Malformation Rates for Midland and Other Selected Michigan Counties Compared
Nationally and Statewlse 1970-1981. Michigan Dept. of Public Health. May 4.
Michigan Department of Public Health. 1983b. Evaluation of Soft and
Connective 1 Issue Cancer Mortality Rates for Midland and Other Selected
Michigan Counties Compared Nationally and Statewide. Michigan Dept. of
Public Health. May 4.
Mlettinen, 0. 1976. Est1mab1l1ty and estimation 1n case referent studies.
Am. J. Ep1dem1ol. 103: 226.
M1lby, T.H., E.L. Hustlng, M.D. Whorton and S. Larson. 1980. Potential
Health Effects Associated with the Use of Phenoxy Herbicides: A Summary of
Recent Scientific Literature. A Report for the National Forest Products
Association from Environmental Health Associated, Inc., Berkeley, CA.
Miller, R.A., L.A. Norrls and C.L. Hawkes. 1973. Tox1c1ty of 2,3,7,8-
tetrachlorod1benzo-p-d1ox1n (TCDD) 1n aquatic organisms. Environ. Health
Perspect. 5: 177-186.
Miller, R.A., L.A. Norrls and B.R. Loper. 1979. The response of coho
salmon and gupples to 2,3,7,8-tetrachlorod1benzo-p-d1ox1n (TCDD) 1n water.
Am. F1sh. Soc. Trans. 108(4): 401-407.
1862A 15-56 03/29/84
-------
MHchum, R.K., G.F. Moler and W.A. Korfmacher. 1980. Combined capillary
gas chromatography/atmospheMc pressure negative chemical 1on1zat1on/mass
spectrometry for the determination of 2,3,7,8-tetrachlorod1benzo-p-d1ox1n In
tissue. Anal. Chem. 52(14): 2278-2282.
Moore, J.A. and R.E. FaHh. 1976. Immunologlc response and factors affect-
Ing Us assessment. Environ. Health Perspect. 18: 125-131.
Moore, J.A. and J.G. Vos. 1974. Evaluating the rodent Immune system as a
focus for tetratogenlc effects. Teratology. 9(3): A-29.
Moore, J.A., B.N. Gupta, J.G. Z1nkl and J.G. Vos. 1973. Postnatal effects
of maternal exposure to 2,3,7,8-tetrachlorod1benzo-p-d1ox1n (TCDD).
Environ. Health Perspect. 5: 81-85.
Moore, J.A., M.W. Harris and P.M. Albro. 1976. Tissue distribution of
(l4C)tetrachlorod1benzo-p-d1ox1n 1n pregnant and neonatal rats. Toxlcol.
Appl. Pharmacol. 37(1): 146-147.
Moore, J.A., E.E. McConnell, O.W. Dalgard and M.W, Harris. 1979. Compara-
tive toxicity of three halogenated dibenzofurans 1n guinea pigs, mice and
rhesus monkeys. Ann. NY. Acad. Sci. 320: 151-163.
Moses, M. and I.J. Selikoff. 1981. Soft-tissue sarcomas, phenoxy herbi-
cides and chlorinated phenols. Lancet. 1(8234): 1370.
I862A 15-57 03/29/84
-------
Mottura, A., G. Zei, F. Nuzzo, et al. 1981. Evaluation of results of
chromosome analyses on lymphocytes of TCDO exposed subjects after the Seveso
accident. Mutat. Res. 85(4): 238-239.
Mulcahy, M.I. 1980. Chromosome aberrations and "Agent Orange". Med. J.
Aust. 2(10): 573-574.
Murray, F.J., F.A. Smith, K.D. NHschke, C.G. Humlston, R.J. Kodba and B.A.
Schwetz. 1979. Three-generation reproduction study of rats given 2,3,7,8-
tetrachlorod1benzo-p-d1ox'm (TCDD) 1n the diet. Toxicol. Appl. Pharmacol.
50: 241-251.
Muscarella, D., D. Dennett, J.G. Bablsh and D. Noden. 1982. The effects of
2,3,7,8-tetrachlorod1benzo~p-d1ox1ns on fetal development In the ferret.
lexicologist. 2: 73.
Nagarkattl, P.S., G.D. Sweeney, J. Gauldle and D.A. Clark. 1984. SensUiv-
ity to suppression of cytotoxlc T cell generation by 2,3,7,8-tetrachloro-
d1benzo-p-d1oxin (TCOD) is dependent on the AH genotype of the murlne host.
Toxicol. Appl. Pharmacol. 72: 169-176.
NAS (National Academy of Sciences). 1977. Drinking Water and Health: Part
II. NAS, Washington, DC.
Nash, R.G. and M.L. Beall, Jr. 1980. Distribution of Sllvex, 2,4-D, and
1CDD applied to turf In chambers and field plots. J. Agric. Food Chem. 28:
614-623.
1862A 15-58 03/29/84
-------
Nau, H. and R. Bass. 1981. Transfer of 2,3,7,8-tetrachlorod1benzo-p-d1ox1n
(TCOD) to the mouse embryo and fetus. Toxicology. 20(4): 299-308.
Nau, H., R. Bass and D. Neubert. 1982. Transfer of 2,3,7,8-tetrachlorodl-
benzo~p-d1ox1n (TCDD) to the mouse embryo, fetus and neonate. Ijn: Chlori-
nated D1ox1ns and Related Compounds. Impact on the Environment, 0.
Hutzlnger, R.W. Fre1, E. Merlan and F. Pocch1ar1, Ed. Pergamon Press, NY.
p. 325-337.
Neal, R.A., P.W. Beatty and T.A. Gas1ew1cz. 1979. Studies of the mecha-
nisms of toxlcity of 2,3,7,8-tetrachlorod1benzo-p-d1ox1n (TCDD). Ann. NY
Acad. Sc1. 320: 204-213.
Neal, R.A., J.R. Olson, T.A. Gas1ew1cz and L.E. Gelger. 1982. The toxlco-
klnetlcs of 2,3,7,8-tetrachlorod1benzo-p-d1ox1n In mammalian systems. Drug
Metab. Rev. 13: 355-385.
Nebert, D.W. 1979. Multiple forms of 1nduc1ble drug-metabolizing enzymes:
A reasonable mechanism by which any organism can cope with adversity. Mol.
Cell. Blochem. 27: 27-46.
Nebert, D.W. 1982. Pharmacogenetlcs and human cancer. Host factors In
human carclnogenesls. IARC Scientific Pub. No. 39. Lyon, France.
Nebert, D.W. and J.E. Glelen. 1972. Genetic regulation of aryl hydrocarbon
hydroxylase Induction 1n the mouse. Fed. Proc. 31: 1315-1327.
1862A 15-59 03/29/84
-------
Nebert, D.W. and N.M. Jensen. 1979. The Ah locus: Genetic regulation of
the metabolism of carcinogens, drugs, and other environmental chemicals by
cytochrome P-450 mediated monooxygenases. CRC Cr1t. Rev. Blochem.
6: 401-437.
Nebert, D.W., F.W. Goujon and J.E. Glelen. 1972. Aryl hydrocarbon
hydroxylase Induction by polycycllc hydrocarbons: Simple autosomal dominant
trait In the mouse. Natl. New. B1ol. 236: 107-110.
Nebert, D.W., et al. 1975. Genetic expression of aryl hydrocarbon hydroxy-
lase activity 1n the mouse. J. Cell. Physlol. 85: 393-414.
Nebert, D., S. Thorglersson and J. Felton. 1976. Genetic differences In
mutagenesls, carclnogenesls, and drug toxldty. _Inj Ir± vitro Metabolic
Activation 1n Mutagenesls Testing, F. de Serres, J. Folets, J. Bend and R.
Philpot, Ed. Elsevler/North Holland B1omed1cal Press, Amsterdam.
p. 105-124.
Nebert, D.W., H.J. Eisen, M. Neg1sh1, M.A. Lang, L.M. Hjelmeland and A.B.
Okey, 1981. Genetic mechanisms controlling the Induction of polysubstrate
monooxygenase (P-450) activities. Ann. Rev. Pharmacol. Toxlcol. 21:
431-462.
Nebert, D.W., M. Neglshi and H.J. Eisen. 1983. Genetic differences 1n
enzymes which metabolize drugs, chemical carcinogens and other environmental
pollutants. In: Human and Environmental Risks of Chlorinated D1ox1ns and
Related Compounds, R.E. Rucker, A.L. Young and A.P. Gray, Ed. Plenum Press,
NY, p. 441-462.
1862A 15-60 04/05/84
-------
Neely, W.B. 1979. Estimating rate constants for the uptake and clearance
of chemicals by fish. Environ. Sd. Technol. 13: 1506.
Neely, W.B. 1983. Letter to C.E. Stephan. Dow Chemical Co., Midland, HI.
June 10.
Nelson, C.J., J.F. Holson, H.G. Green and D.W. Gaylor. 1979. Retrospective
study of the relationship between agricultural use of 2,4,5-T and cleft
palate occurrence In Arkansas. Teratology. 19: 377-384.
Nelson, J.O., R.E. Menzer, P.C. Kearney and J.R. Pllmmer. 1977. 2,3,7,8-
Tetrachlorodibenzo-p-dioxin: Ijri yj^trp binding to rat liver mlcrosomes.
Bull. Environ. Contam. Toxlcol. 18(1): 9-13.
NestMck, I.J., L.L. Lamparski and D.I. Townsend. 1980. Identification of
tetrachlorodibenzo-p-d1ox1n Isomers at the 1 ng level by photolytlc degrada-
tion and pattern recognition techniques. Anal. Chem. 52(12): 1865-1874.
Nestrick, T.J., L.L. Lamparski, W.B. Grummet and L.A. Shadoff. 1982.
Comments on variations in concentrations of organic compounds Including
polychlorinated dibenzo-p~d1ox1ns and polynuclear aromatic hydrocarbons in
fly ash from a municipal incinerator. Anal. Chem. 54: 823-824.
Neubert, D. and I. Dillmann. 1972. Embryotoxic effects in mice treated
with 2,4,5-trichlorophenoxyacetic acid and 2,3,7,8-tetrachlorodibenzo-p-
dioxin. Arch. Pharmacol. 272(3): 243-264.
1862A 15-61 03/29/84
-------
Neubert, D., P. Zens, A. Rothenwallner and H.J. Merker. 1973. A survey of
the embryotoxic effects of TCDO 1n mammalian species. Environ. Health
Perspect. 5: 67-79.
Nlemann, R.A., W.C. Brumley, 0. Firestone and J.A. Sphon. 1983. Analysis
of fish for 2,3,7,8-tetrachlorod1benzo-p-d1ox1n by electron capture gas
chromatography. Anal. Chem. 55: 1497-1504.
Nlenstedt, W., M. Parkkl, P. Uotlla and A. A1t1o. 1979. Effects of
2,3,7,8-tetrachlorod1benzo-p-d1ox1n on hepatic metabolism of testosterone 1n
the rat. Toxicology. 13(3): 233-236.
Nllsson, C.-A., K. Andersson, C. Rappe and S.-O. Westermark. 1974. Chroma-
tographlc evidence for the formation of chlorodloxlns from chloro-2-phenoxy-
phenols. J. Chromatogr. 96: 137-147.
NIOSH {National Institute for Occupational Safety and Health). 1982.
Health Hazard Evaluation Determination Report: Long Island Railroad,
No. 80-039.
Nlsbet, I.C.T. and M.B. Paxton. 1982. Statistical aspects of three-genera-
tion studies of the reproductive toxldty of TCDD and 2,4,5-T. Am. Stat.
Vol. 36(3): 290-298.
N1wa, A., K. Kumakl and O.W. Nebert. 1975. Induction of aryl hydrocarbon
hydroxylase activity In various cell cultures by 2,3,7,8-tetrachlorodlbenzo-
p-d1oxin. Hoi. Pharmacol. 11(4): 399-408.
1862A 15-62 03/29/84
-------
Nolan, R.J., F.A. Smith and J.G. Hefner. 1979. Elimination and tissue
distribution of 2,3,7,8-tetrach1orod1benzo-p-d1ox1n (TCDO) 1n female guinea
pigs following a single oral dose. Toxlcol. Appl. Pharmacol. 48(1): A162.
Norback, D.H. and J.R. Allen. 1973. Biological responses of the nonhuman
primate, chicken, and rat to chlorinated d1benzo-p-d1ox1n 1ngest1on.
