Document ypvOy5pqEd5r01j4G29y0ze4E
United States
Environmental Protection Agency
Office of Health and Environmental Assessment Washington DC 20460
Research and Development
Health Assessment
Document for
Polychlorinated Dibenzo-p-Dioxins
E P A /6 0 0 /8 -8 4 /0 1 4 F September 1985
Final Report
EPA /600/8-84/014F September 1985 Final Report
Health Assessment Document tor
Polychlorinated Dibenzo-p-Dioxins
U.S. ENVIRONMENTAL PROTECTION AGENCY Office of Research and Development
Office of Health and Environmental Assessment Environmental Criteria and Assessment Office
Cincinnati, Ohio 45268
/
NOTICE
This document has been reviewed 1n accordance with U.S. Environmental Protection Agency policy and approved for publication. Mention of trade names or commercial products does not constitute endorsment or recommendation for use.
PREFACE
The Office of Health and Environmental Assessment has prepared this Health Assessment Document on polychlorinated d1benzo--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 toxicity of polychlorinated 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 was reviewed by a panel of expert scientists during the peer review workshop held at the Cincinnati Convent1on/Expos1t1on Center, Cincin nati, OH, on July 27, 28 and 29, 1983. The Environmental Health Committee and the Environmental Effects, Fate and Transport Committee of the U.S. EPA's Science Advisory Board Independentally reviewed the document 1n a public session.
111
AUTHORS, CONTRIBUTORS ANO 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 (ECA0-C1nc1nnat1) had overall responsibility for coordination and direction of the document preparation and production effort (Dr. Debdas Mukerjee, Project Manager, Dr.
Jerry F. Stara, Director, ECA0-C1nc1nnat1). The following Individuals
contributed substantially to portions of various chapters of the document and their assistance 1s greatly appreciated.
Dr. D1pak K. Basu
Life and Environmental Sciences Division
Syracuse Research Corporation Syracuse, NY
Dr. Shane Que Hee Kettering Laboratories
Department of Environmental Health University of Cincinnati Medical School Cincinnati, OH
Dr. Debdas Hukerjee Environmental Criteria and
Assessment Office U.S. Environmental Protection Agency Cincinnati, OH
Dr. Michael W. Neal Life and Environmental
Sciences Division Syracuse Research Corporation Syracuse, NY
Dr. Stephen H. Safe Department of Physiology and
Pharmacology College of Veterinary Medicine Texas A&M University College Station, TX
Dr. Marvin A. Schnelderman Bethesda, MD
Dr. James R. Olson Department of Pharmacology
and Therapeutics School of Medicine
State University of New York
at Buffalo Buffalo, NY
The OHEA Carcinogen Assessment Group (CAG) was responsible for prepara tion of the sections on carcinogenicity. Participating members of CAG are
listed below and the authors for the Carcinogenicity Chapter are Indicated by an asterisk.
Roy E. Albert, M.D. (Chairman) Elizabeth L. Anderson, Ph.D. Larry D. Anderson, Ph.D. Steven Bayard, Ph.D.* David L. Bayllss, M.S.*
Chao W. Chen, Ph.D. Herman J. G1bb, B.S., H.P.H. Bernard H. Haberman, D.V.M., M.S.
Charallngayya B. Hlremath, Ph.D.* James W. Holder, Ph.D.
Robert E. McGaughy, Ph.D. Jean C. Parker, Ph.D. Charles H. R1s, M.S., P.E.
Dharm V. Singh, D.V.M., Ph.D. Todd W. Thorslund, Sc.D.
1v
m e to Mowing individuals were asxed to review tnis document ano earner drafts of this document:
Dr. Bernard H. Haberman Dr. Franklin L. Mink Dr. Charles H. Nauman Dr. Sheila L. Rosenthal
Dr. William E. Pepelko W. Bruce Pelrano David J. Relsman John L. Schaum
U.S. EPA Carcinogen Assessment Group U.S. EPA ECA0-C1nc1nnat1 U.S. EPA Exposure Assessment Group U.S. EPA Reproductive Effects Assessment
Group
U.S. EPA ECA0-C1nc1nnat1 U.S. EPA ECA0-C1nc1nnat1 U.S. EPA ECA0-C1nc1nnat1 U.S. EPA Exposure Assessment Group
The following members of the ECA0-C1nc1nnat1 Technical Services Staff were responsible for document production:
Patricia A. Daunt Erma R. Durden
Judith A. Olsen Bette L. Zwayer
V
POLYCHLORINATED DIBENZO-jS-DIOXINS PEER REVIEW PANEL MEMBERS
July 27, 28 and 29, 1983
Cincinnati, Ohio
Co-Chairmen: Dr. Debdas Mukerjee, ECAO-C1nc1nnat1 Dr. Jerry F. Stara, ECAO-C1nc1nnat1
MEMBERS
Dr. Roy Albert Institute of Environmental Medicine New York University Medical Center
Dr. Donald G. Barnes Office of Pesticides and Toxic
Substances U.S. Environmental Protection Agency
Dr. K. Diane Courtney Health Effects Research Laboratory Research Triangle Park U.S. Environmental Protection Agency
Dr. Rolf Hartung University of Michigan
Dr. Alistair W.M. Hay University of Leeds Leeds, United Kingdom
Dr. Otto Hutzlnger University of Amsterdam Amsterdam, The Netherlands
Dr. R.D. Kimbrough Centers for Disease Control
Dr. Frederick Coulston White Sands Research Center
Dr. Richard J. Koclba Dow Chemical Company
Dr. David Firestone Food and Drug Administration
Dr. S. Garatt1n1 Institute d1 Recerche Farmacologlc "Mario Negri" Milan, Italy
Dr. Frederick Kopfler Health Effects Research Laboratory Cincinnati U.S. Environmental Protection Agency
Dr. Marvin Legator University of Texas Medical Branch
Dr. Dolores Graham Health Effects Research Laboratory Research Triangle Park U.S. Environmental Protection Agency
Dr. Richard Grlesemer Biology Division Oak Ridge National Laboratory
Dr. Ruth L1l1s Mt. S1na1 School of Medicine
Dr. Prab D. LotHkar Temple University School of Medicine
Dr. Fumlo Matsumura Michigan State University
Dr. Lennart Hardell University Hospital Umea, Sweden
Dr. E. McConnell National Institute of Environmental
Health Sciences
Dr. Robert Harless Environmental Monitoring Systems
Laboratory, Research Triangle Park
U.S. Environmental Protection Agency
Dr. W.P. McNulty Oregon Regional Primate Research
Center
v1
Dr. Robert Miller National Cancer Institute
Dr. James R. Olson State University of New York
at Buffalo
Dr. Francesco Pocchlarl Instltuto Superlore d1 Sanlta Rome, Italy
Dr. Shane Que Hee University of Cincinnati Medical
Center
Dr. Chrlstoffer Rappe University of Umea, Sweden
Dr. Stephen H. Safe Texas A&M University
Dr. Marvin Schnelderman Environmental Law Institute
Larry SUbart National Wildlife Federation
Dr. Ellen Sllbergeld Environmental Defense Fund
Dr. David Stalling Columbia National Fisheries Research
Laboratory
Dr. Lewis Thibodeaux University of Arkansas
Dr. Thomas Tiernan Wright State University
v11
SCIENCE ADVISORY BOARD
Environmental Health Committee Chairmen: Dr. Herschel E. Griffin, San Diego State University, Sah Diego, CA
HEHBERS
Dr. Seymour Abrahamson University of Wisconsin Madison, WI
Dr. Marvin Kuschner State University of New York Stony Brook, NY
Dr. Morton Corn The John Hopkins University Baltimore,_MD
Dr. D. Warner North
Decision Focus Inc. Los Altos, CA
Dr. Ronald D. Hood University of Alabama Tuscaloosa, AL
Dr. John Doull University of Kansas Medical Center Kansas City, KS
Dr. Bernard Weiss University of Rochester Rochester, NY
Dr. Ronald Wyzga Electric Power Institute Palo Alto, CA
/
Dr. Stephen H.Safe Texas A&M University College Station, TX
COUNSULTANT
Dr. William Lowrance Rockefeller University New York, NY
Environmental Effects, Transport and Fate Committee, Dioxin Subcommittee
Dr. Robert Huggett Institute of Marine Science College of William and Mary Gloucester Point, VA
, Dr. John Laseter Envlro-Health Systems, Inc. New Orleans, LA
v111
TABLE OF CONTENTS
Page
1. INTRODUCTION....................................................... 1-1
2. SUMMARY AND CONCLUSIONS...........................................2-1
2.1. SUMMARY.................................................. 2-1 2.2. CONCLUSIONS.............................................. 2-7
2.3. NEEDS FOR FUTURE RESEARCH................................ 2-8
3. PHYSICAL AND CHEMICAL PROPERTIES/ANALYTICALMETHODOLOGY ......... 3-1
3.1. INTRODUCTION............ ..................................3-1 3.2. PHYSICAL AND CHEMICAL PROPERTIES.......................... 3-1
3.2.1. Chemical Formula and Synonyms .................. 3.2.2. Physical Properties ..............................
3.2.3. Chemical Properties . . . ........................
3-1 3-3
3-5
3.3. ANALYTICAL METHODOLOGY.................................... 3-5
3.3.1. 3.3.2. 3.3.3. 3.3.4.
General Procedure for the Analysis ofPCDDs . . . . 3-7 Analysis of PCDDs 1n SpecificEnvironmental Media . 3-20 B1oanalys1s of PCDDs.................................3-30 Critique of Sampling and Chemical Analysis.......... 3-30
3.4. SUMMARY..................................................... 3-34
4. PRODUCTION, USE, SYNTHESIS, ENVIRONMENTALSOURCES AND ENVIRONMENTAL LEVELS.............. .................... ..
4-1
4.1. PRODUCTION AND USE........................................ 4-1 4.2. SYNTHESIS........ ........................................ 4-1
4.2.1. ,,
4.2.2. 4.2.3. 4.2.4. 4.2.5. 4.2.6.
Reaction of Dlchlorocatechol Salts with
1,2,4,5-Tetrachlorobenzenes 1n DMSO ............... 4-1
Substitution Reaction ............................ 4-2
Photoproduction .................................. 4-2
Ullmann Condensation Reactions.................... 4-2
Pyrolysis of Chlorophenates .................... .. 4-4
Conversion Through Nitration.............. ..
4-4
4.3. ENVIRONMENTAL SOURCES.............................. ..
4-5
4.3.1. 4.3.2. 4.3.3. 4.3.4. 4.3.5.
Manufacturing Processes .......................... 4-5
Municipal Incinerators...............................4-14
Other Combustion Processes...........................4-15
Chemical Dump Sites .............................. 4-17
Photochemical Process ..................
4-17
4.4. RELATIONSHIP BETWEEN SOURCESAND CONTAMINATION IN ENVIRONMENTAL MATRICES................. .................. 4-18
1x
TABLE OF CONTENTS (cont.)
Page
4.5. ENVIRONMENTAL LEVELS........................................ 4-18
4.5.1. 4.5.2.
4.5.3. 4.5.4.
Water ............................................ 4-20 A i r .............................................. 4-21
Soil.............................................. 4-25 Foods and Biological Samples...................... 4-28
4.6. EXPOSURE.................................................... 4-32 4.7. SUMMARY.................................................... 4-38
5. ENVIRONMENTAL FATE AND TRANSPORT PROCESSES...................... 5-1
5.1. FATE...................................................... 5-1
5.1.1. 5.1.2.
5.1.3. 5.1.4.
Water ............................................ 5-1 A i r .............................................. 5-6
Soil.............................................. 5-7 Food.............................................. 5-11
5.2. TRANSPORT.................................................. 5-12
5.2.1. Water ............................................ 5.2.2. A 1 r ........................................ 5.2.3. Soil........................................
5-12 5-13 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. Absorptionfrom the Gastrointestinal Tract......... 7-1 7.1.2. Absorption Through the Skin ....................... 7-6
7.2.
7.3.
7.4. 7.5.
DISTRIBUTION.............................................. 7-7
METABOLISM.................................................. 7-12
ELIMINATION................................................ 7-16 SUMMARY.................................................... 7-20
x
TABLE OF CONTENTS (cont.)
Page
8 . TOXICOLOGY: ACUTE, SUBCHRONIC AND CHRONIC ...................... 8-1
8.1. EXPERIMENTAL ANIMALS...................................... 8-1
8.1.1. Acute ............................................ 8.1.2. Subchronic. . . . ................................ 8.1.3. Chronic ..........................................
8-1 8-44 8-50
8.2. HUMAN ................................................. .. . 8-60
8.2.1. Acute Exposure............................
8-60
8.2.2. Chronic Studies . ................................... 8-65
8.3. MECHANISM OF TOXICITY.......................................8-69
8.3.1. 8.3.2.
8.3.3.
8.3.4. 8.3.5.
Receptor-Mediated Toxicity.......................... 8-70
Metabolism............................
8-78
Vitamin A Depletion .............................. 8-80
L1p1d Peroxidation...................................8-81
Endocrine Imbalance .............................. 8-82
8.4. SUMMARY . ................................................... 8-84
8.4.1. Experimental Animal Data.............................8-84
8.4.2. Human Data...........................................8-88
8.4.3. Mechanisms of Toxicity................
8-88
9. TERATOGENICITY AND OTHER REPRODUCTIVE EFFECTS .................. 9-1
9.1. STUDIES ON EXPERIMENTAL MAMMALS ............ . . . . . . . 9-1
9.1.1.
9.1.2. 9.1.3. 9.1.4. 9.1.5. 9.1.6. 9.1.7.
2,3,7,8-TCDD Administered as a Contaminant of Other Chemicals.................................. 9-1 2,3,7,8-TCDD Studies InMice....................... 9-6 2,3,7,8-TCDD Studies 1nRats..........................9-13 2,3,7,8-TCDD Studies 1nRabbits and Ferrets . . . . 9-18 2,3,7,8-TCDD Studies 1nNonhuman Primates ......... 9-20
Studies 1n Chickens ............................ 9-22
Studies of the Teratogenic and Reproductive Effects of HxCDD.....................................9-23
9.2. STUDIES ON HUMAN POPULATIONS................................. 9-23
9.3. OTHER REPRODUCTIVE EFFECTS................
9-34
9 ."4. SUMMARY..................................................... 9-35
x1
TABLE OF CONTENTS (coni.)
Page
10. MUTAGENICITY AND OTHER INDICATIONS OF GENOTOXICITY.............. 10-1
10.1. RELEVANT STUDIES.......................................... 10-1
10.1.1. Assays In Microorganisms.......................... 10-1 10.1.2. Interactions with Nucleic Acids .................. 10-7 10.1.3. Cytogenetic Effects of 2,3,7,8-TCDD .............. 10-8
10.2. SUMMARY.................................................. 10-13
11. CARCINOGENICITY................................................ 11-1
11.1. ANIMAL STUDIES............................................ 11-1
11.1.1. Studies Using 2,3,7,8-TCDD........................ 11-1 11.1.2. Studies Using HxCDD .............................. 11-39 11.1.3. Summary of Animal Carcinogenicity ................ 11-51
11.2. CASE REPORTS AND EPIDEMIOLOGICAL STUDIES.................. 11-60
11.2.1. Case Reports...................................... 11-60 11.2.2. Epidemiologic Studies ............................ 11-64 11.2.3. Summary of Case Reports and Epidemiologic Studies . 11-108
11.3. QUANTITATIVE ESTIMATION OF RISKS OF EXPOSURE TO 2,3,7,8-TCDD and HxCDDs .................................. 11-109
11.3.1. Introduction...................................... 11-109 11.3.2. Procedures for the Determination of Incremental
Unit from Animal Data and Description of the Low-Dose Animal Extrapolation Model .............. 11-110 11.3.3. Selection of Data ................................ 11-112 11.3.4. Calculation of Human Equivalent Dosages for An1mal-to-Man Extrapolation ...................... 11-112 11.3.5. Alternative Methodological Approaches ............ 11-113 11.3.6. Interpretation of Quantitative Estimates.......... 11-114 11.3.7. Incremental Unit Risk Estimates for 2,3,7,8-TCDD via the Oral and Inhalation Routes................ 11-115 11.3.8. Incremental Unit Risk Estimates for HxCDDs (1,2,3,6,7,8 and 1,2,3,7,8 ,9) via the Oral and Inhalation Routes ............................ 11-119 11.3.9. Relative Potency.................................. 11-125
11.4. SUMMARY AND CONCLUSIONS .................................. 11-131
11.4.1. Summary .......................................... 11-131 11.4.2. Conclusions ...................................... 11-138
x11
TABLE OF CONTENTS (cont.)
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. W A T E R ............................................
13-1
13.2. AIR . . .................................................... 13-1
13.3. FOOD........................................................ 13-1
13.4. SUMMARY .................................................... 13-2
14. EFFECTS OF MAJOR CONCERN AND HEALTHHAZARD ASSESSMENT ........... 14-1
14.1. PRINCIPAL EFFECTS ........................................ 14-2
14.1.1. Toxicity.......................................... 14-2 14.1.2. Mutagenicity...................................... 14-7
14.2. SENSITIVE POPULATIONS .................................... 14-7 14.3. FACTORS INFLUENCING HEALTHHAZARDASSESSMENT..................14-8 14.4. QUALITATIVE HEALTH HAZARD ASSESSMENT........................14-9
14.4.1. Animal Toxicity Data................................ 14-10 14.4.2. Animal Carcinogenicity..............................14-11
15. REFERENCES........................................................ 15-1
APPENDIX APPENDIX APPENDIX
APPENDIX
A .....................................
A-l
B .........
B-l
C .................................
C-l
D........................................................ D-l
x111
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 7- 1 7-2
LIST OF TABLES
Title
Page
Physical Properties of a Few Selected Polychlorinated Dioxins.................................. ..................3-4
A Few Estimated Physical Parameters of Chlorinated D1benzo-jj-d1ox1ns.......................................... 3-6
Potential Interferences 1n the Determination of TCDDs at m/e Values of 319.8966 and 321.8936...................... 3-12
Some Packed and Capillary Columns Used for the Analysis of PCDDs.................................................... 3-14
The Detection Limit, Resolution and Ions Monitored by a Few Mass Spectrometrlc Systems for the Determination of TCDDs . . 3-17
Some Published Method Validation Data for 2,3,7,8-TCDD Recovered from Fortified Matrices and Determined by GC/MS . . 3-31
Levels of Tetra-, Penta- and Hexa-chlorod1benzo--d1ox1ns Reported 1n Chlorophenols and a Few Pesticides Originating from Chlorophenols............................. 4-7
Locations of Companies that have been Major Producers and Formulators of Chlorophenols andTheir Derivatives........... 4-10
Levels of TCDD 1n Soils and Sediments from Different Locations.................................................. 4-26
Predicted BCFs from Calculated and Measured Values of Kow . . 4-36
Measured B1oaccumulat1on Factor for 2,3,7,8-TCDD 1n Freshwater Aquatic Organisms................................ 4-37
B1oconcentrat1on Factor of TCDD for Several Aquatic Organisms.................................................... 5-16
Effect of Acute Exposure to 2,3,7,8-TCDD on AquaticAnimals . 6-2
Effects of Chronic or Subchronic Exposure to 2,3,7,8-TCDD on Aquatic Animals.......................................... 6-5
Levels of 2,3,7,8-TCDDs 1n Fish and Shellfish................. 6-10
TCDD Levels 1n Wildlife.................................... 6-15
Gastrointestinal Absorption of 2,3,7,8-TCDD ................ 7-2
Liver Accumulation of 2,3,7,8-TCDD 1n Guinea Pigs 30 Days after a Single Intragastrlc Exposure to 2,3,7,8-TCDD........ 7-4
x1v
No. 7-3 7- 4 8- 1 8-2
8-3 8-4 8- 5
9-1
9-2 10-1
11-1
11-2 11-3 11-4
11-5
11-6
11-7
11-8
LIST OF TABLES (cont.)
Title
Page
Distribution of 2,3,7,8-TCDD............................... 7-8
Elimination of 2,3,7,8-TCDD ................................. 7-17
Lethal Doses of 2,3,7,8-TCDD Following Acute Exposure . . . . 8-2
Toxic Responses Following Exposure to 2,3,7,8-TCDD: Species Differences ........................................ 8-11
Estimated Single Oral LD50 - 30 Values for PCDDs.......... 8-12
Immunological Effects of 2,3,7,8-TCDD 1n Animals.......... 8-28
Effects of Chronic Exposure to 2,3,7,8-TCDD on Laboratory Rodents.................................................... 8-52
Studies on the Potential Teratogenic Effects of 2,3,7,8-TCDD Contaminated 2,4,5-T........................................ 9-2
Studies on the Potential Teratogenic Effect of 2,3,7,8-TCDD . 9-7
The Results of Mutagenicity Assays for 2,3,7,8-TCDD 1n Salmonella tvphimurlum...................................... 10-2
2,3,7,8-TCDD Intake and Mortality 1n Male Sprague-Dawley Rats.......................... .............................. 11-3
Benign and Malignant Tumors 1n Rats Ingesting 2,3,7,8-TCDD. . 11-5
Liver Tumors 1n Rats Ingesting 2,3,7,8-TCDD ................. 11-6
Hepatocellular Carcinomas and Hepatocellular Hyperplastic Nodules 1n Female Sprague-Dawley Rats Maintained on Diets
Containing 2,3,7,8-TCDD .................................... 11-9
Tumor Incidence 1n Female Rats Fed Diets Containing 2.3.7.8- TCDD................................................ 11-10
Tumor Incidence 1n Male Rats Fed Diets Containing 2.3.7.8- TCDD................................................ 11-11
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 Substrain (2 years) ................................ 11-13
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 Substrain (2 years) ................................ 11-14
xv
No. 11-9 11-10 11-11 11-12 11-13 11-14
11-15
11-16 11-17
11-18 11-19 11-20 11-21 11-22 11-23
LIST OF TABLES (cont.)
Title
Page
Incidence of Primary Tumors 1n Hale Rats Administered 2.3.7.8- TCDD by Gavage.................................... 11-16
Incidence of Primary Tumors 1n Female Rats Administered 2.3.7.8- TCDD byGavage...........
11-17
Cumulative Data on Tumor Incidence........................... 11-18
Incidence of Primary Tumors 1n Male Mice Administered 2.3.7.8- TCDD byGavage....................................... 11-22
Incidence of Primary Tumors 1n Female Mice Administered 2.3.7.8- TCDD byGavage....................................... 11-23
Promoting Effect of 2,3,7,8-TCDD on Hepatocardnogenesls by a Single Dose of D1ethyln1trosam1ne (DEN) and Partial Hepatectomy (PH). . . .............................. 11-24
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
Effects of Intraperitoneal Administration of 2,3,7,8-TCDD on 3-MC-In1t1ated Subcutaneous Tumors ...................... 11-31
Effect of Intraperitoneal or Subcutaneous Administration of 2,3,7,8-TCDD Given 2 Days Before or Simultaneous With Subcutaneous Administration of 3-MC on Tumorlgenesls 1n
D2 M i c e ..................................................... 11-32
Incidence of Tumors 1n Mice Treated With 3-MC and With 3-MC and 2,3,7,8-TCDD....................................... 11-34
Liver Tumor Incidences 1n Hale and Female Osborne-Hendel Rats Administered HxCDD for 104 W e e k s ....................... 11-41
Liver Tumor Incidences 1n Female Osborne-Hendel Rats Administered HxCDD by Gavage for 104 Weeks................... 11-43
Liver Tumor Incidences 1n Hale and Female B6C3F1 Mice Administered HxCDD by Gavage for 104 Weeks................... 11-44
Liver Tumor Response for HxCDD (Observed) and TCDD Contaminant (Calculated)..................................... 11-46
Carcinogenicity Bioassays of 2,3,7,8-TCDD and HxCDD by Dermal Application to M i c e ............................... 11-49
xv1
No. 11-24 11-25 11-26 11-27
11-28 11-29 11-30 11-31 11-32 11-33 11-34
11-35
11-36
11-37
14-1
LIST OF TABLES (cont.)
Title
Page
Carcinogenicity Bioassays of PCDD Administration by the Oral and Dermal Route .................................... . 11-52
Distribution of Tumor Types 1n Two Case-Controls Studies of Soft-Tissue Sarcoma.................. * ................
Exposure Frequencies 1n Two Case-Control Studies of Soft-Tissue Sarcoma .......... . ........................ . 11-68
Relative Risks of Soft-Tissue Sarcoma 1n Relation to Exposure to Phenoxyacetlc Acids and Chlorophenols 1n Two Case-Control Studies................................. . 11-70
Distribution of Histological Types of Soft-Tissue Sarcomas.................................................. . 11-75
Midland County Soft and Connective Tissue Cancer Deaths 1960-1981.......................................... . 11-84
Other Occupations (Minus Forestry/Agrlculture)............ . 11-95
Other Occupations (Minus Forestry/Agrlculture/Woodworkers . . 11-96
Analysis of Stomach Cancer Mortality 1n a Group of West German Factory Workers Exposed to 2,3,7,8-TCDD............ . 11-101
Reanalysis of Stomach Cancer Mortality 1n a Group of West German Factory Workers Exposed to 2,3,7,8-TCDD . . . . . 11-103
Stomach Cancer Mortality 1n Three Studies of Workers Exposed to Phenoxyacetlc Acid Herbicides and/or 2,3,7,8-TCDD.............................................. . 11-105
NTP HxCDD (Gavage) Bioassay. Osborne-Mendel Rats (2 years) Incidences of Neoplastic Nodules and Hepato cellular Carcinomas ...................................... . 11-120
NTP HxCDD (Gavage) Bioassay. B6C3F1 Mice (104 weeks) Incidences of Adenomas Nodules and Hepatocellular Carcinomas................................................ . 11-123
Relative Carcinogenic Potencies Among 55 Chemicals Evaluated by the Carcinogen Assessment Group as Suspect Human Carcinogens ........................................ . 11-128
No-Observed-Effect Levels and Low-Observed-Effect Levels Obtained from Subchronic and Chronic Oral Toxicity Studies of 2,3,7,8-TCDD ..........................................
'i
xv11
No. 14-2
14-3 14-4
LIST OF TABLES (cont.)
Title
Page
No-Observed-Effect Levels and Low-Observed-Effect Levels Obtained from Subchronic and Chronic Oral Toxicity Studies of HxCDD............................................ 14-5
Carcinogenicity Bioassays of 2,3,7,8-TCDD .................. 14-12
Carcinogenicity Bioassays of a 1:2 Mixture of 1,2,3,6,7,8and 1,2,3,7,8,9-HxCDD ...................................... 14-16
xv111
No. 4-1 4-2
11-1 11-2
LIST OF FIGURES
Title
Page
Ullmann Condensation Reactions.................... ..........4-3
Possible Potential Relationship Between Various Sources of PCDDs and the Environmental Matrices Where PCDDs have been Detected.............................................. 4-19
Time-Dependent Inhibition by 2,3,7,8-TCDD of Tumor Initiation. ................................................. 11-37
Histogram Representing the Frequency Distribution of the Potency Indices of 55 Suspect Carcinogens Evaluated by the Carcinogen Assessment Group ............................ 11-127
x1x
ADI AHH bw BCF BromoPeCDD DCDD DNSO DNA EC/GC
ed50 FEL GC/MS GC/SIM/MS
HPLC HRGC HRHS HxCDDs LC50 lo50 LOAEL LRHS MFO NICI NOAEL NOEL
LIST OF ABBREVIATIONS
Acceptable dally Intake Aryl hydroxycarbon hydroxylase Body weight B1oconcentrat1on factor Bromopentachlorod1benzo--d1ox1n Diehlorod1benzo--d1ox1n Dimethylsulfoxide Deoxyribonucleic acid Electron capture/gas chromatography Median effective dose Frank effect level Gas chromatography/mass spectrometry Gas chromatography/spedflc 1on mon1tor1ng/mass spectrom etry High performance liquid chromatography High resolution gas chromatography High resolution mass spectrometry Hexachloro derivatives of d1benzo--d1ox1ns Concentration lethal to 5054 of recipients Dose lethal to 5054 of recipients Lowest-observed-adverse-effect level Low resolution mass spectrometry Mixed function oxidase Negative Ion chemical Ionization No-observed-adverse-effect level No-observed-effect level
xx
OCDD PCDDs PCP PeCDDs ppb ppm ppt RBC RNA SA TCDDs TrICDD 2,4,5-T TWA UV WCOT
LIST OF ABBREVIATIONS (cont.)
Octachlorlnated d1benzo--d1ox1ns All polychlorinated d1benzo--d1ox1ns Pentachlorophenol Pentachloro derivatives of d1benzo--d1ox1ns Parts per billion Parts per million Parts per trillion Red blood cells Ribonucleic acid Satellite association Tetrachloro derivatives of d1benzo--d1ox1ns Tr1chlorod1benzo--d1ox1n 2,4,5-Tr1chlorophenoxyacet1c acid Time-weighted average Ultraviolet Wall-coated open tubular
xx1
1. INTRODUCTION
Dioxins are a class of compounds that contain the d1benzo-j)-d1ox1n nucleus. In chlorinated dioxins, the dlbenzo-ja-dloxln nucleus Is substi tuted with chlorine at different positions of the fused benzene rings. Depending on the number and position of chlorine substitution, 75 congeners are possible for the chlorinated dioxins. This document deals with the most toxic chlorinated dioxins, namely, 2,3,7,8-tetrachloro-, 1,2,3,7,8-pentachloro-, 1,2,3,6,7,8-hexachloro- and 1,2,3,7,8,9-hexachlorod1benzo--d1ox1n. Of these four congeners, the 2,3,7,8-tetrachlorodlbenzo-jD-dioxln has been studied extensively and Is often described In both popular and technical literature as "TCDD" or simply "dioxin."
A few documents exists at the present time that deal with selected aspects of polychlorinated d1benzo-j)-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 dioxins. The follow ing acronyms will hereafter be used when^dlscusslng the polychlorinated d1benzo--d1ox1ns:
PCDDs 2.3.7.8- TCDD 1.2.3.7.8- PeCDD 1.2.3.6.7.8- HxCDD 1 .2.3.7.8.9- HxCDD
Polychlorinated d1benzo-j>-d1ox1ns
2.3.7.8- Tetrachlorod1benzo--d1ox1n
1 .2.3.7.8- Pentachlorod1benzo-[)-d1ox1n
1 .2.3.6.7.8-
Hexachlorod1benzo-j>-d1ox1n
1 .2.3.7.8.9-
Hexachlorod1benzo--d1ox1n
1-1
2. SUMMARY AND CONCLUSIONS 2.1. SUMMARY
Polychlorinated d1benzo-f>-d1ox1ns are a class of chlorinated tricyclic aromatic hydrocarbons consisting of two benzene rings connected by a pair of oxygen atoms. According to the position and number of chlorine atoms 1t 1s possible to form 75 different congeners of chlorinated dioxins. The word "dioxins" 1s often used to refer to this class of compounds, especially with respect to the highly toxic and environmentally widely distributed 2,3,7,8tetrachlorod1benzo-f>-d1ox1n (TCDD). This class of compounds 1s rather stable toward heat, acids and alkalis. The solubility of 2,3,7,8-TCDD 1n water 1s 0.2 yg/ii. This Isomer and the three other PCDDs discussed 1n this document are soluble 1n certain aromatic and aliphatic solvents. The PCDDs are chemically relatively stable and start to decompose at tempera tures >500C; 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 sulfuric acid and base washes to remove lipids and other Impurities from the solvent extract. The extract 1s then subjected to two liquid chromatographic clean-up procedures. The cleaned-up extract 1s finally analyzed for the PCDDs by the gas chromatographic-mass spectrometrlc methods. Despite the specialized methods used for the determination of PCDDs, the results of analysis at very low levels (possibly <9 ppt 1n biological matrices) can be questionable unless special precautions, Including addition of Internal standard, are made.
None of the PCDDs are either commercially manufactured or have any known use. They are produced as unwanted contaminants primarily during the
2-1
manufacture of chlorophenols and their derivatives. The primary sources of PCDD contamination 1n the environment result from the Industrial manufacture of chlorophenols and their derivatives and the subsequent disposal of wastes from these Industries. Municipal Incineration may also produce some envi ronmental emission of PCDDs. From the available data, 1t 1s difficult to ascertain the comparative Importance of these three sources 1n contributing to environmental emissions. The 1,2,3,7,8-PeCDD found 1n environmental samples has only been reported 1n emissions from Incinerators.
The monitoring data to date Indicate that the maximum level of PCDDs 1s likely to be found 1n soil and drainage sediment samples near chlorophenol manufacturing Industries and chemical waste disposal sites. With the excep tion of air near certain contaminated sites, only very limited attempts have been made to determine the level of PCDDs In air samples. In the United States, the highest levels are reported at certain hazardous waste sites and 1n fish and wildlife tissue from areas contaminated with 2,3,7,8-TCDD.
The environmental fates of the four PCDDs are not known with certainty. Most of the Investigations 1n this field have been conducted with 2,3,7,8TCDD, and the conclusions regarding the environmental fate of the other three PCDDs have been drawn by analogy. Few data exist In the literature that would Indicate significant chemical and biological transformation of these compounds 1n atmospheric, aquatic or soil media. The role of photo chemical transformation 1n determining the fates of these chemicals 1n var ious ambient media 1s not known with certainty, but the PCDDs are suscepti ble to photochemical reactions 1n the presence of hydrogen donors. In the aquatic media, a substantial proportion of the PCDDs may be present 1n the sediment-sorbed state or 1n the biota. In the atmosphere, the PCDDs are expected to be present 1n the vapor-phase and particulate-sorbed states.
2-2
The atmospheric transport of these compounds can be predicted from disper sion modeling equations. In the case of the accidental release of 2,3,7,8TCDD at Seveso, Italy, 1t has been estimated from laboratory experiments that 2,3,7,8-TCDD deposition from air to soil follows an exponential decay pattern along the downward wind direction. The most probable transport mechanisms of the PCDOs from soils are transport to the atmosphere by con taminated dust particles, direct volatilization from the surface or near surface zones (<5 cm), and transport to surface water by eroded soil.
Both the calculated and the experimental results show that the PCDDs will concentrate 1n sediments and biota present 1n aquatic media. It has been shown by static test procedures that, depending on the species, the bioconcentration factor (BCF) for 2,3,7,8-TCDD 1n fish ranges from -2000-30,000. The U.S. EPA's best estimate of the BCF for 2,3,7,8-TCDD 1s 5000 (U.S. EPA, 1984).
In mammals, 2,3,7,8-TCDD 1s readily absorbed through the gastrointesti nal tract, and absorption through Intact skin has also been reported. Absorption may decrease dramatically 1f 2,3,7,8-TCDD 1s adsorbed to particu late matter such as activated carbon or soil. After absorption, 2,3,7,8TCDD 1s distributed to tissues high 1n I1p1d content; however, 1n many species, the Uver 1s a major storage site. Metabolism of 2,3,7,8-TCDD occurs slowly, with the polar metabolites excreted 1n the urine and feces. Unmetabollzed 2,3,7,8-TCDD can be eliminated 1n the feces and 1n the milk. It 1s metabolized by the P-450 monooxygenase system through a reactive epoxide Intermediate. The metabolism of 2,3,7,8-TCDD seems to be a detoxi fication process resulting 1n the production of metabolites that are less toxic than the parent compound. Available scientific data supports the contention that the toxic response to 2,3,7,8-TCDD exposure 1s mediation through cytosolic Ah-receptor site binding.
2-3
The PCDDs discussed In this document are among some of the most toxic compounds known, with the lowest LD^q level for male guinea p1gst the most sensitive species, being 0.6 pg/kg for 2,3,7,8-TCDD. The other congeners are somewhat less toxic; however, the LD5Q values are still 1n the pg/kg range. Although 2,3,7,8-TCDD Is highly toxic In all species tested, there are large species differences 1n sensitivity, with the LD,^ for hamsters being 1157-5051 pg/kg. The characteristic signs and symptoms of lethal poisoning are severe weight loss and thymic atrophy. Death usually occurs many days after the exposure. In rats, rabbits and mice, 2,3,7,8-TCDD pro duces an acute Uver Injury that 1s not observed 1n either monkeys, hamsters or guinea pigs. In mice, the Immune response 1s also suppressed. After subchronic or chronic exposure to 2,3,7,8-TCDD 1n rats or mice, the Uver appears to be the most severely affected organ, although systemic hemor rhage, edema and suppressed thymic 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 1n 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 toxicity 1n many cases are similar to those observed 1n animals, with exposure leading to altered Uver function and I1p1d metabolism, porphyria cutanea tarda, neuro toxicity and pathologic changes In hematologic parameters. In addition, exposure of humans to 2,3,7,8-TCDD produces skin lesions such as chloracne and hyperpigmentation. Although some signs such as chloracne are attributed to the PCDDs, the other signs of toxicity may arise, at least 1n part, from the other chemical of which PCDDs are a minor contaminant.
Animal studies have demonstrated that 2,3,7,8-TCDD 1s teratogenic and fetotoxlc 1n rats, mice, rabbits and ferrets; and fetotoxlc 1n monkeys.
2-4
exposure t o , o , ; , o - i c u u in mice pt ouutes r d t i d i c i e i t s , win ic ca^usui c
rats results In edema, hemorrhage and kidney anomalies; rabbits have a higher Incidence of extra ribs. In rats a reduction 1n the gestation Index, decreased fetal weight, Increased Uver-to-body weight ratio and Increased Incidence of dilated renal pelvis 1n the offspring has been observed. Certain human epidemiology studies have shown positive associations with exposure to chemicals contaminated with 2,3,7,8-TCDD and birth defects and abortions, while others have not.
There 1s a limited data base with conflicting evidence for 2,3,7,8TCDD' s mutagenic potential; therefore, the available evidence 1s judged to be Inconclusive. There are no studies 1n the published literature regarding the mutagenicity of HxCDD or any other congeners of PCDD.
There 1s evidence from chronic animal cancer bioassay studies that 2,3,7,8-TCDD and HxCDD are probable human carcinogens. There are no chronic cancer bioassay studies available that evaluate the carcinogenic potential for other PCDDs. The available data for 2,3,7,8-TCDD and HxCDD come from gavage and feeding studies, there being no studies available for Inhalation exposure. The epidemiologic evidence for the carcinogenicity of 2,3,7,8TCDD alone 1s Inadequate, while the evidence for phenoxyacetlc herbicides and/or chlorophenols with 2,3,7,8-TCDD as an Impurity 1s limited. There have been no epidemiologic evaluations, as yet, for HxCDD as the sole compound of concern.
A number of chronic animal cancer bioassays show that 2,3,7,8-TCDD 1s an animal carcinogen. In rats, oral exposure to 2,3,7,8-TCDD resulted 1n an Increased Incidence of hepatocellular carcinomas, squamous cell carcinomas of the tongue and hard palate/nasal turbinates, and squamous cell carcinomas of the lung. In both male and female mice, Increased Incidences of Uver
2-5
cumors were oDservea. a mixture or tne two isomers of HXUUU, discussed in this document has been tested for carcinogenicity and shows increased inci dences of liver tumors in 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 in dermal tumors when the mixture of HxCDDs was tested. Since both compounds produce statistically signifi cant increased incidences of tumors in two species of animals, there is sufficient evidence, according to the interim EPA weight-of-evidence classi fication criteria, to conclude that both 2,3,7,8-TCDD and HxCDD are animal carcinogens. 2,3,7,8-TCDD has been shown to be a promoter as well as an initiator in rodent test systems. Evidence is available from epidemiologic studies that implicate exposure to herbicides contaminated with 2,3,7,8-TCDD with a significantly elevated risk of soft tissue sarcomas and to a lesser extent non-Hodgkins lymphomas; however, the exposures to 2,3,7,8-TCDD were always compounded with exposures to the herbicide chemicals.