Environ. Health Perspect. 5: 233-240.
Norman, R.L., E.F. Johnson and U. Muller-Eberhard. 1978a. Identification
of the major cytochrome P-450 form transplacentally Induced 1n neonatal
rabbits by 2,3,7,8-tetrachlorod1benzo-p-d1ox1n. Fed. Proc. Am. Soc. Exp.
81ol. 37(6): 1720.
Norman, R.L., E.F. Johnson and U. Muller-Eberhard. 1978b. Identification
of the major cytochrome P-450 form transplacentally Induced In neonatal
rabbits by 2,3,7,8-tetrachlorod1benzo-p-d1ox1n. J. B1ol. Chem. 253(23):
8640-8647.
NorMs, L.A. and R.A. Miller. 1974. The toxldty of 2,3,7,8-tetrachlorodl-
ben?o-p-d1ox1n (TCDD) 1n gupples (Poec111a retlculatus Peters). Bull.
Environ. Contam. Toxlcol. 12(1): 76-80.
Norstrom, A., C. Rappe, R. Llndahl and H.R. Buser. 1979. Analysis of some
older Scandinavian formulations of 2,4-d1chlorophenoxy acetic add and
2,4,5-trlchlorophenoxy acetic add for contents of chlorinated d1benzo-p-
dloxlns and dlbenzofurans. Scand. J. Work Environ. Health. 5(4): 375-378.
1862A 15-63 03/29/84
-------
Norstrom, R.J., D.J. Hallett, M. Simon and M.J. Mulvlhlll. 1982. Analysis
of Great Lakes herring gull eggs for tetrachlorod1benzo-p-d1ox1ns. Irr.
Chlorinated Dloxlns and Related Compounds. Impact on the Environment, 0.
Hutzinger, R.W. Fre1, E. Merlan and E. Pocch1ar1, Ed. Pergamon Press, NY.
p. 173-181.
NRCC (National Research Council of Canada). 1981a. PolychloMnated
D1benzo-p-D1ox1ns: Criteria for their Effects on Man and His Environment.
Publ. No. NRCC 18574, ISSN 0316-0114. NRCC/CNRC Associate Committee on
Scientific Criteria for Environmental Quality, Ottawa, Canada. 251 p.
NRCC (National Research Council of Canada). 1981b. PolychloMnated
D1benzo-p-D1oxins: Limitations to the Current Analytical Techniques. Publ.
No. 18576, NRCC/NRC, Ottawa, Canada.
NTP (National Toxicology Program). 1980a. Bloassay of 2,3,7,8-tetrachloro-
dibenzo-p-dioxin for possible cardnogenlcity (gavage study). OHHS Publ.
No. (NIH) 82-1765. Carcinogenesis Testing Program, NCI, NIH, Bethesda, MD,
and National Toxicology Program, RTP, Box 12233, NC.
NTP (National Toxicology Program). 1980b. Bloassay of 2,3,7,8-tetrachloro-
d1benzo-p-diox1n for possible carcinogeniclty (Dermal study). DHHS Publ.
No. (NIH) 80-1757. Carcinogenesis Testing Program, NCI, NIH, Bethesda, MO,
and National Toxicology Program, RTP, Box 12233, NC.
1862A 15-64 03/29/84
-------
NTP (National Toxicology Program). 1980c. Bloassay of 1,2,3,7,8- and
1,2,3,7,8,9-hexachlorod1benzo~p-d1ox1n for possible carclnogenlclty (dermal
study). DHHS Publ. No. (NIH) 80-1758. Cardnogenes 1s Testing Program, NCI,
NIH, Bethesda, MO, and National Toxicology Program, NTP, Box 12233, NC.
NTP (National Toxicology Program). 1980d. Bloassay of 1,2,3,7,8- and
1,2,3,7,8,9-hexachlorod1benzo-p-d1ox1n (gavage) for possible carclnogenlc-
1ty. DHHS Publ. No. (NIH) 80-1754. Carc1nogenes1s Testing Program, NCI,
NIH, Bethesda, MD, and National Toxicology Program, RTP, Box 12233, NC.
Og1lv1e, D. 1981. D1ox1n found 1n the Great Lakes basin. Ambio. 10:
38-39.
O'Keefe, P.M., M.S. Meselson and R.W. Baughman. 1978. Neutral cleanup
procedure for 2,3,7,8-tetrchlorod1benzo-p-d1ox1n residues 1n bovine fat and
milk. J. Assoc. Off. Anal. Chem. 61(3): 621-626.
O'Keefe, P., C. Meyer and K. Dillon. 1982. Comparison of concentration
techniques for 2,3,7,8-tetrachlorod1benzo-p-d1oxin. Anal. Chem. 54:
2623-26?5.
O'Keefe, P., C. Meyer, D. Milker, et al. 1983. Analysis of 2,3,7,8-tetra-
chlorod1benzo-p-d1ox1n 1n Great Lakes fish. Chemosphere. 12: 325-332.
Okey, A.G. 1983. The Ah receptor: A specific site for action of chlori-
nated dloxlns. In: Human and Environmental Risks of Chlorinated D1ox1ns and
Related Compounds, R.E. Tucker, A.L. Young and A.P. Gray, Ed. Plenum Press,
NY. p. 423-440.
1862A 15-65 03/29/84
-------
Okey, A.B. and L.M. Vella. 1982. Binding of 3-methylcholanthrene and
2,3,7,8-tetrachlorod1benzo-p-dioxin to a common Ah receptor site 1n mouse
and rat hepatic cytosls. Eur. J. Blochem. 127(1): 39-47.
Okey, A.B. and L.M. Vella. 1984. Elevated binding of 2,3,7,8-tetrachloro-
d1benzo-p-d1oxin and 3-methylcholanthrene to the Ah receptor In hepatic
cytosols from phenobarbltal-treated rats and mice. Blochem. Pharmacol. (In
press)
Okey, A.B., G.P. Bondy, M.E. Mason, et al. 1979. Regulatory gene product
of the Ah locus. J. Blol. Chem. 254: 11636-11648.
Okey, A.B., G.P. Bondy, M.E. Mason, et al. 1980. Temperature-dependent
cytosol-to-nucleus translocatlon of the Ah receptor for 2,3,7,8-tetrachloro-
dibenzo-p-dioxin In continuous cell culture lines. J. B1ol. Chem. 255(23):
1 1415-11422.
Ol1e, K., J.W.A. Lustenhouwer and 0. Hutzlnger. 1982. PolychloMnated
d1benzo-p-d1oxins and related compounds 1n Incinerator effluents. In:
Chlorinated Dloxlns and Related Compounds: Impact on the Environment, 0.
Hutzlnger et al., Ed. Pergamon Press, NY. p. 227-244.
Olie, K., M.V.D. Berg and 0. Hutzlnger. 1983. Formation and fate of PCDO
and PCDF from combustion processes. Chemosphere. 12: 627-636.
Oliver, J.E, and J.M. Ruth. 1983. Nitration of two TCDDs and their
conversion to 1,2,3,6,7,8-HCOO. Chemosphere. 12: 1497-1503.
1862A 15-66 03/29/84
-------
Oliver, R.M. 1975. Toxic effects of 2,3,7,8-tetrachlorod1benzo-l,4-d1ox1n
in laboratory workers. Br. J. Ind. Med. 32(1): 49-53.
Olson, J.R. and W.E. Bittner. 1983. Comparative metabolism and elimination
Of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCOD). lexicologist. 3: 103.
Olson, J.R., T.A. Gasiewicz and R.A. Neal. 1980a. Tissue distribution,
excretion, and metabolism of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in
the Golden Syrian hamster. Toxicol. Appl. Pharmacol. 56: 78-85.
Olson, J.R., M.A. Holscher and R.A. Neal. 1980b. Toxicity of 2,3,7,8-
tetrachlorodibenzo-p-dioxin in the Golden Syrian hamster. Toxicol. Appl.
Pharmacol. 55: 67-78.
Olson, J.R., M. Gudzinowicz and R.A. Neal. 1981. The ]m vitro and ijrt vivo
metabolism of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCOD) in the rat.
Toxlcologist. 1: 69-70.
Olson, J.R., Gasiewicz, T.A., L.E. Geiger and R.A. Neal. 1983. The metabo-
lism of 2,3,7,8-tetrachlorod1benzo-p-d1oxin in mammalian systems, in: Acci-
dental Exposure to Dioxins: Human Health Aspects, F. Coulston and f. Poc-
chiari, id. Academic Press, NY. p. 81-100.
Olsson, H. and L. Brandt. 1981. Non-Hodgkin's lymphoma of the skin and
occupational exposure to herbicides. Lancet. 9/12/81: 579.
Orris, P. 1981. Unjustified conclusion? J. Occup. Med. 23(1): 7-8.
1862A 15-67 03/29/84
-------
Ott, M.G., B.B. Holder and R.D. Olson. 1980. A mortality analysis of
employees engaged 1n the manufacture of 2,4,5-tr1chlorophenoxyacet1c acid.
J. Occup. Med. 22(1): 47-50.
Owerts, I.S. 1977. Genetic regulation of UDP-glucuronosyltranferase Induc-
tion by polycycllc aromatic compounds 1n mice. J. B1ol. Chem. 252:
2827-2833.
Parker, C.E., W.A. Jones, H.B. Matthews, E.E. McConnell and J.R. Mass.
1980. Chronic toxldty of technical and analytical pentachlorophenol 1n
cattle. II. Chemical analysis of tissues. Toxlcol. Appl. Pharmacol.
55(2): 359-369.
Parkinson, A., R. Cockerline and S. Safe. 1980a. PolychloMnated blphenyl
Isomers and congeners as Inducers of both 3-methylcholanthrene- and pheno-
barbltone-type mlcrosomal enzyme activity. Chem.-Blol. INteract. 29:
277-289.
Parkinson, A., L. Robertson, L. Safe and S. Safe. 1980b. PolychloMnated
blphenyls as Inducers of hepatic mlcrosomal enzymes: Structure-activity
rules. Chem.-B1ol. Interact. 30: 271-285.
Parkinson, A., R. Cockerline and S. Safe. 1980c. Induction of both
3-methylcholanthrene- and phenobarbltone-type mlcrosomal enzyme activity by
a single polychlorlnated blphenyl Isomer. Blochem. Pharmacol. 29: 259-262.
1862A 15-68 03/29/84
-------
Parkinson, A., L. Robertson, L. Uhlig, M.A. Campbell and S. Safe. 1982.
2,3,4,5',5-Pentachloroblphenyl: Differential effects on C56BL/6J and OBA/2J
Inbred mice. Blochem. Pharmacol. 31: 2830-2833.
Parkinson, A., S. Safe, L. Robertson, et al. 1983. Immunochemlcal quantl-
tatlon of cytochrorne P-450 Isozymes and epoxlde hydrolase 1n liver micro-
somes from polychlorlnated and polybromlnated blphenyls: A study of struc-
ture activity relationships. J. B1ol. Chem. 258: 5967-5976.
Passl, S., M. Nazzaro-Porro, L. Bonlfortl and F. Glanottl. 1981. Analysis
of Uplds and dloxin In chloracne due to tetrachloro-2,3,7,8-p-d1benzod1ox-
1n. Br. J. Dermatol. 105(2): 137-143.
Pazderova-Vejlupkova, J., M. Nemcova, J. Plckova, L. Jlrasek and E. Lukas.
1981. The development and prognosis of chronic Intoxication by tetrachloro-
d1benzo-p-d1ox1n In men. Arch. Environ. Health. 36(1): 5-11.
Pegg, O.G., W.R. Hewitt, K.M. McCormack and J.B. Hook. 1976. Effect of
2,3,7,8-tetrachlored1benzo-rho-d1ox1n on renal function 1n the rat. J.
loxlcol. Environ. Health. 2(1): 55-65.
Peterson, R.E., N. Hamada, K.H Yang, B.V. Madhukar and F. Matsumura. 1979a.
Depression of adenoslne trlphosphatase activities 1n Isolated Hver surface
membranes of 2,3,7,8-tetrachlorod1benzo-p-d1ox1n-treated rats: Correlation
with effects on ouabaln biliary excretion and bile flow. J. Pharmacol. Exp.
Iherap. 210(2): 275-282.
1862A 15-69 03/29/84
-------
Peterson, R.E., N. Hamada, K.H. Yang, B.V. Madhukar and F. Matsumura.