>
Assuming that 2,3,7,8-TCDD and HxCDD are carcinogenic in humans, upper bound Incremental unit cancer risks have been estimated for both ingestion and inhalation exposure. The unit risks have been estimated using a multi stage extrapolation model that is linear at low doses. Available metabo lism and pharmacokinetic data are insufficient to alter typically used assumptions for estimating the human equivalent dose. Since incidence data exist only for oral studies in animal test systems, the inhalation risk estimates are based upon the cancer potency derived from the oral studies along with appropriate conversion assumptions.
Using data from a feeding study with female rats the upper limit incremental cancer risk for 2,3,7,8-TCDD is estimated to be 1.56xl0_1 per ng/kg/day. The upper limit estimate of incremental cancer risk Is
2-6
4.5x1O"3 for a continuous lifetime exposure to 1 ng/l of 2,3,7,8-TCOO In
drinking water and 3.3xl0~5 for a continuous lifetime exposure to 1 pg/m3 of 2,3,7,8-TCDD In ambient air.
Using data from an Ingestion study with female rats and male mice, the cancer potency for HxCDD 1s estimated to be 6.2xl0-3 per ng/kg/day. The upper limit estimate of Incremental cancer risk Is 1.8xl0-4 for a contin uous lifetime exposure to 1 ng/8. of HxCDD 1n drinking water and 1.3xl0-6 for a continuous lifetime exposure to 1 pg/m3 of HxCDD 1n ambient air. 2.2. CONCLUSIONS
The PCDDs, 2,3,7,8-TCDD, 1,2,3,7,8-PeCDD, 1,2,3,6,7,8- and 1,2,3,7,8,9HxCDD, are highly toxic following acute exposure. All animal species administered high levels of these compounds developed weight loss and thymic atrophy. In some species liver damage, edema, hair loss and Immunosuppres sion 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 nonneoplastlc lesion was fatty and necrotic change 1n the Uver.
In the species studied, the fetus has been shown to be highly sensitive to the toxic effects of 2,3,7,8-TCDD. In rats the fetotoxlclty observed Included hemorrhage, edema and kidney anomalies, while In mice the predomi nant lesions were cleft palate and kidney anomalies. The lowest reported exposure 1n rats, 1 ng/kg, produced a significant (by some analyses but not others) effect on the fetus, and was similar to the LOAEL observed 1n chronic studies.
Evidence from oral animal cancer bioassays 1s "sufficient11 (according to EPA and IARC criteria) to conclude that 2,3,7,8-TCDD and a mixture of the two Isomers of HxCDD are animal carcinogens. 2,3,7,8-TCDD has Increased the
2-7
Incidence of a variety of tumors, Including hepatocellular tumors 1n rats and mice, while the mixture of HxCDD tested Increased the Incidence of
hepatocellular tumors 1n both sexes of rats and mice. The available
epidemiologic evidence for the carcinogenicity of 2,3,7,8-TCDD alone 1s Inadequate and there have been no epidemiologic evaluations, as yet, for HxCDD as the sole compound of concern. Considering the animal evidence together with the epidemiologic data, the overall we1ght-of-ev1dence classi
fication for 2,3,7,8-TCDD using EPA's Interim classification scheme 1s cate gory B2 meaning that 2,3,7,8-TCDD should be regarded as a "probable" human
carcinogen. The overall we1ght-of-ev1dence classification for HxCDD 1s also category B2 meaning that 1t should be regarded as a "probable" human car
cinogen. In terms of low dose potency, 2,3,7,8-TCDD and the HxCDD mixture are the two most potent carcinogens evaluated by the EPA's Carcinogen Assessment Group. Epidemiologic studies of workers exposed to chemicals contaminated with 2,3,7,8-TCDD such as 2,4,5-tr1chlorophenoxyacet1c acid and 2,4,5-tMchlorophenol have produced positive findings that are suggestive of
an elevated risk of cancer 1n humans. These epidemiologic findings are not Inconsistent with the premise that 2,3,7,8-TCDD 1s probably carcinogenic for
humans. There are no chronic studies available regarding the carcinogenic ity of 1,2,3,7,8-PeCDD. 2.3. NEEDS FOR FUTURE RESEARCH
The basic physical properties such as water solubilities and vapor pressures of the PeCDDs and HxCDDs need to be deter mined. These parameters are Important 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.
2-8
Isotoplcally labeled Internal standard compounds (37C1 or
13C) should be prepared for PeCDDs and HxCDDs.
More research efforts should be directed to determining the environmental fate of the PeCDDs and HxCDDs. The determina tion of the fate of these chemicals with respect to the possi bility of photochemical transformations 1n different environ mental matrices needs special attention.
Pharmacokinetic studies should be conducted to demonstrate more clearly the degree of absorption of the PCDDs by all routes. In particular, studies are needed on respiratory absorption and on PCDDs adsorbed to environmental media.
Although a number of studies demonstrate that 2,3,7,8-TCDD 1s a teratogen, the other congeners should be tested for terato genic potential.
There 1s no Information on the effects of chronic exposure to 1,2,3,7,8-PeCDD, and studies should be conducted to determine both the toxic effects of this compound and Its carcinogenic potential.
Further epidemiology data on the effects 1n human populations exposed to PCDDs might assist 1n determining which effects observed 1n animals are also present 1n humans. In these studies, careful quantitation of PCDD levels 1n humans and Industrial hygiene samples might provide dose-response data necessary for health assessment.
B1oava1lability 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 stateof-the-art test systems.
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3. PHYSICAL AND CHEMICAL PROPERTIES/ANALYTICAL METHODOLOGY 3.1. INTRODUCTION
D1benzo-j>-d1ox1n 1s a derivative of the basic chemical structure j>-d1oxane. The structure of d1benzo-j>-d1ox1n and the conventional numbering system used for defining substituent positions are shown below:
91
A number of substituents Including nltro, amino, alkyl, alkoxy and halogen can be Introduced at the different positions of the two benzene rings. Most environmental Interest 1n substituted d1benzo-ja-d1ox1ns and most studies of this family of compounds have centered on chlorinated d1benzo-|3-d1ox1ns that are loosely referred to as "dioxins." Theoretically, there are 75 different congeners of chlorinated d1benzo-j)-d1ox1ns. In this document, only four polychlorinated d1benzo-j)-d1ox1ns, namely 2,3,7,8-tetrachlorod1benzo-[)-d1ox1n (2,3,7,8-TCDD), 1,2,3,7,8-pentachlorod1benzo-j)-d1ox1n (1,2,3,7,8-PeCDD), 1,2,3,6,7 ,8-hexachlorod1benzo-|3-d1ox1n (1,2,3,6,7,8HxCDD) and 1,2,3,7,8,9-hexachlorod1benzo-j)-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-j>-d1ox1n (2,3,7,8-TCDD)
3-1
Chem. Abstr. Name: 2,3,7,8-tetrachlorod1benzo[b,e](l,4)-d1ox1n Synonyms: Dioxin; TCDBD; TCDD; 2,3,7,8-tetrachlorod1benzod1ox1n, 2,3,7,8tetrachlorodlbenzo- -1,4-d1ox1n.
1.2,3,7 ,8-Pentachlorod1benzo--d1ox1n (1,2,3,7,8-PeCDD> CI
Chem. Abstr. Name: 1,2,3,7,8-Pentachlorod1benzo[b,e](l,4)d1ox1n Synonym: 1,2,3,7, -Pentachlorod1benzod1ox1n
1.2,3,6. ,8-Hexachlorod1benzo--d1ox1n (1,2,3,6 ,7,8-HxCDD) CI
Chem. Abstr. Name: 1,2,3,6,7,8-Hexachlorod1benzo[b,e](l,4)d1ox1n Synonym: 1,2,3,6, ,8-Hexachlorod1benzod1ox1n
1,2 ,3,7, ,9-Hexachlorodlbenzo--dloxln (1,2,3,7,8,9-HxCDD)
CI O CI
Chem. Abstr. Name: 1,2,3,7,8,9-Hexachlorod1benzo[b,e](l,4)d1ox1n Synonym: 1,2,3,7, ,9-Hexachlorod1benzod1ox1n
3 -2
3.2.2. P h y s i c a l P r o p e r t i e s . The physical properties of the four poly
chlorinated dioxins are given 1n Table 3-1. Although the physical proper
ties of 1,2,3,7,8-PeCDD, 1,2,3,6,7,8-HxCDD and 1 ,2,3,7,8,9-HxCDD have not
been well studied, these properties have been more Intensively studied for
2,3,7,8-TCDD. 2,3,7,8-TCDD 1s lipophilic, exhibiting a higher degree of
solubility 1n fats and oils than 1n water. The solubility of 2,3,7,8-TCDD
1n various solvents (at unspecified temperatures) 1s as follows (Crummett
and Stehl, 1973):
Solvent
Solubility (ppm)
water lard oil benzene o-d1chlorobenzene chloroform acetone n-octanol methanol
2 x 10" 44 570 1400 370 110
50 10
The solubilities of HxCDD (Isomer unspecified) 1n 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., dl-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 polychlori nated dioxins may be comparable 1n chlorinated aliphatic hydrocarbons, namely chloroform.
Because of the ir -* ir* transitions the polychlorinated dioxins have two absorption maxima 1n the near UV region. The absorption coefficients resulting from 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,
3 -3
TABLE 3-1 Physical Properties of a Few Selected Polychlorinated Dioxins
Compound
CAS Reg. No.
Molecular Formula
Molecular Height
Description
Melting Point CC)
*maxa (chloroform)
(nm)
E1 b 1
Reference
2,3,7,8-TCDD 1,2,3,7,8-PeCDD 1,2,3,6,7,8 -HxCDD 1 ,2,3,7,8,9-HxCDD
1746-01-6 40321-76-4 57653-85-7 19408-74-3
C1 2 H4CI4 O2 C1 2 H3 CI5O2 C12H2C162 c 12h 2c 162
321.9 356.5 390.9 390.9
colorless needles NA
NA
NA
305-306 240-241 285-286 243-244
310 308 316 317
173.6 171.4 152 104
Pohland and Yang, 1972
6ray et al., 1976
6ray et al., 1975
6ray et al., 1975
aTh1s Is the wavelength of maximum absorption.
bTh1s Is the absorption coefficient for a IX chloroform solution of substrate In 1 cm cell at the absorption coefficient (IT1 cm"1), multiply by one-tenth of the molecular weight.
NA Not available
To convert this to the molar
7973). V a l u e s f o r o t h e r 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
2,3,7,8-TCDD are available from various sources (Mahle and Shadoff, 1982; Pohland and Yang, 1972; Chen, 1973; Kende and Wade, 1973). The mass spectra of the three other PCDDs are also available (Mahle and Shadoff, 1982; Gray et al., 1975, 1976). The response ratios of electron Impact (El) and nega tive chemical Ionization (NCI) and fragmentation of 11 of the TCOD Isomers have been reported by Rappe et al. (1983a). These spectra, particularly the mass spectra, are very useful 1n Identifying the various homologues/1somers 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-CDD (Albro, 1979). These compounds begin to decompose at 500C, and at a temperature of 800C, virtually complete degradation of 2,3,7,8-TCDD occurs within 21 seconds (Stehl et al., 1973). The PCDDs are susceptible to photo degradation 1n the presence of UV light. They also undergo photoreductlve dechlorination 1n the presence of an effective hydrogen donor. Gamma radiation degrades 2,3,7,8-TCDD 1n organic solvents (FanelH et al., 1978). 3.3. ANALYTICAL METHODOLOGY
Several publications on the analytical methods for the determination of PCDD levels 1n different media are available. The analytical methodologies for the separation of the different Isomers of PCDDs are difficult and expensive. Many Investigators, particularly the earlier ones, failed to characterize the Individual Isomers and 1t Is not always clear whether a
3 -5
TABLE 3-2 A Few Estimated Physical Parameters of Chlorinated D1benzo-p-D1ox1nsa
Parameter
2,3,7,8-TCDD
PeCDDb
HxCDDb
Vapor pressure (mm of Hg) at 25C and 1 atmosphere
Octanol/water partition coefficient at 25C
Sorption partition coefficient (Koc)
Water solubility (ppb) at 25C
1.7 x 10" 1 x 10 c
1.4 x 10 6.9 x 10c 1.9 x 10?d 1.4 x 10e
9.9 x 10s 3.3 x 10c
0 .2f
NA 7 x 10
NA 4.2 x 10^
5 x 10 0.04
3 x 107 0.008
aSource: NRCC, 1981a (unless otherwise stated), based on vapor pressure data (Firestone, 1977a) and the octanol/water partition coefficient value (Kenaga, 1980)
bThese are estimated values for nonspecific Isomers
cMabey et al., 1981
dU.S. EPA, 1984
eTh1s 1s a measured value (Neely, 1979)
^Th1s 1s the experimental value (Crummett and Stehl, 1973)
NA = Not available
3 -6
s p e c i e Isomer or a mixture of Isomers was responsible for the observed
effect(s). However, analytical methods for detecting specific Isomers at
low ppt levels are now available for human samples (Crummett, 1983). In the
case of TCDDs, the specific Isomer 2,3,7,8-TCDD has been more thoroughly
studied than any of Its other isomers because of Its high toxicity. It 1s
not the purpose of this section to review the various analytical methodolo
gies available for PCDDs. Such reviews of recent analytical methods have
been done 1n a Canadian document (NRCC, 1981b), a U.S. EPA (1980a) report
and by Tlernan (1983). Instead, this section will attempt to point out the
various problems that may be encountered 1n the analysis of these compounds
and provide a critique of a few typical analytical methods available for
PCODs.
3.3.1. General Procedure for the Analysis of PCODs. The analysis of
PCDDs can be broadly divided into three basic steps (sample preparation,
sample cleanup and sample analysis). The description of each of these steps
with the associated difficulties that may be encountered are discussed below.
3.3.1.1.
SAMPLE PREPARATION -- In this step, the sample 1s homoge
nized or digested and extracted with a suitable solvent or a solvent mixture
to remove the bulk of the sample matrix and to transfer the PCDD residue
Into the solvent(s). Both the selection of the proper solvent(s) and the
method of extraction can be critical 1n obtaining a satisfactory recovery of
PCDDs from the sample matrix. A number of solvents Including hexane,
hexane-acetone, benzene, toluene, chloroform and methylene chloride gener
ally have been used for extracting PCDDs from sample matrix (Kooke et al.,
1981; Harless et al., 1980; Van Ness et al., 1980). If the sample does not
contain water, as 1s the case with fly ash and atmospheric particulate
samples, either benzene or toluene appears to be the desirable solvent
3 -7
(Kooke et al.f 1981). Toluene should be preferred over benzene, however, because of Its lower toxicity. 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 extractabmty 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 1t produces emulsion with milk contain ing higher lipid (Albro, 1979).
In other sample matrices that contain high amounts of water, such as tissues and food samples, the water may alter the extractabmty 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 2 % water, resulting 1n nonreproducllble extraction (Albro, 1979).
Samples that may contain PCDDs bound to the matrices, such as tissue, food, soil and sediment, may require ac1d/base digestion procedures to release the bound substrate Into the extraction media. The ac1d/base extraction 1s normally done with concentrated acid or an alcoholic base (Toslne, 1981; Harless et al., 1980). Kooke et al. (1981) reported highest extraction efficiencies by acid treatment of fly ash before extraction. The Increase 1n efficiency was hypothesized to be due to opening of some of the pores 1n the fly ash structure, thus making the solvent more accessible to the sorbed PCDDs. Refluxing with alkaline potassium hydroxide, however, may cause decomposition of the higher polychlorinated dioxins and oxidation
3 -8
of some products (Hass and F r l e s e n , 1979; A lb r o , 1979). A n e u t r a l e x t r a c
tion system Is reported to circumvent the possibility of this loss and has
been used by several authors (O'Keefe et al., 1978; Harless et al., 1980).
The extraction efficiency may also depend on the method of extraction.
The extraction efficiencies of PCDDs by simple shaking, ultrasonlcatlon and
soxhlet extraction were studied by a few Investigators (Kooke et al., 1981;
Chess and Gross, 1980). While Chess and Gross (1980) reported no signif
icant Improvement 1n extraction efficiencies of PCDDs from fly ash by
sonlcatlon or soxhlet extraction, Kooke et al. (1981) found soxhlet extrac
tion to be a better procedure than the other two methods. Similarly, Albro
(1979) reported that the nature of the sample matrix Influences the effec
tiveness of extraction. Thus, while 1t may be possible to extract Uver 1n
a Teflon-glass homogenlzer, brain tissues may require a blender, and skin a
powerful disintegrator such as the Polytron for the extraction of residues.
3.3.1.2.
SAMPLE CLEANUP -- The sample cleanup procedure normally
consists of three essential steps. A fourth step 1s usually required 1f an
Isomer specific Identification and quantification 1s required. The first
step In the cleanup procedure consists of the removal of lipids from the
extracted sample matrix. The lipid 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 CCl^) gives excellent results when lipids consist primarily of
triglycerides and/or phospholipids. When the lipid consists of cholesterol
esters, however, sulfuric acid 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 saponifica
tion, hydrolysis or degradation. However, acid wash 1s more commonly used
3 -9
than base wash presumably because of the probability of decomposition
(Albro, 1979) and oxidation (Hass and Frlesen, 1979) of sample components as
a result of base wash. The possibility of decomposition of higher PCDDs by
the base may be the reason for Its less frequent use. It should be men
tioned that some Investigators used chromatographic columns such as silica
gel containing sulfuric acid for the ac1d/base cleanup step Instead of wash
ing off the lipids by simple shaking (Lamparskl et al., 1979; FanelH et
al., 1980a; Langhorst and Shadoff, 1980; Buser, 1978; D1Domen1co et al.,
1980a).
The second step 1n the cleanup procedure consists of removal of common
Impurities such as pesticide residues from the PCDDs. Liquid chromatog
raphy with alumina, Florlsll, silica, foam charcoal or carbon dispersed on
glass fibers has been used for this purpose (Harless et al., 1980; Mltchum
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 AgNOg/slUca column system 1s
claimed to be effective 1n the removal of DDE, chlorinated allphatlcs and
sulfides.
There 1s a difference between the various alumina columns (Lamparskl et
al., 1979; Harless et al., 1980). The separation of PCDDs from PCBs may be
accomplished with acidic, neutral and basic alumina; most authors have
provided no reason for choosing one over the other. However, 1t has been
shown by Albro (1979) that acidic alumina may be better than basic alumina,
which 1n turn may be better than neutral alumina for the separation of
residual lipids from the PCDDs 1n the sample extracts.
3 -1 0
The third step 1n the cleanup procedure 1s used solely as an a d d i t i o n a l cleanup of contaminants and has been used by a few Investigators (Langhorst and Shadoff, 1980; Lamparskl et al., 1979; Mltchum et al., 1980). The removal of these additional Impurities has been obtained by using HPLC with both normal and reversed phase packing materials. Recently, PhllUpson and Puma (1980) reported that chlorinated methoxyblphenyls In fish extract could coelute with TCODs through an alum1na-Flor1s1l cleanup sequence and Inter fere with the determination of TCDDs. A few compounds that may Interfere with the determination of TCDD at m/e values of 319.8966 and 321.8936 are given 1n Table 3-3.
The additional cleanup step using the HPLC separation procedure may be essential for the unequivocal separation of Impurities that may Interfere with the MS analysis of PCDDs.
The fourth and final cleanup step consists of the separation of PCDDs Into several different fractions by means of chromatographic techniques. Both liquid chromatography with alumina columns (Hass et al., 1978; Albro and Corbett, 1977) and HPLC with normal and reverse phases have been used (Toslne, 1981; Ryan and P1lon, 1980; Langhorst and Shadoff, 1980; Mltchum et al., 1980). The separation of PCDDs using HRGC 1s necessary for the unequi vocal separation of 2,3,7,8-TCDD, 1,2,3,7,8-PeCDD and 1,2,3,7,8,9-HxCDD from the other congeners. Buser and Rappe (1983) have shown that this separation can be achieved using a 55 m 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 TCDD Isomers. The various cleanup and analysis procedures have been compared by Brumley et al. (1981).
3-11
7AM h 3 -3
Potential Interferences 1n the Determination of TCDDs at m/e Values of 319.8966 and 321.8936*
Compound
Molecular Formula
Interfering Ion
m/e
Resolution for
Separation
3-12
Heptachlorobiphenyls Nonach lorobiphenyls
letrachloromethoxy biphenyls
Tetrachlorobenzylphenyl ethers
Pentachlorobenzylphenyl ethers
DDT (A Isomers)
DDE (4 Isomers)
Hydroxytetrachlorodlbenzofurans Tetrachlorophenylbenzoqulnones Tetrachloroxanthenes
Ci 2H33SC1v C12H 3SC19 C12H 3SC|8 a/(M Cl3H8 3SC14O C13H8 3SC13 37C10
Ci38 35C140 Cl3H8 35C13 37010
C13H7 35C14 37C10 C13H7 35C13 37C120 C14H9 3SC13 37C12 C14H9 33C12 37C13
C14H8 35C12 37C12 C14H8 35C1 37Cl3
^12^4^*H2
Ci 2H4CI4O2
C13H60 3*C13 37C1 C13H5O 35C12 37C12
`Source: NRCC, 1981b NR = Not resolved by MS
M* -235C1 M* -435C1 M* -3:,,>(:i 37C1
M* M* M* M*
M* -H35C1 H* -H35C1
M* -H35C1 M* -H35C1
H* M* M*
M*
M* M*
321.8678
319.8521 321.8491
319.9.329 321.9299
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
12476
7189 7233
8805 8848
8813 8843
18043 18104
9006 9050
9011 9052
NR NR
NR NR
18043 18104
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, 1t can Introduce error 1n two different
ways. It has been shown by Lamparskl et al. (1979) that concentration of
sample solution with prepurified 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% H2S0^ on
B1o-S1l A, and Carbosleve 8S were required. Secondly, O'Keefe et al. (1982)
have demonstrated that significant losses of 2,3,7,8-TCDD occur when
nitrogen evaporation to dryness 1s done at temperatures >50*0.
3.3.1.3.
SAMPLE ANALYSIS -- The final analysis of PCDDs 1s almost
exclusively performed by 6C/MS. Although some of the earlier Investigators
(Lamparskl e t a l . , 1978; F i r e s t o n e , 1977b) used GC w it h e l e c t r o n c a p tu re
detection, 1t does not have the sensitivity for complex samples containing
low levels (<10 ng kg-1) of PCDDs (Hass and Frlesen, 1979).
The final separation procedure for PCDD analysis uses GC with packed or
capillary columns. A typical 11st of packed and capillary columns used for
the analysis of PCDDs 1s given 1n Table 3-4. Capillary columns are prefer
able over packed columns because they provide better separation of compon
ents 1n a complex mixture than packed columns. There are other advantages
of capillary columns, namely, that the narrow band width of the separated
components enhances MS sensitivity, and the capillary columns with their low
bleed rates enhance MS sensitivity by keeping the background contamination
low. A disadvantage of the capillary columns relative to the packed columns
3 -1 3
TABLE 3-4 Some Packed and Capillary Columns Used for the Analysis of PCDDs
PACKED COLUMNS
1.8 m x 2 mm 1.d., 3% Dexsll 300
Van Ness et al., 1980
0.6-2 m x 2.5 mm 1.d., 3% OV-1, 3% OV-17, 3% OV-61, 2% OV-101
D1Domen1co et al., 1980a
1 .8 m x 2 mm 1.d., 3 % 0V-7
Tlernan et al., 1980
2 m x 2 mm 1.d., 3% 0V-210
Parker et al., 1980
2 m x 2 mm 1.d. specially packed 0 . 2 % carbon wax 20 M (Aue packing)
Elceman et al., 1981
2 m x 2 mm 1.d., 0.6% 0V-17/0.4% Poly S179
Langhorst and Shadoff, 1980
2 m x 4 mm 1.d., 1.2% S1lar 10C
Firestone et al., 1979
1.8 m x 2 mm 1.d., 5% SE-30
Baughman and Meselson, 1973
CAPILLARY COLUMNS
18 m x 0.3 mm 1.d., OV-61 WC0T 22 m x 0.3 mm 1.d., OV-17, 101, S1lar 10C 50 m x 0.36 mm 1.d., OV-17 WC0T 30 m x (1.d. not given), SE-30 WC0T 30 m x 0.25 mm 1.d., 0V-101 WC0T 30 m x 0.25 mm 1.d., SE-30 WC0T 20 m, SP-2100 SCOT 25 m x 0.2 mm 1.d., quartz, methyl silicone WC0T 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 10C 55 m x 0.37 mm 1.d., glas OV-17 55 m x 0.40 mm 1.d., glass 0V-101 60 m x 0.26 mm 1.d., Supelco SP-2330 50 m x 0.4 mm 1.d., 0V-101 fused silica 60 m 0V-101 WC0T (1.d. unspecified)
Buser, 1975 Buser, 1976 Buser and Rappe, 1978 Harless and Oswald, 1978 Harless and Lewis, 1980a Harless et al., 1980 Mltchum 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
1s the problem of easy overload In the presence of other coextracted
Impurities. One group of researchers (Langhorst and Shadoff, 1980} has used a packed column for the unequivocal determination of 2,3,7,8-TCDD 1n the presence of 21 other Isomers. However, this determination was possible because of the prior separation of components through fractionation by HPLC with a combination of a reverse phase Zorbax ODS column and a normal phase silica column. D1Domen1co 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 0V-101 and 0V-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 10C 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 S1lov (Tlernan, 1983). A 50 m length of a S1lar 10C 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 1,2,3,7,8,9HxCDD from the less toxic congeners.
3 -1 5
As previously mentioned, MS Is used almost exclusively for the detection
and quantification of PCDDs. Basically, three MS techniques (LRMS, HRMS and
NICI) have been used. A few different MS systems used for the determination
of TCDDs are shown In Table 3-5. It 1s obvious from Table 3-5 that electron
Impact Ionization In the low resolution mode (resolution <8000, 10% valley)
has been the most widely applied MS method used for the determination of
TCDDs.
The electron-impact mass spectra of PCDDs show strong molecular Ions
(M+ ). Fragmentation occurs through the loss of CO and Cl radicals. Major
Ions are at M*-63 (M+-C0C1) and M*-126 (M+-2C0C1). Doubly charged molecular Ions (M2+ ) and minor fragmentation Ions occur at M+-35
(M+-C1), M+-70 (M*-2C1)
and M+-98 (M+-C0C1-C1). The usual charac
teristic Ion clusterings caused by the chlorine Isotopes are also observed.
Based on molecular Ions and fragmentation pattern, PCDDs can be distin
guished from other chlorinated pollutants. However, this requires monitor
ing multiple Ions. The Ions that are commonlymonitored for 2,3,7,8-TCDD
are M+ and Its chlorine Isotope clusters, that 1s, 320 (3SC1^ CDD),
322 (sscigST-ci CDD) and 324 (35C1237C12 CDD).
In some
Instances, fragment Ions at 257 (320-C035Cl ), 259 (322-C035Cl) and 194
(320-2C0S5Cl) are also monitored. The Intensity ratios In the mass spec-
trometrlc peaks that are due to chlorine Isotope proportions 1n native TCDD
can be used for assessing thedegree of Interference and confirming the
Identity of the TCDDs.
Thus, therelative peak Intensities of
pure
2,3,7,8-TCDD at 320:322:324 are expected to be 77:100:49 (NRCC, 1981a). The
response for the 1on at 257 1s -30% of the response for the 1on at 322
(Glaser et al., 1981). Sometimes Internal standards containing
{C12H487C142)
r
*1>C12H43SC1402 i
USed
fr TCDD
analysis give prominent Ion peaks at 328 and 332, respectively. The primary
3 -1 6
lAULt 3-b
/
The Detection Limit, Resolution and Ions Monitored by a Few Mass Spectrometrlc Systems for the Determination of TCDDs3
3-17
Ionization Method and Reference
TCDD Limit of Detection
(pg)
ELECTRON IMPACT Baughman and Meselson, 1973 Crummett and Stehl, 1973 Hummel, 1977 Hummel, 1977 Mahle et al., 1977 Adamoll et al., 1978 Adamoll et al., 1978 O'Keefe et al., 1978 D1Domen1co 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 Lamparskl and Nestrick, 1980
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
m/e Values Monitored for TCDD 320 322 324 326 328 332 259 257
194
4- 44- 44- T 4- 44- 44- 44- 44- 44- 44- 44- 4-
+4-
4- 44- 44- 44- 4-
4-
444- 44-
44-
4444-
4-
4-
444-
4- -
4- 44- 4-
TABLE 3-5 (cont.)
3-18
Ionization Method and Reference
Norstrom et al., 1982 Tosine, 1981 Ryan and Pllon, 1980 Ternan et al., 1980 Ternan et al., 1980
TCDD Lmt of Detection
(pg)
5-10C 10C 10C ld
100C
H/AHb
unit unit 1,000
350 12,500
m/e Values Monitored for TCDD 320 322 324 326 328 332 259 257
194
+ + f
f +
+
CHEMICAL IONIZATION Hass et al., 1978
Mtchum et al., 1980
50-500 IO
unit NR
323 MNCIe, 252 and 276 for M0NCIf, 176 for ONCI 5or -176 from 320, -182 from 332 by 0NIAPCIh
aSource: NRCC, 1981a ^resolution of mass cng.kg_1
dvg.kg_1 emethane negative chemical Ionization f
methane-oxygen negative Ion chemical Ionization goxygen negative Ion chemical Ionization ^oxygen negative In atmospheric pressure chemical Ionization NR = Not reported
M+ ions for PeCDOs and HxCODs 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 1s not specific (Hummel and Shadoff, 1980). However, an unequivocal Identification and quantification of 2.3.7.8- TCDD 1n the presence of Its Isomers will still require HPLC frac tionation or HRGC 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-1nject1on of sample fortified
with 37C1-TCDD and 2,3,7,8-TCDD standard.
5. I n t e n s i t y o f m o lecu lar Ions 320 and 322 must be > 2 .5 times the noise le v e l.
Supplemental criteria that Harless et al. (1980) suggested for highly contaminated extracts are:
1. C0C1 loss Indicative of TCDD structure. 2. GC/MS peak-matching analysis of molecular Ions 320 and 322 In
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 1s that the former technique requires far less time-consuming cleanup steps than those required for LRMS although this 1s dependent on the nature of the
3 -1 9
sample. With the use of properly selected analytical techniques, the PCDDs can be determined down to sub ppt levels (Crummett, 1983).
The use of chemlcai Ionization techniques has received limited applica tion for the Individual TCDD 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 | + C0C1) based on the concept of MS-MS was used for the analysis of TCDD (Chess and Gross, 1980). Although the method had comparable specificity to GC-HRMS, the precision of the method was not as good. 3.3.2. Analysis of 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 PCDDs 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 1n clean (detergent washed, rinsed with acetone or methylene chloride, and dried) amber glass bottles of 1 a or 1 quart capacity fitted with screw caps lined with Teflon or aluminum foil (U.S. EPA, 1982a). If aluminum foil 1s used as a Uner, 1t should be washed with acetoneand the dull side should face the sample to avoid sample contamination (Albro, 1979). Automatic samplers can also be used for collecting flow proportional composite samples 1n amber glass bottles (U.S. EPA, 1982a). The sample
3 -2 0
containers must be kept refrigerated at 4C and protected from light during
compositing. The grab or the composite-samples should be protected from
light and be kept at 4C 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 8, of water was allowed to pass
through a 12 cm x 1.5 cm i.d. XAD-2 column at a rate of 60 m8,/m1nute. The
XAD-2 columns containing the PCDDs should be protected from light and kept
at 4C during transportation and storage.
3.3.2.1.2.
Analysis -- Most of the methods found 1n the literature
described 2,3,7,8-TCDD analysis Instead of other PCDD analyses in aqueous
samples. The methods used for the analysis of 2,3,7,8-TCDD can be used also
for the analysis of the other PCDDs. However, the recovery of the Indi
vidual PCDDs should be established with added Internal standards.
An a p p r o p r ia t e volume o f w ater (depending on the d e s ire d d e te c t io n
limit) with added Internal standard of either " C ^ or 37C1^
2,3,7,8-TCDD 1n the amount of 2.5-25 ng (Harless et al., 1980; U.S. EPA,
1982a) can be extracted with hexane (D1Domen1co et al., 1980a), methylene
chlorine (U.S. EPA, 1982a; Harless et al., 1980) or petroleum ether (Van
Ness et al., 1980). Judging from the recovery data (U.S. EPA, 1982a;
D1Domen1co 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
3-21
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 6C-HRHS (Van Ness et al., 1980; Harless et al., 1980) or high
resolution 6C-HRMS or LRMS (U.S. EPA, 1982a). If an unequivocal Identifica
tion of 2,3,7,8-TCDD 1s required, the U.S. EPA (1982a) method seems to be
most appropriate since 1t recommends using a 50 m S1lar 10C capillary column
and multiple 1on monitoring MS mode that 1s known to unequivocally Identify
and quantify 2,3,7,8-TCDD 1n the presence of Its other Isomers (Buser and
Rappe, 1980). Harless et al. (1980) reported that TCDD 1n water can be
accurately determined to as low a concentration as 0.03 ppt.
3.3.2.2. AIR --
3.3.2.2.1.
Sampling Method -- Monitoring of PCDDs from point sources
of emission and ambient atmospheric level requires development of sample
collection methods from both sources. The available published work suggests
that the PCDDs are associated primarily with 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 organics 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 >100C to
prevent condensation of water. PCDDs that may escape the glass filters may
be collected 1n a polyurethane foam or XAD-2 trap maintained at room
temperature. The sampling must be performed 1n an Isokinetic manner to
ensure representative sampling. To permit evaluation, the efficiency of the
3 -2 2
collection method must be documented. The sampling methodology for point
sources 1s 1n a developmental stage (NRCC, 1981b) and more work 1s needed 1n this area. The recommendations for sample collection procedure given above follow the general U.S. EPA procedure for collection of air samples from hot point sources. A modified U.S. EPA Method 5 sampling train (Federal Register, 1971) consisting of a filtering unit, a condenser unit, a resin cartridge unit and a series of 1mp1ngers have been used by Stanley et al. (1982) to collect 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 (DIDomenlco et al., 1980b). Dustfall jars were constructed from 10 8, glass vessels topped with metal grldded funnels with a collecting cross section of about 0.11 m2. The top of the funnels were about the human breathing level from the ground. The grid allowed particles <500 pm to be collected. Samples were collected for 1 month or the time required for the vessel to be filled with meterolc water and dust. At the end of the sampling time, the liquid phase was separated from the particles by filtration and the two phases were analyzed separately.
The high volume sampling was performed with high volume samplers equipped with A and E glass fiber filters at a flow rate of 1.5 m3/m1nute (DIDomenlco et al., 1980b). The sampling duration was about 160 hours. The whole sampling unit was assembled Into a protective container. The effi ciency of sample collection by either of the above methods was not estab lished. The high volume sampling can lead to stripping of PCDDs from the filter. A backup filter consisting of polyurethane foam plug may be used to prevent this anticipated loss. Particulate and vapor phase TCDD was also
3 -2 3
collected by polyurethane foam filters (U.S. EPA, 1982b; Nash and Beall,
1980). The collection efficiency with this system was determined to be 86%
by Nash and Beall (1980).
3.3.2.2.2.
Analysis -- The analysis of PCDDs 1n the particulate
matter begins with an extraction process. As has been shown 1n Section
3.3.1.1., the best extraction efficiency 1s obtained with dilute HC'I
pretreated particles, followed by soxhlet extraction with benzene or
toluene. L1bert1 and Brocco (1981) found that xylene was a better solvent
than toluene, while Cutle (1981) found that o-d1chlorobenzene may be better
than any of the other solvents. Various extraction procedures for combus
tion effluent samples have been described by Taylor et al. (1983).
Several methods are available for sample cleanup before analysis.
[Basically, the methods used for the analysis of fly ash can be used for
particulate matter (L1bert1 and Brocco, 1981; Elceman et al., 1980; Tlernan,
1983; Buser et al., 1978)]. In one analytical procedure, Lamparskl and
Nestrlck (1980) added Internal standards of 13C-2,3,7,8-TCDD, 13C-
1 ,2,3,4,7,8-HxCDD and 13C-0CDD to the particulate extract. The extract
was cleaned with acid and base washes. Next, the extract was cleaned by
liquid chromatography with AgN0g/s1l1ca column and basic alumina column,
followed by cleanup and sample fractionation 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.
3 -2 4
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.|2"TCDD, 2,3,7,8-37Cl4-TCDF (tetrachlorodlbenzofuran) and
37Clg-0CDD and treated with 1 M hydrochloric acid. The PCDDs and PCDFs
1n the washed and dried sample were extracted with toluene 1n a soxhlet
extractor and the extract was subjected to column chromatography on silica
gel and basic alumina column. The methylene chlor1de-n-hexane (1:1)
fraction from the second column containing PCDDs and PCDFs was subjected to
HRGC/MS analysis. A 55 m x 0.26 mm 1.d. S1lar column was found to be suit
able for the Isomeric separation of all 22 Isomers of TCDD.
3.3.2.3. SOIL --
3.3.2.3.1.
Sampling Method -- Since similar analytical methods are
used for both soil and sediments, this subsection describes the sampling and
analytical methods for these two sample types.
Whenever possible, the sites for soil samples should be chosen 1n open
areas away from physical obstacles. If the soil 1s suspected to be contami
nated because of fallout from a point source, sampling sites should be
established 1n a grid over a topographical map of the suspected area. Soil
samples may be collected by Inserting a 0.5 m long and 7 cm l.d. steel
cylinder Into the soil to a depth of 7 cm and then retracting the soil and
the cylinder system. The earth core should be removed and stored 1n sealed
plastic bags (D1Domen1co et al., 1980c). The bags should be cooled to 4C
during transportation.
To determine the distribution of PCDDs 1n soil, samples can be taken
from the vertical faces of dug trenches of a maximum depth of 2 m. Suitable
steel core cylinders can be Inserted horizontally Into the trench face from
bottom to top. The Individual samples collected 1n this fashion should be
3 -2 5
stored 1n plastic bags at 4C during transportation them. 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 4C
during transportation.
3.3.2.3.2.
Analysis -- Several methods are available for the analysis
of PCDDs 1n soil samples (Chess and Gross, 1980; Van Ness et al., 1980;
Buser, 1978; Buser and Rappe, 1980; Harless et al., 1980). Although most of
these methods have been used for the analysis of 2,3,7,8-TCDD, they are
applicable for other PCDDs. The methods used for the analysis of soil can
also be used with very little 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),
refluxing (Harless et al., 1980), sonlcatlon and soxhlet extraction (Chess
and Gross, 1980), have been used. However, Chess and Gross (1980) demon
strated that, 1n soil, the results obtained by simple stirring with 1:1
hexane/acetone and the more extensive sonlcatlon or soxhlet extraction with
benzene are consistent.
3 -2 6
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) 1s expected to provide a better resolution of components than the
LRMS. The method of Harless et al. (1980) was suitable for the determina
tion of ppt levels of TCDD 1n soils.
3.3.2.4.