1979b. Reversal of 2,3,7,8-tetrachlorod1benzo-p-d1ox1n-1nduced depression
of ouabain biliary excretion by pregnenolone-!6alpha-carbon1tr1le and
splronolactone in Isolated perfused rat livers. Toxlcol. Appl. Pharmacol.
50(3): 407-416.
Philippi, M., V. Krasnobagew, J. Zeyer and R. Huetter. 1981. Fate of
2,3,7,8-tetrachlorodibenzo-p-d1ox1n (TCOD) 1n microblal cultures and soil
under laboratory conditions. FEMS Symp. 12: 2210-233.
Ph1ll1pson, D.W. and B.J. Puma. 1980. Identification of chlorinated
methoxyblphenyls as contaminants 1n fish and as potential Interferences 1n
the determination of chlorinated d1benzo~p-d1ox1ns. Anal. Chem. 42:
2328-2332.
Piper, W.N., R.Q. Rose and P.J. Gehrlng. 1973. Excretion and tissue
distribution of 2,3,7,8-tetrachlorod1benzo-p-d1ox1n 1n the rat. Environ.
Health Perspect. 5: 241-244.
Pitot, H.C., T. Goldsworthy and H. Poland. 1980. Promotion by 2,3,7,8-
tetrachlorodibenzc-p-dioxin of hepatocardnogenesls from d1ethyln1trosam1ne.
Cancer Res. 40: 3616-3620.
Plimmer, J.R. 1978. Photolysis of TCDO and trifluralln on silica and soil.
Bull. Environ. Contam. Toxlcol. 20: 87-92.
1862A 15-70 03/29/84
-------
PUmmer, J.R., J.M. Ruth and E.A. Woolson. 1973. Mass spectrometMc iden-
tification of the hepta- and octachlorlnated d1benzo-p-d1ox1ns and dlbenzo-
furans 1n technical pentachlorophenol. J. AgMc. Food Chem. 21(1): 90-93.
PocchlaM, F., V. Sllano and A. Zamp1er1. 1979. Human health effects from
accidental release of tetrachlorod1benzo-p-d1ox1n (TCDD) at Seveso, Italy.
Ann. NY Acad. Sci. 320: 311-320.
Pocch1ar1, F., A. DIDomenico, V. Sllano and G. Zapponl. 1983. Environment-
al Impact of the accidental release of tetrachlorod1benzo-p-d1ox1n (TCDD) at
Seveso (Italy), in: Accidental Exposure to D1ox1ns: Human Health Aspects,
F. Coulston and F. Pocch1ar1, Ed. Academic Press, NY. p. 5-35.
Pohland, A.E. and G.C. Yang. 1972. Preparation and characterization of
chlorinated d1benzo-p-d1ox1ns. J. AgMc. Food Chem. 20: 1093-1099.
Poiger, H. and C. Schlatter. 1979. Biological degradation of TCDD 1n rats.
Nature. 281(5733): 706-707.
Poiger, H. and C. Schlatter. 1980. Influence of solvents and adsorbents on
dermal and Intestinal absorption of KDD. Food Cosmet. Toxlcol. 18(5):
477-481.
Poiger, H., H. Weber and C. Schlatter. 1982a. Special aspects of metabo-
lism and kinetics of TCDD 1n dogs and rats. Assessment of toxldty of TCDD-
metabollte(s) 1n guinea pigs. In: Chlorinated D1ox1ns and Related Com-
pounds. Impact on the Environment, 0. Hutzlnger, R.W. Fre1, E. MeMan and
F. Pocchlari, Ed. Pergamon Press, NY. p. 317-325.
1862A 15-71 03/29/84
-------
Polger, H., H.R. Buser, H. Weber, U. Zwelfel and C. Schlatter. 1982b.
Structure elucidation of mammalian TCDD-metabol1tes. Exper1ent1a. 38(4):
484-486.
Poland, A. and E. Glover. 1973a. Chlorinated d1benzo-p-d1ox1ns: Potent
Inducers of <5-am1nolevul1n1c acid synthetase and aryl hydrocarbon
hydroxylase. II. A study of the structure activity relationship. Mol.
Pharmacol. 9: 736.
Poland, A. and E. Glover. 1973b. 2,3,7,8-Tetrachlorod1benzo-p-d1ox1n.
Potent Inducer of delta-am1nolevul1n1c add synthetase. Science.
179(4072): 476-477.
Poland, A. and E. Glover. 1974. Comparison of 2,3,7,8-tetrachlorodlbenzo-
p-d1ox1n, a potent Inducer of aryl hydrocarbon hydroxylase, with Inducer
3-methylcholanthrene. Mol. Pharmacol. 10(2): 349-359.
Poland, A. and E. Glover. 1975. Genetic expression of aryl hydrocarbon
hydroxylase by 2,3,7,8-tetrachlorod1benzo-p-d1ox1n: Evidence for a receptor
mutation In genetically non-responsive mice. Mol. Pharmacol. 11(4):
389-398.
Poland, A. and E. Glover. 1979. An estimate of the maximum jjn vivo cova-
lent binding of 2,3,7,8-tetrachlorod1benzo-p-d1ox1n to rat liver protein,
riboscmal RNA and DNA. Cancer Res. 39(9): 3341-3344.
1862A 15-72 03/29/84
-------
Poland, A. and E. Glover. 1980. 2,3,7,8-Tetrachlorodibenzo-p-dioxin:
Segregation of toxIcHy with the Ah locus. Mol. Pharmacol. 17(1): 86-94.
Poland, A. and J.C. Knutson. 1982. 2,3,7,8-Tetrachlorod1benzo~p-d1ox1n and
related halogenated aromatic hydrocarbons: Examination of the mechanism of
toxldty. Ann. Rev. Pharmacol. Toxlcol. 22: 517-554.
Poland, A.P., D. Smith, G. Metter and P. Possick. 1971. A health survey of
workers in a 2,4-D and ?,4,5~T plant with special attention to chloracne,
porphyrla cutanea tarda, and psychologic parameters. Arch. Environ. Health.
22: 316-327.
Poland, A.P., E. Glover, J.R. Robinson and D.W. Nebert. 1974. Genetic
expression of aryl hydrocarbon hydroxylase activity. Induction of mono-
oxygenase activities and cytochrome PI-450 formation by 2,3,7,8-tetrachloro-
dibenzo-p-dioxin in mice genetically "nonresponsive" to other aromatic
hydrocarbons. J. B1ol. Chem. 249(17): 5599-5606.
Poland, A., E. Glover and A.S. Kende. 1976. Stereospecif1c, high affinity
binding of 2,3,7,8-tetrachlorod1benzo-p-diox1n by hepatic cytosol. Evidence
that the binding species 1s a receptor for induction of aryl hydrocarbon
hydroxylase. J. Biol. Chem. 251(16): 4936-4946.
Poland, A., W.F. Greenlee and A.S. Kende. 1979. Studies on the mechanisms
of action of the chlorinated d1benzo-p-dioxins and related compounds. Ann,
NY Acad. Sci. 320: 214-230.
1862A 15-73 03/29/84
-------
Poland, A., D. Palen and E. Glover. 1982. Tumour promotion by TCDD 1n skin
of HRS/J mice. Nature. 300(5889): 271-273.
Poland, A., J. Knutson and E. Glover. 1983. A consideration of the mechan-
ism of action of 2,3,7,8-tetrachlorod1benzo-p-d1ox1n and related halogenated
aromatic hydrocarbons. J.TK Human and Environmental Risks of Chlorinated
D1ox1ns and Related Compounds, R.E. Rucker, A.L. Young and A.P. Gray, Ed.
Plenum Press, NY. p. 539-559.
Poll, A., G. Grancesdnl, L. Pugl1s1 and C.R. Slrtorl. 1980. Increased
total and high density Upoproteln cholesterol with apoproteln changes
resembling streptozotocln diabetes In tetrachlorodlbenzodloxin (TCDD)
treated rats. Blochem. Pharmacol. 28(5): 835-838.
Poole, C. 1983. Statement of Charles Poole before the United States House
of Representatives Science and Technology Committee, Subcommittee on Natural
Resources, Agricultural Research and Environment. March 23.
Potter, C.L., 1.6. S1pes and D.H. Russell. 1982. Inhibition of ornlthlne
decarboxylase activity by 2,3,7,8-tetrachlorod1benzo-p-d1ox1n. Blochem.
Pharmacol. 31(21): 3367-3371.
Ramsey, J.C., J.G. Hefner, R.J. Karbowskl, W.H. Braun and P.J. Gehrlng.
1982. The jin vitro blotransformatlon of 2,3,7,8-tetrachloroci1benzo-p-d1ox1n
(TCDD) In the rat. Toxlcol. Appl. Pharmacol. 65: 180-184.
1862A 15-74 03/29/84
-------
Rappe, C., S. Marklund, H.R. Buser and H.P. Bosshardt. 1978. Formation of
polychlorlnated d1benzo~p-d1ox1ns and dlbenzofurans by burning or heating
chlorophenates. Chemosphere. 7(3): 269-281.
Rappe, C., H.R. Buser and H.P. Bosshardt. 1979. Oloxlns, dlbenzofurans.,
and other polyhalogenated aromatlcs production use formation and destruc-
tion. Ann. NY Acad. Sci. 320: 1-18.
Rappe, C., S. Marklund, M. Nygren and A. Gari. 1983a. Parameter for
Identification and confirmation 1n trace analyses of polychlorlnated dloxlns
and dlbenzofurans. In: Chlorinated Dloxlns and Dlbenzofurans 1n the Total
Environment, Vol. I, L.H. Keith et al., Ed. Butterworth Publishers.
Rappe, C., S. Marklund, P.A. Bergqvlst and P. Hansson. 1983b. Polychlorl-
nated dloxlns, dlbenzofurans and other polychlorlnated polynuclear aromatlcs
formed during Incinerator and PCB fires. in: Chlorinated Dloxlns and
Dlbenzofurans 1n the Total Environment, Vol. I, L.H. Keith et al., Ed.
Butterworth Publishers.
Regglanl, G. 1980. Acute human exposure to TCDO 1n Seveso, Italy. J.
loxlcol. Environ. Health. 6(1): 27-43.
R11h1mak1, V., S. Asp, A.M. Seppalalnen and S. Herberg. 1978. Symptomatol-
ogy, Morbidity, and Mortality Experience of Chlorinated Phenoxy Add Herbi-
cide (2,4-D and 2,4,5-T) Sprayers 1n Finland: A Clinical and Ep1dem1olog1cal
Study. Working paper for an IARC working group meeting on coordination of
ep1dem1olog1cai studies on the long-term hazards of chlorinated dlbenzo-
dloxlns and chlorinated dlbenzofurans. Lyon, France. Jan. 10-11.
186PA 15-75 03/29/84
-------
R11h1mak1, V., A. S1sko and S. Hernberg. 1982. Mortality of 2,4-d1chloro-
phenoxyacetlc add and 2,4,5-tr1chlorophenoxyacet1c add herbldde applica-
tors 1n Finland. Scand. J. Work Environ. Health. 8: 37-42.
R11h1mak1, V., S. Asp, E. Pukkala and S. Hernberg. 1983. Mortality and
cancer morbidity among chlorinated phenoxy add applicators 1n Finland.
Chemosphere. 12(4/5): 779-784.
Rlkans, L.E., 0.0. Gibson and P.B. McCay. 1979. Evidence for the presence
of cytochrome P-450 1n rat mammary gland. Blochem. Pharmacol. 28(19):
3039-3042.
Rlzzardini, M., M. Romano, F. Turzl, et al. 1983. lexicological evaluation
of urban waste Incinerator emissions. Chemosphere. 12: 559-564.
Robinson, J.R., J.S. Felton, R.C. Levitt, S.S. Thorgelrsson and D.W. Nebert.
1975. Relationship between aromatic hydrocarbon responsiveness and the sur-
vival times In mice treated with various drugs and environmental compounds.
Mol. Pharmacol. 11: 850-865.
Rogers, A.M., M.E. Anderson and K.C. Back. 1982. Mutagenldty of 2,3,7,8-
tetrachlorod1benzo-p-d1ox1n and perfIuoro-n-decano1c add 1n L5178y mouse
lymphoma cells. Mutat. Res. 105: 445-449.
Roll, R. 1971. Studies of the teratogenlc effect of 2,4,5-T In mice. Food
Cosmet. Toxlcol. 9(5): 671-676.
1862A 15-76 03/29/84
-------
Rose, J.Q., J.C. Ramsey, T.H. Wentzler, R.A. Hummel and P.J. Gehrlng. 1976.
The fate of 2,3,7,8-tetrachlorodibenzo-p-d1oxin following single and
repeated oral doses to the rat. Toxlcol. Appl. Pharmacol. 36(2): 209-226.
Ryan, J.J. and J.C. P1lon. 1980. High performance liquid chromatography 1n
the analysis of chlorinated d1benzod1ox1ns and dlbenzofurans 1n the chicken
liver and wood shaving samples. J. Chromatogr. 197: 171-180.
Ryan, J.J., J.C. Pllon, H.B.S. Conacher and D. Firestone. 1983. Inter-
laboratory study on determination of 2,3,7,8-tetrachlorod1benzo-p-d1ox1n 1n
fish. J. Assoc. Off. Anal. Chem. 66: 700-707.
Safe, S., L.W. Robertson, L. Safe, et al. 1982. Halogenated blphenyls:
Molecular toxicology. Can. J. Physlol. Pharmacol. 60: 1057-1064.
SAI (Systems Applications, Inc.) 1980. Human Exposure to Atmospheric Con-
centration of Selected Chemicals. Vol. I. NTIS PB 81-193252.
Sarma, P.R. and J. Jacops. 1981. Thoracic soft-tissue sarcoma In Vietnam
veterans exposed to agent orange. Lancet. 306(18): 1109.
Sawahata, T., J.R. Olson and R.A. Neal. 1982. Identification of metabo-
lites 2,3,7,8-tetrachlorod1benzo-p-dioxin (TCDD) formed on incubation with
isolated rat hepatocytes. Blochem. Biophys. Res. Commun. 105(1): 341-346.
Sawyer, 1. and S. Safe. 1982. PCB Isomers and congeners: Induction of aryl
hydrocarbon hydroxylase and ethoxyresoruf1n 0-deethylaes enzyme activities
In rat hepatoma cells. Toxlcol. Lett. 13: 87-94.
1862A 15-77 03/29/84
-------
Sawyer, 7., S. Bandlera, S. Safe, 0. Hutzlnger and K. Ol1e. 1983. Bio-
analysis of polychlorlnated dlbenzofuran and d1benzo-p-d1oxin mixtures In
fly ash. Chemosphere. 12: 529-535.
Scarpelll, O.G., M.S. Rao, V. Subbarao, M. Beversluls, D.P. Gurka and P.F.
Hollenberg. 1980. Activation of nltrosamlnes to mutagens by post-mitochon-
dria! fraction of hamster pancreas. Cancer Res. 40(1): 67-74.
Schantz, S.L., D.A. Barsottl and J.R. Allen. 1979. lexicological effects
produced 1n nonhuman primates chronically exposed to fifty parts per tril-
lion 2,3,7,8-tetrachlorod1benzo-p-d1ox1n (TCDD). Toxlcol. Appl. Pharmacol.
48: A180.
Schueler, R.L. 1983. Review of Selected Neoplastlc and Non-neoplast1c
Liver Lesions 1n Rats Given Hexachlorod1benzo-p-d1ox1ns. Prepared for the
U.S. EPA under Contract No. 68-02-3763.
Schwetz, B.A., J.M. Norrls, G.L. Sparschu, et al. 1973. Toxicology of
chlorinated d1benzo-p-d1ox1ns. Environ. Health Perspect. 5: 87-99.
Seefeld, M.D. and R.E. Peterson. 1983. 2,3,7,8-Tetrachlorod1benzo-p-
d1ox1n-1nduced weight loss: A proposed mechanism. In: Human and Environ-
mental Risks of Chlorinated Dloxlns and Related Compounds, R.E. Tucker, A.L.
Young and A.P. Gray, Ed. Plenum Press, NY. p. 405-413.
Seefeld, M.O., R.M. Albrecht and R.E. Peterson. 1979. Effects of 2,3,7,8-
tetrachlorod1benzo-p-d1ox1n on 1ndocyan1ne green blood clearance In rhesus
monkeys. Toxicology. 14(3): 263-272.
1862A 15-78 03/29/84
-------
Seefeld, M.D., R.M. Albrecht, K.W. GIlchMst and R.E. Peterson. 1980.
Blood clearance tests for detecting 2,3,7,8-tetrachlorod1benzo-p-d1ox1n
hepatotox1c1ty 1n rats and rabbHs. Arch. Environ. Contam. Toxlcol. 9(3):
317-327.
Seller, J.P. 1973. A survey on the mutagenldty of various pesticides.
Exper1ent1a. 29: 622-623.
Shadoff, L.A., R.A. Hummel, L. Lamparskl and J.H. Davidson. 1977. A search
for 2,3,7,8-tetrachlorod1benzo--p-d1ox1n (1CDD) 1n an environment exposed
annually to 2,4,5-tr1chlorophenoxyacet1c add ester (2,4,5-T) herbicides.
Bull. Environ. Contam. Toxlcol. 18(4): 478-485.
Sharma, R.P. and P.J. Gehrlng. 1979. Effects of 2,3,7,8-tetrachlorodl-
benzo-p-d1oxin (TCDO) on splenic lymphocyte transformation 1n mice after
single and repeated exposures. Ann. NY Acad. Sc1. 320: 487-497.
Shaub, W.M. and W. Tsang. 1983. Physical and chemical properties of
dloxlns in relation to their disposal. _In: Human and Environmental Risks of
Chlorinated Dloxlns and Related Compounds, R. Tucker et al., Ed. Plenum
Publishing Corp., NY. p. 731-748.
Shlverick, K.T. and l.F. Muther. 1982. Effects of 2,3,7,8-tetrachlorodl-
benzo-p-d1ox1n on serum concentrations and the uterotrophlc action of
exogenous estrone In rats. Toxlcol. Appl. Pharmacol. 65(1): 170-176.
1862A 15-79 03/29/84
-------
Sh1ver1ck, K.I. and l.F. Muther. 1983. 2,3,7,8-Tetrachlorod1benzo-p-d1ox1n
(1CDD) effect on hepatic mlcrosomal steroid metabolism and serum estradlol
of pregnant rats. Blochem. Pharmacol. 32: 991-995.
Shum, S., N.M. Jensen and D.W. Nebert. 1979. Ihe Ah Locus. In utero tox-
1c1ty and teratogenesls associated with genetic differences In benzofa]-
pyrene metabolism. Teratology. 20: 365-376.
Sllkworth, J., 0. McMartln, A. DeCapMo, R. Rej, P. O'Keefe and L. Kamlnsky.
1982. Acute toxldty 1n guinea pigs and rabbits of soot from a polychloM-
nated b1phenyl-conta1n1ng transformer fire. Toxlcol. Appl. Pharmacol. 65:
425-439.
Singer, R., M. Moses, J. Valdukas, R. L1l1s and I.J. Sellkoff. 1982.
Nerve conduction velocity studies of workers employed 1n the manufacture of
phenoxy herbicides. Environ. Res. 29: 297-311.
S1rch1a, G. 1978. Report to Lombardy Regional Authority (unpublished).
(Cited 1n Pocch1ar1 et al., 1979)
Smith, J. 1979. EPA halts most uses of herbicide 2,4,5-T. Science.
203(4385): 1090-1091.
Smith, A.M. 1983. School of Public Health, University of California.
Letter to the Carcinogen Assessment Group, U.S. EPA, December 13.
1862A 15-80 03/29/84
-------
Smith, F.A., B.A. Schwetz and K.D. NHschke. 1976. Teratogenlcity of
2,3,7,8-tetrachlorodibenzo-p-dioxin 1n CF-1 mice. Toxlcol. Appl. Pharmacol.
38(3): 517-523.
Smith, A.M., J.E. Francis, S.J. Kay and J.8. Grelg. 1981. Hepatic toxldty
and uroporphyrlnogen decarboxylase activity following a single dose of
2,3,7,8-tetrachlorod1benzo-p-diox1n to mice. Blochem. Pharmacol. 30(20):
2825-2830.
Smith, A.M., 0.0. Fisher, N. Pearce and C.J. Chapman. 1982a. Congenital
defects and miscarriages among New Zealand 2,4,5-T sprayers. Arch. Environ.
Health. 37: 197-200.
Smith, A.H., D.O. Fisher, N. Pearce and C.A. league. 1982b. Do agricultur-
al chemicals cause soft-tissue sarcoma? Initial findings of a case-control
study in New Zealand. Community Health Studies. 6(2): 114-119.
Smith, A.H., 0.0. Fisher, H.J. Giles and N. Pearce. 1983a. The New Zealand
soft tissue sarcoma case-control study: Interview findings concerning
phenoxyacetic add exposure. Chemosphere. 12(4/5): 565-571.
Smith, R.M., P.M. O'Keefe, K.M. Aldous, D.R. Hilker and J.E. O'Brian.
1983b. 2,3,7,8-Tetrachlorodibenzo-p-d1ox1n in sediment and samples from
Love Canal storm sewers and creeks. Environ. Sci. Technol. 17: 6-10.
Sparschu, G.I., Jr., F.L. Dunn, Jr., and V.K. Rowe, Jr. 1971a. Study of
the teratogenicity of 2,3,7,8-tetrachlorod1benzo-p-dioxin in the rat. Food
Cosmet. Toxlcol. 9: 405-412.
1862A 15-81 04/05/84
-------
Sparschu, G.L., F.L. Dunn, R.W. Usowe and V.K. Rowe. 1971b. Effects of
high levels of 2,4,5-tr 1chlorophenoxyacet1c add on fetal development 1n the
rat. Food Cosmet. Toxlcol. 9(4): 527-530.
Spencer, H.W. and W. Woodrow. 1979. Memorandum to D. Relsa. TOAP review
at the University of Wisconsin. TCDO 1n rats. February 8.
Squire, R.A. 1980. Pathologic Evaluations of Selected Tissues from the Dow
Chemical TCDD and 2,4,5-T Rat Studies. Submitted to Carcinogen Assessment
Group, U.S. EPA on August 15 under contract no. 68-01-5092.
Squire, R.A. 1983. An Assessment of the Experimental Evidence for Poten-
tial Cardnogenlclty of Hexachlorod1benzo-p-d1ox1ns. Report prepared for
Vulcan Chemicals and Relchhold Chemicals, Inc. June 29, 1983.
SRI (Stanford Research Institute) International. 1982. 1982 Directory of
Chemical Producers: USA, SRI International, Menlo Park, CA.
Stalling, D.L., L.M. Smith, J.D. Petty, et al. 1983. Residues of poly-
chlorinated d1benzo-p-d1ox1ns and dlbenzofurans In Laurentlan Great Lakes
fish. In: Human and Environmental Risks of Chlor mated D1ox1ns and Related
Compounds, R.E. Tucker et al., Ed. Plenum Press, NY. p. 220-240.
Stanley, J., C. Halle, A. Small and E. Olson. 1982. Sampling and Analysis
Procedures for Assessing Organic Emissions from Stationary Combustion
Sources for EED Studies. Methods-Manual. EPA 580/5-82-014. Office of
Toxic Substances, U.S. EPA, Washington, DC.
1862A 15-82 03/29/84
-------
Stehl, R.H., R.R. Paperfuss, R.A. Bredeweg and R.W. Roberts. 1973. The
stability of pentachlorophenol and chlorinated dloxlns to sunlight, heat and
combustion. Adv. Chem. Ser. 120, Am. Chem. Soc. p. 119.
Stephan, C.E. 1980. Memorandum to Jerry F. Stara, U.S. EPA, Cincinnati,
OH. July.
Stevens, K.M. 1981. Agent Orange toxldty: A quantitative perspective.
Human Toxlcol. 1(1): 31-39.
Stohs, S.J., M.Q. Hassan and W.J. Murray. 1983. L1p1d peroxldatlon as a
possible cause of 1CDO toxldty. Blochem. Blophys. Res. Commun. Ill:
854-859.
Strlk, J.J. and J.M. de W1t. 1980. Health aspects of rabbits 1n a low-TCDO
contaminated area. Int. J. Blochem. 12(5-6): 999-1001.
Sundstrom, G., S. Jensen, B. Jansson and K. Erne. 1979. Chlorinated
phenoxyacetic add derivatives and tetrachlorod1benzo-p-d1ox1n 1n foliage
after application of 2,4,5-tr1chlorophenoxyacet1c add esters. Arch.