BIOLOGICAL MEDIA -- In this section, the sampling and
analysis of PCDDs 1n a number of media, namely, blood, urine, fish, egg,
gelatin, Uver, milk, cream, lean and adipose tissue, grain, grass, leaves,
vegetables and sawdust, will be discussed 1n general.
3.3.2.4.1.
Sampling Methods -- Only a limited systematic study has
been performed on the methods of sample collection for the different biolog
ical media. A review of available literature reveals certain facts that
should be considered during sample collection. The concentration of PCDDs
1n blood 1s ~2-3 orders of magnitude lower than their concentrations 1n
adipose tissue (Firestone et al., 1979). There Is also evidence In several
species that the accumulation of TCDD 1n Uver tissue 1s higher than 1n
adipose tissue (Section 7.2.). Liver 1s also preferable because Its lipid
content 1s lower than adipose tissue (samples with high 11p1d content are
more difficult to extract and clean up). One of the most convenient
sampling media that does not require sacrificing or surgically removing the
tissue 1s milk. Because of the high 11p1d content of milk, PCDDs are
expected to be accumulated 1n this medium (Langhorst and Shadoff, 1980).
3-27
The dry solid samples, such as rice grain, grass, vegetables and sawdust
can be collected 1n polyethylene bags. Samples should be frozen 1n dry Ice
during transportation and should be stored 1n a freezer (-18C) until
analyzed (Jensen et al., 1983). However, 1t has been reported that tissue
samples stored 1n linear polyethylene bottles sorbed ~ 2 % of added 14C-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 4C and frozen until analysis.
3.3.2.4.2.
Analysis -- Numerous analytical methods are available for
the analysis of samples 1n this category (NRCC, 1981a; Crummett, 1983; Rappe
et al., 1984; Smith et al., 1984). The ac1d/base and neutral extractions
procedures are available. Neutral extraction procedures are preferred over
ac1d/base procedures since the latter may decompose the higher PCDDs. The
analytical methods for the determination of PCDDs 1n three typical media,
namely, fish and lean tissue, adipose tissue, and milk, will be discussed
here. In choosing the analytical methods, the results of the study of
Brumley et al. (1981) have been given due consldera- t1on.
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 1s obtained. The mixture should
be packed Into a glass column and extracted with methylene chloride. The
extract should be first cleaned through a dual-column system of silica,
concentrated sulfuric acid In silica, and sodium hydroxide In silica,
followed by a second dual-column system of silver nitrate on silica and
3 -2 8
basic a l u m i n a . The PCDD fractions should then be cleaned up by normal phase silica HPLC, followed by reverse-phase (Zorbax-ODS) HPLC. This extraction and clean-up method 1s a combination of procedures employed by Hucklns et al. (1978) and Lamparskl et al. (1979), and 1s expected to provide a better method for the analysis of PCDDs 1n lean tissue samples.
Recently, an Interlaboratory round robin study to estimate the reliabil ity of data on the determination of 2,3,7,8-TCDD levels 1n fish and other aquatic species was conducted (Ryan et al., 1983). No significant differ ences 1n the determined concentration of 2,3,7,8-TCDD 1n these species occurred from methods differing 1n the use of digestion or extraction technique, HRMS or LRMS, and Isomer specific or nonspecific separation. The relative standard deviations 1n three fish samples analyzed by seven labora tories varied between 14 and 25%. This study Indicated the necessity for the use of an Internal standard to obtain precise results.
Thawed adipose tissue samples should be ground with anhydrous sodium sulfate (8 g Na2S04/g fat) 1n a mortar and pestle to remove excess moisture. The homogenized sample should be extracted with chloroform methanol (2:1) 1n a blender. The methanol should be removed from the extract by adding aqueous KC1. The chloroform layer should then be sub jected to the clean-up procedures. For the cleanup of the chloroform extract, the method described for lean tissue should be followed. The extraction and clean-up method described 1s a combination of procedures employed by Hass et al. (1978) and Lamparskl et al. (1979). However, hexane-acetone (1:2) was used by Ryan and Williams (1983) 1n extracting 2,3,7,8-TCDD from human adipose tissue.
The milk samples should be mixed with sodium oxalate and ethanol and the ^solution extracted with ethyl ether-hexane (1:1.4). The ether-hexane extract should be dissolved 1n hexane and the clean-up procedure described
3-29
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 Lamparskl et al. (1979) may be employed. 3.3.3. B1oanalys1s of PCDDs. There are currently three methods for the bloanalysls of PCDDs, namely, radioimmunoassay (Albro et al., 1979; McKinney 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 1n the developmental stage and are neither specific for PCDDs nor are sensitive enough at low levels. The advantages of these methods are that they are Inexpensive and quick compared with chemical analytical methods. Therefore, these methods have some potential for high volume screening of samples for the presence of PCDDs, but should not be used as substitutes for chemical analysis. 3.3.4. Critique of Sampling and Chemical Analysis. The greatest weak nesses that persist 1n the determination of PCDD levels 1n environmental samples are the lack of data for validating the accuracy of sample collec tion, transportation and storage procedures. The lack of representativeness of samples during collection, loss of sample by sorption on container walls or photodecomposition during transportation and storage, and contaml- 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 1n the sampling procedures.
There are several possible points of weakness 1n the analytical methods as well. Although some validation data are available for the overall recovery of 2,3,7,8-TCDD 1n fortified matrices, these data, as shown In Table 3-6, may not represent the true recoveries, since 1t 1s difficult 1f
3 -3 0
TABLE 3-6
Some Published Method Validation Data for 2,3,7,8-lCDD Recovered from fortified Matrices and Determined by GC/HS'
3-31
m/e Values
320. 322. 335
320. 322. 324 320. 322, 324
320. 322, 324, 335 320, 322, 328
320, 322, 328
320, 322, 328
320, 322, 328, 329 320, 322, 328
320, 322, 328
320, 322, 328, 329 320, 322, 328
Matrix
human milk
soil soil
soil
fish, liver
human milk
water, sediment water, sediment water, sediment water, sediment bovine feed
liver
TODD Level of fortification, na/kq'1
Native
Isotope C, (Cl)
2.6 166
NA 100 10 NA
50 b
0-125
1000
0-5 250
0.01-1000
250
0.7-65
66
2 NA
NA 625
13-200
390-1000
20 1000
Number of Replicates
8 6 28 8 17 13 14 12 3 4 16 9
Mean X Recovery with S.D.
Native
Isotopes
25 7
NA 87 17
99.2 5
15c
38c
16c
85-100 (8-17) 83.3
NA
80-100 (5-18) 34 7
37 19
87 15 NA
59.8
86 15
68
87
71-87 (12-21)
NA
64
77-105 (9-18) 27 5
Reference
Langhorst and Shadoff, 1980
Hummel, 1977
DIDomenlco et al., 1980a
Lamparskt and Nestrlck, 1980
Harless et al., 1980
Harless et al., 1980
Harless et al.,
1980
'
O'Keefe et al., 1978
Hahle et al., 1977
Hahle et al., 1977
O'Keefe et al., 1978
Baughman and Heselson, 1973
TABLE 3-6 (cont.)
3-32
m/e Values
Matrix
TCDO Level of fortification, nq/kq"1
Hatlve
Isotope >*C, (ClJ
Humber of Replicates
Mean X Recovery with S.0.
Hatlve
Isotopes
320, 322, 324 320, 322, 324 320, 322, 324
carrots beets spinach
0.5-1.0 0.5-1.0 0.5-1.0
HA HA HA
20 64.5-66.6
HA
(i18.9-t25.5)d
20 60.8-79.8
HA
Ii17-i!7.7)<l
20 46.6-67.7
HA
(14.2-*24.7)
Indicates publishing author's recovery data was converted from ng to ppt or from ppt to X. bpius Indicates fortified with Isotope but amount not specified clearly. cThese data Indicate the mean X accuracy for TCDO obtained with quality assurance samples. ^Number In the bracket represents the X variation experienced; unclear as to how calculations were made. HA Hot added; SO Standard deviation
Reference
Cavallaro et al., 1980b Cavallaro et al., 1980b Cavallaro et al., 1980b
n o t I m p o s s i b l e t o incorporate the Internal standard 1n the same physical/
chemical form 1n the sample matrix as the PCODs. This situation weakens the reliability of much of the analytical data on PCDO levels 1n various matrices.
The recovery of the overall analytical procedures 1s normally done by measuring the recovery of Internal standards such as 37C14-TCDD and 13C-TCDD. Methods that used Internal standards that exceeded the native TCOO by 50-2500 times are at best questionable. Also, the recovery data based on one Internal standard to correct for another congener or another Isomer, such as 1,2,3,4-TCDD for OCDD or 2,3,7,8-TCDD, may be questionable 1n view of the fact that recovery and response factors may vary between congeners and Isomers. This could cause serious problems with the deter mined detection limits.
Despite some rigorous criteria (Harless et al., 1980) that may be used for positive Identification of 2,3,7,8-TCDD (assuming that the GC column resolves 2,3,7,8-TCDD from other TCDDs), false positive results have been obtained under certain conditions. A collaborative study conducted by the U.S. EPA exemplifies this point. Of the total of 20 unspiked samples 1n this study, 10 gave false positive results (Crummett, 1980). In a recent method validation study by the U.S. EPA (Gross et al., 1981), 2,3,7,8-TCDD levels <9 ppt could not be detected with accuracy. Clearly, there 1s a need for more exhaustive examination for potential Interferences that may cause false positive results.
Another major factor limiting the research 1n the field 1s the shortage or lack of availability of Individual Isomers. Unless the authentic com pounds are available, analytical data developed for one Isomer on the basis of the response factor of another Isomer will remain largely questionable.
3-33
3.4. SUMMARY The solubility of 2,3,7,8-TCDD 1n water 1s 0.2 yg/8,. This congener
and the other three PCDDs are more soluble 1n aromatic solvents than aliphatic solvents. The PCDDs are relatively stable In the environment and they start to decompose at temperatures >500C.
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 lipid and other Impurities from the solvent extract. The extract 1s then subjected to two liquid chromatographic clean-up procedures. The cleaned up extract 1s finally analyzed for the PCDDs by a GC/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.
3-34
4. PRODUCTION. USE. SYNTHESIS. ENVIRONMENTAL SOURCES
AND ENVIRONMENTAL LEVELS 4.1. PRODUCTION AND USE
PCODs 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 PCODs 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 Dichlorocatechol Salts with 1,2,4,5-Tetrachlorobenzenes in DMSO. This general reaction has been used to synthesize 2,3,7.8-TCDD according to the reaction scheme shown below:
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-2,5,6trichlorobenzene as shown below (Gray et al., 1976):
V
^
no2
4.
H O *^
.VDMSO
V Pl
V
4-1
4.2.2. Substitution Reaction. The following substitution reactions have been used for the synthesis of 2,3,7,8-TCDD:
pnta-CDD + trl-C D D
The yield of 2,3,7,8-TCDD by this reaction has been reported to be low (U.S. EPA, 1980a). However, when the chlorination of the unsubstltuted d1benzo-dloxln was conducted without the FeClg, the yield of 2,3,7,8-TCDD was reported to be 40-S0X (U.S. EPA, 1980a). The substitution of d1benzo-j>dloxln with 2,3-d1chlorod1benzo--d1ox1n In the presence of FeCl^ and 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. Photoproduction. Small amounts of mixtures of lower PCDDs have been produced by the UV Irradiation of OCDD (Buser, 1979). For example, a mixture of tri-, penta-, hexa- and hepta-CDD has been produced by this method. 4.2.4. Ullmann Condensation Reactions. The condensation reactions as shown 1n Figure 4-1 have been used for the synthesis of tetra- and hexa-CDO.
The yield of the desired products by the condensation reactions are not always satisfactory because of other competing reactions. Examples of some of these competing reactions are condensation with Cl atoms meta to a hydroxyl group, condensation of Cl atoms para to the hydroxyl group, dechlorination reactions, and Smiles rearrangement (U.S. EPA, 1980a). Although the best conditions for dioxin formation are unknown, It has been speculated that a temperature of 180-400C, a pressure of >1 atmosphere
4-2
FIGURE 4-1 Ullmann Condensation Reactions
4-3
(necessary to retain some precursor compounds in the liquid state to permit dioxin formation), and the presence of some catalyst provide the most suit able conditions for dioxin formation (U.S. EPA, 1980a). However, some of the catalysts, namely, Cu, Fe, Al-salts and 12 , may encourage competing reactions, thereby reducing the yield of the desired product(s) (U.S. EPA, 1980a). 4.2.5. Pyrolysis of Chlorophenates. All 22 TCDD Isomers have been syn thetically prepared from different chlorophenates (dl-, trl- and tetra-) using a simple pyrolysis procedure (Buser and Rappe, 1980). Pyrolyses of these chlorophenates were conducted by placing 1 mg of the chlorophenates 1n a glass reaction tube plugged with glass wool and alumina. They were heated for 30-60 minutes at 300C. The yields of the TCDOs have been reported to be 1n the yg range (Buser and Rappe, 1980). 4.2.6. Conversion Through Nitration. It has recently been shown by Oliver and Ruth (1983) that 1,2,3,6,7,8-hexachlorod1benzo--d1ox1n can be selectively prepared from two synthetic routes each consisting of dlnltrat1on of a tetrachlorod1benzo--d1ox1n, followed by reduction and a Sandmeyer reaction as shown below:
The recovery of 1 ,2,3,6,7,8-HxCDD was excellent by this method. 4-4
4.3. ENVIRONMENTAL SOURCES
The sources of PCDDs and particularly 2,3,7,8-TCDD, 1,2,3,7,8-PeCDD,
1,2,3,6,7,8-HxCDD and 1,2,3,7,8,9-HxCDD 1n 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 In the environment. Each of these categories 1s dis
cussed Individually 1n the following subsections.
4.3.1. Manufacturing Processes. PCDDs are generally produced during the
production of chlorinated phenols, during the production of chemicals uti
lizing the chlorophenols (1.e., 2,4,5-T and 2,4-D) and In various Industrial
Incinerators where materials containing chlorinated phenol and polychlori
nated diphenyl ethers are Incinerated.
4.3.1.1.
PRODUCTION OF CHLOROPHENOLS -- PCDDs are formed as
by-products during the manufacture of chlorophenols. Chlorophenols are
produced by two processes, the 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
Cl
Cl Cl
NaOH
1 ,2,4,5-Tetrachlorobenzene
4-5
Similarly, HxCDDs are formed during the manufacture of tetrachlorophenols by the above reaction process. PCDDs are also expected to be formed during the hydrolytic production of polychlorinated benzenes. The amounts of PCDDs 1n commercial chlorophenols vary according to manufacturing process and condi tions. The levels of TCDDs, PeCDDs and HxCDDs found 1n different chloro phenols have been shown In Table 4-1. It can be seen from Table 4-1 that the specific Isomers of the TCDDs, PeCDDs and HxCDDs have not always been Identified 1n the products. However, 2,3,7,8-TCDD has been Identified 1n commercial trlchlorophenols (Table 4-1). On the other hand, 2,3,7,8-TCDD 1s not produced 1n the manufacture of PCP (Buser and Rappe, 1978). The main HxCDD Isomers produced during the manufacture of PCP are 1,2,4,6,7,9-, 1,2,3,6,8,9- and 1,2,3,6,7,8-HxCDD present 1n a ratio of 1:4:5 (Buser, 1979). However, the composition and quantities of PCDDs 1n PCP may vary widely from batch to batch and manufacturer to manufacturer, depending on the manufacturing processes.
The annual world production of chlorophenols 1s estimated to be ~150,000 tons (Rappe et al., 1979). U.S. production figures for dl- and tetrachloro phenols are not available. However, the 1977 estimated figures Indicate that the annual production capacity for PCP 1n the United States was 53 million pounds (U.S. EPA, 1980a). Canadians manufacture ~4000 tons of chlorophenols annually with the total release Inventory to the environment estimated at >1365 tons/year (Environmental Canada, 1984). The chloro phenols are used as fungicides, herbicides, sllmaddes, bactericides and Intermediates 1n the production of chlorinated phenoxy acid herbicides 1n agriculture and forestry. The antiseptic, hexachlorophene, 1s also prepared from 2,4,5-trlchlorophenol (Rappe et al., 1979). Therefore, the use or presence of contaminated chlorophenols 1n facilities such as chlorophenol
4-6
-v
Compound
o-Chlorophenol
2,4-D1chlorophenol 2,6-D1ch1oropheno1 2,4,5-TCPc 2,4,6-TCP 2,4,5-TCP (Na salt) TCP (unspecified) 2,3,4,6-Tetrachlorophenol
Tetrachlorophenol (unspecified) PCP
TABLE 4-1
Levels of Tetra-, Penta- and Hexa-chlorod1benzo-[>-d1ox1ns Reported 1n Chlorophenols and a Few Pesticides Originating from Chlorophenols
Ch1orod1benzo-p-d1ox1n (-CDD) level. DDm
Tetra-
Penta-
Hexa-
No. Contain.3 No. Tested
NO 0.037b
NO
NO NO NR NR
NO NO
0/1
several samples
0/1
NO
NO NO
0/1
ND-6.2 (2,3,7,8-)d N0-0.3 (1,3,6,0-) 49 (1,3,6,8-)
1.40 (2.3,7,8-)
NO-1.5 NO NO
NO NO NO
3/4
1/1 1/2
NO
NR
ND-<10
4/6
NO NO HD-29 2/3
0.7
5.2 9.6
NA
NR
NR 6
1/1
NO
NR
ND-<100
3/3
NO
NO
0.17-39
6/6
NO
NR
ND-clOO
10/11
NR
NR 9
1/1
NO
NR
9-27
several
samples
NO
NR
0.02-42
2/2
NO
NO
0.03-10
12/13
Reference
Firestone et al., 1972 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
Vlllaneuva et al.. 1973 Buser and Bosshardt, T9'
8-fr
Compound
TABLF 4-1 (cont.)
Chlorodlbenzo-o-dloxln (-C0D) level. DDm
Tetra-
Penta-
Hexa-
No. Contam.a No. Tested
PCP (cont.)
PCP (Na salt)
2,4-0 (-08, -DP)f
2,4-0 and 2,4,5-T mixtures (formulated products) 2,4-0 (acid, esters, and amines) 2,4-0 (acid, esters, and amines) 2,4,5-Tb
NR
ND 0.06-0.4
ND
ND
ND-0.739 (1,3,6,8-/ 1.3.7,9-)
0 (1,3,6,8-)
ND-<100
NR ND ND-0.08 ND ND NR NR NR
ND-2 14-20 ND-6.8 ND--<10 ND NR NR ND-<100
several samples
2/2 6/6
1/2B
0/10
28/58
2/30
23/42
2,4,5-T (acid, esters, and formulated products)
SI1vex*
0.010-0.080 NR NR (2.3,7,8-)
ND--<10
NR ND
12/30 1/7
Agent Orange (1:1 mixture of butyl esters of 2,4-0 and 2,4,5-T)
Agent Purple (5:3:2 mixture of n-butyl 2,4-0, n-butyl 2,4,5-T and Iso-butyl 2,4,5-T)
1.901 (2,3,7,8-)
32.01 (2,3,7,8-)
NR NR NR NR
490/490 NR
aThese are the ratios of the number of samples contaminated with any chlorodloxlns to the number of samples tested.
b2,3,7,8-Isomer detected but not quantified "TCP: trlchlorophenol "These Indicate specific dioxin concentrations. *PCP: pentachlorophenol 'These are dlchlorophenoxy-acetlc, -butyric acid and -propionic acid. 9The Isomers could not be separated. hTh1s Is 2,4,5-trlchlorophenoxy acetic acid. Uhls Is an average value. NO * Not detected; NR - Not reported; D - detected; NA = Not available
Reference
Dow, 1978
Firestone 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 a l . , 1972 Young, 1983
Young, 1983
and pestldde/herblclde plants, cooling towers, pulp a n d p a p e r i n d u s t r y .
Incinerators and disposal sites are potential exposure areas for PCDDs
(Josephson, 1983).
The locations of current and former producers and formulators of
chlorophenols are presented 1n Table 4-2. The Inclusion of the locations of
the former producers has been judged necessary for the Identification of
past sources of contamination that may present an environmental hazard 1n
the future (1.e., airborne contaminated dust particles) because of the
environmental persistence of 2,3,7,8-TCDD (Chapter 5).
4.3.1.2.
PRODUCTION OF CHLOROPHENOL DERIVATIVES -- PCDDs have been
detected also as contaminants produced during the manufacture of commonly
used chlorophenol derivatives, such as 2,4-D, 2,4,5-T and hexachlorophene by
mechanisms hypothesized to be similar to those discussed 1n the case of
chlorophenols. The amounts of 1,3,6,8- and 1,3,7,9-TCDD 1n commercial Iso
octyl-, mixed butyl- and propylene glycol butyl ether ester of 2,4,-D varied
from nondetectable to 8 .7 mg/kg (Cochrane e t a l . , 1 98 1 ). Agent Orange,
which 1s a 1:1 mixture of the butyl esters of 2,4-D and 2,4,5-T, has been
shown to contain 2,3,7,8-TCDD 1n quantities 1n the range of 0.1-47 yg/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 1n 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 1960s were shown to be 1.98 and
32.8 ppm, respectively (Young, 1983). Efforts were made during the 1970s to
control and minimize the formation of 2,3,7,8-TCDD and, at the present time,
all the producers claim that their products contain <0.1 yg/g of 2,3,7,8-
TCDD (Rappe et al., 1979).
4-9
TABLE 4-2
Locations of Companies that have been Major Producers and Formulators of Chlorophenols and Their Derivatives3
Chemical 2,4-D Acid and Esters
2,4,5-T
Producer*
Aleo Chemical Corp., Philadelphia, PA *Amvac-Chem1cal Corp., Los Angeles, CA&
Chempar, Portland, OR ^Diamond 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 City, KS *PBI-Gordon Corp., Kansas City, KS Rhodla, Inc., Portland, OR
St. Paul, MN St. Joseph, MO *Rhone-Poulenc, Inc., Portland, OR *R1verdale Chemical Co., Chicago Heights, IL Rorer-Amchem, Fremont, CA St. Joseph, MO Thompson Chemical, St. Louis, MO Union Carbide Corp., Ambler, PA *Vels1col Chemical Corp., Beaumont, TX
Bayport, TX Vertac, Inc., Jacksonville, AR Woodbury, Orlando, FL
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 City, 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
4-10
TABLE 4 - 2 ( c o n t . )
Chemical
Producer
2,4,5-T (cont.)
2,4,5-T derivatives Sllvex esters and salts Ronnel Erbon Hexachlorophene
2,4,5-TCP and salts
2,3,4,6-Tetrachlorophenol PCP and salts
Rorer-Amchem, St. Joseph, HO Jacksonville, AR
Thompson Chemical, St. Louis, MO Union Carbide Corp., Fremont, CA
St. Joseph, HO Ambler, PA Vertac, Inc., Jacksonville, AR
Dow Chemical U.S.A., Midland, MI Hercules, Inc., Jacksonville, AR North American Phillips Corp., Kansas City, KS *R1verdale Chemical Co., Chicago Hts., IL Vertac, Inc., Jacksonville, AR *Dow Chemical U.S.A., Midland, HI *Dow Chemical U.S.A., Midland, MI Glvaudan Corp., Clifton, NJ
Diamond Shamrock Corp., Cleveland, OH Dow Chemical, U.S.A., Midland, MI GAF Corp., Linden, NJ Hercules, Inc., Jacksonville, AR Hooker Chemical, Niagara Falls, NY Merck and Co., Inc., Rahway, NJ Nalco Chemical Co., Chicago, IL North Eastern Pharmaceuticals, Verona, MO Roberts Chemical, Inc., Nitro, 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.
4-11
As can be seen from Table 4-1,, 2,4-D, 2,4,5-T and their formulated
products may contain other PCDDs 1n addition to TCDDs. It has also been
reported that Agent Orange and 2,4,5-T samples used during the Vietnam con
flict contain other PCDDs at levels similar to that of 2,3,7,8-TCDD. Agent
Orange and European 2,4,5-T formulations from the 1960s, on the other hand,
may contain primarily 2,3,7,8-TCDD and only minor amounts of other PCDDs
(Rappe et al., 1979). The average 2,3,7,8-TCDD contents 1n Agent Orange and
Agent Purple given 1n Table 4-1 refer to these materials manufactured 1n the
1960s.
Hexachlorophene 1s prepared from the same starting material as 2,4,5-T,
namely, 1 ,2,4,5-tetrachlorobenzene. Because of additional purification,
however, the level of 2,3,7,8-TCDD In this product has been reported to be
<0.03 yg/g (Rappe et al., 1979).
The locations of current and former producers of chlorophenol deriva
tives have been shown 1n Table 4-2.
4.3.1.3.
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-T, and the equipment remained contami
nated with 2,3,7,8-TCDD (Federal Register, 1980a).
4-12
4 .3 .1 .4 . DIPHENYL ETHER HERBICIDES ~ The presence of TCDDs, PeCDDs and HxCDDs as contaminants 1n diphenyl ether herbicides was reported by Yamag1sh1 et al. (1981). The source of PCDDs 1n these herbicides was specu lated to be the trlchlorophenol used 1n their production. The concentra tions of the two major Impurities, TCDDs and PeCDDs, 1n commercial formula tions were -150 and 30 ppm, respectively. The Isomeric distribution of TCDDs showed that the major components were 1,3,6,8- and 1,3,7,9-Isomers. The Isomer 2,3,7,8-TCDD was not detected 1n the commercial products.
4.3.1.5. INCINERATION OF SELECTED INDUSTRIAL HASTES -- The combus tion of a variety of chlorinated hydrocarbons has been shown to produce PCDDs (Tlernan et al., 1982a). The formation of PCDDs would likely occur 1n Incinerators operating at 750-900C; chlorophenols are probably the precur sors of PCDD formation. At temperatures >1200-1400C and residence time of <1 second, PCDDs are likely to decompose and these compounds are not expected to form (lunk and Richard, 1981). From kinetic and thermodynamical considerations, Shaub and Tsang (1983) estimated that 99.99% gas phase dis sociation of tetrachlorod1benzo--d1ox1ns at 727C may require -15 minutes, while the same decomposition at 977C may require <1 second.
In an Industrial boiler 1n the United States where PCP was known to have been burned, Rappe et al. (1983b) reported -5 ppm PCDDs 1n the bottom and baghouse ash. More than 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 1n Binghamton, NY, 1n February, 1981, revealed that 2,3,7,8-TCDD (0.6 ppm) and 1,2,3,7,8-PeCDD (2.5 ppm) were the dominating Isomers of the PCDDs formed (Buser and Rappe, 1983; Rappe et al., 1983b). The origin of the PCDDs was probably the chlorobenzenes 1n the transformer oil (Buser, 1979). The analysis of wipe tests from a garage
4-13
adjacent to this site did reveal the presence of PCDDs before cleaning the garage. Following the cleanup, no contamination was found (Hernan et al., 1982b; Hernan, 1983). Therefore, 1t 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 particulate matter from municipal Incinerators by several Investigators 1n Canada, Europe and the United States. The particulate matter forming the emissions (air particulates) 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 In the fly ash from a variety of municipal Incinerators 1n Canada, Europe and the United States have been studied by several authors (Elceman et 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 (1.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 Tn 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 In the Immediate vicinity (CEQ, 1981). Evaluation of stack emissions of PCODs have to be based on the amount of dioxins 1n both the flue gas condensate followed by an effective absorption or adsorption step (Ballschmlter et al., 1984). PCDDs have been
4-14
detected 1n the emissions of some municipal waste Incinerators 1n Europe (G1zz1 et al., 1982; Benfenatl et al., 1983; Taylor et al., 1983; O U e et al., 1982, 1983; Lustenhouwer et al., 1980; Barnes, 1983). Observations on PCDD emissions from an Industrial boiler have been discussed 1n Section 4.3.1.5. (Rappe et al., 1983b).
In a study of municipal fly ash conducted between a single Incinerator 1n the United States and one In Europe, Lamparskl and Nestrlck (1980) detected at least 14 of the 22 possible TCDD Isomers. Although the ratio of Isomers to the total present were similar 1n both fly ashes, their absolute amounts varied by a factor >10. It has been demonstrated by Rappe et al. (1979) that minor amounts of the highly toxic PCDD congeners, 1,2,3,7,8PeCDD, 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 particulate matter from most combustion sources. These fin d in g s led to a hypothesis which suggested that PCDDs may be formed 1n trace amounts from chemical reactions during the com bustion of many chlorinated hydrocarbons (Bumb et al., 1980; Crummett et al., 1981). These Investigators detected PCDDs Including TCDDs and HxCDDs 1n particulate matter from municipal and Industrial Incinerators, 1n mufflers from diesel truck and passenger vehicles, from home wood-burning fireplaces and from soot and cigarette smoke. Since the trace chemistries of fire hypothesis was presented, several Investigators have attempted to test 1t. Tlernan (1982) reported the detection of 0.65 ppb TCDD 1n soot from a wood-burning fireplace. Although there 1s general agreement regard ing the production of PCDDs from the burning of wood with additional HC1 and from Incinerators burning chlorinated products or wastes (Tlernan et al.,
4-15
1982a, Tlernan, 1983), production from the combustion of coal and hydro carbons (such as occurs 1n gas burners, and auto and truck engines) has not been confirmed (NRCC, 1981a). For example, Rappe et al. (1979) concluded from their pyrolysis experiments that PCDDs are produced by the burning of very specific chemicals, such as chlorinated phenols, polychlorinated benzenes and polychlorinated diphenyl ethers. Wood pregnated with these compounds might produce PCDDs during Incineration and the history of wood to be burned 1n fireplaces 1s often unknown. Junk and Richard (1981) and Kimble and Gross (1980) failed to measure TCDD above the detection limits of 1 or 1.2 ppt, respectively, from their analysis of one fly ash sample from stack emissions of a low sulfur and h1gh-ash coal burning power plant. Recent Investigations (Halley et al., 1983; Stanley et al., 1982) also failed to detect (detection limit: flue gas, 100-700 pg/m3; fly ash, 10-70 pg/g) PCDD homologues 1n any sample from four coal-fired power plants. Independent confirmation of "trace chemistries of fire" as proposed by Dow, U.S.A., 1s not yet available.
Czuczwa and Hites (1984) and Czuczwa et al. (1984, 1985) analyzed for the PCDDs and PCDFs 1n sediments from the Great Lakes Including the sediment core from S1sk1w1t Lake of Isle Royale 1n northern Lake Superior. The sediment that came from S1sk1w1t Lake was used because 1t received only atmospheric Inputs. In all cases the authors detected the flux of PCDDs and PCDFs, which began at about 1940. When this "1940 horizon" was compared with combustion trends 1n the last century, the authors found evidence that the combustion of synthetic chlorinated organic chemicals 1s the primary source of PCDDs and PCDFs. Furthermore, the authors responded that the flux of PCDDs and PCDFs to three Swiss lakes, where combustion has been extensive during the last century, Increased only after the development of the
4-16
chlorinated organic chemical industry. The authors also addressed the debate regarding 2,3,7,8-TCDD in coal fly ash. Reaffirming similar find ings, no 2,3,7,8-TCDD was found above a detection limit of 100 ppt. These results strongly suggest that coal combustion is not a significant source of 2,3,7,8-TCDD contamination to the environment. 4.3.4. Chemical Dump Sites. At present, other potential sources of PCDDs are chemicals known to be contaminated with PCDDs but withdrawn from use and awaiting disposal, and disposal sites where chemical wastes containing PCDDs have been dumped. It has been estimated that --11,600 metric ton/year of hazardous wastes are produced in the manufacture of chlorophenols and ~79,000 metric ton/year are produced in the manufacture of phenoxy compounds (Jett, 1982). Process wastes from the manufacture of chlorophenols and phenoxy compounds are landfilled, or injected into deep-well. Treatment wastes are frequently subjected to on-site impoundment (Jett, 1982). Recent Canadian environmental data indicate that 2,3,7,8-TCDD may be leaking into the Great Lakes from t o x ic dump s i t e s (Hallett, 1984). 4.3.5. Photochemical Process. Photochemical processes can also lead to formation of PCDDs. For example, the dimerization of chlorophenols to OCDD has been studied by Crosby and Wong (1976). Lamparski et al. (1980) also reported that photolysis of PCP-treated woods may lead to the formation of PCDDs. Similarly, photochemical cyclization of predioxins (chlorinated 2-phenoxyphenols, precursors of PCDDs) can also produce PCDDs. Since pre dioxins are common impurities (1-5%) in commercial chlorophenols, exposure of chlorophenols containing those impurities to light may produce PCDDs (Nilsson et al., 1974).
Another photochemical process of potential environmental importance 1s the formation of highly toxic TCDD and PeCDD congeners from the dechlori-
4-17
nation of higher PCDDs. However, photolysis of 1,2,3,6,7,8-HxCDD and 1 ,2,3,7,8,9HxCDD produced only 13% of the toxic 1,2,3,7,8-PeCDD and no 2,3,7,8-TCDD (K1m et al., 1975), while the photolysis of octa-CDD was shown to produce mainly 1,4,6,9-TCDD, 1,2,4,6,9-PeCDD and 1,2,4,6,7,8-HxCDD. Con sequently, 1t was concluded that the most toxic Isomers are not likely to be formed from the photolysis of the higher PCDDs (Buser and Rappe, 1978).
Formation of tetra- and pentachlorod1benzo-p-d1ox1ns has been observed by the photolysis of 1,2,3,6,7,8- and 1,2,3,7,8,9-HxCDDs (Buser, 1979). There seems to be a preferential dechlorination of the HxCDDs occurring at the lateral positions flanked on both sides by adjacent chlorines (Choudhry and Hutzlnger, 1984). However, formation of trace amounts of 2,3,7,8-TCDD were also observed from the photolysis of the above two Isomers of HxCDDs (Buser, 1979). 4.4. RELATIONSHIP BETWEEN SOURCES AND CONTAMINATION IN ENVIRONMENTAL
MATRICES The potential relationship between various sources of PCDDs and the environmental matrices where these compounds have been detected (NRCC, 1981a) Is depicted In Figure 4-2, which has been modified from the original reference to Indicate the possible Inhalation exposures from these sources. 4.5. ENVIRONMENTAL LEVELS The detection of PCDD residues, particularly the residue of the four toxic PCDDs under discussion, 1n various environmental matrices 1s Indica tive of the potential Impact that the various sources could have on the environment. However, the monitoring efforts for the determination of the levels of these compounds 1n the environment are extremely limited for sev eral reasons. The primary reasons are the nonavailability of standardized sampling methods and the specialized analytical techniques that must be used for the determination of traces of these difficult to separate compounds 1n
4-18
wood preservation plants
effluents
receiving waters
biota
chlorophenol-treated woods
food stored in CP tre ate d bins wood shaving for bedding -------
food food
industrial plants
a i r -------------- mammals receiving waters
biota
a ir;
mammals
municipal incinerators
precipitated fly ash in receiving w aters
fly ash disposal site
biota
biocide formulated products
mammals receiving w aters ------------ biota
kraft pulp m ill----------- - effluents ---------- receiving w a te rs ----------- biota
^ ^ ^ ^ . s k i n scrap in g s------- --------------- - enimals leather ta n n e ry ------------ - e fflu e n ts ------------ - receiving w a te rs ------------ biota
unidentified ----------------- - receiving w aters ----------------- - biota.
FIGURE 4-2 Possible Potential Relationship Between Various Sources of PCDDs
and the Environmental Matrices Where PCDDs have been Detected Source: Modified from NRCC, 1981a
4-19
the presence of a large number of Interfering compounds. Measurable quanti ties of these compounds have been detected 1n the environment under special circumstances, that 1s, after accidents 1n factories producing chlorophenols and their derivatives, 1n the environment after certain herbicide use, and 1n the environment near certain dumpsltes. In other words, the current available data demonstrate that the major sources of PCDDs 1n the environ ment are those associated with the production, use and disposal of chloro phenols and their derivatives. Choudhary (1983) 1n a review paper provided a 11st for some of the potential workplaces where occupational exposure to PCDDs may occur. It should also be recognized that most of the environ mental monitoring Investigations measured 2,3,7,8-TCDD levels, whereas mon itoring data for other PCDDs are even more limited. 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 1n various environmental media have been presented 1n the following subsections. 4.5.1. Water. NAS (1977) reported that no 2,3,7,8-TCDD has ever been detected 1n drinking water using methods with limits of detection In the ppt range. Other PCDDs Including PeCDD and HxCDD have not been detected 1n drinking water. However, TCDD, Including the 2,3,7,8-Isomer, has been reported 1n aqueous Industrial effluent samples and leachates from hazardous waste disposal sites. For example, Van Ness et al. (1980) analyzed eight effluents from a trlchlorophenol manufacturing plant site and detected TCDD 1n two of these effluents (detection limit 10-30 pg/g). The concentrations of TCDD 1n the two samples with detectable TCDD concentrations were 17 and 100 pg/g. Although the specific Isomer was not routinely separated, the authors concluded from their study that a significant portion of the TCDD was apparently the 2,3,7,8-Isomer.
4-20
T h e a n a l y s i s o f leachate samples from two waste disposal sites for the analysis of TCOO 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-TCDD (Thibodeaux, 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-TCDD. 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-T. 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 1n the atmosphere 1s the field spraying of the herbicide 2,4,5-T. The spraying of 2,4,5-T containing 2,3,7,8-TCDD Impurity may lead to a concomitant exposure to 2,3,7,8-TCDD. However, the measurement of air concentration at any particular time after spraying may not be a representative sample because of spray drift to non target sites and the Intermittent nature of spray application. From micro agroecosystem chamber and field studies, Nash and Beall (1980) determined
4-21
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 TCDD Impurity. Using trltlated 2,3,7,8-TCDD, these authors found that atmospheric concentrations of 2,3,7,8-TCDD decreased with time either at an exponential rate (granular formulation) or at a log log rate (emulsiflable formulation) 1n chambers. The emulslf1able formulation resulted 1n considerably higher TCDD concentrations (~1000-fold or more) 1n air than 1n granular formulation Initially, but with time (200 days) approached the concentrations 1n air similar to the granular formulation (10 fg/m3; fg = 10_ls g). In a small field trial, with a nonshaded plot, TCDD concentrations 1n air from the application of 2 kg/ha of emulsiflable Sllvex containing 15 ppm TCDD were about twice (620 fg/m3) that of a shaded plot (270 fg/m3) on the treatment day, but only -33% of the amount from the shaded plot on the second day. Presumably, this was a result of the lesser quantities (<50%) of TCDD remaining on the grass for volatiliza tion during the second day.
A1r filter samples collected from Elizabeth, NJ, after an Industrial fire on April 22, 1980, were analyzed for TCDD by Harvan et al. (1981). Coll1s1on-1nduced-d1ssoc1at1on mass-analyzed 1on kinetic energy spectrometry was used for the confirmation of the presence of TCDD. Of the nine samples analyzed by these authors, one contained 20 pg of TCDD, four contained <9 pg of TCDD, and four others probably contained 5-12 pg of TCDD. However, the concentration of TCDD 1n the air cannot be given for these samples because the air volumes corresponding to the filters analyzed were not specified by the Investigators.
The atmospheric concentrations of TCDD near two hazardous waste sites have been monitored. In one study, U.S. EPA (1982b) failed to detect
4-22
(detection limit 1-20 ppt) any 2,3,7,8-TCDD 1n the atmosphere at the Love Canal, NY, area. In another study of a waste disposal site near Jackson ville, AR, an average concentration of 1100 ppt of TCDD 1n two air particu late samples collected near the disposal site was reported (Thibodeaux, 1983).
The levels of 2,3,7,8-TCDD 1n atmospheric dust were monitored 1n the Seveso, Italy, area between 1977 and 1979. The concentrations of 2,3,7,8TCDD 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 (D1Domen1co 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 (G1zz1 et al., 1982; Benfenatl et al., 1983; Taylor et al., 1983; O U e et al., 1982, 1983; Lustenhouwer et al., 1980; Barnes, 1983). The concentrations of TCDD, PeCDD and HxCDD 1n fly ash from Canadian municipal Incinerators have been studied extensively by Elceman et al. (1980, 1981). Elceman et al. (1979) also determined the TCDD levels 1n fly ash from Incinerators 1n Japan and the Netherlands. The average concentrations of the PCDDs 1n the Canadian studies (Elceman et al., 1979, 1980, 1981) were estimated with the assumption that the SIM response factors for all the PCDDs were the same as the response factor from 1,2,3,4-TCDD used as a standard. However, the analytical method used by these authors has been criticized by Nestrlck et al. (1982). Recently, Karasek et al. (1982) also determined the total TCDD, PeCDD and HxCDD levels 1n a French municipal Incinerator to be none detect ed, 7.8 and 21.8 ng/g, respectively. It was also concluded by these authors that the PCDDs tend to concentrate 1n particles of lower mean size (30 ym vs. >850 ym).
4-23
In another study, Bumb et al. (1980) studied the PCDD level 1n fly ash from a municipal Incinerator 1n Nashville, TN, several European municipal Incinerators, and the Industrial Incinerators of the Dow Chemical Co. facil ity 1n Midland, MI. The TCDD concentrations were determined to be 7.7 ng/g (0.4 ng/g of 2,3,7,8-TCDD), 2-20 ng/g and 0-38 ng/g (2,3,7,8-TCDD not detected), respectively. The corresponding values of HxCDD were reported to be 14, 30-200 and 1-20 ng/g. However, the analytical method used by these Investigators has been criticized by other Investigators (Hay, 1979). Buser and Rappe (1983) and Buser and Bosshardt (1978) also analyzed the fly ash from Incinerators 1n Switzerland and Canada. In one such study (Buser and Bosshardt, 1978), the total amount of PCDDs 1n the fly ash from a Swiss municipal and Industrial Incinerator were found to be 0.2 and 0.6 ppm, respectively. The dioxin Isomers known to be most toxic, namely 2,3,7,8TCDD, 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 dioxins found. In another study (Buser and Rappe, 1983), the presence of TCDDs (3 ppb), PeCDDs (20 ppb) and HxCDDs (50 ppb) was Indicated 1n the fly ash from a municipal Incinerator 1n Zurich, Switzerland. The TCDD, PeCDD and HxCDD Isomers with substitution at 2,3,7,8- positions, such as 2,3,7,8-TCDD, 1,2,3,7,8-PeCDD and 1,2,3,6,7,8-, 1,2,3,7,8,9- and 1,2,3,4,7,8-HxCDD were present only as 2, 14 and 24% of the total TCDDs, PeCDDs and HxCDDs. A fly ash sample from Ontario, Canada, was also found to contain TCDDs (150 ppb), PeCDDs (550 ppb) and HxCDDs (900 ppb). Although the sample was reported to contain significantly higher levels of PCDDs 1n comparison with the Swiss fly ash sample, It showed similar proportions of 2,3,7,8-substltuted PCDDs (4, 12 and 27% of the total TCDDs, PeCDDs and HxCDDs, respectively). It Is not yet known whether the
4-24
h i g h e r levels of PCDDs result from different Incinerator operating condi
tions, different feed stock or different fly ash collection conditions (Buser and Rappe, 1983). Similarly, the fly ash from a municipal Incinera tor 1n the United States showed the presence of at least 11 TCOD Isomers, but 2,3,7,8-TCDD was found to be a minor product (U.S. EPA, 1980a).
The U.S. EPA evaluated the magnitude and significance of TCDO emissions from combustion processes. In 1981, the U.S. EPA sampled five municipal waste combustors and concluded that emissions from these waste combustors do not present a public health hazard for residents living 1n the Immediate vicinity (CEQ, 1981). In view of the recent data of Pocchlarl et al. (1983) reporting the presence of 1,3,6,8- and 1,3,7,9-TCDD (0.4-2 ppt) 1n eplgeal parts of a large number of plants grown 1n the proximity of municipal Incinerators, and the toxicological evaluation of TCDD 1n ashes from urban Incinerators (Bronzettl et al., 1983; R1zzard1n1 et al., 1983), the question of health hazard for residents living 1n the Immediate vicinity of municipal Incinerators needs further evaluation. Another reason for the presence of 1,3,6,8- and 1,3,7,9-TCDD 1n eplgeal parts of plants may also be due to contamination by TCDD-conta1n1ng herbicide or pesticide application, as observed by Yamag1sh1 et al. (1981). 4.5.3. Soil. The levels of 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 TCDD, the specific Isomer Identification was not performed 1n many cases. The levels of TCDD 1n different soil, sediment and dust samples are shown 1n Table 4-3.
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Sample Type Soils Sediments
Soils Sediments Sediments Soils Soils Soils Sediments Soils Soils
Sampling Site
TABLE 4-3
Levels of TCDD 1n Soils and Sediments from Different Locations
Sample History
Concentration 1n Sample
Total TCDD
2,3,7,8-TCDD
Love Canal, NY Love Canal, NY
Love Canal, NY
Love Canal, NY Love Canal, NY NR
Eastern Missouri, U.S.A. Seveso, Italy
canal north of Amsterdam Seveso, Italy
Jacksonville, AR
waste disposal site
sediments from storm sewers and creeks near water disposal site
soils collected away from source of contamination
sediments from storm sewers
sediments from sump
sample originated from an Industrial site
sample originated from contaminated horse arena
sample originated from ICMESA plant accident site
sample originated from a dump site
sample originated from ICEMSA plant accident site
waste disposal site
<0.0025-6.7 ppb NR
NR 0.9-312 ppb
NR
NR NR ND (20-2300 ppt)a559 ppb NR
NR
NR NR
NR
ND (1-20 ppt)a
ND (1-20 ppt)a672 ppb
ND (1-20 ppt)a9570 ppb NR
detected**
detected**
55-5062 ppt <5-20,000 jig/ma
ND-2.9 ppb
Reference
Smith et al., 1983b Smith et al., 1983b
U.S. ERA, 1982b
U.S. ERA, 1982b
U.S. ERA, 1982b
Van Ness et al., 1980
Buser and Rappe, 1980
8user and
Rappe, 1980
Heida, 1983
DIDomenlco et al., 1980c
Thibodeaux, 1983
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Sample Type
Sampling Site
TABLE 4-3 (cont.)
Sample History
Concentration 1n Sample
Total TCDD
2,3,7,8-TCDD
Sediments
Jacksonville, AR
Soll/sludge Soils
Soll/dust So11/dust
Love Canal, NY
unspecified Midwestern community In U.S.A
Midland, MI
Urban U.S. areas
Soils Soils
Northwest Florida Eastern Missouri
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
NR
0.3-199 ppb
NO (<3 ppt)a -
0.021 ppb
1-120c ppb
1-4d ppb NO (1-10 ppt)a -
0.03 ppbc NO (1-10 ppt Ia-
0.04 ppbd 0.010-0.70 ppb*
12.3 ppb9 31.8-33.0 ppm
NO-22.1 ppb
NR NR
0.3-100c ppb 0.7-3d ppb
NRe
NR NR
aNot detected and the detection limit Indicated within parentheses bVa1ue not quantified cVa1ue for soil dVa1ue for dust eDust sample from St. Louis, MO, area showed 0.12 ppb 2,3,7,8-TCDD. ^Thls Is the soil residue after 10 years of periodic aerial spraying of 2,4-D and 2,4,5-T.
9Th1s 1s the soil residue Immediately after spraying.
NR = Not reported; NO = Not detected
Reference
Thibodeaux, 1983
Tlernan, 1982
Hryhorczuk et al., 1981
Bumb et al., 1980 Bumb et al., 1980
Cockerham et al.. 1980 Carter et al., 1975
It 1s 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 1s reflected by the high level of 2,3,7,8-TCDD found 1n sediments from sump and 1n sediments from storm sewers and creeks near waste disposal sites. The reported levels of 2,3,7,8-TCDD 1n soil and sediment samples near the Jacksonville, AR, waste disposal site are such that this site requires care ful reexamination. 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 1n foods could result from the following: 1) spraying of certain grain crops with PCDD-contam1nated herbicides, such as Sllvex and 2,4^5-T; 2) consumption by livestock of PCDD-contam1nated 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 1n human tissues (1.e., urine and milk). The detection of these compounds 1n 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 1n locally grown garden fruit and vegetables following the ICMESA accident 1n Seveso, Italy, In 1976 (FanelH et al., 1982; Cocuccl et al., 1979; Pocchlarl et al., 1983; W1pf et al., 1982). Studies with either the seeds or the mature plants of soybeans or oats showed that 2,3,7,8-TCDD was neither absorbed by the seeds after
4-28
s p r a y i n g n o r t a k e n u p from the s o n 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 yg/m2 of 2,3,7,8-TCDD In soil) Seveso area 1n Italy, Cocuccl 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 In unpolluted soils. However, the study of Cocuccl et al. (1979) contradicts the Investi gations of W1pf et al. (1982) In 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 In these rice grains nor were any TCDDs found 1n 30 samples of rice purchased In retail stores throughout the United States. Contamination of fruits, vegetables or grains In the United States with TCDD has never been reported.
The contamination of a large number of vegetables grown 1n the proximity of municipal Incinerators has been reported by 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
4-29
to the ICMESA plant accident. This finding suggests the possibility of human exposure of TCDD from edible vegetables grown 1n areas close to mu nicipal Incinerators.
Different Investigators have reported the presence of PCDDs 1n the fat of cattle that had grazed on pasture experimentally treated with 2,4,5-T (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-TCDD at low levels (3-4 ppt).
Results of a collaborative program to analyze a selected beef sample by the U.S. EPA, Dow Chemical Company, Wright State University and Harvard University showed that TCDD could be detected 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 FanelH et al. (1980b). The concentration of 2,3,7,8-TCDD was found to vary from none detected (detection limit <40 ppt) to as high as 7.9 ppb. Other Investigators have failed to detect either 2,3,7,8-TCDD (detection limit 1 ppt) or HxCDD (detection limit 25 ppt) 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).
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TCDDs Including 2,3,7,8-TCDD, PeCDDs Including 1,2,3,7,8-PeCDD and HxCDDs Including 1,2,3,6,7,8-HxCDD have been detected 1n fish from a few PCDD-contam1nated areas. This 1s discussed 1n detail 1n Section 6.2.
PCDDs have been detected 1n gelatin samples obtained from supermarkets and 1n 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 1n any sample. The origin of PCDDs 1n gelatin was speculated to be PCP and trlchlorophenol that are routinely used 1n the leather-tanning Industry. The use of by-product fat materials from PCP-treated hides as animal feed constituents led to widespread outbreaks of chick edema disease 1n the late 1950s (Firestone, 1973).
Bumb et al. (1980) analyzed charcoal-broiled steak under conditions representing rare, well-done and overdone samples and failed to detect either TCDD (detection limit 1-10 ppt) or HxCDD (detection limit 10-50 ppt) 1n the cooked meat of selected meat samples.
The analysis of human milk and urine for 2,3,7,8-TCDD has been per formed. A study of 103 samples of breast milk from mothers living 1n areas within the United States that were sprayed with 2,4,5-T/sllvex revealed no TCDD at a detection limit of 1-4 ppt and 0.1-10 ppt (U.S. EPA, 1980a; Heath et al., 1985). The control samples that were also negative for 2,3,7,8TCDD were derived from mothers living 1n areas where no records for 2,4,5-T or sllvex exposure exist (Heath et al., 1985).
In an Interlaboratory collaborative analytical study on adipose tissue 1t was revealed that tissues from three Vietnam veterans heavily exposed to
4-31
Agent Orange contained 2,3,7,8-TCDD residues with levels ranging from 20-173 ppt (Gross et al., 1984). A survey of 72 autopsy tissue materials from across Canada found 2,3,7,8-TCDD averaging between 5 and 10 ppt, OCDD averaging between 600 and 800 ppt, and Intermediate levels for penta-, hexa- and hepta-d1ox1ns (Ryan et al., 1985). Results on the distribution of tetra- and octa-chlorlnated dioxins 1n autopsy tissues from the general population, supported by the above observations, Indicate that there 1s substantial contamination of the general population In the United States and Canada with 2,3,7,8-chlorlne substituted tetra- through octa-d1ox1ns (Schecter et al., 1985). Rappe et al. (1984, 1985) also reports the pre sence of PCDDs 1n human adipose tissue. In the same study Rappe et al. (1985) reported their analytical results on a survey of mothers' milk from Sweden, Germany, Denmark and Vietnam. All the samples contained different levels of PeCDD, HxCDD, HpCDD and OCDD residues. The most toxic Isomer, 2,3,7,8-TCDD, was found only 1n the milk samples of mothers from Sweden and Germany but not Vietnam. This Isomer was not analyzed 1n milk samples of mothers from Denmark (Rappe et al., 1985).
The monitoring of urine samples from two people Involved with spray application (2,4,5-T) showed no detectable level of TCDD at a detection limit of ~2 ppt (Lavy et al., 1980). 4.6. EXPOSURE
The exposure of the general United States population to the four PCDDs cannot be estimated because the levels of these compounds 1n air, drinking water and foods have not been established. In fact, no PCDD contamination of any United States drinking water has ever been reported. Although the local atmosphere near a few chemical disposal sites and municipal Incinera tors has been reported to be contaminated with TCDD and HxCDD, no comprehen-
4-32
slve study Is available to demonstrate the atmospheric levels of these com
pounds in areas farther away from the point sources. Similarly, some of these compounds have been detected 1n edible aquatic species. Again, these fish contaminations have been reported 1n areas near a limited source where effluents contaminated with these compounds may have been discharged Into surface waters. One of the consumer products that has been found to be contaminated with HxCDD 1s gelatin. However, 1t 1s difficult to estimate the contribution of food to human exposure of PCDDs from such limited data. It seems more prudent to try to estimate the exposure of these compounds to populations 1n certain localized areas (e.g., dump sites and known sources of Industrial pollution) and certain special population groups (1.e., occupational) when adequate data are available.
The concentrations of 2,3,7,8-TCDD 1n bottom sediments of a drainage canal passing through a dump area (wastes from 2,4,5-T production) 1n northern Amsterdam, Holland, were reported by Helda (1983). The concentra tions of 2,3,7,8-TCDD 1n sediments within the dump area varied from 844-5062 ppt and outside the dump area from 55-611 ppt. Analysis of the eel revealed that only two samples originating from shallow ponds adjacent to the main drainage canal contained between 1.0 and 1.1 ppt of 2,3,7,8-TCDD. 2,3,7,8TCDD was not detected 1n other eel samples collected farther away from the dump site. This study demonstrates the possibility of TCDD contamination near dump sites.
The results of analysis for 2,3,7,8-TCDD and HxCDD 1n human milk samples were reported by Langhorst and Shadoff (1980). About 6 of the 9 samples showed 2,3,7,8-TCDD at levels slightly higher than the detection limits (0.2-0.7 ppt). All nine samples showed HxCDD at levels slightly higher than
4-33
the detection limit (0.2-0.5 ppt). However, these results remain uncon firmed because of the lack of validation of the precision and accuracy of data. Investigations of 103 breast milk samples from mothers living 1n areas 1n the United States sprayed with 2,4,5-T revealed no TCDO at a detection limit of 1-4 ppt (U.S. EPA, 1980a).
The monitoring of urine samples from two people Involved with spray application (2,4,5-T) showed no detectable level of TCOO at a detection limit of ~2 ppt (Lavy et a!., 1980).
In one Polish study (Gorskl, 1981), 1,2,3,6,7,8-HxCDD was detected 1n latex nipples at a concentration of 20-400 ppt. However, no TCDD or PeCDD was detected. The origin of PCDDs 1n the latex was speculated to be the result of y-1rrad1at1on of latex (for crosslinking) containing PCP during Its manufacturing process.
A BCF relates the concentration of a chemical In aquatic species to the concentration 1n water. The steady-state BCFs for a Upld-soluble compound 1n the tissues of various aquatic species seem to be proportional to the percent 11p1d 1n the tissue. Thus, the per capita Ingestion of a Upldsoluble chemical can be estimated from the per capita consumption of fish and shellfish, and a steady-state BCF for the chemical.
Data from a recent survey on fish and shellfish consumption 1n the United States were analyzed by SRI International (U.S. EPA, 1980b). These data were used to estimate that the per capita consumption of freshwater and estuarine fish and shellfish 1n the United States 1s 6.5 g/day (Stephan, 1980). In addition, this Information was used with data on the fat content of the edible portion of the same species to estimate that the weighted average percent lipids for consumed freshwater and estuarine fish and shell fish 1s 3.0%.
4-34
Severa] equations have been developed for predicting the steady-state BCF for an organic compound from its octanol-water partition coefficient (Kenaga and Goring, 1980; Veith et al., 1980; Veith 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 2,3,7,8TCDD. Use of six equations with four values for the partition coefficient, Kqw, results in the following predicted BCFs (Table 4-4). The predicted BCFs range from 7000-900,000 using the calculated values of the partition coefficient and from 3000-68,000 using the one measured value.
Several measured BCFs have been reported for 2,3,7,8-TCDD (Table 4-5), but none can be considered definitive values. Many were determined 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 in Table 5-1 . U n til fu rth er Information is a v a ila b le , the
U.S. EPA's best current estimate for the BCF of 2,3,7,8-TCDD in 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% lipids on which the equation is based to the 3.0% lipids that is the weighted average percent lipids consumed per capita from fish and shellfish (U.S. EPA, 1980b). The weighted average BCF for 2,3,7,8-TCDD in the edible portion of all freshwater and estuarine aquatic organisms consumed by Americans is calculated to be 5000x0.395=1975. Uptake by fish from lower tropic levels may add to uptake from water, so this BCF may underestimate concentrations in wild aquatic organisms.
4-35
TABLE 4-4 Predicted BCFs from Calculated and Measured Values of Kow a
Equation
6.84
log Kow
Calculated
7.14
7.28
Measured** 6.15
log BCF = 0.542 log Kow + 0.124 log BCF = 0.76 log Kow - 0.23 log BCF = 0.79 log Kow - 0.40 log BCF = 0.635 log Kow + 0.7285 log BCF = 0.85 log Kow - 0.70 BCF = 0.048 Kow
6,780 93,000 101,000 118,000 130,000 332,000
9,860 157,000 174,000 183,000 234,000 663,000
11,700 201,000 224,000 225,000 308,000 915,000
2,870 27,800 28,740 43,000 33,700 67,800
aSources: Kenaga and Goring, 1980i; Veith et al., 1980; Veith and Kosian, 1983
**This measured value has been reported by Neely, 1979
4-36
TABLE 4-b Measured Bioaccumulation Factor for 2,3,7,8-TCDD In Freshwater Aquatic Organisms
Species
Tissue
Percent Lipid
Duration (days)
Bioconcentration Factor
Reference
4-37
Alga, Oedoqonlum cardlacum
Alga, Oedoqonlum cardlacum
Snail, Phvsa sp.
Snail, Phvsa sp.
Cladoceran, Daphnla maqna
Cladoceran, Daphnla maqna
Catfish, Ictalurus punctatus
Mosqultoflsh, Gambusla afflnls
--
--
NR NR whole body whole body whole body whole body whole body whole body
-
NR NR NR NR NR NR NR NR NR NR
33 32 33 32 30 32 28 14
-
aThese are arithmetic mean of several values given bThese are values at equilibrium tissue concentrations Calculated as ratio of uptake and clearance rate constants NR = Not reported
3094a
207 5b 2083 5471a
2095b 3731 3895a
7070b 7125 2000
4850b 4875 9080C 5400
Isensee, 1978
Isensee, 1978 Yocklm et al.. 1978 Isensee, 1978
Isensee, 1978 Yocklm et al.. 1978 Isensee, 1978
Isensee, 1978 Yocklm et al., 1978 U.S. EPA, 1983a>; Thomas, 1983 Isensee, 1978 Yocklm et al., 1978 Neely, 1979 Kenaga, 1980
The BCF for 2,3,7,8-TCDD 1n the earthworm, Allobophora caUglnosa or rosea, from soil with Initial 2,3,7,8-TCDD concentration 1n the range of 0.06-9.2 ppb has been determined to be ~ 10 (FanelH et al., 1982).
The BCFs for other PCDDs cannot be estimated because of the lack of solubility data.
Finally, the levels of TCDD 1n wildlife have been determined by various authors and are discussed 1n detail 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-T and SHvex. At the present time, there 1s no known manufacturer of trlchlorophenol In the United States. Its derivatives distributed 1n the market before banning, however, continue to be used as pesticides 1n the United States. The level of 2,3,7,8-TCDD contaminants 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 1n the environment probably are Industrial manufacturers of chlorophenols or their derivatives, and chemical disposal sites containing the wastes from these Industries. Municipal waste Inciner ation also may produce some environmental emission of PCDDs. The signifi cance of this source of emission compared with Industrial emission and prob able contamination from chemical disposal sites cannot be assessed with the available data. The 1,2,3,7,8-PeCDD now found 1n environmental samples has only been reported 1n emissions from Incinerators.
PCDDs, particularly TCDD and Its specific Isomer 2,3,7,8-TCDD, have been monitored 1n a number of environmental media, Including air, water, soil,
4-38
food and biological media. The monitoring data to date I n d i c a t e t h a t the maximum level of PCDDs is likely to be found in soil and drainage sediment samples near chlorophenol manufacturing industries and chemical waste disposal sites. PCDDs have rarely been monitored in United States air samples. Small amounts of PCDD contamination have been found in fish and wildlife in the United States in areas around chlorophenol manufacturing industries and chemical waste disposal sites.
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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 biodegradation. Only 5 of -100 microbial 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 biodegradation of 14C-labeled 2,3,7,8-TCDD by using lake
waters and sediments from Wisconsin. The observed half-Hfe of 2,3,7,8-TCDD
1n sediment-containing lake waters was found to be 550-590 days. In lake
water alone, -7056 of the 2,3,7,8-TCDD remained after 589 days. Using an
outdoor pond as a model aquatic ecosystem and dosing 1t with 14C-labeled
2.3.7.8- TCDD, Tsushlmoto et al. (1982) and Matsumura et al. (1983) estimated
the apparent half-Hfe of 2,3,7,8-TCDD to be -1 year. Although biodegrada
tion may have been responsible for part of the degradation, 1t 1s almost
Impossible to estimate the biodegradation half-life of 2,3,7,8-TCDD In
aquatic systems from this experiment. It 1s likely that the apparent biode
gradation loss was due to volatilization through a1r/water Interface. Other
Investigators (Huetter and Philippi, 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 biodegradation than 2,3,7,8-TCDD.
The biodegradation half-11fe 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 biotransformation rate
5-1
constant of lxlO-1 ms. cell-1 hour-1 (Mabey et al., 1981) and the
concentration of microorganisms capable of degrading TCDD as 5x10s cell
ms.-1 (Burns et al., 1981), the half-life of biodegradation can be esti
mated to be >1 year. It should be emphasized that the role that biodegrada
tion plays 1n the removal of PCDDs from water Is not clear.
5.1.1.2.
PHOTOTRANSFORMATION -- 2,3,7,8-TCDD has a UV absorption
maximum at 310 nm with an extinction coefficient of 5590 M-1 cm-1 (NRCC,
1981a). 2,3,7,8-TCDD 1n a pure state Is photochemically stable but It will
photolyze 1n 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 In distilled water remained unchanged when Irradi
ated with a sunlamp. Similarly, a thin dry film of 2,3,7,8-TCDD on a glass
plate or 2,3,7,8-TCDD on dry and wet soils showed negligible photodegrada
tion after Irradiation with sunlamps (Crosby et al., 1971). In contrast,
2.3.7.8- TCDD 1n methanol solution or benzene solution of 2,3,7,8-TCDD In
water stabilized by surfactant underwent substantial photodegradation under
sunlamp or sunlight Irradiation (Pllmmer et al., 1973; Crosby et al., 1971).
Botre et al. (1978) demonstrated that cationic surfactants, namely 1-hexade-
cylpyrldlnlum chloride, act as an energy transfer agent 1n facilitating the
photodecomposition of TCDD In aqueous solutions. These laboratory studies
may not be applicable to the ambient environments. To explain the longer
half-Hfe of 2,3,7,8-TCDD 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-medlated photosens1t1zat1on of 2,3,7,8-TCDD may cause some photode
composition of this compound. Nestrlck et al. (1980) estimated the photo
5-2
lytic half-life of 2,3,7,8-TCOD 1n n-hexadecane under sunlamp ir r a d ia tio n to
be ~57 minutes. From the available Information, 1t 1s difficult to predict the fate of 2,3,7,8-TCDD 1n aquatic media under environmental photolytlc conditions. In the presence of hydrogen atom donating substrate(s) 1n surface waters, photolysis may be a significant fate process.
An Increase 1n chlorine substitution 1s expected to decrease the rate of photodegradation (Nestrlck et al., 1980; Helling et al., 1973). For example, Crosby et al. (1971) showed that although complete decomposition of 2,3,7,8-TCDD 1n methanol occurred 1n 24 hours under UV Irradiation, >80% OCDD 1n methanol remained unreacted during the same period under similar Irradiation conditions.
Although the degree of photolysis may be related to the extent of chlorination, positional Isomerization also plays a critical and perhaps dominant part 1n the photolysis of higher PCDDs. In higher PCDDs, there appears to be preferential loss of chlorine from the 2, 3, 7 and 8 positions ( N e s t r lc k e t al., 1980; Buser and Rappe, 1978; Choudhry and Hutzlnger, 1984). However, Buser (1979) observed the formation of 2,3,7,8-TCDD 1n trace quantities, and PeCDD form photolysis of 1 ,2,3,6,7,8-HxCDD and 1,2,3,7,8,9-HxCDD. PCDD compounds with chlorine substitutions 1n positions 2, 3, 7 and 8 are likely to photodegrade faster than compounds not having these positional substitutions. According to such a predicted rule, 1t 1s not likely that photodegradation of OCDD and other higher PCDDs will yield a high quantity of 2,3,7,8-TCDD as the stable end product. For example, the photolysis half-Hfe of 1,2,3,7,8-PeCDD has been estimated to be 7.6 hours 1n n-hexadecane solution under sunlamp Irradiation (Nestrlck et al., 1980). Similarly, the photolytlc half-Uves of 1,2,3,7,8-PeCDD, 1,2,3,6,7,9-HxCDD and 1 ,2,4,6,7,9-HxCDD 1n hexane solutions under sunlight Irradiation have been determined to be 5.4, 17 and 47 hours, respectively (Dobbs and Grant,
5-3
1979). Nestrlck et al. (1980) reported a half-Hfe value of 6.8 hours for
1.2.3.6.7.8- HxCDD 1n n-hexadecane under sunlamp Irradiation. The Interme
diates of the photodegradation of higher PCDDs are probably lower chlori
nated dioxins, but the pathways of degradation are not known with certainty
(NRCC, 1981a).
From the preceding discussions of the photolysis of PCDDs 1n the
presence of organic hydrogen donating substrates, 1t 1s difficult to predict
the photolytlc fate of these compounds 1n natural aquatic media where suf
ficient organic hydrogen donating substrate(s) may or may not be available.
The situation Is 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 (R02*.
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 microbial studies, no quantitative
Information regarding the volatilization of 2,3,7,8-TCDD from aquatic media
1s available (Ward and Matsumura, 1977; Matsumura et al., 1983; Huetter and
Phi 11ppl, 1982). 2,3,7,8-TCDD may undergo some water-mediated evaporation
1n aquatic media (Matsumura et al., 1983). Using the formulas of L1ss and
Slater (1974), a vapor pressure value of 1.7xl0"6 torr (0.2 m Pa) and a
5-4
solubility value of 6.2xlO"10 mole/Jt, the volatilization half-life for
2.3.7.8- TCDD was 6 minutes from water of 1 cm depth and 10 hours from water of 1 m depth (NRCC, 1981a). The limitations of this theory to predict the rate of volatilization have been discussed 1n the NRCC (1981a) document. The L1ss-Slater model does not consider terrestrial matrices (suspended solids, sediments, biota, etc.) normally encountered 1n natural surface water and thus Ignores the effects of these parameters on the volatilization rate. Employing a computerized EXAMS model for two standardized aquatic ecosystems (lake and pond; see NRCC, 1981a, for definitions) and the Input parameters for 2,3,7,8-TCDD given 1n NRCC (1981a), volatilization has been estimated to account for 100% of the fraction lost; biodegradation 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 (Thibodeaux, 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 1n 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 PeCDDs and HxCDDs from aquatic media could not be found 1n the available literature. However, these compounds with higher molecular weight and more chlorine substitution are expected to volatilize more slowly than 2,3,7,8-TCDD from aquatic media.
5.1.1.5. SORPTION -- Data from microcosm experiments Indicate that 2.3.7.8- TCDD 1s highly sorbed to sediments and biota (Isensee and Jones, 1975; Ward and Matsumura, 1978). More than 90% of 2,3,7,8-TCDD 1n an, aquatic medium may be present 1n the adsorbed state (Ward and Matsumura,
5-5
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 Karlckhoff et al. (1979) predicts a sorption partition co efficient value of 104 for 2,3,7,8-TCDD 1n sediments containing 2 % organic carbon. Similarly, the higher PCDDs are likely to be present predominantly 1n the sediment-sorbed state 1n aquatic media. 5.1.2. A1r. A number of PCDDs, Including TCDDs, PeCDDs and HxCDDs, have been detected 1n the dust and fly ash from municipal Incinerators (Cavallaro et al., 1980b; Clement and Karasek, 1982; Elceman et al., 1981). Size fractionations of fly ash from municipal Incinerators have shown that larger concentrations of 2,3,7,8-TCDD and PeCDDs occurred on the larger (550 jim) particles, while the 30 ^m particles had greater relative concentrations of OCDD (Clement and Karasek, 1982). Hernan et al. (1982b) also reported higher concentrations of TCDDs on larger particles (3-10 urn) from a refuse-fueled municipal Incinerator effluent than on smaller particles (<1 urn). 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 noncombustion sources, such as herbicide-treated soils and vaporized PCDDs from aquatic media (Thibodeaux, 1983), are also likely to be associated with air particulate matter. Cupltt (1980) presented mathematical descriptions of physical removal mechanisms for the fate of toxic and hazardous materials 1n the air environment. For the adsorption of chemicals on aerosol particles he developed a general model based on aerosol surface area and chemical saturation vapor pressure. H1s results suggest that adsorption will be a reasonable vapor-phase removal mechanism from air only for materials with saturation vapor pressures of 10"' torr (mm) or less. 2,3,7,8-TCDD has an estimated vapor pressure of 1.7xl0-6 torr.
5-6
Photodegradation and wet and dry de p o sitio n of p a rtic u la te -b o u n d PCDDs are probably the most Important fate-determ1n1ng processes for the atmos pheric PCDDs. The available data relating the photodegradation of these compounds 1n the sorbed phase or as films are conflicting. For example, experiments of earlier Investigators Involving photoreactivity of 2,3,7,8TCDD as films or sorbed on solid surfaces and exposed to the atmosphere yielded negligible photodegradation 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 in the condensed phase (1.e., coated on glass plate or on silica) may take place. In the condensed phase, photodecomposition 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-TCDD to light of wavelength >290 nm and observed 9254 decomposition 1n 7 days. The half-Hfe for photodegradation of 2,3,7,8-TCDD film on glass surfaces has been estimated to be 5.8 days under Irradiation with sunlamps (Nestrlck et al., 1980). It 1s not known whether a similar photodegradation of particle-bound 2,3,7,8-TCDD will occur 1n 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 1n the
OX
atmosphere 1s unknown. 5.1.3. Soil.
5.1.3.1. SORPTION -- From the empirical correlation of Karlckhoff et al. (1979), 1t 1s possible to predict a soll/water partition coefficient of 4.8xl04 for a soil containing 1054 organic matter.
5-7
Because of their high affinity toward soils, particularly those with significant organic content, and because of their extremely low water solubilities, 2,3,7,8-TCDD (and presumably other PCDDs) tend to remain on or near the surface of soils (U.S. EPA, 1984). With time, 2,3,7,8-TCDD bound to soil becomes more difficult to desorb (Philippi et al., 1981; Huetter and Philippi, 1982).
Several authors have shown that vertical movement of 2,3,7,8-TCDD 1n soil 1s negligible, although movement of 2,3,7,8-TCDD may occur by hori zontal transfer (eroded soil transported by water) and through contaminated airborne dust particles (U.S. EPA, 1984; Helling et al., 1973). Therefore, underground water supplies are unlikely to be contaminated with 2,3,7,8TCDD. However, as the organic content of soil decreases, the likelihood of vertical movement of PCDDs 1n soil Increases. In areas of heavy rainfall and sandy soil, vertical migration of 2,3,7,8-TCDD and 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 (D1Domen1co et al., 1980d,e). The monitoring of Seveso soil 1 year after the accident showed that the . highest 2,3,7,8-TCDD levels were not present 1n the topmost soil layer (0.5 cm), but very often 1n the second (0.5-1.0 cm) or third (1.0-1.5 cm) layers. In view of the low water solubility of 2,3,7,8-TCDD, probable explanations of this vertical distribution could be due to volatilization through the a1r/so1l Interface or solvation of 2,3,7,8-TCDD by organic solvents (NRCC, 1981a), or biotic mixing by earthworms or other soil Invertebrates. It 1s, therefore, possible that 2,3,7,8-TCDD may appear 1n the air above and 1n normal water leachate of soils, particularly after multiple PCDD application or accidental release of 2,3,7,8-TCDD on soil.
5-8
5.1.3.2. PHOTOTRANSFORMATION -- The photodecomposltlon of 2,3,7,8TCDD on wet or dry soil under artificial and natural sunlight was studied by Crosby et al. (1971). The photodecomposltlon was found to be negligible 1n soils. Similarly, PUmmer et al. (1973) determined that photodecomposltlon of TCDD on soils was too slow to be detected. In a later experiment, PUmmer (1978) found that although TCDD decomposed significantly from precoated silica plate (-22%) 1n 8 hours of sunlight Irradiation, practic ally no decomposition of TCDD was observed from TCDD sorbed on soil under similar conditions.
The photodegradation of TCDD 1n combination with other pesticide mixtures was studied by Crosby and Wong (1977). When Agent Orange contain ing 15 ppm of TCDD was applied on the surface of glass plates (5 mg/cm2), rubber plant, Hevea brasHlensIs (6.7 mg/cm2 ), and on the surface of sieved Sacramento loam soil (10 mg/cm2 ) and exposed to sunlight, TCDD was found to photodecompose. The loss of TCDD 1n 6 hours was >50% from glass p l a t e , -100% from the s u rfa c e of leaves and -10% from the surface of soil. The rapid photolysis of TCDD from these surfaces Indicates that the herbi cide formulation provided a hydrogen donor that probably allowed the photolysis to occur. The authors attributed the slower photolysis of 2.3.7.8- TCDD 1n soil to a shading effect by lower layers of soil particles.
5.1.3.3. BIODEGRADATION -- Polger and Schlatter (1980) noted that 2.3.7.8- TCDD absorbs strongly onto soil particles, thereby reducing Its b1oava1labH1ty. Young (1983) also noted that 2,3,7,8-TCDD 1s not likely to metabolize readily by soil microorganisms. It can be concluded from the following discussions that the biodegradation half-Hfe 1n soil 1s likely to be >1 year.
5-9
The overall half-life of 2,3,7,8-TCDD 1n soil has been reported to be 1-3 years by Kearney et al. (1972). Studies performed by the U.S. A1r Force (Young et al., 1976; IARC, 1977) suggested that soil bacteria may biodegrade TCDD. The half-Hfe of this chemical 1n soils under relatively dry condi tions (Utah test area) was found to be -330 days and 1n more moist soils and under warm conditions (Florida test area) was found to be -190 days. This 1s consistent with the biodegradation half-Hfe of ~0.5 year for TCDD determined by Commoner and Scott from the soil 1n rural Missouri after the accidental spraying of TCDD-contam1nated oil (IARC, 1977). However, these half-Hfe estimates may greatly underestimate the true value, since 1t has recently been shown that radiolabeled TCDD adsorbed to soil becomes progres sively more resistant to extraction (Philippi et al., 1981; Huetter and Philippi, 1982).
The rate of disappearance of 2,3,7,8-TCDD following an accidental 2.3.7.8- TCDD release from a trlchlorophenol manufacturing plant at Seveso, Italy, was studied by D1Domen1co et al. (1980d, 1982). The disappearance of 2.3.7.8- TCDD from the topmost soil layer after 1 year was speculated to be due to photodegradation, 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; Wlpf and Schmid, 1983) Indicate that the half-life of 2,3,7,8-TCDD 1n soil 1s about 10-12 years. Since most of the other PCDDs are no more susceptible to transformat1on/degradat1on than TCDDs, their half-lives 1n soil are presumed to be similar to that postulated for TCDDs.
5-10
5.1.4. Food. Isensee and Jones (1971) conducted experiments to study the possibility of absorption and translocation 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. Cocuccl 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 yg/m2 of 2,3,7,8-TCDD. 2,3,7,8TCDD was found to be 3-5 times higher 1n foliage than 1n fruits. The fact that the highest 2,3,7,8-TCDD content was found adjacent to the conductive tissue was Interpreted as evidence of translocation of 2,3,7,8-TCDD from roots to the outer parts of the plants. The Investigation of these authors also suggested that 2,3,7,8-TCDD may be eliminated from the mature plants. W1pf et al. (1982), however, failed to detect any measurable 2,3,7,8-TCDD 1n the flesh of fruits and vegetables collected from the contaminated area 1n Seveso during 1977-1979, although the soil 2,3,7,8-TCDD concentration was ~10 ppb. These authors concluded that 2,3,7,8-TCDD may not be translocated from soil to the plants. A similar conclusion was reached by Pocchlarl et al. (1983) from their uptake experiments with plants. It can be concluded from these studies that 2,3,7,8-TCDD 1s not likely to concentrate In plants grown 1n contaminated soils.