Environ. Contam. Toxlcol. 8(4): 441-448.
Sweeney, G.D. and K.G. Jones. 1978. On the mechanism of porphyrla due to
chlorinated hydrocarbons. Int. Congr. Ser. Excerpta Med. 440: 229-231.
1862A 15-83 03/29/84
-------
Sweeney, G.G. and K.G. Jones. 1983. Studies of the mechanism of action of
hepatotoxlclty of 2,3,7,8-tetrachlorod1benzo-p-d1ox1n (TCDD) and related
compounds. Iji: Human and Environmental Risks of Chlorinated Dloxlns and
Related Compounds, R.E. lucker, A.L. Young and A.P. Gray, Ed. Plenum Press,
NY. p. 415-422.
Sweeney, G.O., K.G. Jones, P.M. Cole, 0. Basford and F. Krestynski. 1979.
Iron deficiency prevents liver toxldty of 2,3,7,8-tetrachlorodlbenzo-p-
dloxln. Science. 204(4390): 332-335.
Swift, L.L., T.A. Gas1ew1cz, G.O. Dunn, P.O. Soul'e and R.A. Neal. 1981.
Characterization of the hyper!1p1dem1a 1n guinea pigs Induced by 2,3,7,8-
tetrachlorod1benzo-p-d1ox1n. Toxlcol. Appl. Pharmacol. 59(3): 489-499.
Taylor, J.S. 1979. Environmental chloracne: Update and overview. Ann. NY
Acad. Sci. 320: 295-307.
Taylor, M.L., T.O. Tier nan, J.H. Garrett, G.F. Van Ness and J.G. Solch.
1983. Assessments of Incineration processes as sources of supertoxlc chlo-
rinated hydrocarbons: Concentrations of polychlorlnated d1benzo-p-d1ox1ns/
dlbenzofurans and possible precursor compounds In Incinerator effluents.
ln_: Chlorinated 01ox1ns and Dlbenzof urans In the Total Environment, Vol. I,
L.H. Keith et al., Ed. Butterworth Publishers, p. 125-164.
leltelbaum, P.J. and A.P. Poland. 1978. Studies of the hepatic uptake of
2,3,7,8-tetrachlorod1benzo-p-d1ox1n 1n the mouse. Fed. Proc. Fed. Am. Soc.
Exp. B1ol. 37(3): 692.
1862A 15-84 03/29/84
-------
Thlbodeaux, L.J. 1979. Chemodynam1cs-Env1ronmental Movement of Chemicals
In A1r, Water and Soil. John Wiley and Sons, Inc., NY. p. 176-177.
Ihlbodeaux, L. 1983. Off-site transport of 2,3,7,8-tetrachlorodlbenzo-p-
dloxln from a production disposal facility. In: Chlorinated D1ox1ns and
Dlbenzofurans 1n the Total Environment, Vol. I, L.H. Keith et al., Ed.
Butterworth Publishers, Woburn, MA.
Ihless, A.M. and R. Frentzel-Beyme. 1977. Mortality Study of Persons
Exposed to D1ox1n Following an Accident which Occurred 1n the BASF on 13,
November, 1953. Proceedings of MEDICHEM Congress V, San Francisco, Sept. 5.
Ihless, A.M. and R. Frentzel-Beyme. 1978. Mortality Study of Persons
Exposed to Dloxln Following an Accident which Occurred In the BASF on 13,
November, 1953. Working Papers, Joint NIEHS/IARC Working Group Report,
Lyon, France, June, 1978.
Ihless, A.M., R. Frentzel-Beyme and R. Link. 1982. Mortality study of
persons exposed to dloxln 1n a trlchlorophenol-process accident that
occurred In the BASF AG on November 17, 1953. Am. J. Ind. Med. 3: 179-189.
Ihlgpen, J.E., R.E. Faith, E.E. McConnell and J.A. Moore. 1975. Increased
susceptibility to bacterial Infection as a sequela of exposure to 2,3,7,8-
tetrachlorod1benzo-p-d1ox1n. Infect. Immun. 12(6): 1319-1324.
Thomas, N.A. 1983. Memorandum to C.E. Stephan, U.S. EPA, Duluth, MN.
July 22.
1862A 15-85 03/29/84
-------
Thomas, P.E., J.J. Mutton and B.A. Taylor. 1973. Genetic relationship
between aryl hydrocarbon hydroxylase 1nduc1b1lHy and chemical carcinogen
Induced skin ulceratlon 1n mice. Genetics. 74: 655-659.
Thomas, P.I. and R.D. H1nsd1ll. 1979. The effect of perinatal exposure to
tetrachlorod1benzo-p-d1ox1n on the Immune response of young mice. Drug
Chem. Toxlcol. 2(1,2): 77-98.
Thomas, H.F. 1980a. Internal memo to P. Cohn, Office of Toxic Substances,
U.S. EPA, Washington, DC.
Thomas, H.F. 1980b. 2,4,5-T use and congenital malformation rates 1n
Hungary. Lancet. 11: 214-215.
Thunberg, T., U.G. Ahlborg and H. Johnsson. 1979. Vitamin A (retlnol)
status In the rat after a single oral dose of 2,3,7,8-tetrachlorod1benzo-p-
dloxin. Arch. Toxlcol. 42(4): 265-274.
Thunberg, T., U.G. Ahlborg, H. Hakansson, C. Krantz and M. Monler. 1980.
Effect of 2,3,7,8-tetrachlorod1benzo-p-d1ox1n on the hepatic storage of
Retlnol 1n rats with different dietary supplies of vitamin A (Retlnol).
Arch. Toxlcol. (Berl). 45(4): 273-285.
Tlernan, T.O. 1982. Chlorod1benzod1ox1ns and chlorodlbenzofurans: An
overview, in: Detox1cat1on of Hazardous Wastes, J.H. Exner, Ed. Ann Arbor
Science Publishers, Inc., Ann Arbor, MI. p. 243-260.
1862A 15-86 04/05/84
-------
Hernan, T.O. 1983. Analytical chemistry of polychlorlnated d1benzo-p-
dloxlns and dlbenzofurans: A review of the current status. In: Chlorinated
01ox1ns and Olbenzofurans 1n the Total Environment, Vol. I, L.H. Keith et
al., Ed. Butterworth Publishers, p. 211-237.
Hernan, 1.0., M.L. Taylor, S.D. Erk, J.G. Solch and G. Van Ness. 1980.
D1ox1ns. Vol. II. Analytical Method for Industrial Wastes. Final Report
Industrial Environmental Research Lab., Cincinnati, OH. EPA 600/280-57.
80 p.
Hernan, T.O., M.L. Taylor, J.G. Solch, G.F. Van Ness, J.H. Garrett and M.O.
Porter. 1982a. Incineration of chemical wastes containing polychlorlnated
blphenyls: Assessment of tests conducted at Rollins Environmental Sciences,
Deer Park, TX, and Energy Systems Co., El Dorado, AR. Iin: Detoxification of
Hazardous Waste, J.H. Exner, Ed. Ann Arbor Science (Butterworth).
p. 143-184.
Tlernan, T.O., M.L. Taylor, J.G. Solch, G.F. Van Ness and J.H. Garrett.
1982b. Characterization of toxic components 1n the effluent from a refuse-
fired Incinerator. Resour. Conserv. 9: 343-354.
ToHlon, P.J., P.G. Peters, R.P. Clement, D.M. Hardwlcke and W.N. Piper.
1980. Depressed guinea pig testlcular mlcrosomal cytochrome P-450 content
by 2,3,7,8~tetrachlorod1benzo-p-d1cx1n. Life Sd. 27(10): 871-876.
Tognonl, G. and A. Bonaccorsl. 1982. Ep1dem1olog1cal problems with
2,3,7,8-tetrachlorod1benzo-p-d1oxi.n (TCDD). Drug Metab. Rev. 13: 447-469.
1862A 15-87 04/05/84
-------
Toslne, H. 1981. Method used by Ontario Ministry of the Environment
primarily for the analysis of fish tissues and raw and untreated waters.
in: Polychlorlnated D1benzo-p-D1ox1ns: Limitations to the Current Analytical
Techniques, NRCC/NRC, Ottawa, Canada, p. 129-137.
Toth, K., J. Sugar, S. Somfal-Relle and J. Bence. 1978. Carcinogenic bio-
assay of the herbicide, 2,4,5-trlchlorophenoxyethanol (TCPE) with different
2,3,7,8-tetrachlorod1benzo-p-d1ox1n (dloxln) content 1n Swiss mice. Prog.
Blochem. Pharmacol. 14: 82-93.
Toth, K., S. Somfal-Relle, J. Sugar and .1. Bence. 1979. Carc1nogen1c1ty
testing of herbicide 2,4,5-trlchlorophenoxyethanol containing dloxln and of
pure dloxln 1n Swiss mice. Nature (Lond). 278(5704): 548-549.
Townsend, J.C., K.M. Bodner, P.F. Van Peenen, R.D. Olsen and R.R. Cook.
1982. Survey of reproductive events of wives of employees exposed to
chlorinated dloxlns. Am. J. Ep1dem1ol. 115(5): 695-713.
Tsushimoto, G., F. Matsumura and R. Sago. 1982. Fate of 2,3,7,8-tetra-
chlorod1benzo-p-d1ox1n (TCOD) 1n an outdoor pond and 1n model aquatic
ecosystem. Environ. Toxlcol. Chem. 1: 61-68.
Tulp, M.T.M. and 0. Hutzlnger. 1978. Rat metabolism of polychlorlnated
d1benzo-p-d1ox1ns. Chemosphere. 9: 761-768.
1862A 15-88 04/05/84
-------
Turner, J.N. and D.N. Collins. 1983. Liver morphology 1n guinea pigs
administered either pyrolysls products of a polychlorInated blphenyl trans-
former fluid or 2,3,7,8-tetrachlorod1benzo-p-d1ox1ns. Toxlcol. Appl.
Pharmacol. 67: 417-429.
United Kingdom Ministry of Agriculture, Fisheries and Food. 1983. Advisory
committee on pesticides: Report on phenoxy add herbicides. Whitehall
Place, London, February 7.
U.S. EPA. 1978. Report of the ad hoc study broup on pentachlorophenol and
contaminants. EPA/SAB/78/001. p. 170. (Cited in NRCC, 1981b)
U.S. EPA. 1979a. Chemistry Laboratory Manual for Bottom Sediments and
Elutriate Testing. EPA 903/4-79-014. NTIS PB 294 596, Springfield, VA.
U.S. EPA. 1979b. Report of the FIFRA Scientific Advisory Panel on Proposed
Section 6(b) (2) Notices for 2,4,5-T and Sllvex. Memorandum from Dr. H.
Wade Fowler, Jr., Executive Secretary FIFRA Scientific Advisory Panel to the
Deputy Assistant Administrator for Pesticide Programs. Sept. 27, 1979.
U.S. EPA. 1979c. Report of Assessment of a Field Investigation of Six-year
Spontaneous Abortion Rates 1n Three Oregon Areas 1n Relation to Forest
2,4,5-T Spray Practice. OTS, U.S. EPA.
U.S. EPA. 1980a. D1ox1ns. Industrial Pollution Control Division, U.S.
EPA, Cincinnati, OH. EPA 600/2-80-197.
1862A 15-89 04/05/84
-------
U.S. EPA. 1980b. Seafood Consumption Data Analysis, SRI International,
Menlo Park, CA. Final Report, Task 11. EPA Contract No. 68-01-3887.
U.S. EPA. 1980c. Carcinogen Assessment Group's Risk Assessment on (2,4,5-
Tr1chlorophenoxy)acet.1c add (2,4,5-T), (2,4,5-Tr1chlorophenoxy)prop1on1c
add (Sllvex), and 2,3,7,8-Tetrachlorod1benzo-p_-d1ox1n (TCDD). September 12.
U.S. EPA. 1982a. Test Method: 2,3,7,8-Tetrachlorod1benzo-p-d1ox1n-Method
613. Storet No. 34675. Environmental Monitoring and Support Laboratory,
Cincinnati, OH.
U.S. EPA. 1982b. Environmental Monitoring at Love Canal. Vol. I. EPA
600/4-82-030a, Office of Research and Development, U.S. EPA, Washington, DC,
May, 1982. NTIS PB 82-237330.