With respect to potential 2,3,7,8-TCDD exposure through aerial parts of plants, when an aqueous suspension of pure 2,3,7,8-TCDD was exposed to either artificial light or sunlight, photodecomposition was negligible. However, 1n conjunction with other pesticides, 2,3,7,8-TCDD rapidly degraded when exposed to light (Crosby and Wong, 1977). This 1s consistent with the observations that TCDD was found not to persist on foliage (Sundstrom et al., 1979; Crosby and Wong, 1977) after application with other pesticides
5-11
(2,4,5-T, Agent Orange). The half-life of 2,3,7,8-TCDD disappearance from grass 1n 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 TCDD were monitored for TCDD content 1n the body fat. TCDD from the body fat presumably dis appeared with a half-Hfe of ~16.5 weeks (Jensen et al., 1981). Similarly, cattle fed rations fortified with 500 ppt 2,3,7,8-TCDD showed a maximum level of 90 ppt of 2,3,7,8-TCDD 1n cows' milk. On withdrawal of 2,3,7,8TCDD 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 1n aquatic media are volatilization and sorption onto suspended particulates 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 microbial studies (Ward and Matsumura, 1977; Huetter and Philippi, 1982). There 1s a very wide difference 1n the calculated values of half-11fe of volatilization for 2,3,7,8-TCDD. For example, calculation based on the L1ss and Slater (1974) equation gives a half-Hfe for evaporation of 10 hours from water of 1 m depth (see Section 5.1.1.4.). Calculation based on a reaeration rate ratio of 0.373 (Mabey et al., 1981) and an oxygen reaeration rate constant of 0.19 day"1, 0.96 day"1 and 0.24 day"1 (Mabey et al., 1981) for pond, river and lake water, respectively, gives half-11fe values of 10, 2 and 8 days for 2,3,7,8-TCDD 1n pond, river and lake water, respectively. These wide variations are conceivable when examined with the volatilization
5-12
models for half-Hfe (Thibodeaux, 1979). Evaporation half-life Is shown to be proportional to water depth and Inversely proportional to the masstransfer coefficient. A more realistic calculation based on EXAMS predicts half-Hfe values for TCDD of 5.5 and 12 years from pond and lake water, respectively (see Section 5.1.1.4.). The EXAMS calculation routine contains an added element that accounts for the sorption of TCDD both on the suspended and on-bottom sediment. For substances with high sorption coef ficients such as TCDD, the evaporation rate 1s reduced significantly. A comparison of calculated transport rates from an Industrial site Indicates that evaporation of TCDD from a contaminated cooling water pond sediment 1s negligible 1n comparison with other contaminated areas on the site (Thibo deaux, 1983). It will also become apparent from the following discussion that volatilization may be Insignificant compared with sorption processes for the transport of TCDD and presumably other PCDDs from aquatic media.
It has already been shown (see Section 5.1.1.5.) that 2,3,7,8-TCDD 1s highly sorbed to sediments and b i o t a (Is e n s e e and Jones, 1975) and >90% of 2,3,7,8-TCDD 1n aquatic media may be present 1n the sorbed state (Ward and Matsumura, 1978). This Is consistent with the sorption partition coeffic ient value of this compound. Although the sorption effects of the higher PCDDs have not been studied, based on their expected higher octanol/water partition coefficient values, these compounds are likely to be present predominantly 1n the sediment-sorbed state 1n aquatic media. 5.2.2. A1r. All the PCDDs are believed to be transported 1n the vaporphase and In particulate bound form 1n the atmosphere (see Section 5.1.2.). The transport of these compounds from stationary point sources (1.e . stack emission) and area sources (waste disposal sites) can be theoretically predicted from dispersion modeling (Oosephson, . 1983). Although such
5-13
dispersion modeling has been performed for 2,3,7,8-TCDD (SAI, 1980), the correlation between the theoretical value and experimental monitoring data has never been performed. In the case of accidental release of toxic clouds containing TCDD at Seveso, Italy, Cavallaro et al. (1982) determined the transport pattern and the ground deposition of the TCDD from the cloud. They determined that the TCDD deposition from air to soil should follow an exponential decay pattern along the downwind direction and follow a Gauss1an-d1str1but1on along the cross-section of the downwind direction. From regression equations, these Investigators determined that the aerial deposi tion y (vg/m2) should be y = 2900 e-3,3x for x<2 km and y = 45 e-0 *5x for 2 km<x<6 km. It Is doubtful whether this equation can be used In the general case of accidental release of TCDD because of the varying meteorological conditions. 5.2.3. Soil. The probable media and modes of transport of PCDDs from soils are the following: 1) to air through contaminated airborne dust particles; 2) to surface water by eroded soil transported by water; 3) to groundwater by leaching; and 4) to air by volatilization. Movement of particulate matter containing sorbed PCDDs Is considered to be a much more Important transport mechanism than leaching because of the low water solu bility of these compounds (Josephson, 1983). However, one year following the Seveso accident the highest 2,3,7,8-TCDD levels In soil were very often detected 1n the second (0.5-1.0 cm) or third (1.0-1.5 cm) layers but not 1n the top most soil layer (0.5 cm). This disappearance of at least a part of the 2,3,7,8-TCDD from the topmost soil layer was speculated to be due to volatilization or vertical movement down through the soil (D1Domen1co et al., 1980d). Results of off-s1te transport calculations from contaminated soil surfaces are available (Thibodeaux, 1983). The calculations show that
5-14
between T2 0 and T2 0 0 g/year of TCDD were volatilized from a highly contami
nated soil surface between 1978 and 1979 before the Implementation of remedial measures. Over the same period 1t was estimated that 28-37 g/year left the site by wind-blown particle entrainment, 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 1n 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 biotic 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 bioconcentration of TCDD 1n various aquatic species has been studied under controlled laboratory conditions using static test chambers. The results of these Investigations have been discussed 1n Section 4.6. and are given 1n Table 5-1. In all these experiments, the total amounts accumulated were found to be related to the Initial TCDD concentrations 1n aquatic phase. The Investigation of Ph 111 pp 1 et al. (1981) made 1t clear that bloaccumulatlon would be significantly affected by the physical form (sorbed or 1n solution) 1n which TCDD occurs 1n the environment. Isensee (1978) reported that the concentration 1n the tissues of the tested species reached equilibrium 1n 7-15 days. In the absence of any experimental BCFs derived under dynamic test conditions, the values of Isensee (1978) reported 1n Table 5-1 probably represent the best experimental values available (1n species other than fish) since these values were derived from equilibrium concentrations of TCDD 1n the tested tissues. The BCF for 2,3,7,8-TCDD 1n the earthworm, Allobophora caliginosa 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 (Fanelli et al., 1982).
5-15
TABLE 5-1 B1oconcentrat1on Factor of TCDD for Several Aquatic Organisms3
Species
Initial Aquatic Concentration (ppt)
Bioconcentration Factor
Reference
5-16
Alqae. Oedoqonlum cardlacum Alqae. Oedoqonlum cardlacum Alqae. Oedoqonlum cardlacum Ostracod Duckweed. Lemna minor Snail. Phvsa s d . Snail. Phvsa so. Snail. Helosoma so. Daohnlds. Daphnia maqna Daphnlds. Daphnia maqna Daphnlds. Daphnia maqna
0.05-1300 0.05-1300
0.1c
2.6 0.05-1300 0.05-1300 0.05-1300
0.1c
0.05-1300 0.05-1300 0.4
2,075 9,000b 2,080
110 3,625b 2,095 20,000b 2,080 7,070 26,000b 2,200
Isensee, 1978
Isensee and Jones, 1975
Yocklm et al., 1978
Matsumura and Benezet, 1973
Isensee and Jones, 1975
Isensee, 1978
Isensee and Jones, 1975
Yocklm et al., 1978
Isensee, 1978
Isensee and Jones, 1975
Matsumura and Benezet, 1973
Species
TABLE 5-1 (cont.)
Initial Aquatic Concentration (ppt)
Bioconcentration Factor
Reference
5-17
Mosquito fish. Gambusla afflnls Mosquito fish. Gambusla afflnls Mosquito fish. Gambusla afflnls Mosquito larvae. Aedes aeqvptl Brine shrimp. Artlmla sallna Catfish. Ictalurus ounctatus Catfish. Ictalurus ounctatus
Brook Sllverslde. Laludesthes slcculus Pond Weed. Elodea nuttall and
CeratoDhvllon demersum
0.05-1300 0.05-1300 0.1c 0.45 0.1c 0.05-1300 0.05-1300 1.3 53.7
4,850 26,000b
4,875 9,200 1,570 9,000b 4,875
545d 30,300
Isensee, 1978
Isensee and Jones, 1975
Yocklm et al., 1978
Matsumura and Benezet, 1973
Matsumura and Benezet, 1973
Isensee and Jones, 1975
Yocklm et al., 1978
Matsumura and Benezet, 1973
Tsushlmoto et al., 1982
aBCF values derived by Isensee and Jones (1975) were based on dry weight for all biological and sediment materials.
^Average of several values
cThese are Initial concentrations of TCDD In soil added to water
dError In the original publication corrected In the value reported here.
5.4. SUMMARY The four transformation processes (photoreaction, biotransformation,
hydrolysis and radical oxidation) that control the fate of a chemical In aquatic media do not appreciably transform TCDD and possibly other PCDDs In aquatic media. However, the two former processes may be more Important for the transformation of 2,3,7,8-TCDD 1n aquatic media. The transport of these compounds to the atmosphere by volatilization from surface water may take place through a water-mediated process, particularly 1n the case of 2,3,7,8TCDD, 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 tropospheric free radicals Is not known. Although appreciable photolysis of TCDD coated on glass plate or sorbed onto silica has been observed, 1t 1s not known whether a similar photodegradation of particle-bound TCDD and other PCDDs will occur 1n the atmosphere. The transport of vapor phase and particle-bound PCDDs may be theoretically predicted from dispersion modeling equations. In the case of accidental release of toxic clouds containing TCDD at Seveso, Italy, 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 biodegradation reactions 1n soil. The half-life of 2,3,7,8-TCDD 1n soils may be >10 years. These compounds are likely to be transported from soil through movement of particulate matter containing sorbed PCDDs. The most probable transport mechanisms are transport of these compounds to the atmosphere by contami nated airborne dust particles, evaporation, and transport to surface water
5-18
via eroded soil transported by water. Leaching is a less likely transport
process for these chemicals except for very sandy soils. Both the calculated and experimental results show that these compounds
will bioaccumulate 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 bioaccumula tion factors for these compounds in different aquatic species.
5-19
6. ECOLOGICAL EFFECTS
6.1. EFFECTS ON ORGANISMS
6.1.1. Aquatic Life Toxicology. Almost all of the available Information
concerning the toxicity of PCDOs to wildlife pertains to aquatic species,
and most of the aquatic Information 1s based on acute exposure to calcu
lated, rather than measured concentrations of 2,3,7,8-TCDD.
6.1.1.1.
ACUTE TOXICITY -- The effects of acute exposure to 2,3,7,8-
TCDD have been reported for four species of freshwater fish and one species
of amphibians (Table 6-1). In almost all of these studies, toxic effects
were observed only after the acute exposure period ended. Miller et al.
(1973, 1979) exposed juvenile coho salmon, Oncorhvnchus klsutch. to a range
of 2,3,7,8-TCDD concentrations for up to 96 hours. Concentrations were
expressed as ng/g wet bw and as ng/l of water, based on the amount of
2,3,7,8-TCDD added to the water 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
t1mes/week. Experiments were conducted with two groups of fish that dif
fered 1n Initial mean wet weight (3.51 and 6.63 g). Food consumption,
growth and survival of smaller fish were measured until 60 days after expo
sure and were found to be significantly reduced at 5.4 yg/kg bw (0.0056
yg/lt), but not at 0.54 yg/kg bw (0.00056 yg/l) or lower. Growth
and survival of larger fish were measured until 114 days after exposure and
were significantly reduced at 5.4 yg/kg bw (0.0105 yg/l) but not at
0.54 yg/kg bw (0.00105 yg/l) or lower. The actual concentrations 1n
fish and water were undoubtedly lower than the calculated values, because
much of the added 2,3,7,8-TCDD would be adsorbed to all containers.
6-1
TABLE 6-1
Effect of Acute Exposure to 2,3,7,8-TCOD on Aquatic Anlnals
6-2
Species
Life Stage, Weight or Length
Duration of Exposure
(hours)
Duration of Test
(days)
LC50 (ug/t)
LT5oa Lowest Effect (days) Concentration
(ug/D
No Effect Concentration
(wg/i)
Effect
Reference
Coho Salmon,
3.S g
Oncorhvnchus klsutch
96
64 0.0056
60 0.0056
0.00056
reduced growth, food
Hiller et al., 1973,
consumption, survival 1979
Coho Salmon,
6.6 g
Oncorhvnchus klsutch
96
114
0.0105b
114 0.0105
0.00105
reduced growth, food consumption, survival
Hiller et al., 1973, 1979
Rainbow Trout, Salmo aalrdnerl
eggs and larvae
96 72 NR
NR 0.0001
NO
temporary growth Inhibition
Helder, 1981
Rainbow Trout, Salmo aalrdnerl
eggs and larvae
96 164 NR
NR 0.001
0.0001
teratologic effects, decreased survival and growth
Helder, 1981
Rainbow Trout, Salmo aalrdnerl
0.8S g
96 72 NR
NR 0.010
NR
decreased survival
and growth, histo logical effects
Helder, 1981
Guppy, Poecllla reticulata
9-40 mm
120 37 NR
21.7 0.1
NO
100X mortality by 3.7 days after
beginning exposure
Hiller et al 1973; Norris and Hiller, 1974
Guppy, Poecllla reticulata
8-12 mm
24 69 NR
NR 0.0001
0.00001
higher Incidence of fin necrosis
Hiller et al., 1979
Northern Pike, Esox luclus
eggs and larvae
96 23 NR
NR 0.0001
NO
temporary Inhibition of egg development
Helder. I960
Northern Pike, Esox luclus
eggs and larvae
96
23 0.001
23 0.001
0.0001
decreased survival and growth
Helder, I960
frog, Rana catesblana
larvae
l.p. Injection
50 NR
NR NO
1000 ug/kg bw
no effect on survival metamorphosis, histology
Beatty et al.. 1976
Frog, Rana catesblana
adults (150-250 g)
l.p. Injection
35 NR
NR 500 ug/kg bw
250 ug/kg bw
temporary decrease In food consumption, but no effects on survival or histology
Beatty et al.. 1976
alTjq = median lethal time In days after beginning exposure b47X mortality NR * Not reported; NO - Not determined
Acute exposure experiments were also conducted by these researchers
(Miller et al., 1973, 1979; Norris and Miller, 1974) with guppies, PoeclHa reticulata. Miller et al. (1973) and Norris and Miller (1974) reported the effects of exposing guppies to nominal concentrations of 0.1, 1.0 and 10.0 vg/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; smaller fish gener ally died first. F1n necrosis was observed 1n all fish surviving more than 10 days. In a later study, Miller et al. (1979) measured the Incidence of fin necrosis 1n guppies exposed for 24 hours to much lower nominal concen trations of 2,3,7,8-TCDD and then maintained for 69 days. The Incidence of fin necrosis was significantly greater 1n fish exposed to >0.8 vg/kg bw (0.0001 vg/8-) than In controls or 1n fish exposed to 0.08 vg/kg bw (0.00001 vg/&).
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 qalrdnerl. were reported by Helder (1980) and Helder (1981), respectively. In both studies, newly fertilized eggs were exposed for 96 hours to a range of nominal 2,3,7,8-TCDD concentrations (0.0001, 0.0010, 0.010 ug/8.) followed by transfer to clean water. There was no significant Increase 1n egg mortality up to the highest nominal test concentration of 0.010 vg/8- tor either species. Significantly greater mortality occurred after hatching and during yolk sac absorption In both species at concentrations as low as 0.0010 vg/il. Total mortality of pike fry reached 99% at 0.010 vg/8- and 50% at 0.0010 vg/. by 23 days after fertilization. Total mortality of trout fry was 26% at 0.010 vg/l and 12% at 0.0010 vg/l. Although cumula tive mortality was not significantly Increased at the lowest test concentra-
6-3
t1on (0.0001 yg/a), sublethal effects occurred 1n both species. At this
concentration, growth was significantly, but temporarily, retarded 1n both
species.
Helder (1981) also exposed juvenile trout to nominal concentrations of
0.100 and 0.010 yg/a for 96 hours and followed growth and survival for
72 days. Growth was significantly reduced 1n both groups. Mortality
reached 10054 by 27 days at the highest concentration, but was only 754 at the
lowest concentration.
The only other study regarding the effects of acute exposure on aquatic
animals 1s that of Beatty et al. (1976), who Investigated the effects of
single 1ntraper1toneal Injections of 2,3,7,8-TCDD 1n larval and adult frogs,
Rana catesblana. Groups of 15 tadpoles and 5 adults were Injected with
2,3,7,8-TCDD 1n olive oil at maximum nominal dosages of 1000 and 500 yg/kg
bw, respectively. There were no effects on survival and metamorphosis of
larvae through 50 days after Injection, or on survival of adults for 35 days
after Injection. There was a slight, temporary decrease 1n food consumption
by adults at the highest dose. H1stopatholog1cal examination revealed no
significant lesions 1n metamorphoslzed or adult frogs. The lack of toxicity
1n this amphibian species 1s 1n sharp contrast to the results previously
described with fish. Although the difference may be due, 1n part, to the
different routes of exposure, 1t 1s probable that some fish are actually
more sensitive, because toxic effects occurred 1n coho salmon at an Internal
dose of 5.4 yg/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, Phvsa sp., adult ollgochoete worms, Paran-
a1s sp., and mosquito larvae Aedes aegyptl to a nominal Initial concentra-
6-4
TABLE 6-2
Effects of Chronic or Subchronic Exposure to 2,3,7,8-TCDD on Aquatic Animals
6-5
Species
Life Stage, Weight or
Length
Duration of Exposure
(days)
Duration of Test
(days)
Lowest Effect Concentration
(t>9/t)
No Effect Concentration
(i* g / D
Effect
Mosquito, Aedes aeavotl
Ollgochaete Worm, Parana1s so.
Snail, Phvsa sp.
Snail, Helosoma so.
Waterflea, Daohnla magna
Mosqultoflsh, (ambus1a afflnls
Channel Catfish, Ictalurus ounctatus
Rainbow Trout, Salmo aalrdnerl
larvae adult adult adult adult NR flngerllngs 7.8 cm
17 55 36 32 32 15
20
105
30 ND
55 0.2 48 0.2
0.2 no effect on pupation
ND reduced reproduction ND reduced reproduction
46 ND
0.003
no apparent effects
32 ND
0.003
no apparent effects
15 0.003
20 0.003
ND ND
100X mortality 10036 mortality
105
2300 t>g/kg
2.30 t>g/kg
reduced survival,
In 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 Norris, 1977
NO c Not determined
t1on of 0.20 pg/fi, for 36, 55 and 17 days, respectively. There was no significant difference In total pupation or pupation rate between exposed and control mosquito larvae during the 17-day exposure period or for the 30-day total test period. Exposure of adult snails to 0.20 pg/i, 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) In 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 In a significant decrease 1n the total number of worms at 55 days. Total and mean dry weight were also reduced, but the variation among replicates reduced the statistical significance of this effect to p=0.057, Indicating that 0.20 pg/st 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 Norris (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 pg/kg had no effect on survival, food consumption, growth and fin morphology. In contrast, fish fed the highest dose showed reduced food consumption after 10 days, reduced growth by 7 days, fin erosion by 14 days, and mortality that began on day 33 and reached 50% by day 61 and 88% by day 71.
The only other Information concerning subchronic toxicity to aquatic animals was provided by Yocklm et al. (1978), who exposed channel catfish, Ictalurus punctatus. mosqultoflsh, Gambusla afflnls. waterfleas, Daphnla
6-6
magna. snails, Helosoma sp., and algae, Oedogonlum cardlacum. to 14C-
labeled 2,3,7,8-TCDD 1n a recirculating aquatic model ecosystem. Soil was
treated with 100 yg/kg and flooded with water, and organisms were added 1
day after flooding. Organisms were removed periodically for measurement of
tissue residues. The mean concentration (yg/l) 1n the water, measured
by liquid scintillation counting, was 0.0034 at day 1, 0.0029 at day 3,
0.0024 at day 7, 0.0026 at day 15 and 0.0042 at day 32. The mean concentra
tion through the 32-day period was 0.0031 yg/l. No effects over the
32-day exposure period were observed 1n algae, waterfleas or snails as
measured by reproductive activity, feeding and growth. All unharvested
mosqultoflsh died by day 14, with a mean tissue concentration of 7.2 yg/kg
bw. A second group of mosqultoflsh added at day 15 were all dead after
15-20 days. Channel catfish added at day 32 all died after 15-20 days of
exposure, with a mean tissue concentration of 4.4 yg/kg bw. These results
Indicate that 15-20 days of exposure to ~0.003 yg/8, was lethal to fish,
but had no effects on snails, waterfleas and algae.
6.1.1.3.
AQUATIC PLANT EFFECTS -- As mentioned earlier, Yocklm et al.
(1978) did not observe any obvious effects of 0.003 yg/8, on the growth
of the freshwater algae, 0. cardlacum, over a 32-day period. The only other
Information concerning toxicity to aquatic plants was provided by Zullel and
Benecke (1978), who conducted contact Inhibition studies with filamentous
algae, Phorm1d1um sp. Filter paper was spotted 1n three places with 1 yg
of 2,3,7,8-TCDD. Disks (5mm diameter) of filtered algae were placed on the
spots, and the filter paper was placed 1n a petrl dish containing nutrient
media. The motility of the algae filaments outward from the disks was
measured over a 3-hour period with a photoelectric cell. Relative to
controls, 1 yg of 2,3,7,8-TCDD caused a significant Inhibition of
6-7
motility. Although the exposure concentration 1s unknown, these results Indicate that this algal species may be affected by contact with contami nated substrates (1.e., sediment).
Jackson (1972) studied the progression of mitosis 1n the African blood Uly, Haemanthus katherlnae. endosperm cells. In this study, cells were exposed during prophase, prometaphase, metaphase and anaphase to 2,3,7,8TCDD at nominal levels of either 0, 0.1 or 0.5 yg/l, and the ability of the cells to progress to the next stage of cell division within a 2-hour period was evaluated. Regardless of the stage of cell division during which exposure occurred, the treatment resulted 1n an Inhibition of progression to the next stage. The authors noted that 2,3,7,8-TCDD strongly adsorbs to glass and speculated that the concentrations. 1n the test chamber were actually lower than reported. It was estimated that the higher concentra tion may possibly be approaching 0.2 yg/Jl, the solubility of 2,3,7,8TCDD 1n water. 6.2. TISSUE RESIDUES
Levels of 2,3,7,8-TCDD 1n several species of commercial fish taken from eastern Lake Ontario, Lake Erie and the Welland Canal ranged from 0.002 0.039 yg/kg 1n those fish with positive test results (Josephson, 1983). Rock bass showed no detectable levels. Highest concentrations generally occurred 1n eels (0.006-0.039 yg/kg), followed by smelt and catfish. The high fat content 1n these species (37, 13 and 3.5%, respectively) may explain, 1n part, the higher 2,3,7,8-TCDD concentrations.
Analysis by the NYS Department of Health showed levels of 2,3,7,8-TCDD 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,
6-8
white sucker, brown bullhead, rainbow trout, coho and Chinook salmon, and brown trout. The Ontario Ministry of the Environment (NRCC, 1981a) reported
i concentrations oi1 2,3,7,8-TCDD ranging between 0.010 and 0.019 yg/kg In fillet samples of lake trout, brown trout, white bass, white perch and smelt 1n Lake Ontario, but no detectable (<0.010 yg/kg) levels 1n fish from the Niagara River, Lake Erie, Lake Huron or Lake Superior. Other fish residue data summarized by NRCC (1981a) Included 2,3,7,8-TCDD concentrations 1n positive samples ranging from 0.020-0.230 yg/kg 1n Tlttabawassee River, Saginaw Bay and other locations near Midland, MI; 0.015-0.480 yg/kg 1n the Arkansas River; and 0.019-0.102 yg/kg 1n Lake Ontario and Niagara River. OCDD concentrations 1n fish ranged from 0.040-0.150 yg/kg near Midland, MI, and from 0.004-0.078 yg/kg 1n the Honesatonlc River. The levels of 2,3,7,8-TCDD 1n fish and shellfish as determined by various authors are given 1n Table 6-3.
Levels ranging from 0.004-0.695 yg/kg were cited by the U.S. EPA (1984) for the edible portion of channel catfish, carp, yellow perch, smallmouth bass, sucker and lake trout from Tlttabawassee, Grand and Saginaw Rivers, Lake Michigan and Saginaw Bay. The highest concentrations were detected 1n bottom-feeding catfish and carp, and the lowest concentrations were detected 1n bass, perch and suckers (Harless and Lewis, 1980b).
Young et al. (1976) measured 2,3,7,8-TCDD residue levels 1n terrestrial and aquatic animals from contaminated areas of Eglln A1r Force Base, FL, which had received massive amounts of herbicides, one of which (2,4,5-T) was contaminated with 2,3,7,8-TCDD. Beach mice from contaminated areas contained 0.540-1.30 yg/kg 1n the Uver and 0.130-0.140 yg/kg 1n pelts. Residues 1n racerunner lizards trapped from the most highly contaminated
6-9
T A B L E .6-3 Levels of 2,3,7,8-TCDDs 1n Fish and Shellfish
6-10
Type/Sect1on 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 0ntar1o/Lake Erie/ Welland Canal Lake 0ntar1o/Lake Erie/ Welland Canal Bergholtz Creek, Love Canal 2,4,5-T contaminated watershed 1n Arkansas and Texas; Tlttabawassee and Saginaw Rivers Lake Ontario Lake Ontario Lake Ontario Lake Ontario Lake Ontario Lake Ontario Lake Ontario
Concentration (PPt)
480 ND (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
Mltchum et al., 1980 MHchum et al., 1980 Mltchum et al., 1980 MHchum et al., 1980 Yamaglshl et al., 1981 Josephson, 1983
Josephson, 1983
Smith et al., 1983b Shadoff et al., 1977; U.S. EPA, 1980a; Buser and Rappe, 1980
O'Keefe et al., 1983 O'Keefe et al., 1983 O'Keefe et al., 1983 O'Keefe et al., 1983 O'Keefe et al., 1983 O 'Keefe et al., 1983 O'Keefe et al., 1983
11-9
Type/Sect1on of Fish
Smallmouth bass Brown bullhead Carp/Goldflsh Northern pike Pumpkin seed Rock bass Coho salmon Mall eye pike Smallmouth bass Carp/Goldf1sh Lake trout Carp Channel catfish Sucker Yellow perch Coho salmon Rainbow trout Perch/sucker Catfish Carp
inU LL u - j
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-<3.5 2.6 1.6-<2.4 ND (2.6) 21 26 20 25 ND (8.7) ND (3.8) 1.0 ND (3.8)-25 14-37 23-47
Reference
O'Keefe et 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 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 O'Keefe et al., 1983 O 'Keefe et al., 1983 O'Keefe et al.. 1983 Niemann et al.. 1983 Niemann et al., 1983 Niemann et al., 1983
6-12
TABLE 6-3 (coni.)
Type/Sectlon of Fish
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
Sampling Site
Bayon Meto/Arkansas River Bayon Meto/Arkansas River Lake Ontario Lake Ontario Atlantic Ocean Lake Huron Saginaw Bay Bay Port THtabawassee River Lake Michigan Lake Ontario Woods Pond, MA THtabawassee River, Saginaw River and Grand River THtabawassee River, Saginaw River and Grand River THtabawassee River and Saginaw River Grand River
Concentration (PPt)
NO (3.8) ND (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
Reference
Niemann et a!., 1983 Niemann et al., 1983 Niemann et al., 1983 Niemann et al., 1983 Niemann et al., 1983 Stalling et al., 1983 Stalling et al., 1983 Stalling et al., 1983 Stalling et al., 1983 Stalling et al., 1983 Stalling et al., 1983 Buser and Rappe, 1983 Harless and Lewis, 1982
Harless and Lewis, 1982
Harless and Lewis, 1982
Harless and Lewis, 1982
TABLE 6-3 (cont.)
Type/Sectlon of Fish
Sampling Site
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 ND (5)c 61.2 (3.6) 32.3 (3.6)
30.4 (3.6)
Harless and Lewis, 1982 Harless and Lewis, 1982 Ryan et al., 1983 Ryan et al., 1983
Ryan et al., 1983
aNot detected and the detection limit Is Indicated within the parentheses. b0nly the GC/MS results of these authors are Included 1n tabulation cThese are the mean concentrations 1n samples showing detectable levels of 2,3,7,8-TCDD. ND = Not detected
6-13
areas contained 0.36-0.37 yg/kg 1n the visceral mass and trunk, respec tively. Residues were also found 1n three fish species taken from a stream and pond 1n the contaminated area. Residue levels of 0.012 yg/kg were found 1n the viscera of sallfln shiners and 1n 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 yg/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 yg/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 Saginaw Bay and 1n Lake Ontario gull eggs (Table 6-4), Norstrom et al. (1982) Indicated the possibility of Industrial contamination since the former 1s near a major 2,4,5-T manufac turing plant on the Sag1naw/T1ttabawassee River, and the latter 1s down stream from a 2,4,5-TCP plant at Niagara Falls, NY. 6.3. ECOSYSTEM EFFECTS
Investigations concerning the ecosystem effects of 2,3,7,8-TCDD are restricted to the field studies of Young et al. (1975) at the Eg! 1n A1r Force Base. A 1-square mile area was sprayed with massive amounts of herbi cides over an 8-year period (1962-1970). In particular, a 92-acre test area was sprayed from 1962-1964 with 87,186 pounds of 2,4,5-T that was contami nated with 2,3,7,8-TCDD. Analysis 1n 1974 of surface soils 1n this area showed 2,3,7,8-TCDD levels of 0.010-0.710 yg/kg. Large numbers of beach mice were trapped from contaminated and control sites and evaluated for differences 1n organ weights and hlstopathology. The only significant differences 1n organ weight were Increased Uver weight 1n females and
6-14
IMDLC D-t
TCDD Levels 1n Wildlife
6-15
Type of Animal
Rabbit Field mouse Hare Toad Snake Snake Earthworm Eagle Herring gull
Tissue
llyer whole body 11ver whole body 11ver adipose tissue whole body carcass egg
Sampling Site
Seveso, Italy Seveso, Italy Seveso, Italy Seveso, Italy Seveso, Italy Seveso, Italy Seveso, Italy throughout U.S. Saginaw Bay, Lake Ontario
2.3.7.8-TCDD Concentration (ppb)
Average3
Range
31 1-<1024
4.5 0.07-49
7.7 2.7-13
0.2 LS
2.7 LS
16 LS
12 LS
<50 ppb
NR
NR 0.043-0.093
Reference
Fanelli et al., 1980a
Fanelli et al., 1980c
Fanelli et al., 1980c
Fanelli et al., 1980c
Fanelli et al., 1980c
Fanelli et al., 1980c
Fanelli et al., 1980c
HelUng et al., 1973
Ogllvle, 1981
TABLE 6-4 (cont.)
6-16
Type of Animal
Herring gull Herring gull Herring gull Herring gull Herring gull Herring gull Herring gull Turtle Snake Muskrat
Tissue
egg egg egg egg egg egg egg egg and Uver Uver and muscle Uver
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
2.3.7.8-TCDD Concentration (ppb)
Average3
Range
0.011
NR
0.009
NR
0.009
NR
0.043
NR
0.086
NR
0.011
NR
0.059
NR
0.15
LS
0.060
LS
ND (40 ppt) b
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
Mltchum et al., 1980
Mltchum et al., 1980
Mltchum et al., 1980
TABLE 6-4 (cont.)
6-17
Type of Animal
Tissue
Sampling Site
2.3.7.8-TCDD Concentration (ppb)
Average3
Range
Racoon Frog Horse
Horse
liver
liver and muscle fat
Uver
Bayou Heto/ Arkansas River
Bayou Heto/ Arkansas River
Hldwest wire reclamation Incinerator
Hldwest wire reclamation Incinerator
NO (10 ppt)b >10 0.04S
NO (<6 ppt)b
LS LS LS
LS
aThese are averages of samples that had above detectable levels of TCDD. ^Not reported and the limit of detection Indicated In parentheses NR = Not reported; LS = Limited samples
Reference
Hltchum et al., 1980 Hltchum et al., 1980 Hryhorczuk et al., 1981
Hryhorczuk et al., 1981
Increased spleen weight In males and females taken from the contaminated sites; however, no hlstopathologlcal effects could be attributed to the collection sites. Similar studies on racerunner lizards showed no signifi cant difference 1n relative or total body weight of animals collected from contaminated and control sites. Sweep net surveys of the contaminated sites for terrestrial Insects 1n 1971 and 1973 Indicated that there was a signifi cant Increase 1n the number of families and total number of Insects In the contaminated test site, which was correlated with the Increase In vegetation after herbicide spraying. Aquatic species diversity studies were conducted 1n 1969, 1970, 1973 and 1974 on a stream In the contaminated area and a con trol stream. As mentioned before, 2,3,7,8-TCDD was detected In sediments and fish from the contaminated stream; however, there was no significant difference 1n Ichthyofauna diversity 1n the two streams, and no significant change 1n diversity through time In either stream. As a result, the only effects that can be attributed to 2,3,7,8-TCDD contamination were Increased Uver and spleen weight 1n beach mice. The ecological significance of this effect 1s unknown, especially since no obvious detrimental effects were observed 1n this or other species from contaminated sites.
Korfmacher et al. (1984) analyzed fat tissue and eggs from snakes for 2.3.7.8-TCDD. Water snakes were selected as a possible marker for 2.3.7.8- TCDD contamination. Three snakes were collected from Lake Dupree, Arkansas In 1983. This lake 1s a site of 2,3,7,8-TCDD contaminated sediment and fish (Arkansas Dept, of Pollution Control and Ecology, 1983). Two snakes were collected from a lake evidently not contaminated with 2.3.7.8- TCDD from any Industrial source. Eggs were derived from one of the snakes obtained from Lake Dupree. 2,3,7,8-TCDD concentration 1n the fat material of three snakes from contaminated lake varied from 500-730 ppt, In
6-18
the snake eggs varied from 151-294 ppt, and 1n the fat material from two snakes from noncontamlnated lake varied from 38-378 ppt.
The only other Information pertinent to ecosystem level effects was provided by Bollen and Norris (1979), who Investigated the effects of 2.3.7.8- TCDD on respiration (C02 production) In forest Utter and soil samples. Litter and soil samples were air dried, placed In biometer flasks, moistened and treated with 2,3,7,8-TCDD. Concentrations as high as 0.031 vg/kg dry weight 1n Utter had no effect on respiration. Concentrations as high as 0.052 yg/kg dry weight 1n soil caused a slight but significant stimulation of C02 production. Because higher concentrations were not tested, 1t 1s unknown whether 2,3,7,8-TCDD would have Inhibitory effects on soil microbial populations, carbon metabolism or nutrient cycling at the higher levels of soil contamination found 1n such contaminated areas as the Egl1n A1r Force Base test site. 6.4. SUMMARY
Almost all of the available Information concerning the toxicity of PCDDs to wildlife deals with aquatic species. Acute exposure to Initial nominal 2.3.7.8- TCDD concentrations as low as 0.0001 yg/a has been shown to cause delayed sublethal effects 1n early life stages of northern pike and rainbow trout (Helder 1980, 1981) and In adult guppies (Miller et al., 1979). Decreased growth, food consumption and survival have been reported In these and other fish species after acute exposure to >0.001 yg/a. 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 1s possible that constant acute or chronic exposure to dissolved concentrations <0.0001 yg/a would produce toxic effects 1n sensitive aquatic organisms.
6-19
Several studies provide evidence that 2,3,7,8-TCDD 1s less toxic to aquatic Invertebrates and amphibians than to the tested fish species. Subchronic exposure to an Initial nominal concentration of 0.20 yg/a, had no effect on mosquito population and caused a 30-50% decrease 1n reproduc tion of snails and ollgochoete worms (Miller et al., 1973). In contrast, acute exposure to 0.1 yg/i caused 100% delayed mortality 1n guppies (Norris and Miller, 1974) and juvenile rainbow trout (Helder 1981). Similarly, exposure to relatively constant, measured, dissolved concentra tions of ~0.002-0.004yg/8, In aquatic model ecosytems killed all exposed mosqultoflsh and channel catfish 1n 15-20 days, but had no discernible effects on snails and waterfleas over a total test period of 32-46 days (Yocklm et al., 1978). The dying mosqultoflsh and catfish had mean wholebody 2,3,7,8-TCDD concentrations of 7.2 and 4.4 yg/kg, respectively. In contrast, single 1ntraper1toneal Injections of 2,3,7,8-TCDD at maximum doses of 500 or 1000 yg/kg bw, respectively, had no effects on adult frogs over a 35-day period or on frog larvae over a 50-day period (Beatty et al., 1976).
Chronic feeding studies with groups of rainbow trout showed that dally feeding of 2300 yg/kg 1n the diet was lethal to all but two fish (88%) 1n 71 days, but no significant effects were seen 1n fish fed dally a diet containing 2.3 yg/kg for 105 days (Hawkes and Norris, 1977). Residue analysis of single fish sampled at the end of the tests showed 2,3,7,8-TCDD levels of 1380 yg/kg bw 1n one high dose fish and 1 .573 yg/kg 1n one low dose fish.
Although only limited Information was found concerning the effects of 2,3,7,8-TCDD on aquatic plants, 1t 1s probable that they are less sensitive than fish. Using model ecosystems, Yocklm et al. (1978) observed no obvious effects on algae at concentrations (0.002-0.004 g) that killed fish. Zullel
6-20
and Benecke (1978) observed contact Inhibition of filamentous algae placed 1n contact with 1 yg quantities of 2,3,7,8-TCDD spotted on filter paper.
The only available Information concerning the effects of low level environmental exposure to 2,3,7,8-TCDD on terrestrial wildlife was reported by Young et al. (1975), who Investigated tissue residues and several bio logical parameters 1n mice and lizards from contaminated and control sites at Eglln A1r Force Base, FL. The concentrations of 2,3,7,8-TCDD 1n contami nated soils were 0.010-0.710 yg/kg. Mice trapped from the contaminated site contained 0.540-1.30 yg/kg 1n the Uver and had significantly higher spleen and Uver weights than mice from control sites. No other differences (hlstopathology, weights of other organs, Incidence of abnormal fetuses, etc.) were observed. Racerunner lizards from the contaminated site con tained 0.36-0.37 yg/kg 1n the viscera and trunk and showed no differences 1n body weight or hlstopathology compared with lizards from control sites. Residues of 2,3,7,8-TCDD 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 yg/kg. PCDD residues have been reported for numerous other fish species and other snakes from contaminated water bodies. The PCDD concentrations (primarily 2,3,7,8-TCDD) 1n positive fish tests ranged from 0.002-0.695 yg/kg.