U.S. EPA. 1982c. Tolerances and Exemptions from Tolerances for Pesticide
Chemicals 1n or on Raw Agricultural Commodities. Food Drug Cosmetic Law
Reporter, 40 CFR 180.302.
U.S. EPA. 1983a. Dow Chemical Company -- Midland Plant Wastewater Charac-
terization Study. Preliminary Summary of Results. March 28.
U.S. EPA. 1983b. Memorandum from U.S. EPA Cancer Assessment Group, to Judy
BeHin, OSW, October 19.
U.S. EPA. 1984. Ambient Water Quality Criteria for 2,3,7,8-Tetrachlorodl-
benzo-p-d1ox1n. Environmental Criteria and Assessment Office, U.S. EPA,
Cincinnati, OH. EPA 440/5-84-007.
1862A 15-90 04/05/84
-------
USITC (United States International Trade Commission). 1982. Synthetic
Organic Chemicals. United States Production and Sales, 1981. USITC Publ.
No. 1292, Washington, DC.
Van der Berg, M., K. Olio and 0. Hutzlnger. 1983. Uptake and selective
retention )n rats of orally administered chlorinated dloxlns and dlbenzo-
furans from fly ash and fly ash extract. Chemosphere. 12(4/5): 537-544.
van Logten, M.J., B.N. Gupta, E.E. McConnell and J.A. Moore. 1980. Role of
the endocrine system In the action of 2,3,7,8-tetrachlorod1benzo-p-d1ox1n
(TCDD) on the thymus. Toxicology. 15(2): 135-144.
van Logten, M.J., B.N. Gupta, E.E. McConnell and J.A. Moore. 1981. The
Influence of malnutrition on the toxldty of 2,3,/,8-tetrachlorod1benzo-p-
dloxln (TCDD) In rats. Toxicology. 21(1): 77-88.
Van Miller, J.P., R.J. Marlar and J.R. Allen. 1976. Tissue distribution
and excretion of trltlated tetrachlorod1benzo-p-d1ox1n 1n non-human primates
and rats. Food Cosmet. Toxlcol. 14(1): 31-34.
Van Miller, J.P., J.J. Lallch and J.R. Allen. 1977a. Increased Incidence
of neoplasms 1n rats exposed to low levels of 2,3,7,8-tetrachlorod1benzo-p-
dloxln. Chemosphere. 6(10): 625-632.
Van Miller, J.P., J.J. Lallch and J.R. Allen. 1977b. Increased Incidence
of neoplasms In rats exposed to low levels of 2,3,7,8-tetrachlorod1benzo-p-
dloxln. Chemosphere. 6(9): 537-544.
1862A 15-91 04/05/84
-------
Van Ness, G.F., J.G. Solch, M.I. laylor and T.O. Tlernan. 1980. Tetra-
chlorod1benzo~p-d1ox1ns 1n chemical wastes, aqueous effluents and soils.
Chemosphere. 9: 553-563.
Veccht, A., A. Mantovanl, H. S1ron1, W. Lu1n1, F. Spreaflco and S.
Garattlni. 1980. The effect of acute administration of 2,3,7,8-tetra-
chlorod1benzo-p-d1ox1n (1CDD) on humoral antibody production and cell-
mediated activities 1n mice. Arch. Toxlcol. (Suppl.) 4: 163-165.
Vecchi, A., M. S1ron1, M.A. Canegratl, M. Recchla and S. Garattlni. 1983.
Immunosuppresslve effects of 2,3,7,8-tetrachlorod1benzo-p-d1ox1n 1n strains
of mice with different susceptibility to Induction of aryl hydrocarbon
hydroxylase. Toxlcol. Appl. Pharmacol. 68: 434-441.
Velth, G.D. and P. Koslan. 1983. Estimating bloconcentratlon potential
from octanol/water partition coefficients. In: Physical Behavior of PCBs 1n
the Great Lakes, D. Hackay et al., Ed. Ann Arbor Science, Ann Arbor, MI.
p. 269.
Velth, G.D., K.J. Macek, S.R. Petrocelll and J. Carroll. 1980. An evalua-
tion of using partition coefficients and water solubility to estimate bio-
concentration factors for organic chemicals 1n fish. In: J.G. Eaton et al.,
Ed., Aquatic Toxicology. ASTM STP 707. American Society for Testing and
Materials, Philadelphia, PA. p. 116-129.
Vlllaneuva, E.G., R.W. Jennings and V.W. Burse. 1973. Chlorod1benzo-p-
dloxln contamination of two commercially available pentachlorophenols. J.
AgMc. Food Chem. 21(4): 739.
186PA 15-92 04/05/84
-------
Vlnopal, J.H. and J.E. Caslda. 1973. Metabolic stability of 2,3,7,8-tetra-
chlorod1benzo-p-d1ox1n 1n mammalian Hver mlcrosomal systems and 1n living
mice. Arch. Environ. Contam. Toxlcol. 1(2): 122-132.
Vos, J.G. 1977. Immune suppression as related to toxicology. Cr1t. Rev.
Toxlcol. 5(1): 67-101.
Vos, J.G. and R.B. Beems. 1971. Dermal toxldty studies of technical
polychlorlnated blphenyls and fractions thereof 1n rabbits. Toxlcol. Appl.
Pharmacol. 19: 617-633.
Vos, J.G. and J.H. Koeman. 1970. Comparative toxlcologlc study with poly-
chlorinated blphenyls 1n chickens with special reference to prophyrln, edema
formation, liver necrosis and tissue residues. Toxlcol. Appl. Pharmacol.
17: 656-668.
Vos, J.G. and J.A. Moore. 1974. Suppression of cellular Immunity 1n rats
and mice by maternal treatment with 2,3,7,8-tetrachlorod1benzo-p-d1ox1n.
Int. Arch. Allergy Appl. Immunol. 47(5): 777-794.
Vos, J.G., J.A. Moore and J.G. Z1nkl. 1973. Effect of 2,3,7,8-tetrachloro-
d1benzo-p-d1ox1n on the Immune system of laboratory animals. Environ.
Health Perspect. 5: 149-162.
Vos, J.G., J.A. Moore and J.G. Zlnkl. 1974. Toxldty of 2,3,7,8-tetra-
chlorod1benzo-p-d1ox1n (TCDD) In C57B1/6 mice. Toxlcol. Appl. Pharmacol.
29: 229-241.
1862A 15-93 04/05/84
-------
Vos, J.G., J.G. Kreeftenberg and L. Kater. 1978a. Immune Suppression by
TCDD. UK D1ox1n: Tox1colog1cal and Chemical Aspects. SD Medical and
Scientific Books, NY. p. 163-175.
Vos, J.G., J.G. Kreeftenberg, H.W. Engel, A. Mlnderhoud and J.L.M. Van
Noorle. 1978b. Studies on 2,3,7,8-tetrachlorod1benzo-p-d1ox1n Induced
Immune suppression and decreased resistance to Infection: Endotoxln hyper-
sensitivity, serum zinc concentrations and effect of thymos 1n treatment.
Toxicology. 9(1-2): 75-86.
Walker, A.E. and J.V. Martin. 1979. L1p1d profiles 1n dloxln-exposed
workers. Lancet. 1: 446-447.
Walsh, J. 1977. Seveso: The questions persist where dloxln created a
wasteland. Science. 197(4308): 1064-1067.
Ward, A.M. 1982. Investigation of the Immune capability of workers
previously exposed to 2,3,7,8-tetrachlorod1benzo-para-d1oxin (TCDO). Iru
The Chemical Scythe: Lessons of 2,4,5-T and D1ox1n, A. Hay, Ed. Plenum
Press, NY. p. 117-118.
Ward, C. and F. Matsumura. 1977. Fate of 2,4,5-T contaminant, 2,3,7,8-
tetrachlorod1benzo-p-d1ox1n (TCDD) 1n aquatic environments. NTIS PB-264187.
28 p.
Ward, C.T. and F. Matsumura. 1978. Fate of 2,3,7,8-tetrachlorodlbenzo-p-
dloxln (TCDD) 1n a model aquatic environment. Arch. Environ. Contam.
Toxlcol. 7: 349-357.
1862A 15-94 04/05/84
-------
Wassom, J.S., J.E. Huff and N. LopMeno. 1978. A review of the genetic
toxicology of chlorinated d1benzo-p-d1ox1ns. Mutat. Res. 47(3,4): 141-160.
Weber, G., P. Luz1, L. Res1, P. TanganelH, M.R. Lovatl and A. Poll. 1983.
Natural history of KDD-1nduced Hver lesions 1n rats as observed by trans-
mission electron microscopy during a 32-week period after a single 1ntra-
perHoneal Injection. J. Toxlcol. Environ. Health. 12: 533-540.
Weber, H., H. Polger and C. Schlatter. 1982. Fate of 2,3,7,8-tetrachloro-
d1benzo-p-d1ox1n metabolites from dogs 1n rats. Xenoblotlca. 12(6):
353-357.
Welssberg, J.B. and J.G. Zlnkl. 1973. Effects of 2,3,7,8-tetrachloro-
d1benzo-p-d1ox1n upon hemostasls and hematologlc function 1n the rat.
Environ. Health Perspect. 5: 119-123.
Wlpf, H.K. and J. Schmld. 1983. Seveso — An environmental assessment.
In; Human and Environmental Risks of Chlorinated D1ox1ns and Related
Compounds, R.E. Tucker et al., Ed. Plenum Publishing Corp., NY. p. 255-274.
W1pf, H.K., E. Homberger, N. Nelmer, U.B. Ranalder, W. Vetter and J.P.
Vullleumelr. 1982. TCDD levels 1n soil and plant samples from the Seveso
area. ITK Chlorinated D1ox1ns and Related Compounds: Impact on the Environ-
ment, 0. Hutzlnger et al., Ed. Pergamon Press, NY. p. 115-126.
Woods, J.S. 1973. Studies of the effects of 2,3,7,8-tetrachlorod1benzo-p-
dloxln on mammalian hepatic 6-am1nolevul1n1c add synthetase. Environ.
Health Perspect. 5: 221.
1862A 15-95 04/05/84
-------
Woolson, E.A., R.F. Thomas and P.O.J. Ensor. 1972. Survey of polychloro-
d1benzo-p-d1ox1n content 1n selected pesticides. 20(2): 350-354.
Yamag1sh1, T., T. M1yazak1, K. Aklyama, et al. 1981. PolychloMnated
d1benzo-p-d1oxins and dlbenzofurans 1n commercial dlphenyl ether herbicides,
and 1n freshwater fish collected from the application area. Chemosphere.
10(10): 1137-1144.
Yamamoto, H., H. Yoshimura, M. FujHa and T. Yamamoto. 1976. Metabolic and
toxlcologlc evaluation of 2,3,4,4',4'-pentachlorob1phenyl 1n rats and mice.
Chem. Pharm. Bull. 24: 2168-2174.
Yang, K.H. and R.E. Peterson. 1977. Differential effects of halogenated
aromatic hydrocarbons on pancreatic excretory function 1n rats. Fed. Proc.
Fed. Am. Soc. Exp. Blol. 36(3): 356.
Yang, K.H., W.A. Croft and R.E. Peterson. 1977. Effects of 2,3,7,8-tetra-
chlorod1benzo-p-d1ox1ns on plasma disappearance and biliary excretion of
foreign compounds 1n rats. Toxlcol. Appl. Pharmacol. 40: 485-496.
Yang, K.H., E.J. Choi and S.Y. Choe. 1983. Cytotoxldty of 2,3,7,8-tetra-
chlorod1benzo-p-d1ox1n on primary cultures of adult rat hepatocytes. Arch.
Environ. Contam. Toxlcol. 12: 183-188.
Yocklm, R.S., A.R. Isensee and G.E. Jones. 1978. Distribution and toxldty
of 1CDD and 2,4,5-T 1n an aquatic model ecosystem. Chemosphere, 7(3):
215-220.
1862A 15-96 03/29/84
-------
Yoshlhara, S., K. Nagata, H. Yoshlmura, H. Kurokl and Y. Masuda. 1981.
Inductive effect on hepatic enzymes and acute toxldty of Individual poly-
chlorinated dlbenzofuran congeners 1n rats. Toxlcol. Appl. Pharmacol. 59:
580-588.