6-21
7. COMPOUND DISPOSITION AND RELEVANT PHARMACOKINETICS 7.1. ABSORPTION
Data are available regarding the absorption of 2,3,7,8-TCDD 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 1s 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 1n Table 7-1. The GI absorption of 2,3,7,8-TCDD has been Investigated more extensively 1n the rat than 1n other species. When 2,3,7,8-TCDD was administered 1n the diet at 7 or 20 ppb for 42 days, 50-60% of the consumed dose was absorbed (Fries and Marrow, 1975). Administration of 2,3,7,8-TCDD by gavage 1n acetone:corn oil (1:25 or 1:9) as a single dose or as repeated doses (5 days/week x 7 weeks) resulted 1n absorption of a larger percentage (70-86%) of the dose (Rose et al., 1976; Piper et al., 1973). It would appear, therefore, that the GI absorption of 2,3,7,8-TCDD may vary, depending upon the vehicle used. The Influence of vehicle or adsorbent on GI absorption has been Investigated by Polger and Schlatter (1980), using hepatic concentrations 24 hours after dosing as an Indicator of the amount absorbed. They found a linear rela tionship between ng 2,3,7,8-TCDD administered by gavage 1n 50% ethanol (for doses of 12-280 ng, equivalent to 0.06-1.4 pg/kg) and the percentage of the dose 1n hepatic tissues (36.7-51.5%). At the next higher dose of 1070 ng the percentage was 42%. Administration of 2,3,7,8-TCDD 1n an aqueous suspension of soil resulted 1n a decrease 1n the hepatic levels of 2,3,7,8TCDD as compared with hepatic levels resulting from administration of
7-1
TABLE 7-1 Gastrointestinal Absorption of 2,3,7,8-TCDD
Species Vehicle
Dose Schedule (vg/kg)
% Absorption Mean + SD
Reference
Guinea pig Rat
Rat
Rat
Rat
NR
7 ppb, 1n diet
20 ppb, 1n diet
A:C, 1:25
A:C, 1:25
Rat Hamster
A:C, 1:9
olive oil
NR single dose
0.5 yg/kg/day x 42 days
1.4 yg/kg/day x 42 days
1.0 yg/kg, single dose
0.1 or 1.0 yg/kg/day, 5 days/week x 7 weeks
50.0 yg/kg, single dose
650 yg/kg, single dose
50 50 - 60 50 - 60 84 + 11* 86+12*
70 74 + 23*
*Mean + standard deviation NR = Not reported; A:C = Acetone:corn oil, v:v
Nolan et al., 1979 Fries and Marrow, 1975 Fries and Marrow, 1975 Rose et al., 1976 Rose et al., 1976
Piper et al., 1973 Olson et al., 1980a
s
7-2
2.3.7.8- TCDD in 50% ethanol. The extent of the decrease was directly pro
portional to the length of time the 2,3,7,8-TCDD had been 1n contact with the soil. McConnell et al. (1984) observed a dose-response relation of liver accumulation of 2,3,7,8-TCDD as a result of 1ntragastr1c exposure of young male Hartley guinea pigs to 2,3,7,8-TCDD 1n corn oil or 1n soil (Table 7-2). In Sprague-Dawley female rats, they found as high as 40.8 ppb and 20.3 ppb liver accumulation of 2,3,7,8-TCDD by 1ntragast1c exposure to 2.3.7.8- TCDD 1n corn oil and 1n soil, respectively. Ph111pp1 et al. (1981) and Huetter and Philippi (1982) have shown that radiolabeled 2,3,7,8-TCDD becomes progressively more resistant with time to extraction from soil. Polger and Schlatter (1980) also demonstrated that 2,3,7,8-TCDD mixed 1n an aqueous suspension of activated carbon was very poorly absorbed (<0.07% of the dose 1n hepatic tissues). In addition, Sllkworth et al. (1982) observed an Increase 1n the LD^q value for female guinea pigs from 2.5 to 19 yg/kg when the 2,3,7,8-TCDD was administered by gavage 1n corn oil or aqueous methyl cellulose, respectively.
A comparative study on 4he biological uptake 1n the rabbit of 2,3,7,8TCDD 1n different formulations, Including accident-contaminated Seveso soil, was conducted by Bonaccorsl et al. (1983). On the whole, the results Indicated that soil-borne 2,3,7,8-TCDD had a b1oava1labH1ty lower than that of free (solvent-borne) 2,3,7,8-TCDD.
The feeding of fly ash containing PCDDs to rats 1n the diet for 19 days resulted 1n 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 a.l., 1983). The PCDDs were tentatively Identified as 2,3,7,8TCDD, 1 ,2,3,7,8-PeCDD, 1,2,3,6,7,8-HxCDD and 1,2,3,7,8,9-HxCDD. The
7-3
TABLE 7-2
Liver Accumulation of 2,3,7,8-TCDD in Guinea Pigs 30 Days after a Single Intragastric Exposure to 2t3,7,8-TCDDa
7-4
Group
1 2
3 4 5
6 7 8
9 10
No. of Animals
6 6
6 6 5d
5 6 6
6 5e
Composition of the Haterial Gavaged
Corn oil TCDD in corn oil
TCDD in corn oil Time Beach soil Time Beach soil
Time Beach soil Hinker Stout soil Hinker Stout soil
Hinker Stout soil Time Beach soil (uncontaminated)
Total Quantity Gavaged
0.1 ma/100 g 0.1 ma/100 g
0.1 ma/100 g 0.35 g 1.07 g
3.60 g 0.26 g 0.80 g
2.67 g 3.60 g
Dosage of TCDD (vg/kg bw)
0 1
3 1.3 3.8
12.8 1.1 3.3
11.0 0
Average Liver Concentration of TCDD^
ppt + SEH
ND
1.6+0.2 4.1e
13.3+2.3
<1.0
1.0+0.1 3.2e
34.3+6.0
<1.0
1.4+0.3 2.0+0.Ie
25.7+5.2
ND
TABLE 7-2 (coni.)
7-5
Group
No. of Animals
Composition of the Material Gavaged
Total Quantity Gavaged
11
6
Time Beach soil
2.71 g
(uncontaminated
but TCDD added)
aSource: McConnell et al. (1984) bDetect1on limit 100 ppt cAn1mal/an1mals which died before 30 days done animal died 2 days after dosing (not Included) e0ne animal died at the time of dosing SEM = Standard error of the mean ND = Not detected
Dosage of TCDD Ug/kg bw)
10
Average Liver Concentration of TCDDb
ppt i SEM
45.4+8.4
difference 1n 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 1n the hamster, the species most resistant to the acute toxicity of this toxin. Olson et al. (1980a) administered a single, sublethal, oral dose of [1,6-3H]-2,3,7,8TCDD 1n olive oil (650 yg/kg) to hamsters and reported that 74% of the dose was absorbed, while Nolan et al. (1979) reported that absorption 1n the guinea pig, the most sensitive species, was ~50% following administration of an unspecified amount of 2,3,7,8-TCDD. The vehicle and method for calculat ing the absorbed dose were not given 1n this report. 7.1.2. Absorption Through the Skin. Information on the absorption of 2,3,7,8-TCDD through the skin 1s extremely limited. Polger and Schlatter (1980) administered 26 ng 2,3,7,8-TCDD 1n 50 yl methanol to the skin of six rats. After 24 hours, the Uver contained 14.8+2.6% of the dose. By comparing with hepatic levels obtained (1n the same study) after oral administration 1n 50% ethanol (see Section 7.1.1.), assuming that hepatic levels are valid estimates of the amount absorbed from both oral and dermal routes and that absorption from methanol 1s equivalent to absorption from 50% ethanol, the amount absorbed from a dermal application can be estimated at ~40% of the amount absorbed from an equivalent oral dose. As compared with dermal application 1n methanol, dermal application of 2,3,7,8-TCDD to rats 1n vaseline or polyethylene glycol resulted 1n hepatic tissue concen tration of 1.4 and 9.3% of the dose, respectively, but had no observable effect on the concentration of 2,3,7,8-TCDD required to Induce skin lesions (~1 yg) 1n the rabbit ear assay (Polger and Schlatter, 1980). Application of 2,3,7,8-TCDD In a soll/water paste decreased hepatic 2,3,7,8-TCDD to ~2% of the administered dose and Increased the amount required to produce skin
7-6
lsions to 2-3 yg 1n rats and rabbits, respectively. Application 1n an activated carbon/water paste essentially completely eliminated absorption, as measured by percent of dose 1n the Uver, and Increased the amount of 2,3,7,8-TCDD required to produce skin lesions to ~160 yg. 7.2. DISTRIBUTION
The tissue distribution of 2,3,7,8-TCDD 1n a number of species 1s summarized 1n Table 7-3. As would be predicted from the 11pophl11c nature of this compound, accumulation fends to occur 1n tissues with a high I1p1d content. In rats and mice, 2,3,7,8-TCDD residues are localized 1n the Uver and adipose tissue. In the rat, hepatic levels of 2,3,7,8-TCDD accounted for ~38-52% of the administered dose during the first week following oral administration of a single dose ranging from 0.07-50 yg/kg (Piper et al., 1973; Polger and Schlatter, 1979). The latter dose 1s within the LD^q 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-TCDD to rats; 43% of the total dose was localized In the Uver (Van Hiller et al., 1976). In two strains of mice, the Uver contained ~35% of an admin istered 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 1n adipose tissue were similar to or slightly lower than levels 1n the Uver, and were considerably higher than concentrations 1n other tissues (Piper et al., 1973; Rose et al., 1976; Van Miller et al., 1976; Manara et al., 1982), Including the thymus (Rose et al., 1976; Van Miller et al., 1976).
In a 7-week gavage study and a 2-year dietary study of 2,3,7,8-TCDD 1n rats, 2,3,7,8-TCDD was present 1n the Uver at 3-5 times the concentration 1n adipose tissue when the dally dose or Intake of the compound was >0.01 yg/kg/day (Rose et al., 1976; Kodba et al., 1976) and was present at
7-7
TABLE 7-3 Distribution of 2,3,7,8-TCDD
Species
Route of Administration
Rat Rat Rat Rat Rat Rat House Mouse Rhesus monkey Golden Syrian hamster Guinea pig Guinea pig
oral oral oral oral oral 1.p. oral l.p. l.p. l.p. or oral
oral 1.p.
l.p. = Intraperitoneal
Principal Organ Depots
liver liver > fat liver > fat liver > fat liver > fat liver > fat liver > fat > kidney > lung liver > fat > kidney > lung > spleen fat > skin > liver > adrenals = thymus liver > fat
fat > liver > adrenals > thymus > skin fat > liver > skin
Reference
Fries and Harrow, 1975 Rose et al., 1976 Piper et al., 1973 Koclba et al., 1978a Allen et al., 1975 Van Hiller et al., 1976 Hanara et al., 1982 Manara et al., 1982 Van Hiller et al., 1976 Olson et al., 1980a
Nolan et al., 1979 Gaslewlcz and Neal, 1979
7-8
about the same concentration as 1n adipose tissue when the dally Intake was
0.001 yg/kg/day (Kodba et al., 1976). As 1n the single-dose studies, 2.3.7.8- TCDD levels were considerably lower 1n other tissues, Including the thymus, than 1n Uver or adipose tissue (Rose et al., 1976).
There 1s 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 1n the diet, -85% of the total body residue of male rats was located 1n the Uver, as compared with 70% 1n females (Fries and Harrow, 1975). This small difference 1n distribution patterns may have resulted from sex differences 1n relative adipose tissue content.
The ability of mouse Uver to sequester 2,3,7,8-TCDD Increases with prolonged exposure (Teltelbaum and Poland, 1978). The hepatic uptake of [3H]2,3,7,8-TCDD 1n Swlss-Webster mice was maximal 12 hours after Intra 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 1s consistent with other data that Indicate that 2,3,7,8TCDD 1s a potent Inducer of hepatic microsomal mixed-function oxidase (Section 8.1.1.5.) and that >90% of the hepatic 2,3,7,8-TCDD 1s localized 1n the mlcrosomes (Allen et al., 1975). The toxicity 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 Uver seems to have much less of a role 1n 2.3.7.8- TCDD accumulation. Van Hiller et al. (1976) have compared the tissue distribution of [3H]2,3,7,8-TCDD 1n adult rhesus monkeys, Infant rhesus monkeys, and Sprague-Dawley rats 7 days after a single Intraperiton eal Injection of 400 y g 2,3,7,8-TCDD/kg bw. They found that while 43% of the administered dose was localized 1n the livers of the rats, only 10.4%
7-9
was found 1n the livers of adult monkeys and 4.5% 1n the livers of Infant monkeys. This difference cannot be explained by differences 1n absorption or excretion, since these parameters were observed to be similar 1n both species. In monkeys, larger percentages of the dose were found 1n adipose tissue, skin and muscle than was the case for rats.
McNulty et al. (1982) reported that 2 years after administration of a single oral dose of 1 yg/kg of 2,3,7,8-TCDD to an adult rhesus macaque monkey, tissue levels of the compound were 1000 ppt 1n adipose tissue and 15 ppt 1n the Uver. These results Indicate that prolonged retention of 2.3.7.8- TCDD may occur 1n this species. The tissue distribution of 2,3,7,8TCDD 1n the guinea pig appears to be similar to the monkey, with the highest concentration of the toxin being found 1n adipose tissue (Gas1ew1cz and Neal, 1979; Nolan et al., 1979). The Interspecies difference 1n the tissue distribution of 2,3,7,8-TCDD may be related to the relative adipose tissue content of a given species and the affinity of 2,3,7,8-TCDD for the hepatic microsomal fraction; however, the significance of these differences remains 1n doubt. For example, the hepatotoxlclty of 2,3,7,8-TCDD 1n a given species does not appear to be related to the hepatic concentration of the toxin (Neal et al., 1982).
Very limited data are available on the tissue distribution of 2,3,7,8TCDD 1n humans. Facchettl et al. (1980) reported tissue concentrations of 2.3.7.8- TCDD at levels of 1-2 ng/g 1n adipose tissue and pancreas, 0.1-0.2 ng/g 1n Uver 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 adenocarcinoma (which was not considered related to the accident) that Involved the pancreas, Uver and lungs.
7-10
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 A1r Force officers with known heavy exposure to Agent Orange during disposal operations and 10 control veterans with no known herbicide exposure. In the first group, 10 of the 20 had measurable levels of 2,3,7,8-TCDD (5 with 5-7 ppt, 3 with 9-13 ppt, 1 with 23 and 35 ppt and another with 63 and 99 ppt). In the second group, only two officers had
i
measurable 2,3,7,8-TCDD levels that did not exceed 3 ppt. In the 10 control veterans, 4 had 2,3,7,8-TCDD levels between 6 and 14 ppt. Levels of 2.3.7.8- TCDD 1n adipose tissue did not appear to be associated 1n this study with 111 health or any particular symptom; however, 1t was considered that Information on background levels of 2,3,7,8-TCDD 1n adipose tissue was too limited to draw any firm conclusions.
2,3,7,8-TCDD has been demonstrated to be fetotoxlc 1n the rat (Section 9.1.). The ability of 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- TCDD 1n the fetus at gestation days 14, 18 or 21. The radioactivity appeared to be evenly distributed throughout the fetus on days 14 and 18; however, Increased levels of radioactivity were detected 1n fetal Uver on day 21.
Nau and Bass (1981) (more recently reported by Nau et al., 1982) Investigated the fetal uptake of 2,3,7,8-TCDD 1n NMRI mice following oral, Intraperitoneal or subcutaneous administration of 5, 12.5 or 25 yg/kg In
7-11
DMSO:corn oil or acetonercorn oil. The chemical was usually administered as a single dose 2 days before sacrifice. Embryonic 2,3,7,8-TCDD concentra tions were maximal on gestational days 9 and 10; however, low levels were found 1n the embryo and fetus between gestational days 11 and 18. This sharp decrease 1n 2,3,7,8-TCDD concentration coincides with placentatlon. 2,3,7,8-TCDD concentrations 1n the placenta were an order of magnitude greater than 1n the fetus Itself. The affinity of fetal Uver for 2,3,7,8TCDD was relatively low, as compared with maternal Uver; however, 2,3,7,8TCDD levels 1n fetal livers were 2-4 times higher than the levels 1n other fetal organs. An attempt was made to correlate 2,3,7,8-TCDD levels 1n the fetuses with the observed Incidence of cleft palate, but no clear relation ship was observed (1.e., 5 minutes to 61 days after Injection).
Autoradiographic studies of tissue localization following Intravenous administration of [14C]2,3,7,8-TCDD 1n DMSO to three strains of mice Indi cated that the Uver had the highest concentration and longest retention of
\
radioactivity 1n the body, followed by the nasal mucosa (Appelgren et al., 1983). In pregnant mice, the concentration of radioactivity 1n the fetuses was lower than 1n the dams, but a similar, selective labelling of the Uver and the nasal mucosa was seen In the fetuses at day 17 of gestation. In the adult animals, labelling of the adrenal cortex was about equal to that of the Uver at 1 hour after dosing, but thereafter was much lower than 1n the Uver. Labelling of the thymus, lymph nodes, bone marrow and prostate were low at all observation times. 7.3. METABOLISM
Vlnopal and Caslda (1973) found no evidence of water soluble metabolites of 2,3,7,8-TCDD following Incubation with mammalian Uver mlcrosomes or
7-12
I n t r a p e r i t o n e a l 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, 1n contrast to free 2,3,7,8-TCDD, was not ethyl acetate extractable. This binding was found to result from oxidative metabolism, as Indicated by a requirement for NADPH, and could be Induced by phnobarbital pretreatment. Binding was not covalent, because the bound radioactivity could be extracted with chloroform:methanol (9:1); this extracted radioactivity cochromato graphed with the 2,3,7,8-TCDD standard.
Ramsey et al. (1982) detected five distinct radioactive compounds 1n the bile of rats given dally oral doses of 15 yg [14C]2,3,7,8-TCDD. Incuba tion of the bile with <p-glucuron1dase resulted 1n an Increase 1n the amount of [14C] extracted, Implying the existence of conjugated [14C]2,3,7,8-TCDD metabolites. All of the 2,3,7,8-TCDD-der1ved radioactivity 1n the bile corresponded to metabolized 2,3,7,8-TCDD. JUi vivo metabolism has also been detected 1n the Golden Syrian hamster (Olson et al., 1980a) and 1n dogs (Polger et al., 1982a). In urine and bile from 14C-TCDD treated rats, hamsters and guinea pigs, all of the radioactivity corresponded to metabolites of TCDD, as assessed by HPLC (Neal et al., 1982). Enzymatic hydrolysis of the TCDD metabolites present 1n urine and bile produced alterations 1n their HPLC profiles that Indicated the presence of glucuron1de conjugates 1n bile and sulfate conjugates 1n urine (Olson and Bittner, 1983).
7-13
The ability of 1,6-3H-2,3,7,8-TCDD derived radioactivity to bind to rat hepatic macromolecules Iji vivo was Investigated by Poland and Glover (1979). They found maximum levels of 60 pmol 2,3,7,8-TCDD/mole of amino acids 1n protein, 12 pmol 2,3,7,8-TCDD/mole of nucleotide 1n rRNA, and 6 pmol of 2,3,7,8-TCDD/mole of nucleotide 1n DNA. According to the authors this corresponds to one 2,3,7,8-TCDD-DNA adduct/35 cells (Poland and Glover, 1979). Similar results were obtained using a mouse Uver microsomal system (Guenthner et al., 1979a). [3H]2,3,7,8-TCDD was found to bind to micro somal protein 120-2640 times more readily than to deprotelnlzed salmon sperm DNA. They estimated the rate of 2,3,7,8-TCDD metabolism to be between 9000 and 36,000 times lower than the rate of P-450-med1ated benzo[a]pyrene metabolism.
Tulp and Hutzlnger (1978) studied the metabolism of a variety of PCDDs, Including 1 ,2,3,4-TCDD, 1n the rat. In d1- and higher substituted dioxins, only mono- and dihydroxy derivatives were detected. Primary hydroxylatlon occurred exclusively at the 2-, 3-, 7- or 8-pos1t1on, so the significance of this study for the metabolism of 2,3,7,8-TCDD 1s not clear. Sawahata et al. (1982) Investigated the metabolism of 2,3,7,8-TCDD 1n Isolated rat hepatocytes. The major product was deconjugated with <p-glucuron1dase, derlvatlzed with diazomethane, and separated Into two compounds by HPLC. These metabolites were subsequently identified as l-hydroxy-2,3,7,8-TCDD and 2-hydroxy-3,7,8-tr1chlorod1benzo-f>-d1ox1n.
Polger et al. (1982a) Identified six metabolites 1n the bile of dogs that were given [3H]2,3,7,8-TCDD. The major metabolite was 1,3,7,8-tetrachloro-2-hydroxyd1benzo--d1ox1n. 2-Hydroxy-3,7,8-tr1chlorod1benzo--d1ox1n
7-14
and l,2-d1chloro-4,5-d1hydroxybenzene were also Identified as minor metabo
lites. The structures of the three remaining metabolites were not deter mined; however, two appeared to be tr1chloro-d1hydroxyd1benzo-})-d1ox1ns and the third was apparently a chlorinated 2-hydroxyd1phenyl ether. The pres ence of these metabolites 1s consistent with a 1,2-arene oxide Intermediate.
Isolated rat hepatocytes 1n suspension have been used as an in vitro system for assessing 2,3,7,8-TCDD metabolism under various conditions. Data Indicate that the rate of 2,3,7,8-TCDD metabolism 1n rat hepatocytes correlates directly with drug induced changes 1n hepatic cytochrome P-450 monooxygenase activity, suggesting that 2,3,7,8-TCDD 1s metabolized by this enzyme (Olson et al., 1981).
Beatty et al. (1978) found a correlation between hepatic mixed-function oxidase (MFO) activity and the toxicity of 2,3,7,8-TCDD 1n rats. Both 1n naturally occurring age- and sex-related differences 1n MFO activity and following the administration of Inducers and Inhibitors of MFO enzyme systems, hepatic MFO activity was Inversely related to toxicity that corre sponds to direct relationship between the 20-day LD5Q and MFO activity.
The fate of 2,3,7,8-TCDD metabolites from dogs has been examined In rats by Weber et al. (1982). 2,3,7,8-TCDD metabolites were extracted from the bile of 2,3,7,8-TCDD-treated dogs and administered by gavage to female Sprague-Dawley rats. The 2,3,7,8-TCDD metabolites were rapidly cleared from the bodies of b1le-duct-cannulated rats, with >85% of the dose recovered 1n 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.
7-15
Polger et al. (1982a) Investigated the toxicity 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 yg of parent compound/kg bw. Other groups of guinea pigs received bile extract from untreated dogs or 2,3,7,8-TCDD Itself. A comparison of the mortality data at 5 weeks after dosing Indicated that the acute toxicity of 2,3,7,8TCDD to guinea pigs was at least 100 .times higher than was the acute toxicity of Its metabolites.
Olson and Bittner (1983) reported that the rate of metabolite formation In vitro was considerably higher In hepatocytes from the hamster than In hepatocytes from the rat. Qualitative evaluation of in vivo and in vitro metabolites by HPLC also suggested major Interspecies variability. The authors suggested that such differences 1n metabolism may partially explain the differences 1n toxicity 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 (Gas1ew1cz and Neal, 1979), elimination data yield a straight line on a sem1logar1thm1c plot, Indicating a first order process. H1les and Bruce (1976) pointed out that the studies of Allen et al. (1975) and Piper et al. (1973) can be Interpreted equally well by either zero or first order kinetics. The majority of the data, however, seem to support the assumption of a first order elimination process.
2,3,7,8-TCDD 1s slowly excreted from the bodies of all species tested (Table 7-4), with a half-Hfe 1n the body of 10-43 days. In the GolderT Syrian hamster, the least sensitive mammalian species to the acute toxicity of 2,3,7,8-TCDD, excretion occurs readily through both the urine (41%) and
7-16
Species
Single Treatment yg/kg (route)
Elimination of 2,3,7,8-TCDD
Half-L1fe for Elimination (days)
Relative % of TCDD-Derlved Radioactivity
Feces
Urine
Reference
CM
7-17
Guinea pig
Guinea pig
Rat
Rat
Rat
Rat
Monkey (adult)
Monkey (Infant)
Monkey
Mouse C57BL/65 DBA/2J B6D2Fi/3*
Hamster
Hamster
2 (i.p.) 1.45 (oral) 1.0 (oral) 50 (oral) 50 (oral) 400 (I.p.) 400 (I.p.)
400 (1.p.)
1 (oral)
10 (I.p.) 10 (I.p.) 10 (I.p.) 650 (I.p.) 650 (oral)
30.2 5.8 22 - 43 31 t 6 17.4 t 5.6 21.3
NT NT
NT
365
11.0 + 1.2 24.4 1.0 12.6 t 0.8 10.8 t 2.4 15.0 2.5
94.0 NT >99 80.0 95.5 91.0 78.0
39.0
NR
72.0 54.0 72.0 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
^Offspring of C57BL/6J and DBA/2J that are heterozygous at the Ah locus NT = Not tested; NR = not reported
Gaslewlcz and Neal, 1979 Nolan et al., 1979 Rose et al., 1976 Piper et al., 1973 Allen et al., 1975 Van Miller et al., 1976 Van Miller et alT, 1976
Van Miller et al., 1976
McNulty et al., 1982
Gaslewlcz et al., 1983a,b Gaslewlcz et al., 1983a,b Gaslewlcz et al., 1983a,b Olson et al., 1980a Olson et al., 1980a
feces (5954) (Olson et al., 1980a). The high levels found 1n the urine of
Infant monkeys were probably due to the Incomplete separation of urine and
feces (Van Hiller et al., 1976). Ip all the other species so far tested,
excretion occurs mainly through the feces (80-10054) with only minor amounts
of 2,3,7,8-TCDD metabolites found 1n the urine (Piper et al., 1973; Allen et
al., 1975; Rose et al., 1976; Gas1ew1cz and Neal, 1979).
Rose et al. (1976) Investigated the elimination of [14C]2,3,7,8-TCDD
1n rats given repeated oral doses of 0.01, 0.1 or 1.0 yg/kg/day Monday
through Friday for 7 weeks, or a single dose of 1.0 yg/kg. In these
studies, no 14C was excreted 1n the urine following a single dose; how
ever, the urine contained 3-1854 of the cumulative dose by 7 weeks. This
study Indicated that steady-state concentrations will be reached 1n the
bodies of rats In ~13 v/eeks. The rate constant defining the approach to
steady-state concentrations was Independent of the dosage of 2,3,7,8-TCDD
over the range studied. This 1s consistent with the observations of Fries
and Harrow (1975), who found that the total retention 1n the bodies of rats
was proportional to total Intake. When rats were maintained on a diet
containing either 7 or 20 ppb TCDD, the amount of TCDD retained 1n the body
was 5.5 times the dally Intake of TCDD at 14 days, 7.5 times the dally
Intake at 28 days, and 10.0 times the dally Intake at 42 days.
The data 1n Table 7-4 suggest some 1nterspec1es differences 1n the half
life for elimination
2,3,7,8-TCDD. In the hamster, the least
sensitive species to the acute toxicity of 2,3,7,8-TCDD, a mean
10.8 days was observed (Olson et al., 1980a,b), and 1n the guinea pig, the
most sensitive species to the acute toxicity of 2,3,/,8-TCDD, the mean
t ^ g was 30.2 days (Gas1ew1cz and Neal, 1979). The observed Interspecies
differences 1n the t ^ 2 of 2,3,7,8-TCDD may 1n part be related to the
7-18
relative sensitivity of a given species to the a c u t e t o x i c i t y o f 2.3.7.8- TCDD.
The 1ntrastra1n differences 1n the t ^ ^ f 2,3,7,8-TCDD 1n three mouse strains may be due to the finding that the DBA/20 strain possesses ~2-fold greater adipose tissue stores than the C57B1/63 and B6D2F^/J strains (Gas1ew1cz et al., 1983b). The sequestering of the lipophilic toxin 1n adiposetissue 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 7-4, urinary and fecal elimina tion were monitored for a period of only 20-22 days, and from these data 1t was assumed that elimination followed a single component, first order kinetic model. Recently, Olson and Bittner (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 yg 3H-2,3,7,8-TCDD/kg. They observed first order kinetics for elimination, with a fast component having a t ^ 2 of 7 days (represents 13% of total elimination) and a slow compo nent having a t ^ 2 f 75 days (87% f 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 In the fat of a monkey to be ~1 year.
Studies 1n the rat, guinea pig, hamster and mouse have found that all of the 2,3,7,8-TCDD-der1ved radioactivity excreted 1n the urine and bile corre sponds to metabolites of 2,3,7,8-TCDD (Neal et al., 1982, 1984). The apparent absence of 2,3,7,8-TCDD metabolites 1n liver and fat suggests that
7-19
once formed, the metabolites of 2,3,7,8-TCDD are readily excreted. Thus, urinary and biliary elimination of 2,3,7,8-TCDD Is apparently dependent upon metabolism of the toxin. Although urine and bile appear to be free of unmetabollzed 2,3,7,8-TCDD, data from the hamster and rat Indicate that a significant amount (10-40%) of unchanged 2,3,7,8-TCDD may be excreted Into the feces. Unmetabollzed 2,3,7,8-TCDD thus appears to enter the Intestinal lumen by some route other than bile for a number of days following treat ment. These data suggest that the In. vivo half-life for elimination of 2.3.7.8- TCDD may not directly reflect the rate of 2,3,7,8-TCDD metabolism 1n a given animal (Neal et al., 1982, 1984). These data are consistent with the observation of Manara et al. (1982) that the lethal effects of 2,3,7,8TCDD were decreased in C57B1/6J mice regardless of whether the compound was administered by gavage or 1ntraper1toneal Injection 1f the animals were given diets containing activated carbon. 7.5. SUHMARY
Exposure to 2,3,7,8-TCDD occurs by Inhalation, dermal or GI absorption. Inhalation exposure to detectable levels of 2,3,7,8-TCDD 1s less likely because of low vapor pressure of this compound; however, Inhalation exposure could result from Inhalation of mist, dust or other contaminated particulate matter. Monitoring of atmospheric dust 1n the Seveso area detected 2,3,7,8TCDD levels ranging from 0.06-2.1 ng 2,3,7,8-TCDD/g airborne dust (D1Domen1co et al., 1980b). This corresponds to an estimated 24-hour Inhalation exposure of 1.4 pg assuming an average Intake of 10 m3 air containing 0.14 mg dust/m3. No studies on the systemic absorption of 2.3.7.8- TCDD have been performed, so the significance of this route of exposure 1n contaminated areas cannot be assessed.
7-20
2,3,7,8-TCDD As readily absorbed under experimental conditions (vide ante) and following environmental contamination (Cockerham et al., 1980; Fanelll et al., 1980c; Walsh, 1977). Afteliding absorbed, 2,3,7,8-TCDD 1s rapidly distributed to tissues with a high I1p1d content (fat, skin, adrenals). In most species studied, the major storage site for 2,3,7,8-TCDD 1s the Uver (see Table 7-3). 2,3,7,8-TCDD exposure results 1n Induction of MFO activity and a proliferation of smooth endoplasmic reticulum, the major subcellular storage site for 2,3,7,8-TCDD (Section 8.1.1.5.). The ability of 2,3,7,8-TCDD to produce this effect has been correlated with the sensi tivity of various strains of mice to 2,3,7,8-TCDD toxicity (Van Miller et al., 1976; Poland and Glover, 1980).
2,3,7,8-TCDD appears to be distributed throughout the body and stored largely as the parent compound (Olson et 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; Neal et al., 1984). Studies have also Indicated that 2,3,7,8-TCDD was metabolized by the hepatic cyto chrome P-450 monooxygenase system. The structures of six metabolites In the dog (Polger et al., 1982b) and two 1n the rat (Sawahata et al., 1982) have been elucidated; however, the structure of the metabolites of 2,3,7,8-TCDD have not been determined for the other species studied. Although some [1,6-3H]-2,3,7,8-TCDD-der1ved radioactivity was capable of binding cova lently to cellular macromolecules (Guenthner et al., 1979b; Nelson et al., 1977; Poland and Glover, 1979), metabolism of 2,3,7,8-TCDD seems to be predominantly a detoxification process (Beatty et al., 1978; Polger et al., 1982a).
7-21
2,3,7,8-TCDD and Its metabolites are excreted from the body by a variety of mechanisms. Lactatlng rats excrete 2,3,7,8-TCDD In the milk (Moore et al., 1976). 2,3,7,8-TCDD, 1,2,3,7,8-PeCDD and 1,2,3,4,6,7,8-HpCDD and OCDD have been detected 1n human milk samples from Swedish and German mothers (Rappe et al., 1985). These Investigators could detect 1,2,3,4,7,8-, 1,2,3,6,7,8- and 1,2,3,7,8,9-HxCDDs only 1n the mothers' milk from Sweden. Piper et al. (1973) reported the excretion of [l4C]2,3,7,8-TCDD-der1ved radioactivity 1n the feces, urine and expired air of rats given a single oral dose of 50 yg/kg. Over a 21-day period, 53, 13 and 3% of the admin istered radioactivity was eliminated through the feces, urine and expired air, respectively. This pattern of excretion seems typical of most species studied, with the exception of the hamster, which was observed to excrete 41% of the 2,3,7,8-TCDD-der1ved radioactivity In the urine (Olson et al., 1980a). In all species so far studied, metabolism and excretion are rela tively slow processes, with the observed Initial half-Hves 1n experimental animals on the order of a few weeks (see Table 7-4).
7-22
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
2.3.7.8- TCDD. A summary of the single dose LD^q data for 2,3,7,8-TCDD 1s
presented 1n Table 8-1. The dose that results 1n death varies extensively
with species, with the male guinea pig being the most sensitive species
tested (LDgg of 0.6 yg/kg) (Schwetz et a!., 1973), and the male hamster
the least sensitive species tested (LDcn of 5051 yg/kg) (Henck et al.,
1981). The rat and monkey appear to be the second most sensitive species,
with LD5Qs between 22 and 70 yg/kg (Schwetz et al., 1973; McConnell et
al., 1978a), while other species tested (rabbit and mouse) had LDggS
between 114 and 283 yg/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-TCDD, while Beatty et al. (1978) found adult female and weanling
male rats more sensitive than adult male rats (Table 8-1). In C57B1/10
mice, Smith et al. (1981) reported adult males to be far more sensitive to
the acute toxicity of 2,3,7,8-TCDD than adult females. Thus, data on sex
differences 1n sensitivity to the acute toxicity of 2,3,7,8-TCDD are con
flicting and may depend on the species or strain examined.
Harris et al. (1973) studied the toxic effects of 2,3,7,8-TCDD 1n rats,
mice and guinea pigs with regard to single or multiple exposures. Similar
effects were observed after a single exposure to 2,3,7,8-TCDD as were
observed when multiple exposures totaled the same dose as received 1n the
single exposure. As Illustrated most clearly 1n rats, a single dose of 25
yg/kg, 6 weekly doses of 5 yg/kg, or 30 dally doses of 1 yg/kg were
8-1
TABLE 8-1
Lethal Doses of 2,3,7,8-TCDD Following Acute Exposure
8-2
Specles/Straln Sex/No./Group
Route/ Vehicle
Dose Tested (vg/kg)
Duration of Observation
Guinea pigs/ Hartley
Guinea pigs/ Hartley
Guinea pigs/ Hartley
H/NR H/NR M/9
gavage/corn oil-acetone (9:1)
gavage/corn o11-acetone (9:1)
gavage/ corn oil
NR NR NR
2-8 weeks 2-8 weeks 30 days
Guinea pigs/ Hartley
F/6
Guinea pigs/ Hartley
F/6
Rats/ Sherman
M/5-10
Rats/ Sherman
Rats/SpragueDawley
Rats/SpragueDawley
F/NR H/6 F/6
gavage/ corn oil
gavage/ methyl cellulose
gavage/corn oil-acetone (9:1)
gavage/corn o1l-acetone (9:1) l.p./ol1ve oil
1.p./olive oil
0.1
0.5 2.5 12.5 20.0
0.1
0.5 2.5 12.5 20.0
8 16 32 63
NR
NR
NR
42 days
12 days
2-8 weeks 2-8 weeks 20 days 20 days
LOSO (vg/kg)
Comments
Reference
0.6 (0.4-0.9)*
Time to death was 5-34 days, the 2,3,7,8-TCDD was 91X pure
Schwetz et al., 1973
2.1 (1.5-3)*
Time to death was 9-42 days, the 2,3,7,8-TCDD was 99X pure
Schwetz et al., 1973
2
2.5 (1.2-5.4, 95X confidence)
Median time to death was 17-20 days, marked weight loss, thymus atrophy, Intestinal hemorrhage, no porphyria and only mild liver Injury
Time to first death was 32 days In the 2.5 vg/kg group, with 50X mortality by day 42
McConnell et al., 1978b
Sllkworth et al.. 1982
19 (15-23, 95X confidence)
Time to first death was 12 days 1n the 20.0 vg/kg group, with 67X mortality by day 42
Sllkworth et al., 1982
22
Time to death was 9-27 days, the
Schwetz et al.,
2,3,7,8-TCDD was 91X pure
1973
45 (30-66)*
60
25
Time to death was 13-43 days, the 2,3,7,8-TCDD was 91X pure
LD$o (vg/kg, mean + SE) adult male, 60.2 7.8; weanling male, 25.2 1.4
Adult female had a mean SE of 24.6 f 2.0 vg/kg
Schwetz et al., 1973 Beatty et al., 1978
Beatty et al., 1978
TABLE 8-1 (coni.)
8-3
Spedes/Straln Sex/No./Group
Route/ Vehicle
Dose Tested (wg/kg)
Duration of Observation
Honkey/rhesus
F/3
N1ce/C57B1
H/14
gavage/ corn oil
gavage/corn oil-acetone (9:1)
0 70 350
0 100 150 200
>35 days 60 days
ld50 (wg/kg) <70
114
H1ce/C57B1
H/9
gavage/ corn oil
NR 30 days
283.7
H1ce/C57B1/10
N/5
N1ce/C57B1/I1)
F/5
H1ce/C57B1/6J mce/DBA/?J H1ce/B6D2Fi/J
n/NR M/NR n/NR
gauge/ arachls oil
gauge/ arachls oil
1.p./ol1ve oil 1 .p./olive oil 1.p./olive oil
85 107 135 170 213
85 107 135 170 213 269 338 426 536
NR
NR
NR
45 days 45 days
30 days 30 days 30 days
146 >450
132 620 300
Comments
Reference
Weight loss, edema, severe thymus atrophy, loss of hair, mild liver damage
Time to death 1n the high dose group was 15-20 days, bw loss, edema In 25% of treated animals, severe thymic and spleen atrophy, hemor rhage In the region of the eye and small Intestine, liver necrosis In the centrllobular region
Redlan time to death was 22-25 days, dose-related bw loss, thymic atrophy, Increased liver weight and porphyria, gross and historic liver alterations, subcutaneous edema. Intestinal hemorrhage
95% confidence limits of 111-211 tig/kg. Host deaths occurred from 22-26 days after dosing. Signs of porphyria, edema, hemorrhage.
HcConnell et al., 1978a Vos et al., 1974
RcConnell et al., 1978b
Smith et al., 1981
1 of 4 animals died at dose of 426 ug/kg
Smith et al., 1981
BG02Fy/J mice are the offspring of C57B1/6J and DBA/2J.
The BGD2j/J mice are heterozygous at the Ah locus.
No comment
6as1ew1cz et al., 1983a,b
Gaslewlcz et al., 1983a,b
Gaslewlcz et al., 1983a,b
TABLE 8-1 (cont.)
8-4
Specles/Straln Sex/No./Group
Route/ Vehicle
Oose Tested (pg/kg)
Duration of Observation
LO50 (ug/kg)
Comments
Reference
Rabbits/ New Zealand Rabbits/ New Zealand
Rabbits/ New Zealand
Hamster/ golden Syrian
Hamster/ golden Syrian
Hamster/ golden Syrian
Dogs/Beagle
Dogs/Beagle
M&F/NR M.F/5
gavage/corn oil-acetone (9:1)
I.p./ corn oil
M F /NR
dermal/ acetone
H/6 gavage/corn oil-acetone
(9:1)
MF/5-6
I.p./ olive oil
H/5 gavage/ olive oil
H/2 gavage/corn oil-acetone (9:1)
F/2 . gavage/corn oil-acetone (9:1)
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
2-8 weeks 4 weeks
115 (38-345)
NR
3 weeks
275 (142-531)*
Time to death was 6-39 days, the 2,3,7,8-TCOD was 91X pure
Time to death was 6-23 days, 2-3 anlmals/group died In all but the low exposure group
Time to death was 12-22 days
Schwetz et al., 1973
Schwetz et al., 1973
Schwetz et al., 191?
55 days
50 days 50 days 2-8 weeks 2-8 weeks
5051 (3876-18,487, 95X confidence)
Time to death was 26-43 days, the liver and thymus appeared to be the primary target organs, only 1 death occurred In the 300 and 3000 ug/kg group
Henck et al., 1981
>3000 1157 NA
Significant, dose-related decrease In thymus weight starting at 500 ug/kg, only 2 deaths occurred out of 11 hamsters In the 3000 ug/kg group.
Death generally occurred between 24 and 45 days, decrease In bw above 2000 ug/kg, proliferative Ileitis with mild to severe Inflammation
All animals died
Olson et al., 1980b
Olson et al.. 1980b
Schwetz et al., 1973
NA All animals survived
Schwetz et al.. 1973
`The number In parentheses appears to Indicate the range of lethal doses; however, the article did not specify what these numbers represented. I.p. = Intraperitoneal; NR * Not reported; NA Not applicable
a l l t h e t h r e s h o l d d o s e f o r observing a decrease 1n body weignt. in general, other endpoints, Including lethality, decrease 1n thymus weight, and a no effect level for body weight change 1n rats, mice and guinea pigs required a specific threshold level regardless of whether this level was achieved through a single exposure or a small number of multiple exposures.
Although 2,3,7,8-TCDD has over a 103-fold difference 1n toxicity depending upon the species tested, some of the signs of lethal toxicity were the same regardless of species. One of the most characteristic observations after acute lethal exposure to 2,3,7,8-TCDD was the protracted time between exposure and death (see Table 8-1). In determining the LD5Q 1n the least sensitive animal, the hamster, the test animals died between 24 and 45 days after a single acute exposure (Olson et al., 1980b), and similar observa tions were made 1n all other species tested Including the most sensitive species, the guinea pig, 1n which animals died up to 42 days after treatment (Schwetz et al., 1973).
During this extended period between treatment and death the animals had poor weight gain or loss of weight resulting 1n a "wasting syndrome" that resembled starvation. Though weight loss 1s the primary general feature observed 1n adult rats, 1n the young animals depletion of body fat results 1n lean tissue formation (Peterson et al., 1984) In female Wlstar rats Intubated with 2,3,7,8-TCDD at a dose of 100 yg/kg, the weight loss was blphaslc (Courtney et al., 1978). The Initial weight loss occurred rapidly during the first 7-10 days after treatment and was associated with decreased food and water consumption. This Initial phase of weight loss was reversed with the resumption of normal food Intake for 4 or 5 days, only to be fol lowed by a second, more gradual, decline 1n food and water Intake and weight until death. Providing animals with an adequately nutritious liquid diet
8-5
by Intubation did not appreciably alter the pattern of weight loss nor affect survival. In contrast, Gaslewlcz et al. (1980) observed that provid ing rats with total parenteral nutrition would prevent some of the weight loss Induced by 2,3,7,8-TCDD; however, there was no protection from the lethal effects of 2,3,7,8-TCDD. Seefeld and Peterson (1983) and Seefeld et al. (1984) found that a reduction In food Intake caused by 2,3,7,8-TCDD 1s primarily responsible for the loss of body weight or depressed growth rate of rats. Pair-fed control rats lost weight at the same rate and to the same extent as their weight-matched 2,3,7,8-TCDDtreated partners (25 or 50 yg/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 yg/kg groups was 33 and 75%, respectively, while 1n the corresponding pair-fed groups the mor tality was 0 and 15%. The authors proposed a hypothesis that 2,3,7,8-TCDD lowers a regulated level or "set-point" for body weight control 1n the rat. The ensuing change 1n food Intake was thought to occur secondarily to the change 1n set-point (Seefeld and Peterson, 1983; Seefeld et al., 1984; Peterson et al., 1984). Vitamin A or E did not protect or Inhibit the decrease 1n body weight, respectively. Further, these vitamins provided little protection against 2,3,7,8-TCDD-1nduced lethality 1n rats (Hassan et al., 1985).
Also, severe thymic atrophy Is universally observed In all species given lethal doses of 2,3,7,8-TCDD, and since weight loss and thymic atrophy are both associated with malnutrition, van Logten et al. (1981) Investigated the effects of dietary protein on the toxicity of 2,3,7,8-TCDD. Groups of female Fischer 344 rats administered 2,3,7,8-TCDD (20 yg/kg) and main tained on low (3.5%), normal (26%) or high (55%) protein diets maintained
8 -6
approximately the same amount of weight (-0.2+3, 7+6 and 7+3 g for each dietary group, respectively) during the subsequent 10-day period. The weight gain In treated animals was 10-18 g less than that 1n the respective control rats. Dietary protein also had no effect on preventing or enhancing the 2,3,7,8-TCDD Induced thymic atrophy. Although weight loss and thymic atrophy were present In most species tested, there were other symptoms that were characteristic of toxicity In only some species.
In the guinea pig, besides thymic atrophy, no gross changes were observed 1n Internal organs after a lethal oral or l.p. dose of 2,3,7,8-TCDD (Grelg et al., 1973, Gupta et al., 1973). Hemorrhages were observed 1n a number of organs Including the adrenal gland, urinary bladder, GI tract and mesenteric 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 1n the thymus, spleen and lymph nodes, and hemorrhages observed 1n many organs. In addition, marked hyperplasia of the urinary bladder was observed. Of particular Interest was the absence of severe toxic effects on the Uver. Gross observation under UV light Indicated no excess of porphyrin, while histologic examinations revealed diffuse single cell necrosis. Identical observations were made by McConnell et al. (1978b) 1n guinea pigs administered lethal doses of 2,3,7,8-TCDD, with the additional observation that the sternal bone marrow was hypocellular 1n all types of blood-forming cells.
Turner and Collins (1983) described some histologic changes In the Uver of guinea pigs treated with 2,3,7,8-TCDD. Groups consisting of 4-6 female Hartley guinea pigs were treated with 2,3,7,8-TCDD at doses of 0.0, 0.1, 0.5, 2.5, 12.5 or 20 vg/kg, and 1 male guinea pig each was treated with a dose of 0.1 or 0.5 vg/kg. The 2,3,7,8-TCDD was administered by gavage as
8-7
an aqueous suspension 1n 0.75% 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 polychlorinated biphenyls and chlorinated benzenes (1, 10, 100 and 500 mg/kg). The histologic observations as described were applied 1n general to both treatment groups and there was no apparent relationship between dose and response. At the light microscope level, hepatocellular hypertrophy, steatosis, focal necrosis, cytoplasmic degeneration and acido philic hyal1n-Hke cytoplasmic Inclusion bodies were observed. Even though there was no dose-response relationship for these Uver lesions, the doses spanned a range that resulted 1n the lowest dose being nonlethal (none of the 4 female guinea pigs died during the study), while 1n the high dose group 4 of 6 animals died before 42 days post-treatment. The L0gQ for female guinea pigs was determined 1n this study to be 2.5 or 19 yg/kg bw depending on whether the compound was administered by gavage 1n corn oil or 1n aqueous methyl cellulose (Sllkworth et al., 1982).
The greatest difference at necropsy 1n the gross and histologic effects In rats and mice of exposure to lethal doses of 2,3,7,8-TCDD was pathologic alterations In the Uver, as compared with guinea pigs. An early report by Buu-Ho1 et al. (1972) described alterations 1n the architecture of the Uver of rats within 5 days of receiving a low dose of 2,3,7,8-TCDD (10 yg/kg by 1.p. Injection). At higher oral doses of 100 or 50 yg/kg, which killed 43 and 7% of the animals, respectively, Gupta et al. (1973) also observed marked distortion of Uver architecture 1n rats; however, only mild regener ative changes of the Uver were observed at the sublethal dose of 5 yg/kg administered weekly for 6 weeks. Liver toxicity appeared to develop slowly In the rat with no change 1n Uver function, as Indicated by plasma protein
8 -8
and b i l i r u b i n l e v e l s , or a l k a l i n e phosphatase, g lu ta m lc -o x a la c e tlc tra n s
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 (Grelg et al., 1973). Bilirubin levels were, however, markedly elevated from 0.33 yg/100 ml 1n control animals to 10.97 yg/100 ml 1n treated animals 21 days after exposure (the other parameters were not measured at this time, although plasma protein was slightly but significantly decreased when determined 9 days post-treatment). As 1n rats, the livers of mice exposed to lethal levels of 2,3,7,8-TCDD had signs of necrotic changes (Vos et al., 1974); however, Jones and Grieg (1975) reported that the centrllobular necrosis, bile duct proliferation and Upld accumulation were more extreme 1n mice than 1n rats. Examination of mouse livers using long wave UV light showed fluorescence suggestive of excess porphyrin accumulation (McConnell et al., 1978b). Although excess porphyrins may be present 1n the livers from 2,3,7,8-TCDD-exposed rats, fluorescence Is not usually observed.
Besides effects on the liver, 2,3,7,8-TCDD exposure produced other toxic effects 1n rats and mice that were not observed or were observed to a lesser extent 1n guinea pigs. In rats that died from 2,3,7,8-TCDD exposure, there were extensive hemorrhages of the heart, Uver, 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 necrotic spermatocytes and spermatozoa present (McConnell et al., 1978b; Vos et al., 1974). Death 1n mice was frequently attributed to terminal hermorrhages (Vos et al., 1974).
In monkeys exposed to lethal levals of 2,3,7,8-TCDD, McConnell et al. (1978a) reported clinical and histologic signs of toxicity, some of which
8-9
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 1n guinea pigs, Uver Injury appeared to be mild; however, Increased serum GOT and aldolase activity and decreased albumin levels Indicative of Uver 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 toxicity, v/h1ch were observed 1n other species as well, monkeys had progressive loss of hair, toenails 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) summarized the toxic response of various species to 2,3,7,8TCDD 1n 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 LDgQ for nine congeners of PCDD following a single treatment by gavage 1n mice and guinea pigs. A comparison of the LDC_ expressed as ymol/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 1n a chlorine from any one of these posi tions. It also appears that addition of one or more chlorines to 2,3,7,8TCDD results 1n a decrease 1n lethality. Although the congeners vary 1n effective dose between mice and guinea pigs, the relative order of toxicity of these congeners did not change. Also, similar effects of toxicity were observed for all congeners as described above for 2,3,7,8-TCDD when the com parison was made within a single species.
8-10
TABLE 8-2
Toxic Responses Following Exposure to 2,3,7,8-TCDD: Species Differences8
8-11
M o n k e y Guinea Cow** Rat M o u s e R a b b i t b Chicken** H a m s t e r Pig
Hyperplasia and/or metaplasia
Gastric mucus Intestinal mucosa Urinary tract Bile duct and/or gall Lung: focal alveolar Skin
bladder
f+C
f 4-
ff
0 f0
44* f f 04
0
0 *d 0
0
0
0
4*4-
0 4-4-
0
0
Hypoplasia, Atrophy or Necrosis
Thymus Bone marrow Testicle Other Liver lesions Porphyria Edema
4-
40
fff 4 f 4-
4-4- f 0 f 4-40 0f
ff
4f f
f 4+f
4-
f 0 4-
References:
monkey (McConnell et al., 1978b; Norback and Allen, 1973; Allen et al., 1977); guinea pig ( M c C o n n e l l et a l ., 197 8 b ; M c C o n n e l l , 1980; M o o r e et a l . , 1 9 7 9 ; T u r n e r a n d C o l l i n s , 1 9 8 3 ) ; cow (McConnell, 1980); rat (McConnell, 1980; Koclba et al., 1978a; Koclba et al., 1979); mouse (Schwetz et al., 1973; McConnell et al., 1978b; Vos et al., 1973); rabbit (Klmmlg and Schultz, 1957; Schwetz et al., 1973; Vos and Beems, 1971); chicken (Schwetz et al., 1973; Norback and Allen, 1973; Allen and Lalich, 1962; Vos and Koeman, 1970); hamster (Olson et al., 1980b; Henck et al., 1981).
^ R e s p o n s e s f o l l o w e d e x p o s u r e to 2 , 3 , 7 , 8 - T C D D or s t r u c t u r a l l y r e l a t e d c h l o r i n a t e d a r o m a t i c h y d r o c a r b o n s .
c S y m b o l s : 0, l e s i o n no t o b s e r v e d ; +, l e s i o n o b s e r v e d ( n u m b e r of
denote severity); , lesion observed
to a very limited extent; blank, no e v i d e n c e r e p o r t e d In literature.
^Skln lesions 1n cattle are observed, but they differ f r o m the skin lesions o b s e r v e d 1n other species.
Adapted from Poland and Knutson, 1982.
TABLE 8-3 Estimated Single Oral LD50 - 30 Values for PCDDs3
Chlorination of PCDDs
Guinea Pigs (ymol/kg)b
2.8 2,3.7 2 ,3,7,8 1 ,2 ,3,7,8 1 ,2 ,4,7,8 1 ,2 ,3,4,7,8 1,2,3,6 ,7,8 1,2,3,7,8,9 1 ,2 ,3,4,6 ,7,8
>1180 120.41 0.006 0.009 3.15 0.185
0.178-0.255c 0.153-0.255c
>1.400
aSource: McConnell et al., 1978b bSpearman-Karber method cEst1mated range due to variability 1n replicates NR = Not reported
Mice (ymol/kg)b
NR >10
0.88 0.94 >14 2.11 3.19 >3.67 NR
8-12
0 . I . I..
errc o i a u n m e l i li\
I 11 C
I I I <) V V v y I V U I
changes 1n the Uver 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
2.3.7.8- TCDD at 0.0, 5 and 25 yg/kg by gavage. The animals were killed In
groups of 5 on days 1, 3, 6, 9, 16 and 28 after treatment and the livers
were prepared for histologic examination. The major ultrastructural change
observed was a dose-related Increase 1n the smooth and rough endoplasmic
reticulum (ER) In cells near the bile canallcull. 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 1n Uver cells following
2.3.7.8- TCDD treatment would be consistent with the Induction of protein and
RNA synthesis.
Transmission electron microscopic observations revealed that single 1.p.
administration of 20 yg/kg of 2,3,7,8-TCDD 1n Sprague-Dawley male rats
produces necrotizing hepatic lesions that become progressively worse up to
the 16th week postexposure followed by gradual Improvement of the condition
and disappearance of the lesions (Weber et al., 1983).
At higher doses of 200 yg/kg, Jones and Butler (1974) observed necro
sis and proliferative changes 1n the Uver of rats to be the predominant
lesions. After treatment by gavage, groups of 4 male and 4 female rats were
killed and examined on a weekly basis for 10 weeks. By the first week,
degenerating cells were observed near the central vein and these lesions
progressed to areas of focal necrosis by the sixth week. Superimposed on
8-13
the necrotic changes were hyperplasia of the viable cells with multlnucle*'
ated cells common by the ninth week. At week 10 central vein fibrosis and scattered necrosis remained. Fine structure observed after this large dose of 2,3,7,8-TCDD also revealed Increases 1n smooth ER; however, the most striking effect was degeneration of the plasma membrane with the resulting fusion of parenchymal cells. In a study of similar design, Jones (1975) followed the distribution with time after treatment of membrane associated ATPase activity by hlstochemlcal 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 mldzonal 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-TCDD at 0.0, 10 or 25 iig/kg were used for deter mination of Na+ , K+-ATPase and Mg++-ATPase activity. The activity of Na+ , K+-ATPase was depressed to the same extent for both doses of 2,3,7,8-TCDD from day 2-40 after treatment, while a similar depression of the Mg++-ATPase activity was observed only 1n the high dose group. In the low dose group, there was a decrease 1n Mg*+-ATPase at 20 days, but recovery to normal levels occurred by 40 days post-treatment. It was demonstrated that the effect of 2,3,7,8-TCDD on ATPase activity was not the result of 2,3,7,8-TCDD Induced food deprivation and in vitro studies Indi cated that the loss of activity was not due to the direct Interference of
8-14
2 , 3 , 7 , 8 - T C D D with the enzyme. Q u a n tita tiv e changes (both Increases and decreases) have been reported for the protein composition of plasma membranes Isolated and analyzed by electrophoresis from Sprague-Dawley rats 10 days after an 1.p. Injection of 2,3,7,8-TCDD, Indicating that exposure was actually affecting membrane components (Brewster et al., 1982).
Peterson et al. (1979a) observed a positive correlation between the levels of LSM ATPase activity and both In vivo cumulative biliary excretion of ouabain and bile flow (yi/mln/g Uver). Using perfused Uver, however, Peterson et al. (1979b) reported a segregation between LSM ATPase activity and biliary excretion of ouabain when 2,3,7,8-TCDD rats were exposed to the protective agents pregnenolone-l6a-carbon1tr1le or splrenolactone. It was concluded that LSM ATPase did not directly participate 1n ouabain transport.
Additional studies have described the effect of 2,3,7,8-TCDD on the bil iary excretion of a variety of xenoblotlcs. Early studies by Hwang (1973) Investigated 2,3,7,8-TCDD Inhibition of biliary excretion 1n male CD rats given a single dose of 2,3,7,8-TCDD at 25 or 5 yg/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 1n the 25 yg/kg dose group at 16 days. Even with this Increased bile flow, however, the cumulative biliary excretion of ICG was decreased 1n a dose-dependent manner with the greatest depression observed 7 and 16 days after the exposure to 2,3,7,8-TCDD. The levels of ICG 1n the plasma and Uver was higher 1n treated animals than 1n control animals, while the concentration 1n the bile was lower, reflecting the decrease 1n total excretion of ICG.
8-15
Yang and Peterson (1977) compared the effect of 2,3,7,8-TCDD on the bil iary excretion of the organic neutral compound, ouabain, with that of the organic anions phenol-3,6-d1bromophthale1n (DBSP) and sulfobromophthaleln (BSP) 1n male Holtzman rats. Animals were Intubated with 2,3,7,8-TCDD at doses of 10 or 25 vg/kg and excretion was evaluated periodically between 2 and 4 days postexposure. The biliary excretion of ouabain was depressed In a dose-related manner starting on the second day post-treatment, with maxi mum depression developing between 10 and 20 days, and some recovery observed by day 40. Decreases 1n bile flow followed a pattern similar to that observed for ouabain. The pattern of biliary excretion was different for DBSP and BSP In which only a transient small decrease was observed 10 days after exposure 1n the high-dose group. In the low-dose animals there was actually an Increase at days 10 and 25 1n the excretion of the anions. The results obtained for DBSP and BSP differ sharply from those for the organic neutral ouabain or those reported by Hwang (1973) for the organic anion ICG, In which a dose-related decrease In biliary excretion was observed. The authors concluded that the effects of 2,3,7,8-TCDD on the multiple pathways Involved 1n biliary excretion depend on the specific compound being studied.
In the guinea pig and rhesus monkey, which develop little liver pathol ogy after exposure to 2,3,7,8-TCDD, there was also little change In ICG blood clearance rates; 1n the rabbit, which develops 2,3,7,8-TCDD-lnduced Uver 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 sorbitol dehydrogenase and glutamic pyruvic transaminase activity as further Indications of 2,3,7,8-TCDD-produced Uver damage. In the monkey, which received 2,3,7,8-TCDD by gavage at doses of 5, 25 or 75 yg/kg, there was an Initial slight Increase In the blood clearance of ICG at 2 days post
8-16
treatment, followed In the two higher-dose groups by a dramatic decrease a few days before death. Although some serum enzymes (sorbitol dehydrogenase and glutamic pyruvic transaminase) Indicative of Uver damage were elevated, the hlstopathology of the Uver was within normal limits. It appears that major effects on biliary excretion occur only 1n species that are sensitive to the hepatotoxlc effects of 2,3,7,8-TCDD.
Other gross signs of the hepatotoxlc effects of 2,3,7,8-TCDD observed 1n some species Included fatty degeneration and porphyria. Early observations by Cunningham and Williams (1972) described a decrease 1n Ui vivo (1 hour pulse) Incorporation of 3H sodium acetate Into Uver 11p1ds 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 yg/kg followed 1n either 3 or 7 days by the assessment of 11p1d synthesis. At 3 days Incorporation decreased from 258 to 98 dpm/mg 11p1d 1n the control and treated animals, respectively. There was an approximately similar decrease observed 7 days postexposure. When Individual classes of lipids were examined, there was a decrease 1n the synthesis of triglycerides, dlglycerldes and phospholipids. Although Cunningham and Williams (1972) observed that 2,3,7,8-TCDD decreased 11p1d synthesis, Albro et al. (1978) reported an Increase 1n total 11p1ds 1n the livers of rats 13 days after treatment with 2,3,7,8-TCDD at a lethal dose of 50 yg/kg. For Individual classes of lipids there was an Increase 1n free fatty acids and cholesterol esters; no change occurred 1n the content of phospholipids, free cholesterol or triglycerides. The fatty changes 1n the Uver were confirmed by ultrastructural examination of Uver specimens. At a sublethal dose of 10 yg/kg there was a different pattern of lipid accu mulation; triglycerides and fatty acids Increased and cholesterol esters decreased. The changes 1n the 11p1d profile of the Uver was attributed to
8-17
2.3.7.8- TCDD Induced mobilization of body fat, a decrease 1n lysosomal acid Upase (74% decline 1n this enzyme 10 days after a 50 yg/kg dose of 2.3.7.8- TCDD) and an Increase In lipid peroxidation as Indicated by a sharp Increase 1n the production of Upofuscln pigments.
Porphyria was Initially characterized quantitatively 1n mice by Goldstein et al. (1978). Groups of 12 male C57B1 mice received 4 weekly Intubations of 2,3,7,8-TCDD at doses of 0.0, 1, 5 or 25 yg/kg, or a single dose of 150 yg/kg followed 21-25 days after treatment by analysis of the Uver for porphyrins. Porphyrin levels were unchanged except 1n the 25 and 150 yg/kg groups where the levels were Increased 2000- and 4000-fold, respectively. The difference 1n responsiveness to the development of por phyria was studied by Smith et al. (1981) 1n C57B1 mice that were sensitive to, and DBA/2 mice that were Insensitive to, the toxicity of 2,3,7,8-TCDD. Hale and female C57B1 mice had a dose-related Increase 1n hepatic porphyrins 1n the two high dose groups 3 weeks after a single exposure to 2,3,7,8-TCDD at 0.0, 5, 15, 50 or 75 yg/kg; however, only minimal nondose-related changes 1n hepatic porphyrin were observed 1n DBA/2 mice exposed to up to 1200 yg/kg. In the sensitive C57B1 mice there was only a small difference 1n hepatic porphyrin between the sexes even though males were >3 times as sensitive to the toxic effects of 2,3,7,8-TCDD than females (see Table 8-1). Results similar to those above were reported for urinary porphyrin levels 1n male C57B1 and DBA/2 mice given 6 weekly doses of 2,3,7,8-TCDD 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 (Cantonl et al., 1981). Female CD rats were
8-18
administered weekly o ra l doses of 2 ,3 ,7 ,8-TCDD at levels of 0.01, 0.1 and
1.0 yg/kg for 45 weeks. The Initial Increase was observed 1n the high dose group at 3 months, and In the other two groups at 4 months, after the start of exposure. Not only did the absolute amount of porphyrin Increase, but the relative distribution also changed to compounds containing more carboxyl groups. Only 1n the high dose group did the livers, at the ter minal necropsy, show signs of excess porphyrin under examination by UV light.
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 porphyrin have been studied. Goldstein et al. (1978) showed that -aminolevulinic acid synthetase, a rate-limiting enzyme 1n porphyrin synthesis, was slightly Increased (2-fold) 1n male C57B1 mice given 4 weekly doses of 2,3,7,8-TCDD at 25 yg/kg. This dose of 2,3,7,8TCDD Increased Uver porphyrin levels 2000-fold. Catabolism of porphyrin by uroporphyrinogen decarboxylase (UD) also appeared to be decreased 1n 2,3,7,8-TCDD treated mice. Smith et al. (1981) reported a decrease 1n UD activity from ~25 to 7 n moles/hr/g Uver In male and female C57B1 mice 3 weeks after a single oral exposure to 2,3,7,8-TCDD at a dose of 75 yg/kg. No effect of 2,3,7,8-TCDD on UD activity was observed 1n DBA/2 mice that were Insensitive to the Induction of porphyria. A time course of changes 1n UD activity with length of time after exposure to 2,3,7,8-TCDD Indicated a steady decline In activity starting 3 days after exposure to 2,3,7,8-TCDD, which continued until day 21 when the study was terminated. Sweeney and Jones (1978) reported similar results after 5 weekly doses of 2,3,7,8-TCDD at 25 yg/kg. In this study the UD activity declined ~48% 1n C57B1 mice
8-19
and only 4% 1n DBA/2 mice. Other factors besides the Increase 1n -amino levulinic acid synthetase and the decrease 1n UD activity may also partici pate 1n the dramatic Increase 1n Uver porphyrin In mice associated with exposure to near lethal doses of 2,3,7,8-TCDD.
As a result of the protracted time observed between exposure to 2,3,7,8TCDD 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-TCDD on DNA synthesis 1n the Uver. Grelg et al. (1974) measured the in vivo Incorporation of 3H-thy-
i
midine (1 hour pulse) Into Uver DNA of male and female Porten strain rats after a single exposure to 2,3,7,8-TCDD at doses of 10 and 200 yg/kg. When the 2,3,7,8-TCDD was given either 0, 24 or 72 hours before a 3/4 partial hepatectomy there was only a slight, but not significant, decrease 1n thymidine Incorporation observed when DNA synthesis was measured 24 hours after the operation.
Although 2,3,7,8-TCDD had no effect on 1_n vivo DNA synthesis, similar studies by Conway and Matsumura (1975) and Dickens et al. (1981) demon strated an Increase 1n thymidine Incorporation when determined in vitro. Conway and Matsumura (1975) administered male Sprague-Dawley rats 2,3,7,8TCDD at a dose of 5 yg/kg followed 1n 10 days by removal of the Uver and the in vitro determination of DNA synthesis 1n Uver slices. Incorporation of thymidine Into the nuclei Increased from 29 cpm/mg 1n control animals to 45 cpm/mg 1n treated animals. A similar near doubling of DNA synthesis was observed by Dickens et al. (1981); however, when DNA synthesis was stimu lated by a 1/3 partial hepatectomy, thymidine Incorporation Into liver slices was Increased 10-fold 1n rats treated 5 days earlier with 2,3,7,8TCDD as compared with hepatectomlzed controls. The onset of DNA synthesis
8-20
after partial hepatectomy (~20 hours) was the same In both 2,3,7,8-TCDD treated and control animals; however, the treated animals had a more rapid and extensive increase in DNA synthesis between 20 and 32 hours after the partial hepatectomy. The rates of DNA synthesis were again the same in 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 1m viltro and in vivo measurements of DNA synthesis in the liver after exposure to 2,3,7,8-TCDD.
Extensive hepatic necrosis in the rabbit may be responsible for death in
this species (Poland and Knutson, 1982). j
Besides the effects on the liver of 2,3,7,8-TCDD exposure described above, it Is known that 2,3,7,8-TCDD is a potent inducer of microsomal enzymes. These studies will be discussed in Section 8.1.1.5., which describes the ability of this xenobiotic to induce microsomal enzymes in a number of tissues and organs.
8 .1.1.3. EFFECTS ON OTHER ORGAN SYSTEMS -- The most noticeable feature of 2,3,7,8-TCDD toxicity 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-arginine and L-histidine using the everted intestinal sac technique 1n CD-I mice exposed to 2,3,7,8-TCDD. In measurements made 7 days after treatment with doses of 0.0, 10, 25, 75, 150, 200 or 300 yg/kg, D-glucose was absorbed to a lesser degree at all doses than in control animals. The two low doses produced a dose-related decrease in absorption; however, at doses of >75 yg/kg the decrease was
8-21
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 malabsorption. Treatment with 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 malabsorption of D-glucose. In this study, however, absorption of leucine was also decreased. The decrease in leucine absorp tion took longer to manifest itself; a significant decrease was observed only after 2 weeks treatment with 2,3,7,8-TCDD.
In contrast to the results observed for D-glucose, intestinal iron transport was shown to be elevated by exposure to 2,3,7,8-TCDD. Manis and Kim (1979a) examined the effect of prior treatment of male Sprague-Dawley rats on the 30-minute transport of 59Fe out of a duodenal loop created by ligating a 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 yg/kg the increase was ~10054. The time after treatment at which serosol transfer was greatest was 1 day, with rapid decline in 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 i.p. injection. In similar experiments calcium transport was decreased, and glulactose and proline transport were unaffected by prior
8-22
e x p o s u r e to 2 , 3,7,8-TCDD. Manls and K1m (1979b) had Identical results when
the everted Intestinal sac was used to assess Iron transport. It was Interesting to note that only duodenal sacs were stimulated, with no effect of 2,3,7,8-TCDD exposure observed 1n the adjacent distal segment of the Intestine. Increased Iron transport was also observed by Manls and K1m (1979a) 1n an unidentified strain of mice. Increased Iron transport may be one of the earliest effects of 2,3,7,8-TCDD; however, at present the toxico logic relevance of this transient disturbance 1n Iron transport 1s unknown.
One of the common gross observations of 2,3,7,8-TCDD toxicity 1s severe edema, suggestive of a breakdown 1n salt and water homeostasis. 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 In 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 yg/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 1n the high dose group; cation transport was lower at both dose levels tested. The decrease 1n anion transport was con firmed 1n an 1_n vivo study. Ammon1ogenes1s and gluconeogenesls were not affected 1n 2,3,7,8-TCDD treated rats, even when the animals were made acldotlc, which suggests no effect on the kidneys' ability to maintain acid base balance. Also, sodium reabsorption was shown In vivo to be within normal range. Since decreases 1n cation and anion transport were the only effects observed, and since these compounds are transported by a different mechanism, the authors concluded that the effect of 2,3,7,8-TCDD was merely
8-23
a general decrease 1n kidney functon reflecting the poor condition of the treated animals (animals 1n all treated groups had decreased weight gain), and not a cause of debilitation.
Although kidney function was only minimally affected by exposure to 2.3.7.8- TCDD, Grelg et al. (1974) demonstrated that pre-exposure to 2,3,7,8TCDD could reduce the ability of the rat kidney to respond to stimuli of DNA sythesls. Folate-stimulated DNA synthesis measured 1_n vivo 1n Porten strain rats was decreased between 67 and 25% 1n animals receiving 2,3,7,8-TCDD at a dose of 10 yg/kg on day 0-9 before administration of folic acid. No sig nificant difference 1n folate-stimulated DNA synthesis was observed 1f 2.3.7.8- TCDD was given 23 hours after folic acid. The lack of effectiveness of administering 2,3,7,8-TCDD shortly after treatment with folic acid sug gested that 2,3,7,8-TCDD did not directly Interact with cellular DNA, nor Inhibit the protein synthesis necessary to support folate-stimulated 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 1s not known, although 1t was demonstrated that another agent capable of Inducing microsomal enzymes, 3-methylcholanthrene (3-HC), had similar effects on the kidney.
Additionally a number a hematologic and clinical chemistry changes have been observed 1n the blood of laboratory animals after exposure to 2,3,7,8TCDD. Many of these changes, as described by Z1nkl et al. (1973), reflect damage to previously described organ systems. In female CD rats given 30 dally doses of 2,3,7,8-TCDD at levels of 0.1, 1.0 or 10 yg/kg, the clini cal chemistry of the serum reflected Uver damage. In the high-dose group, serum GOT and serum GPT were elevated starting 13-17 days after Initial
8-24
t r e a t m e n t . T h e r e w a s a marginal change 1n GPT 1n the mid-dose group and
lactic dehydrogenase (LDH) 1n the high-dose group, but the Increases were only transitory. Serum cholesterol was Increased 1n the high-dose animals starting at day 10, with a transitory Increase again observed 1n the mid dose group. Conversely, there was a decrease 1n serum protein from day 24 on 1n the high-dose animals. Along with these clinical chemistry changes Indicative of liver damage, the only other major effect observed 1n the blood was thrombocytopenia. The decrease 1n platelet count was detected early, by day 3, 1n the 10 and 1 yg/kg groups; 1n the 0.1 yg/kg group a significant decrease was not observed until day 17. Thrombocytopenia was also observed 1n female guinea pigs after 8 weekly oral doses of 2,3,7,8TCDD at 0.2 yg/kg, and 1n mice (administered a single dose of 1.0, 10 or 50 yg/kg). In guinea pigs lymphopenia was also observed. Other hemato logic changes were attributed to hemoconcentratlon.
In a more extensive Investigation of 2,3,7,8-TCDD-1nduced hyperlipidemia In male Sprague-Dawley rats. Poll et al. (1980) treated animals with a single 1.p. Injection of 2,3,7,8-TCDD at 2 doses of 2.5, 5, 10 and 20 yg/kg. At day 21 after treatment there was a dose-related Increase 1n total plasma cholesterol and high density lipoprotein cholesterol, while no change was observed In triglycerides or very low and low density lipopro teins (VLDL and LOL, respectively). At a dose of 20 yg/kg the maximum Increase 1n HDL cholesterol and total cholesterol occurred 30 days after treatment, and a significant elevation was still present at 60 days after treatment when the study was terminated. Slight changes 1n the apoprotein of HDL from 2,3,7,8-TCDD rats and control rats were Indicative of new apo protein synthesis. Although the Increases 1n HDL cholesterol may be 1n response to eliminating excess lipids, the exact function has not been
8-25
clearly shown. There 1s some evidence from studies of workers exposed to 2.3.7.8-TCDD that there were reduced levels of blood HDL cholesterol and raised total cholesterol as compared with a matched control group (Walker and Martin, 1979).
In contrast to rats, male Hartley strain guinea pigs given a single l.p. Injection of 2,3,7,8-TCDD at a dose of 2 yg/kg had Increased hyperlipide mia characterized by Increases 1n VLDL and LDL (Swift et al., 1981). In animals sacrificed 7 days after exposure to 2,3,7,8-TCDD, there was an Increase 1n total serum lipid, cholesterol esters, triglycerides and phos pholipids, when comparison was made with pair-fed, weight-paired or ad libitum 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 acids that were abundant 1n the adipose tissue of guinea pigs. Anaylsls of lipoproteins revealed a 19-fold Increase 1n VLDL, a 4-fold Increase In LDL, and no change observed In the levels of HDL. The VLDL was also qualitatively different 1n the 2,3,7,8-TCDD treated animals, containing less cholesterol ester and an altered C apoprotein. The Import ance of these qualitative changes 1s unclear. The hyperlipidemia may result from the 2,3,7,8-TCDD mobilization of free fatty acids, which are then used 1n the synthesis of VLDL and are subsequently formed Into LDL. The rela tionship of the changes 1n serum lipid levels to the mechanism of 2,3,7,8TCDD toxicity needs further study.
Elovaara et al. (1977) observed some changes 1n blochemlcals of the brain of male Wlstar and heterozygous Gunn rats given a single Intubation of 2.3.7.8- TCDD at a dose of 20 yg/kg. At 7 days post-treatment, there was a small but significant decrease as compared with vehicle treated control animals 1n both the protein and RNA content of the Wlstar rats, while levels
8-26
of a c id p r o te in a s e and D T-dlaphorase (an enzyme Induced by 2 ,3 ,7 ,8 -T C D D In
the liver) had a small but significant Increase 1n the heterozygous Gunn rats. There were no significant changes observed 1n homozygous rats given 2.3.7.8-TCDD at 20 yg/kg. The authors noted that acid proteinase may participate 1n chemically Induced degeneration of the brain.
8 .1.1.4. IMMUNOLOGICAL EFFECTS -- During acute toxicity studies with 2.3.7.8- TCDD, thymic atrophy was noted as a consistent effect 1n all species that have been Investigated. This finding suggested that 2,3,7,8-TCDD may alter the Immune response, and Initiated 1mmunotox1c1ty studies 1n exposed animals. In guinea pigs treated with 8 weekly oral doses of 2,3,7,8-TCDD (0, 0.008, 0.04, 0.2 or 1.0 yg/kg bw), body weight, spleen weight and thymus weight were depressed, adrenal weight was Increased and leukocyte and lymphocyte counts were elevated (Vos et al., 1973). Upon histological examination, 2,3,7,8-TCDD-exposed rats had a severe depletion of lymphocytes from the thymic cortex (Vos and Moore, 1974). Hematological changes were
noted 1n r a t s exposed to 10 and 14 d a l l y doses o f 10 y g /k g 2 ,3 ,7 ,8 -T C D D
(Welssberg and Z1nkl, 1973). Increased red blood cell count, decreased platelet count, Increased neutrophil count and Increased packed cell volumes were reported 1n 2,3,7,8-TCDD-exposed rats. A summary of the data available on the 1mmunotox1c effects of 2,3,7,8-TCDD 1n animals 1s presented 1n Table 8-4. A review of 1mmunotox1c1ty and Immunosuppression was reported by Vos (1977).
Vos et al. (1973) Investigated the humoral and cell-mediated Immune response 1n Hartley guinea pigs, CD rats and B6D2F1 mice. The humoral Immune response was tested 1n 2,3,7,8-TCDD-treated hamsters by Injecting tetanus toxoid (subcutaneously) Into the footpad and later testing for the concentration of tetanus antitoxin from the serum by an Immunodiffusion
8-27
TABLE 8-4 Immunological Effects of 2,3,7,8-TCDD In Animals
8-28
Species/ Strain
Sex Exposure Route
Dose(s)
Duration of Exposure
N1ce/B6D2F1 N gavage
0. 0.2, 1.0, 5.0, 4 weeks 25.0 ,,g/kg bw/week
N1ce/C57Bl/6 F.N
maternally administered (gavage)
0, 1.0, 2.0, 5.0, 25.0 vg/kg
4 or 6 weeks (3 or 5 administrations)
Nice/ C57B1/6JH
N gavage
0, 0.5, 1. 5, 10. 20 vg/kg bw/week
4 weeks
Hlce/Swlss
N gavage
0. 1.5, 5, 15. 50 t>g/kg bw/week
4 weeks
Nlce/B6C3f1 F
In vitro
0.5, 5.0, 50 {ig/mt 5-60 seconds
(spleen cells)
Hlce/Swlss- F.N maternally
Hebster
administered
(diet)
0, 1, 2.5, 5, 10. 20 ppb (dietary)
10 weeks (pregestation
and 3 weeks postparturltlon)
Nlce/CB
N gavage
Nlce/CO
N In vitro
0. 0.01, 0.1, 1.0, up to 8 weeks 10.0 t>g/kg bw/week
10 -10' N
single
Nlnlmum Effective Dose
Parameter
Effect
Reference
NA 5.0 t>g/kg bw/week 5.0 t>g/kg bw/week
1.0 t>g/kg bw/week 25.0 vg/kg bw/week
2.0 vg/kg bw/week
1.0 vg/kg bw/week
1.5 vg/kg bw/week
50 t>g/mft
2.5 ppb 2.5 ppb 5 ppb 1 ppb
NA 0.01 t>g/kg bw/week 1.0 vg/kg bw/week
10 N
bw thymus weight graft-versus-
host response
thymus weight PHA response skin graft
rejection
Salmonella Infection
endotoxin (E. coll) susceptibility
protein, DNA, and RNA synthesis
antigenic RBC reaction
thymic cortex contact sensitivity
to DNFB endotoxin
(Salmonella) susceptibility Listeria Infection
serum Immunoglobln level
serum Immunoglobln level
lymphocyte blasto gnie transformatlon
no change decreased decreased
Vos et al., 1973
decreased decreased prolonged
Vos and Hoore, 1974
Increased mortality and decreased time to death
Thigpen et al.. 1975
Increased Vos et al., mortality 1978a
decreased Luster et al., 1979a,b
decreased
atrophy decreased
Thomas and Hlnsdlll, 1979
Increased mortality
no change
Increased decreased
Sharma and Gehrlng, 1979
Increased Sharma and Gehrlng, 1979
TABLE 8-< (cont. )
8-29
Species/ Strain
Sex Exposure Route
Dose(s)
Ouratlon of Exposure
Minimum Effective Oose
Parameter
Effect
Reference
Mlce/Swlss- F oral (diet) Webster
Mice/ C57B1/6J
H l.p.