Young, A.L. 1983. Long-term studies on the persistence and movement of
1COD 1n a natural ecosystem. Jjn: Human and Environmental Risks of Chlori-
nated Dloxins and Related Compounds, R.E. Tucker et al., Ed. Plenum Pub-
lishing Corp., NY. p. 173-190.
Young, A.L., C.E. lhalken and W.E. Ward. 1975. Studies of the ecological
Impact of repetitive aerial applications of herbicides on the ecosystem of
test area C-52A, Eglln AFB, Florida. A1r Force Armament Laboratory, Eglln
A1r Force Base, Florida. A1r Force Report No. AFA7L-TR-75-142. 142 p.
NTIS AD-A03P773.
Young, A.L., C.E. lhalken, E.L. Arnold, J.M. Cupello and L.G. Cockerham.
1976. Fate of 2,3,7,8-tetrachlorod1benzo-p-d1ox1n (TCOD) 1n the environ-
ment: Summary and decontamination recommendations. Air Force Tech. Report
No. USAFA-TR-76-18, U.S. Air Force Academy, CO. p. 41.
Young, A.L., H.K. Kang and B.M. Shepard. 1983. Chlorinated dloxlns as
herbicide contaminants. Environ. Sc1. Technol. 17: 530A-540A.
Zack, J.A. and R.R. Susklnd. 1980. The mortality experience of workers
exposed to tetrachlorod1benzod1ox1n 1n a tMchlorophenol process accident.
J. Occup. Med. 22(1): 11-14.
186PA 15-97 03/29/84
-------
Zelger, E. 1983. Memorandum from Dr. Zelger of Dr. E.E. McConnell on the
results of test performed for the Environmental Mutagenesls Development
Program. NTP, HIEHS.
Zimmermann, E.F. and D. Bowen. 1972. Distribution and metabolism of trlam-
dnolone acetonlde 1n Inbred mice with different cleft palate sensitivities.
Teratology. 5: 335-344.
Z1nkl, J.G., J.G. Vos, J.A. Moore and B.N. Gupta. 1973. Hematologlc and
clinical chemistry effects of 2,3,7,8-tetrachlorod1benzo-p-d1ox1n In labora-
tory animals. Environ. Health Perspect. 5: 111-118.
Zullei, N. and G. Benecke. 1978. Application of a new bioassay to screen
the toxicity of polychlorlnated blphenyls on blue-green algae. 20(6):
786-792.
1862A 15-98 03/29/84
-------
APPENDIX A
A-l
-------
TABLE A-l
Cumulative Mortality of Male 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
yg/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
62.0
74. Ob
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
44.0
50.0
56.0
60.0
68.0
74.0
76.0
78.0
^Source: Koclba et al., 1977
blnterval of greatest difference, 0, In cumulative mortality curves of
controls and treatment group. None of the differences were statistically
significant (Kolmogorov-Smlrnov test, p>0.05).
1865A
A-2
03/09/84
-------
TABLE A-2
Cumulative Mortality of Female Rats3
Time
(end of 30-day period)
0-5
6-8
9
10
11
12
13
14
15
16
1 /
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
yg/kg/day
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
2,3,
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
30.0
36.0
t.
46.0
60.0
66.0
72.0
72.0
7,8-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
34.0
36.0
' 44.0
52.0
58.0
66.0
68.0
^Source: Kodba et al., 1977
blnterval of greatest difference, D, 1n cumulative mortality curves of
controls and treatment group. The mortality curve for the rats fed 0.1
yg/kg/day differed significantly from that for controls (D = 30.4, p<0.01,
Kolmogorov-Smirov test). The other two groups did not differ significantly
from controls (p>0.05).
1865A
A-3
03/09/84
-------
TABLE A-3
Males: 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 yq/kq/day
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/day
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 yg/kq/day
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 (X2=2.54, n.s.
451-540 10/72 vs. 8/41(X2=0.37, n.s.) 10/43(X2=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 (X2=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.)
1865A
02/09/84
-------
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 pg/kg/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 pg/kg/day
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 yg/kg/day
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.)
1865A
A--5
02/09/84
-------
APPENDIX B*
*Taken from 2,4,5-T and 2,3,7,8-TCOO Risk Assessment Document
B-l
-------
oo
TABLE 8-1
DOW (Dr. Koclba) 2,3,7,8-TCDD Oral Rat Study (1978) with Dr. R. Squire's Review
Male Sprague-Dawley Rats - Spartan Substrain (2 yrs)*
CD
I
r-0
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 (0%)
Total 0/76 (0%)
Dose Levels (yg/kq/day)
0.001 0.01
1/49 (2%) 1/49 (2%)
1/34 (3%) 0/27 (0%)
2/49 (4%) 1/49 (4%)
0.1
3/42 (7%)
(p=0.043)
4/30 (13%)
(p-0.016)
7/42 (17%)
(p=5.12x!0~4)
R. Squire's Review
1 . Tongue
Squamous cell carcinoma
2. Nasal turblnates/hard palate
Squamous cell carcinoma
0/77 (0%)
0/55 (0%)
1/44 (2%)
1/34 (3%)
1/49 (2%)
0/26 (0%)
3/44 (7%)
(p=4.60xlO~2i
6/30 (20%)
(p=1.36x!0~3
« Total (1 or 2 above)
o (each rat had at least
one tumor above)
to
oo
0/77 (0%)
2/44 (5%)
1/49 (2%)
9/44 (20%)
(p=6.28xlO~s;
*Average body weight of male rat = 600 g
-------
CD
TABLE B-2
DOW (Dr. Koclba) 2,3,7,8-TCDD Oral Rat Study (1978) with Dr. R. Squire's Review
Female Sprague-Dawley Rats - Spartan Substraln (2 yrs)*
Tissue and Diagnosis
(control)
Dose Levels (pg/kg/day)
0.001
0.01
0.1
CD
I
Dow (Kociba) Analysis
1. Lung
Kerat1n1z1ng 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%)
1/54 (2%)
9/86 (10%)
0/50 (0%)
0/30 (0%)
3/50 (6%)
0/49 (0%)
1/27 (4%)
18/50 (36%)
(2 had both)
(p=4.37xlO~«)
7/49 (14%)
(p=6.21xlO~«)
5/24 (21%)
(p=9.46xlO~3)
34/48 (71%)
(p=9.53xlO"13)
Total (1, 2, or 3 above)
(each rat had at least one
tumor above)
9/86 (10%)
3/50 (6%)
18/50 (36%)
(p=4.37x!0"«)
34/49 (69%)
(p=2.13x!0~12)
03
-------
oo
TABLE B-2 (cont.)
Tissue and Diagnosis
0
(control)
Dose Levels (yg/kg/day)
0.001
0.01
0.1
R. Squire's Review
1. Lung
Squamous cell carcinoma
0/86 (0%)
0/50 (0%)
0/49 (0%)
8/47 (17%)
(p=1.61x!0~4)
CD
I
JT-
2. Nasal turblnate/hard palate
Squamous cell carcinoma
3. Liver
Neoplastlc nodules/hepato-
cellular carcinoma
0/54 (0%)
16/86 (0%)
0/30 (0%)
8/50 (16%)
1/27 (4%)
27/50 (54%)
;p=2.42x10-*)
5/22 (23%)
(p=1.43xlO~3)
33/47 (70%)
(p=4.92x!0~9)
Total combined (1, 2 or 3 above)
(each animal had at least
one tumor above)
16/86 (19%) 8/50 (16%)
27/50 (54%)
(p=2.42x!0~5)
34/47 (72%)
(p=l.20x10-*)
o
r\3
O
LD
*Average body weight of female rat = 450 g
CD
-------
CO
en
en
TABIE B-3
NCI 2,3,7,8-TCOD (Gavage) Bloassay (No. 80-1765)
Osborne-Mendel Female Rats (2 years; weight ^ 450 g)
Dose Levels (yg/kq/week)
Tissue and Diagnosis Vehicle Control Low Medium
0 0.01 0.05
1. Liver
Neoplastlc nodule or 5/75 (7%) 1/49 (2%) 3/50 (6%)
hepatocellular carcinoma
2. Adrenal*
Cortical adenoma, or 11/73 (15%) 9/49 (18%) 5/49 (10%)
carcinoma
High
0.5
14/49 (28%)
(p=0.001)
14/46 (30%)
(p=0.038)
*The biological significance of this tumor 1n old rats 1s questionable, since 1t 1s commonly observed 1n
control rats and associated with the aging process.
CO
-------
CO
CD
I
TABLE B-4
NCI 2,3,7,8-TCDD (Gavage) Bloassay (No. 80-1765)
B6C3F1 Male Mice (2 years; weight = 48 g)
Tissue and Diagnosis
Liver
Hepatocellular adenoma
or carcinoma
Hepatocellular cardnomab
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%)
(pg/kq/week)
Medium
0.05
13/49 (26%)
8/49 (16%)
High
0.5
27/50 (54%)
(p=l. 31x10"*)
17/50 (34%)
(p=0.002)
aCochran-Arm1tage test for linear trend
bUsed for Unit Risk Estimate
CD
-------
03
TABLE B-5
NCI 2,3,7,8-TCDD (Gavage) Bloassay (No. 80-1765)
B6C3F1 Female Mice (2 years)3
CD
I
1 .
2.
3.
4.
Tissue and Diagnosis
Subcutaneous tissue
Fibrosarcoma
Hematopo1et1c system
Lymphoma or leukemia
Liver
Hepatocellular adenoma
or carcinoma
Hepatocellular carcinoma
Thyroid
FolUcular cell adenoma
Total (1, 2, 3 or 4 above)
(each mouse had at least
one tumor above)
Vehicle Control
0
1/74 (1%)
18/74 (24%)
3/73 (4%)
(p=0.0050)
1/73 (1%)
(p=0.008)&
0/69
22/74 (30%)
Dose Levels (yq/kq/week)
Low Medium
0.04 0.2
1/50 (2%) 1/48 (2%)
12/50 (24%) 13/48 (27%)
6/50 (12%) 6/48 (12%)
2/50 (4%) 2/48 (4%)
3/50 (6%) 1/47 (2%)
20/50 (40%) 19/48 (40%)
High
2.0
5/47 (11%)
(p=0.032)
20/47 (43%)
(p=0.028)
11/47 (23%)
(p=1.84xlO"3)
6/47 (13%)
(p=0.014)
5/46 (11%)
(p=8.93xlO~3)
31/47 (66%)
(p=8.94x!0~5)
CO
aAverage body weight of female mouse = 40 g
bCochran-Armltage test for trend
-------
os
c^
cr>
3>
TABLE B-6
Curve FU 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-spec 1es Male rat
Weight (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 - Kodba
CO
Exposure level (mg/kg/day) 0 1 x 10 6 1 x 10 5
+r/n 0/76 2/49 1/49
1 x 10"4
7/42
+r = number of animals with one or more of the tumors
n = total number of animals examined
Estimated
multistage parameters qrj Ql Q2 93 91*
When all dose groups
are used 1.40 x 10~2 1.10 x 103 0 5.86 x 1010 3.01 x 103
Goodness of fit
X2
3.34 (d.f. = 2)
When the highest dose
group 1s not used Above fH 1s satisfactory
CO
\
CO
q-|* = the maximum linear component from the model with adequate goodness of fit (p>0.01) = 3.01xl03
(mg/kg/day)'1
qn* = q-|* (70/wa)1/3 = 1.47x10* (mg/kg/day)'1, the upper 95% limit slope factor associated
with human dose response.
-------
CD
I
TABLE B-7
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-spedes Male 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 6 1 x 10~5
+r/n 0/77 2/44 1/49
1 x 10"4
9/44
+r = number of animals with one or more of the tumors
n = total number of animals examined
Estimated
multistage parameters qg qi q2 Q3 Qi*
When all dose groups
are used 0.015 1.05 x 103 0 109.40 x 109 3.53 x 103
Goodness of fit
X*
3.90 (d.f. = 1)
When the highest dose
group 1s not used Above fit 1s satisfactory
qi* = the maximum^ linear component from the model with adequate goodness of fit (p>0.01) = 3.53xl03
(mg/kg/day)'1
o qn* = q-|* (70/wa)1/3 = 1.73x10" (mg/kg/day)"1, the upper 95% limit slope factor associated
\ with human dose response.
CO
-------
CD
TABLE B-8
Curve FH of the Multistage Model Parameters to Experimental Data by Study
and Pathologist Linear Parameter q-| , Maximized to Give Upper 95% L1mH q]*
Compound ..................... 2,3,7,8-TCDO
Study ........................ Dow
Sex-spedes .................. Female rat
Weight (wa) .................. 450 g
Tumor sites (one or more) ---- Liver, lung, hard palate, or nasal turblnates (ref. Table B-2)
Pathologist - Koclba
CD
I
CO
Exposure level (mg/kg/day) 0
+r/n 9/86
+r = number of animals with one or more of the
n = total number of animals examined
Estimated
multistage parameters qg q]
When all dose groups 0.12 1.23 x 104
are used
When the highest dose
group 1s not used 0.09 0
When the two highest dose
groups are not used
1 x 10"6 1 x 10'5
3/50 18/50
tumors
Q2 Q3 0.25
* =
Qh =
the maximum linear component from the model with adequate goodness of fH (p>0.01) = 1.67xl04 -
4.69xl04 (mg/kg/day)"1
91* (70/wa)l/3 = 8.98xl04 - 2.52x10* (mg/kg/day)~i. 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 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 - Kociba {Eliminating first year's data to adjust for high early
mortality in the high-dose group.)
Exposure level (mg/kg/day) 0 1 x 10 6 1 x 10 s 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 qg q^ qp q3 q-|* X2
When all dose groups 0.11 2.08 x 10« 0 0 2.82 x 10« 3.38 (d.f. = 2)
are used 0.25 < p < 0.10
When the highest dose
group is not used Above fit 1s satisfactory p > 0.25
q-j* = the maximum linear component from the model with adequate goodness
of fit (p>0.01) -- 2.82xl04 (mg/kg/day)'1
qn* = qi* (70/wa)1/3 =- 1.51xlOs (mg/kg/day)"1, the upper 95%
limit slope factor associated with human dose response.
1866A B-ll 04/13/84
-------
CD
CT>
3>
TABLE B-9
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 Kociba - 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
CD
1
o
-p.
CO
-p.
Exposure level (mg/kg/day) 0 1 x 10 6 1 x 10 5
+r/n 16/86 8/50 27/50
1 x 10~4
34/47
+r = number of animals with one or more of the tumors
n = total number of animals examined
Estimated
multistage parameters q0 q-| q2 Q3 Ql*
When all dose groups 0.26 1.25 x 10" 0 0
are used
When the highest dose
group 1s not used 0.19 0 5.83 x 109 7.90 x 104
Goodness of fit
X2
9.8 (d.f. = 2)
p<0.01
0.209 (d.f. = 1)
When the two highest dose
groups are not used Above fit 1s satisfactory
q-|* = the maximum linear component from the model with adequate goodness of fH (p>0.01) = 7.90xl04
(mg/kg/day)"1
qn* = q-|* (70/wa)1/3 = 4.25xlOs (mg/kg/day)"1, the upper 95% limit slope factor associated
with human dose response.
-------
TABLE B-9A
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 Kodba - Dow
Sex-spedes 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 10~6 1 x 1(T5 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
Estlnitilpd Goodness of fit
multistage parameters qg q^ qp qg q-j* X2
When all dose groups
are used 0.24 2.12 x 10" 0 0 3.00 x 104 6.41 (d.f.= 2)
0.025 < p < 0.05
When the highest dose
group 1s not used Above fit 1s satisfactory
* _
the maximum linear component from the model with adequate goodness
of fit (p>0.01) =- 3.00x10" (mg/kg/dayr1
qi* (70/wa)1/3 - 1.61xlOs (mg/kg/day)"1, the upper 95%
limit slope factor associated with human dose response.
1866A B-13 04/13/84
-------
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 q]*
co
CT>
01
*• Compound ..................... 2,3,7,8-TCDD
Study ........................ NCI
Sex-spec 1es .................. Female rat
Weight (wa) .................. 450 g
Tumor sites (one or more) ... .Liver neoplastlc nodules or hepatocellular carcinoma (ref. Table B-3)
Pathologist - NCI Reviewed
ro
Exposure level (mg/kg/day) 0 1.43 x 10 6 7.14 x 10"6
4-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-| q? qs q-|*
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 1s satisfactory
q-|* = the maximum linear component from the model with adequate goodness of fit (p>0.01) = 6.09xl03
(mg/kg/day)"1
O
* Qh* = Ql* (70/wa)1/3 = 3.28xl04 (mg/kg/day)'1, the upper 95% limit slope factor associated
Z» with human dose response.
\
co
-------
TABLE B-ll
__ Curve Fit of the Multistage Model Parameters to Experimental Data by Study
|> and Pathologist Linear Parameter q-], Maximized to Give Upper 95% Limit q]*
3>
Compound 2,3,7,8-TCDD
Study NCI
Sex-specles Male mice
Weight (wa) 48 g
Tumor sites (one or more) Hepatocellular carcinomas (ref. Table B-4)
Pathologist - NCI Review
Exposure level (mg/kg/day) 0 1.43 x 10"6 7.14 x 10~6
+r/n 8/73 9/49 8/49
7.14 x 10"5
17/50
30 +r = number of animals with one or more of the tumors
1 n = total number of animals examined
,T
Estimated
multistage parameters qn q] q2 qs q-|*
When all dose groups
are used 0.15 3.80 x 103 0 0 6.63 x 103
Goodness of fit
X2
2.43 (d.f. = 2)
When the highest dose
group 1s not used Above fH 1s satisfactory
q-|* = the max1mum_ linear component from the model with adequate goodness of fH (p>0.01) = 6.63xl03
o (mg/kg/day) *
w qn* = q-|* (70/wa)1/3 = 7.52xl04 (mg/kg/day)"1, the upper 95% limit slope factor associated
oo with human dose response.
-------
CD
CTi
TABLE B-12
Curve FH 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-TCOD
Study ........................ NCI
Sex -species .................. Female mice
Weight (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
2
CD
Exposure level (mg/kg/day) 0 5.71 x 10 6 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 qi q2 Q3 Ql*
When all dose groups
are used 0.41 2.38 x 103 0 0 3.78 x 103
Goodness of fit
X2
1.20 (d.f. = 2)
When the highest dose
group 1s not used Above fit 1s satisfactory
q-j* = the maximum linear component from the model with adequate goodness of fit (p>0.01) = 3.78xl03
(mg/kg/day)'1
qh* = q-,* (70/wa)1/3 = 4.56x10* (mg/kg/day )~i, the upper 95% limit slope factor associated
with human dose response.
-------
CD
TABLE B-13
Summary of Human Slope Estimates
OJ
I
o
-ta.
CO
•x
CO
Compound Species
2,3,7,8-TCDD Rat
Rat
Rat
Rat
Rat
2,3,7,8-TCDD Mice
Mice
Study Sex
Dow Male Kocl
Squl
Female Kod
Female Squ1
NCI Female NCI
NCI Male NCI
Female NCI
Pathologist
ba
re
ba - unadjusted
- adjusted
for early
deaths
re - unadjusted
- adjusted
for early
deaths
- Reviewed
- Reviewed
- Reviewed
Human Slope Estimate qn*
1n (mg/kg/day)'1
1.47
1.73
8.98
2.52
1.51
4.25
1.61
3.28
7.52
4.56
x 104
x 10*
x 104
x 10s
x 10s
x 105'''
x 104
x 104
x 104
Ref. Table
No.
86
87
88
B8A
89
B9A
810
811
812
"rvalues used to determine geometric mean of 1.56 x 10s (mg/kg/day)
-------
APPENDIX C
COMPARISON OF RESULTS BY VARIOUS EXTRAPOLATION MODELS
The estimate of unit risk from animals presented In the body of this
document Is calculated by the use of the linearized multistage model, for
the reasons given herein. The use of this nonthreshold model Is 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 1n 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 log-Problt, and the Welbull models. The one-hit model Is
characterized by a continuous downward curvature, but Is linear at low
doses. Because of Its 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 It 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-Problt and the Welbull models, because of their general "S"
curvature, are often used for the Interpretation of toxlcologlcal data In
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 log-Problt model was originally used In biological
assay problems such as potency assessments of toxicants and drugs, and Is
1867A C-l 03/02/84
-------
generally used to estimate such values as percentlle lethal dose or percen-
tlle effective dose. The development of the model occurred along strictly
empirical lines, I.e., 1t was observed In 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 t (1-c) § (a+blog D) a.b > 0 < C < 1
where P 1s the proportion responding at dose D, c 1s an estimate of the
background rate, a 1s an estimate of the standardized mean of Individual
tolerances, and b 1s 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-(atbd) 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 Is 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 > 0, this model yields low-dose estimates of risks usually
significantly lower than either the multistage or one-hit models, which are
1867A C-2 02/09/84
-------
linear at low doses. 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 eliminating early mortality. The results are
Identical for the multistage and one-hit models, as shown 1n 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. As discussed 1n
the text and shown 1n Tables B-8 and B-9, dropping the highest dose resulted
In 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.
1867A C-3 02/09/84
-------
as
cr>
TABLE C-l
Estimates of Low-Dose Risk to Humans Exposed to 2,3,7,8-TCDD Based on Female Sprague-Dawley Rats
From the Dow Chemical Co, Feeding Study Derived from Four Different Models
Data - Koclba Analysis, Eliminating Early Mortality (Ref. TABLE B-8A)
o
to
o
10
O3
Maximum likelihood estimates
additional risks
Dose Multistage/One-hit Weibull
(yg/kg/day) Model Model* Model
10'5 l.lxlO'6 l.SxlO'5
10"4 l.lxlO'5 l.lxlO'4
10~3 l.lxlO'4 7.1xlO~4
10'2 l.lxlO'3 4.5xlO~3
10'1 l.lxlO'2 2.9xlO'2
1 l.lxlO'1 1.7X10"1
of
Log-Probit
Model
0
1.2xlO~13
4.9xlO'9
1.7xlO~5
5.2x10'3
1.7X10'1
95% upper confidence limit
additional risks
Multistage/One-hit
Model Model*
1.5xlO'6
1.5xlO~5
l.SxlO"4
1.5xlO"3
,.5«10-.
1.4x10'*
Weibull
Model
9.7xlO'5
5.3xlO'4
2.9xlO'3
l.SxlO'2
7.2xlO~2
S.OxlO'1
of
Log-Probit
Model
7.7xlO"1B
3.0xlO'12
7.5xlO~8
l.SxlO"4
2.3xlO"2
S.lxlO"1
*Both models gave Identical results
Human equivalent dose (ng/kg/day): 0 0.186 1.86 18.6
Animal tumors/number examined: 9/85 3/48 18/48 34/40
Human equivalence conversion: 1 ng/kg/day (oral) = 25.0 ng/m3 in air
-------
CD
O>
—I
TABLE C-2
Estimates of Low-Dose Risk to Humans Exposed to 2,3,7,8-TCDD Based on Female Sprague-Dawley Rats
From the Dow Chemical Co. Feeding Study Derived from Four Different Models
Data - Squire Analysis, Eliminating Early Mortality (Ref, TABLE B-9A)
o
I
en
O
CO
co
•t*
Maximum likelihood estimates
additional risks
Dose
(yg/kg/day)
10~5
10"4
10"3
10~2
10"1
1
Multlstage/One-hit
Model Model*
l.lxKT6
l.lxlO"5
l.lxlO"4
l.lxlO"3
l.lxlO"2
l.lxlO"1
Weibull
Model
S.OxlCT4
1.2xlO"3
4.9xlO~3
2.0xlO"2
7.8xlO~2
2.8X10"1
of
Log-Probit
Model
2.2xlO~12
7.6x!0~*
5.8xlO"6
9.2xlO"4
3.3xlO"2
2.9X10"1
95% upper confidence limit
additional risks
Multlstage/One-hi
Model Model
1.6xlO"6
1.6xlO~5
1.6xlO~4
1.6xlO"3
1.6xlO"2
1.5X10"1
t Weibull
* Model
1.3xlO"3
4.4xlO~3
1.5xlO"2
S.lxlO"2
1.6X10"1
4.3X10'1
of
Log-Probit
Model
4.4X10"1
l.lxlO"7
5.3xlO"5
5.8xlO"3
l.lxlO"1
4.8X10"1
*Both models gave identical results
Human equivalent dose (ng/kg/day): 0 0.186 1.86 18.6
Animal tumors/number examined: 16/85 8/48 27/48 34/40
Human equivalence conversion: 1 ng/kg/day (oral) = 25.0 ng/m3 in air
------- |