H1ce/B6C3F1 N.F maternally administered
H1ce/C5781/6 N l.p.
H1ce/C57Bl/6 H l.p.
Rat/CO
F oral
Rat/CD
F oral
0, 10, 100 ppb
5 weeks (or more)
0, 1, 2. 6, 30 pg/kg bw
single Injection
0. 1.0, 5.0, 15.0 pg/kg bw/day
4 days during gestation and lactation
0, 0., .0, 40 pg/kg bw/week
4 weeks
0, 0.004, 0.04, 0.4 pg/kg bw/week
4 weeks
0, 0.2, 1.0, 5.0 pg/kg bw/week
6 weeks
0, 10 pg/kg bw/day 10, 14 days
10 ppb 10 ppb 10 ppb 10 ppb 10 ppb
1 pg/kg
1 pg/kg
1.0 pg/kg bw/day 1.0 pg/kg bw/day 5.0 pg/kg bw/day
4.0 pg/kg bw/week 0.4 pg/kg bw/week
0.004 pg/kg bw/week
5.0 pg/kg bw/week 5.0 pg/kg bw/week NA
10 pg/kg bw/day 10 pg/kg bw/day 10 pg/kg bw/day
tetanus response antigenic RBC
response sensitization to
ONFB resistance to
Salmonella resistance In
Listeria
macrophage and natural killer cell activity
macrophage and natural killer cell number
antibody production
L. monocytogenes susceptibility
PYB6-tumor suscep tibility
bone marrow hypocellularlty
thymus atrophy cytotoxic T-cell
response
In vitro aeneratlon of cytotoxic T-cells
bw thymus weight tuberculin hyper
sensitivity
erythrocyte count platelet count neutrophil count
decreased H1nsd1ll, decreased et al., 19B0
decreased
Increased mortal 1ty Increased mortality
no change Mantovani et al.. 19B0
decreased
decreased
Increased Increased Increased
Luster et al., 1980
Increased Clark et al., decreased 1981
decreased Clark et al., 1981
decreased decreased no change
Vos et al., 1973
Increased decreased Increased
Welssberg and Zlnkl, 1973
TABLE 8-4 (coni.)
8-30
Species/ Strain
Sex Exposure Route
Oose(s)
Duration of Exposure
H1n1mum Effective Dose
Parameter
Effect
Reference
Rat/F-344
F.H maternally administered
Rat/F1scher
F.H maternally administered
(NR)
Rat/F1scher- F.H Ulstar
maternally administered (NR)
Rat/Sprague- H Dauley
1.V.
Guinea pig/ F gavage Hartley
0, 1.0, 5.0 pg/kg bw/dose
4 or 6 weeks (3 or 5 administrations)
NR 4-6 weeks (during ges tation and neonatally)
0, 5 pg/kg bw/dose
3 or 4 applications during gestation and neonatally
0. 1 pg/kg bw
single Injection
0, 0.008, 0.04, 0.2, 1.0 pg/kg bw
8 weeks
1.0 pg/kg bw/dose 5.0 pg/kg bw/dose 5.0 pg/kg bw/dose 5.0 pg/kg bw/dose 5.0 pg/kg bw/dose NA
NR NR
5 pg/kg bw/dose
5 pg/kg bw/dose 5 pg/kg bw/dose
1 pg/kg bw
0.04 pg/kg bw/week 0.04 pg/kg bw/week 0.04 pg/kg bw/week 0.2 pg/kg bw/week
bw and thymus weight spleen weight
PHA response graft-versus-host
response skin graft
rejection pseudorabies
virus Infection
Con A and PHA response
oxazolone skin hypersensitivity
antibody production to bovine gamma globulin
PHA and Con A response
thymus and bw
thymic RNA synthesis
thymic RNA polymerase activity
bw thymus weight tuberculin hyper
sensitivity tetanus antitoxin
decreased
decreased decreased decreased
Vos and Hoore, 1974
prolonged
no change
decreased decreased
Hoore and Faith, 1976
no effect Faith and Luster, 1979
decreased
decreased until 128 days
decreased decreased
Kurl et al., 1982
decreased decreased decreased
decreased
Vos et al., 1973
H = male; F = female; 1.p. * Intraperltoneal l.v. = Intravenous; PHA = Phytohemagglutinin; Con A = Conconavalln A; RBC = red blood cell; DNFB = 2,4-d1n1tro, 1-fluorobenzene; NA = Not applicable; NR = Not reported
technique. Cell-mediated Immunity was tested by Injecting Mycobacterium tuberculosis (subcutaneously) Into guinea pigs on day 35 of 2,3,7,8-TCDD treatment (during a schedule of 8 weekly doses). Intradermal tuberculin hypersensitivity was determined by measurements of skin thickening on days 47 and 54. Decreased skin hypersensitivity was noted 1n hamsters treated with 0.04 g 2,3,7,8-TCDD/kg and higher doses. Decreased tetanus antitoxin levels were evident 1n guinea pigs treated with 0.2 yg 2,3,7,8-TCDD/kg, but not at lower dose levels. Vos et al. (1973) also tested the cellmediated Immunity 1n rats exposed to 2,3,7,8-TCDD (0, 0.2, 1.0 or 5.0 yg/kg, once weekly for 6 weeks). M. tuberculosis was Injected Into rats by day 28 of the treatment period, followed by Intradermal hypersensitivity testing on day 42. No changes 1n the thickness of skin were noted 1n 2,3,7,8-TCDD treated rats when compared with controls.
Mice were used to test the effect of 2,3,7,8-TCDD on cell-mediated Immunity by use of the "graft-versus-host" experiment (Vos et al., 1973).
In th is t e s t , spleen c e lls from 2,3,7,8-TCDD-exposed mice (0 , 0 . 2 , 1 .0 or
5.0 yg/kg once weekly for 4 weeks) of the C57B1/6 strain were Injected Into the right footpad of a hybrid recipient mouse (C57B1/6 x DBA-2). Donor cells possessing sufficient activity will respond to the DBA-2 antigen on the host cells, resulting 1n the enlargement of the popliteal lymph node. Host cells are tolerant of the donor cells since both have C57B1/6 anti gens. In this test Vos et al. (1973) noted a significant (p<0.01 ) doserelated decrease 1n the activity of 2,3,7,8-TCDD-treated spleen cells (as measured by the degree of popliteal lymph node enlargement on the site of the spleen cell Injection). Lymph node enlargement was significantly less (p<0.01) 1n hybrid recipient mice receiving spleen cells from mice treated with 5 yg 2,3,7,8-TCDO/kg/week than from donor cells of untreated mice.
8-31
Studies continued 1n an attempt to Identify the mechanism of 2,3,7,8TCDD-1nduced Immunodeficiency. Rats (F-344) exposed pre- and postnatally by maternal dosing (1 or 5 yg 2,3,7,8-TCDD/kg administered to dams on days 11 and 18 of gestation and 0, 7 and 14 postnatally) had prolonged times until graft rejection, decreased spleen cell graft-versus-host activity and decreased binding response to phytohemagglutinin (PHA) (Vos and Moore, 1974; Moore and Vos, 1974). Response to conconavalln A (Con A), a humoral Immune response, was actually Increased.
Since thymus-derived lymphocytes (T-cells) play a central role 1n cellmediated 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 Thigpen et al. (1975) 1n male pathogen-free mice (C57B1/6Jfh). 2,3,7,8-TCDD was admin istered to mice at 0.5, 1, 5, 10 or 20 yg/kg once weekly for 4 weeks fol lowed by Inoculation with Salmonella bern 2 days after the final 2,3,7,8TCDD administration. Mortality rates and "time until Infection" were used to determine the Immunological effect of 2,3,7,8-TCDD. A significant (p<0.05) Increase 1n mortality and decrease 1n time of Infection were noted 1n groups treated with 1 yg/kg or higher doses of 2,3,7,8-TCDD when compared with controls. 2,3,7,8-TCDD at 0.5 yg/kg did not alter these parameters and was regarded as a no effect level. The Immune-resistance of mice to S. bern 1s therefore reduced by treatment with 1 yg 2,3,7,8-TCDD/ kg/week (for 4 weeks).
Pretreatment with 2,3,7,8-TCDD greatly enhances the susceptibility of mice to E.. coll endotoxin (Vos et al., 1978a). Injection of 250 yg of endotoxin to mice pretreated with 0, 1.5, 5 and 15 yg 2,3,7,8-TCDD/kg
8-32
r e s u lte d In 0 / 5 , 1 / 5 , 6 /6 and 6 /6 d eaths, r e s p e c t i v e l y . Mice p r e tr e a te d
with 15 and 50 yg 2,3,7,8-TCDD/kg and Injected with 10 yg of endotoxin 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 endotoxin. Increased mortality (2/6) 1n a control group was noted only when 500 yg of endotoxin was administered; however, 10 yg of endotoxin was sufficient to cause similar mortality (2/5) 1n mice treated with 50 yg 2,3,7,8-TCDD/kg.
The Immunocomptence of 5-week-old offspring of Swlss-Webster mice fed diets containing 1, 2.5, 5, 10 or 20 ppb 2,3,7,8-TCDD was tested by several means (Thomas and H1nsd1ll, 1979). The number of cells reactive to anti genic RBC, differential white blood cell counts, organ weights, hlstopathologles, hypersensitivity to 2,4-d1n1tro-l-fluorobenzene (DNFB) and the resistance to E.. coll lipopolysaccharide (LPS), Listeria monocytogenes and Salmonella typh1mur1um LPS were all measured for mice exposed to different levels of 2,3,7,8-TCDD. Adult female mice were exposed to 2,3,7,8-TCDD for 4 weeks before mating, throughout gestation and for 3 weeks postparturlt1on. Young mice being tested for 1mmunotox1c1ty were therefore exposed to 2.3.7.8- TCDD only ^n utero and through lactation. The typical decrease 1n thymus weight was noted 1n mice exposed to 2.5 and 5.0 ppb but was not evi dent 1n the 1.0 ppb group. A decrease 1n the number of plaque-forming cells (PFC) reactive to sheep RBCs was significantly reduced 1n the 2.5 and 5.0 ppb 2,3,7,8-TCDD-exposed groups. (Because of the poor survival of young 1n the 10 and 20 ppb 2,3,7,8-TCDD-exposed groups, results and comparisons were usually reported for the three lower dose groups). The humoral content of ant1-RCD antibodies, however, was not lower 1n 2,3,7,8-TCDD-exposed groups when compared with controls. A decrease 1n the skin hypersensitivity
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to DNFB following sensitization was noted 1n all 2,3,7,8-TCDD-treated groups
(only the 5-ppb group was statistically reduced from controls). 2,3,7,8-
TCDD caused an Increased susceptibility (Increased mortality level) to S.
typh1mur1um 1n a dose-related fashion. The response to E.. coll LPS and L.
monocytogenes was not different from controls. 2,3,7,8-TCDD exposure did
not alter the response of lymphocytes (Band T-cells) .In vitro to Con A, nor
was mitogen-induced lymphocyte proliferation affected (Thomas and HInsdlll,
1979).
Similar findings were reported 1n F1scher/W1star rats exposed to
2.3.7.8- TCDD during gestation (18th day) and neonatally, or neonatally alone
(on days 0, 7 and 14) (Faith and Luster, 1979). Dams were treated with 5
g/kg 2,3,7,8-TCDD on each dose day. Typically, body weight and thymic
weights were decreased 1n progeny, which lasted until 135 days of age. The
thymic- and splenic-cell response to PHA and Con A was decreased 1n all
2.3.7.8- TCDD-treated animals and did not return to normal until day 270.
Delayed hypersensitive reaction was also suppressed until 270 days of age.
The production of antibodies to bovine gamma globulin, which requires
T-helper cell function, was not affected by 2,3,7,8-TCDD exposure during rat
development (Faith and Luster, 1979).
Neonatal B6C3F1 mice, exposed to prenatal (maternal dosing on day 14 of
gestation) and postnatal (days 1, 7 and 14 after birth) doses of 0, 1.0, 5.0
or 15.0 yg/kg 2,3,7,8-TCDD, were studied for 1mmunotox1c effects and host
susceptibility (Luster et al., 1980). At the 15.0 y g 2,3,7,8-TCDD/kg dose
level, 70% of the neonates died with overt toxic effects (decreased body
weight, Uver weight, spleen weight and thymus weight). Bone marrow hypo-
cellularlty and depressed macrophages-granulocyte progenitor cells and
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pleuMpotent stem cells were associated with 2,3,7,8-TCDD exposure at the 5.0 and 15.0 yg/kg dose levels. Hematological changes, such as decreased RBC count, hematocrit and hemoglobin, and lymphocyte count showed a doserelated response. Host susceptibility to L. monocytogenes and PYB6-tumor cells was tested 1n the 2,3,7,8-TCDD-exposed neonates. Death occurred-1n 73 and 40% of the L. monocytogenes Inoculated (1.2x10* viable organisms) mice 1n the 5.0 and 1.0 yg/kg dose groups, respectively, compared with 28% of controls. Tumor development occurred 1n 44, 60 and 22% of the neonates Inoculated with 5xl04 tumor cells from the 5.0 yg 2,3,7,8-TCDD/kg, 1.0 yg 2,3,7,8-TCDD/kg and control groups, respectively.
H1nsd1ll et al. (1980) reported that 2,3,7,8-TCDD administered 1n the diet of Swlss-Webster mice at 100 ppb for 5 weeks caused a marked suppres sion of total serum protein, gamma globulin and albumin, but an Increase 1n B-globulins. At 10 ppb 1n the diet, 2,3,7,8-TCDD caused decreased Immune response to tetanus toxoid, sheep RBC, S. typh1mur1um and L. monocytogenes,
and lowered c o n ta c t s e n s i t i v i t y to DNFB. This study a ls o sugggested t h a t
although young animals are more susceptible to 2,3,7,8-TCDD, older animals are still Immunosuppressed and exposure In. utero and neonatally Is not more crucial than 1n other periods. Vos and Moore (1974) had previously reported that 1-month-old mice were more sensitive to 2,3,7,8-TCDD than were 4-monthold mice (C57B1/6). Decreased body weight and thymus weight and spleen cell response to PHA were evident at lower doses 1n 1-month-old mice than 1n 4-month-old mice.
The effect of single 1.p. doses of 2,3,7,8-TCDD (1, 2, 6 and 30 yg/kg) on peritoneal macrophage and splenic natural killer cell function 1n mice (C57B1/6J) was studied by Mantovanl et al. (1980) and Vecchl et al. (1980).
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2.3.7.8- TCDD treatment at all dose levels did not decrease the cytostatic and cytocldal activity of macrophages or natural killer cells on a per cell basis. The total number of macrophages and splenic natural killer cells recovered from 2,3,7,8-TCDD-treated animals, however, was reduced when com pared with untreated controls. Harked hypocellularlty noted In the bone marrow of 2,3,7,8-TCDD-treated mice may account for the decrease 1n peri pheral cell counts (McConnell et al., 1978b). The lack of macrophages and natural killer cells was suggested as being Instrumental In the decreased resistance to Infection common to 2,3,7,8-TCDD-exposed animals (Mantovanl et al., 1980). Although 2,3,7,8-TCDD was a strong Immunosuppressant, animals given a lethal dose of 2,3,7,8-TCDD did not appear to die from Infections, nor did a germ-free environment protect them from death (Grelg et al., 1973).
The actual mechanism of 2,3,7,8-TCDD 1mmunotox1c1ty 1s unknown but several Investigators have tested various hypotheses. Vos et al. (1973, 1978a,b) attempted to address the Indirect causes for decreased thymic growth and altered T-lymphocyte activity following 2,3,7,8-TCDD treatment. Vos et al. (1973) measured serum cortisol and cortlcosteron levels 1n guinea pigs exposed to 2,3,7,8-TCDD to evaluate the possible Indirect Immunosup pression by these hormones. There was, however, no significant difference 1n the level of these hormones between treated and control animals. Indirect Immunosuppression of this type was unlikely. Later studies (Vos et al., 1978a,b) Investigated the role of thymic hormones (thymosin) on the atrophy of the thymus during 2,3,7,8-TCDD treatment. Thymosin administered 1n conjunction with 2,3,7,8-TCDD did not protect mice from the typical 2.3.7.8- TCDD-1nduced 1mmunotox1c alterations. Thymus weight was maintained but not Increased by thymosin, and thymus-derived cells continued to show
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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 Immunosuppression.
van Logten et al. (1980) Investigated the possible Influence of the
adrenal gland, hypophysis and pituitary, and growth hormone on thymic
atrophy and Immunosuppression following 2,3,7,8-TCDD exposure 1n female
F-344 rats. Adrenalectomy and exogenous growth hormone had no preventative
action on thymic Involution. Hypophysectomlzed rats showed advanced thymic
atrophy.
Sharma and Gehrlng (1979) noted that 2,3,7,8-TCDD caused stimulation of
lymphocyte transformation to blast form cells (mltotlcally active precur
sors) when no mitogens were present 1n the culture system. This represents
a phenomenon similar to actual antigenic challenge. At low doses (0.01 and
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 yg/kg/week) resulted 1n a decrease 1n the serum Immunoglobulin
level. It was suggested that 2,3,7,8-TCDD may elicit an antigenic response
either by combining with a body protein or by causing cellular or biochemi
cal damage that releases antigenic proteins. Sharma and Gehrlng (1979) also
noted that thymic atrophy was observed after 2 and 4 weeks of treatment but
not after 8 weeks. There may be a recovery of thymic tissue, either by
Immune tolerance or Immune unresponsiveness' as a sort of adaptat\ion to \
2,3,7,8-TCDD-exposure and Its possible antigenic complex.
\
Luster et al. (1979a,b) reported that 2,3,7,8-TCDD affects the Immune
system directly by altering lymphocyte function. The function of T-helper
cells was not altered, since no change 1n response to bovine gamma globulin
8-37
\ \
(requires T-helper cell cooperation) was noted 1n W1star/F1scher and Fischer rats exposed to 2,3,7,8-TCDD. In vitro. 2,3,7,8-TCDD (100 ng/mit) sup pressed DNA, RNA and protein synthesis 1n splenic lymphoid 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 2,3,7,8TCDD, measured by specific mitogen binding assays, than B-lymphocytes. These authors (Luster et al., 1979a) suggested that 2,3,7,8-TCDD may bind directly to the lymphocyte cell membrane and alter Its function. Faith and Luster (1979) reported that lymphocytes from the spleen, thymus, bone marrow and lymph nodes of Fischer rats exposed to 2,3,7,8-TCDD showed abnormal homing patterns within the body. 2,3,7,8-TCDD exposure apparently altered the cell surface markers so that spleen lymphocytes were taken up by the thymus of recipient rats. These authors (Faith and Luster, 1979) suggested that 2,3,7,8-TCDD may change cellular metabolism, which alters the cell mem brane constituents or may Insert directly Into the membrane. Kurl et al. (1982) reported that 2,3,7,8-TCDD causes changes 1n thymic transcription and RNA synthesis that may lead to cell surface changes. Cell surface changes could presumably result 1n altered antigen recognition and cell-to-cell recognition, causing Immunosuppression and thymic atrophy.
Clark et al. (1981) reported that 2,3,7,8-TCDD treatment (0.4, 4.0, 40 yg/kg weekly for 4 weeks by l.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
8-38
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 1n vitro) but did not appear directly toxic to the cytotoxic T-cells. At present, the mechanism of Immunosuppression 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 1n DBA/2 mice as compared with C56B1/6 mice. This Indicates that susceptibility to 2,3,7,8TCDD 1mmunotox1c1ty segregates with the Ah locus, which 1s consistent with a receptor mediated mechanism. The receptor mediated mechanism was further supported by the susceptibility of the C57B1/6 x DBA/2J hybrid mouse to 2.3.7.8- TCDD suppression of the cytotoxic T-cells, which 1s again consistent with the dominant Inheritance of Ah (Nagarkattl et al., 1984).
Few reports are available 1n which the Immunological effects of 2,3,7,8TCDD exposure were studied 1n humans. Regg1an1 (1980) reported that the 1mmunocapab1!1ty of 17 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 1mmunoglob1n 6, A, M, D and E, as well as lymphocytes, T-cells, B-cells, PHA response and blood cell counts. These determinations were made 10 years after workers had developed 2.3.7.8- TCDD-1nduced chloracne. In this group of workers, there was a sig nificant Increase 1n the proportion of cases with reduced IgD and IgM. It was suggested that the 2,3,7,8-TCDD-exposed group had a reduced Immune capability and a deficiency In T-cell and B-cell cooperation. The Immunotoxlclty of 2,3,7,8-TCDD 1n humans cannot be properly assessed because of the paucity of data recorded soon after exposure. The most prominent effects 1n animals (1.e., humoral responses) were not measured 1n humans.
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8 .1.1.5. ENZYME INDUCTION BY TCDD --
8 .1.1.5.1.
In Cell Cultures -- Although 2,3,7,8-TCDD has a very low
toxicity to cells In culture (Beatty et al., 1975; Bradlaw et al., 1976;
Knutson and Poland, 1980; Yang et al., 1983), 1t 1s an extremely potent
enzyme Inducer 1n these systems (Kour! et al., 1974; N1wa et al., 1975;
Bradlaw et al., 1976; Malik and Owens, 1977; Malik et al., 1979; Bradlaw et
al., 1980). This enzyme Induction 1s so sensitive that 1t has been proposed
as a bioassay for detecting planar polychlorinated organic compounds
(Bradlaw et al., 1975, Bradlaw and Casterllne, 1979; N1wa et al., 1975).
Kourl et al. (1974) found that 2,3,7,8-TCDD Induced aromatic hydrocarbon
hydroxylase (AHH) activity 1n cultured human lymphocytes to the same extent
as 3-MC; however, the concentration of 2,3,7,8-TCDD necessary for maximal
enzyme Induction was 40-60 times less than that of 3-MC. N1wa et al. (1975)
compared AHH Induction by 2,3,7,8-TCDD among cell cultures (H-4-II-E, VERO,
HTC, LB82, MA, Hepa-1, TRL2, JRL-2, NRKE and Chang). ED5Q values ranged
from 0.12 nM 1n the Hepa-1 cell line to >100 nM In the VERO and HTC cell
lines. 2,3,7,8-TCDD did not Induce AHH activity 1n LB82 cells. The respon
siveness of AHH Induction to 2,3,7,8-TCDD was 250-900 times greater than to
3-MC. In addition, cell cultures derived from C57B1/6N mice were 16 times
as sensitive to 2,3,7,8-TCDD as cell cultures derived from DBA/2N mice. The
responsiveness of cell cultures to enzyme Induction by 2,3,7,8-TCDD 1s thus
similar to the effects seen 1_n vivo. The Inductive effect of 2,3,7,8-TCDD
was blocked by actlnomydn D and cyclohexlmlde, 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).
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In all of these studies, there was no correlation between cytotoxicity
and enzyme Induction. This Implies that, despite the correlation in vivo
(Section 8.3.5.), there may be no direct connection between enzyme Induction
and the 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 1n rats and mice have been Investigated extensively. 2,3,7,8-TCDD
ha$ been found to alter many enzyme activities 1n a wide variety of organ
systems (vide Infra). This alteration primarily results 1n 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-demethylat1on 1n male, but not female, rats; however, cytochrome
P-450 and benzpyrene hydroxylase activity were Increased. The suppression
of N-demethylase activity was undetectable for 73 days following a single
oral dose of 25 yg 2,3,7,8-TCDD/kg bw. The suppression of N-demethylase
activity was seen only 1n adult animals. In 10-day-old rats, 2,3,7,8-TCDD
had an Inductive effect on this activity.
The Inductive effects of 2,3,7,8-TCDD have been demonstrated to be organ
specific. A1t1o and Parkkl (1978) Investigated the effects of 2,3,7,8-TCDD
on the activities of AHH, ethoxycoumarln deethylase, cytochrome C reductase,
epoxide hydratase, UDP glucuronosyltransferase, and glutathione S-trans-
ferase 1n the Uver, kidney, lung, small Intestine and testes of male Wlstar
rats. Honooxygenase activity was stimulated 1n the Uver, lung and kidney,
but not 1n any other tissue Investigated. UDP glucuronosyltransferase
activity Increased by a factor of 7 1n the Uver, by a factor of <2 1n the
kidney, and not at all 1n any other tissue. Epoxide hydratase and gluta
thione S-transferase activities were not affected 1n any of the tissues
studied, although stimulation of hepatic glutathione S-transferase has been
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reported by other Investigators (Manis and Apap, 1979). Enzyme Induction has also been reported 1n rat mammary gland (Rlkans et al., 1979), mouse testes (Mattlson and Thorgelrsson, 1978), and rat prostate gland (Lee and Suzuki, 1980), but the rat adrenal gland 1s apparently Insensitive to Induc tive effects of 2,3,7,8-TCDD (Guenthner et al., 1979b).
In the Uver of rats and mice, 2,3,7,8-TCDD affects a wide range of enzymatic activities, Including DT-d1aphorase (Beatty and Neal, 1976a,b), bilirubin catabolism (Kap1tuln1k and Ostrow, 1978), ornithine decarboxylase (Potter et al., 1982), 7-ethoxycoumar1n 0-demethylase (Greenlee and Poland, 1978), glutathione S-transferase (Baars et al., 1978; Manis and Apap, 1979), aldehyde dehydrogenase (Lindahl et al., 1978; De1tr1ch et al., 1977), uro porphyrinogen decarboxylase (Jones and Sweeney, 1977), -aminolevulinic acid synthetase (Goldstein et al., 1982a; Woods, 1973), UDP-glucuronosyl transferase (Marselos et al., 1978) and a number of microsomal oxidative enzyme systems (vide Infra).
2,3,7,8-TCDD 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 In the absorption maximum of the carbon monoxide difference spectrum from 450 to 448 nm. In a number of studies, Increased AHH activity and cytochrome P-448 synthesis have been separated (Chhabra et al., 1976); however, other researchers report an apparent connection between cytochrome P-448 and AHH Induction (K1tch1n and Woods, 1977, 1978a,b). Thus, 2,3,7,8-TCDD not only stimulates AHH activity by Inducing cytochrome P-450 formation, but may enhance AHH activity by other mechanisms as well.
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8 .1.1.5.3. In Rabbit -- The response of the rabbit 1s quite different from that observed 1n rats and mice (Hook et al., 1975a). The only changes In hepatic enzyme activities observed were suppression of benzpyrene hydroxylase and benzphetamlne N-demethylase. In the same study, biphenyl 4-hydroxylase was Induced 1n the lung and benzpyrene hydroxylase was Induced 1n the kidney. In a similar study, a hepatotoxlc dose of 2,3,7,8-TCDD (30 yg/kg) failed to alter prostaglandin synthetase activity 1n hepatic or renal tissue {Kohl 1 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), L1em et al. (1980) and Dees et al. (1982) Isolated a series of cytochromes P-450 from rabbit Uver mlcrosomes. These cytochromes were Immunologlcally distinct, functioned In different catalytic pathways, and responded differently to Induction by polycyclic aromatic hydrocarbons. 2,3,7,8-TCDD was found to Induce two cytochromes, designated as form 4 and form 6. Form 4 1s the major cyto
chrome Induced 1n a d u l t r a b b i t l i v e r by 2 ,3 ,7 ,8 -T C D D ; however, form 6 1s the
major cytochrome Induced 1n newborn rabbit Uver (Norman et al., 1978b), adult rabbit lung, and adult rabbit kidney (Liem et al., 1980; Dees et al., 1982).
8 .1.1.5.4. Other Species -- The guinea pig, the species most sensi tive to the toxic effects of 2,3,7,8-TCDD, 1s similar to the rabbit In Its response to 2,3,7,8-TCDD. Biphenyl 4-hydroxylase was Induced In the Uver, lung and kidney, biphenyl 2-hydroxylase was suppressed In the Uver, 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 (TofUon et al., 1980). Testicular microsomal
8-43
heme levels and -aminolevulinic acid synthetase activity were unaffected by this treatment. In contrast to the rat, 2,3,7,8-TCDD did not Induce DT-d1aphorase 1n brain, spleen, kidney, lung, heart or Uver of male guinea pigs (Beatty and Neal, 1978).
Aryl hydrocarbon hydroxylase and 5-am1nolevul1n1c acid synthetase In the chick embryo have been reported to be extremely sensitive to the Induc tive effects of 2,3,7,8-TCDD (Poland and Glover, 1973a,b), with maximal Induction occurring with 155 pmoles/egg. This Induction 1s relatively long lasting, with 70% of the maximum Induced activity present 5 days following a single dose of 2,3,7,8-TCDD. Structure-activity studies demonstrated a per fect correspondence between the toxicity and Induction potency of a series of d1benzo--d1ox1n congeners (Poland and Glover, 1973a). 8.1.2. Subchronic. Four laboratory studies described the systemic toxic effects of subchronic exposure to 2,3,7,8-TCDD 1n rodents. Also, one semi controlled study evaluated the toxic effects to rabbits after confinement to an area containing soil contaminated with 2,3,7,8-TCDD. No Information was found 1n the literature searched on the effects of subchronic exposure to 1,2,3,7,8-PeCDD, and only one preliminary study was available describing the effects of subchronic exposure to a mixture of two HxCDDs 1n rats and mice.
Kodba et al. (1976) exposed Sprague-Dawley rats to 2,3,7,8-TCDD for 13 weeks. The animals 1n groups of 12 males and 12 females received the com pound suspended 1n acetone-corn oil (1:9) by gavage 5 days/week at doses of 0.0, 0.001, 0.01, 0.1 or 1.0 yg/kg bw. At the end of the treatment period 5 rats of each sex were killed for histopathologic examination, and the remaining animals were continued for postexposure observation. This report on gross, hematologic, clinical chemistry and histopathologic (on animals terminated at the Interim kill or killed when moribund) observations was
8-44
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-TCDD. During the study there were five treatment-related deaths in the high-dose group females, with three 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 yg/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 bilirubin concentrations, urinary copropor phyrin excretion, and changes in relative thymus or liver weight to body weight ratio occurred in the two high-dose female groups, but only in the 1.0 yg/kg male group. Although male rats had significantly decreased hematologic values (packed cell volume, RBC count and hemoglobin) in the two high-dose groups, and these values were normal in all female rats, the 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 1n the size of testes and uteri, and a decrease 1n the number of corpora lutea. Histologic examination revealed Involution of the thymus, decreased number of thymocytes, and focal necrosis and pigment accumulation in the Uver. 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 Uver in the group fed 0.1 yg/kg. Although histologic evidence from animals killed during the Interim sacrifice was consistent with the Uver and thymus being
8-45
the primary target organs, 1n an animal that died during the study there were signs of aortic thrombosis and adrenal hemorrhage, and 1n a second animal there was severe anemia, suggesting possible Involvement of the hema topoietic system near the time of death.
Liver toxicity was the only effect of treatment observed during histo logic examination of rats (Osborne-Mendel) and mice (B6C3F1) administered 2,3,7,8-TCDD for 13 weeks 1n a preliminary subchronic toxicity study designed to define an acceptable dose for a chronic toxicity study (NTP 1980a). The animals 1n groups of 10 males and 10 females were administered the compound In corn o1l-acetone (9:1) twice a week at doses for rats of 0.0, 0.5, 1, 2, 4 and 8 yg/kg/week, and for mice at doses of 0.0, 1, 2, 5, 10 and 20 yg/kg/week. Deaths occurred at the two high-dose levels 1n rats, with 4 females 1n the 8 yg/kg/week and 1 1n the 4 yg/kg/week group dying, while only 2 male rats 1n the 4 yg/kg/week group died. Deaths were accompanied by severe toxic hepatitis. Hepatic lesions were observed 1n all other rats examined 1n groups administered 1-8 yg/kg/week; however, not all animals 1n each group were submitted to necropsy. Normal Uver his tology was observed in the 2 male rats examined from the low-dose groups and only threshold toxic effects occurred 1n the low-dose female rats.
Similar effects of treatment were observed 1n mice, with a single death occurring 1n each sex at the high-exposure level, along with reports of hepatic lesions on histologic examination. In contrast to rats, female mice were less sensitive to the hepatotoxlc effect of 2,3,7,8-TCDD than were the male mice. Hepatic lesions were observed 1n all dose groups of male mice, while the 1 and 2 yg/kg/week dose groups of female mice had normal livers. Although the group sizes were small, making conclusions tenuous, 1t
8-46
appeared that sex differences 1n the sensitivity to the toxic effects of 2,3,7,8-TCDD occurred, and that the more sensitive sex may vary with species tested.
In a more extensive subchronic study in rats, King and Roesler (1974) followed the development of toxicity by a series of interim sacrifices during 28 weeks of exposure to 2,3,7,8-TCDD and a 12-week post-treatment recovery period. Groups of 35 male and 35 female Sprague-Dawley rats were intubated twice weekly with 2,3,7,8-TCDD 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 histologic changes at necropsy.
Besides a d o s e - r e la te d decrease in body w eig h t gain in male r a t s and a
decrease in body weight gain in the high-dose female rats, the only effect of exposure to 2,3,7,8-TCDD was histologic changes in the liver. Liver pathology was normal in 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 lipid droplets in a few centrilobular hepatocytes to lipid droplets in all centrilobular hepatocytes with extension into the midzonal hepatocytes. No clear dose-response pattern was observed in this study; however, it did appear that the severity of fatty changes was greater in male rats. During the recovery period, fatty changes progressively decreased in severity but were still present in some treated animals 12 weeks after cessation of exposure. Other histologic
8-47
changes observed 1n the 11ve!r predominantly 1n the animals killed at 28 weeks Included necrosis, Increased nuclear size, subtle distortion of Uver architecture, and hyperchromatlc nuclei. All of these lesions were consid ered to be slight or mild, and less toxlcologlcally relevant than the fatty changes. The data suggested that the Uver was the most sensitive organ to the toxic effect of 2,3,7,8-TCDD, and although recovery occurred after termination of treatment, the recovery process was slow.
The recovery time was also demonstrated to be long 1n a subchronic study by Goldstein et al. (1982b) of 2,3,7,8-TCDD Induced porphyria. Groups of 8 female Sprague-Dawley rats were given 2,3,7,8-TCDD 1n corn o1l-acetone (7:1) weekly by gavage for 16 weeks at doses of 0.0, 0.01, 0.1 or 10.0 ng/kg/ week and killed 1 week after the last treatment. Additional groups of rats received doses of 0.0 or 1.0 yg/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 toxicity was a decrease 1n body weight gain 1n the group receiving 1.0 ng/kg/week. After 16 weeks of exposure to 2,3,7,8-TCDD, Uver porphyrins were elevated ~1000-fold 1n 7 of 8 animals receiving 1.0 yg/kg/week, but only 1 of 8 animals 1n the 0.1 vg/kg/week group had elevated porphyrin levels. No effect was observed 1n the low-dose animals. After a 6-month recovery period the porphyrin level 1n animals exposed to 1 yg/kg/week was still 100-fold higher than values 1n the control group. A similar pattern was observed for urinary excretion of uroporphyrin. The rate-Hm1t1ng enzyme 1n heme synthesis, -aminole vulinic acid synthetase, was also elevated at both the time of termination of treatment and at the end of the recovery period; however, other enzymes that were Increased after 10 weeks of treatment, cytochrome P-450, AHH and glucuronyl transferase, returned to near normal levels by 6 months. It was
8-48
clear that a 6-month recovery period from subchronic exposure to 2,3,7,8TCDD at a dose of 1.0 yg/kg/week was not sufficient for complete reversal of 2,3,7,8-TCDD Induced porphyria.
In addition to the above laboratory studies, Str1k and de W1t (1980) attempted to Investigate the toxicologic effect on rabbits of exposure to a natural environment that was contaminated with 2,3,7,8-TCDD. Groups of 20 female rabbits and 1 .male rabbit were housed for 5 months 1n pens, located 1n five separate areas, on soil that had been contaminated with 2,3,7,8TCDD. The soil had been cleaned by replacement or cultivation before Initiation of the study. The levels of 2,3,7,8-TCDD before cleaning were from 0.8-23.2 yg/m3; however, the levels of contamination after cleaning were not determined. At the end of 5 months liver histology, Including the localization of porphyrin, was examined, and the levels of cytochrome P-450 and P-420 were determined along with urinary levels of total porphyrin, creatinine 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 subchronic laboratory studies already described.
Information on the subchronic toxicity of HxCDD was provided 1n a pre liminary range-finding study for a chronic bioassay conducted by NTP (1980b) on a 1-2 mixture of 1,2,3,6,7,8- and 1 ,2,3,7,8,9-HxCDD. Osborne-Mendel rats and B6C3F1 mice 1n groups of 10 males and 10 females were administered the HxCDD mixture 1n corn oil-acetone (9:1) by gavage twice a week for 13 weeks. The total weekly doses given rats were 0.0, 2.5, 5, 10, 50 and 100 yg/kg; mice received weekly doses of 0.0, 1.25, 2.5, 5, 10 and 50 yg/kg. At week 10 of the study, the body weight 1n rats was decreased In a dose-related manner to a maximum of ~20% 1n the high-dose group. In mice, body weight
8-49
was also decreased 10-2054 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 Uver pathology was observed, with threshold to moderate hepatotoxlclty occurring at doses of 5 and 10 yg/kg/week for male and female rats, respectively, and at 10 yg/kg/week for both sexes of mice. At higher exposures, splenic hyperplasia and cortical atrophy of the thymus were also detected 1n rats. In rats It was unclear whether the low-dose animals were free of any pathologic findings or none were subjected to necropsy. In mice 1t was stated that no changes were observed 1n males exposed to 2,3,7,8-TCDD at 1.25 yg/kg/week or 1n females exposed to 1.25 or 2.5 yg/kg/week. Although the data are limited, 1t appears that the same target organs are sensitive to the toxic effects of both 2,3,7,8-TCDD and this mixture of HxCDD.
In addition, a second subchronic range finding study conducted by NTP (1980c) evaluated the dermal toxicity of the above mixture of HxCDD. Groups of 10 male and 10 female Swlss-Webster mice were treated by dermal applica tion 3 t1mes/week for 13 weeks. The doses used were from 0.01-50 yg/ application with the test compound dissolved 1n acetone. There was 10054 mortality 1n the 25 and 50 yg/appl1cat1on groups and 8054 mortality 1n the 10 yg/appl1cat1on group. On histologic examination, there were signs of Uver damage at the lowest dose tested 1n both sexes; however, the Incidence and degree of damage were not well correlated to the dose applied. 8.1.3. Chronic. The toxic effects, other than neoplasia, of long-term exposure to 2,3,7,8-TCDD have been studied 1n rats and mice. The primary purpose of many of the studies 1n rodents was to assess the carcinogenicity of 2,3,7,8-TCDD. The observation of non-neoplast1c systemic toxic effects
8-50
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-TCDD. Long-durat1on toxicity assays were also conducted 1n monkeys. Many of the same organs 1n monkeys
\
as 1n rodents were adversely affected by long-term exposure to 2,3,7,8-TCDD; however, the monkeys also developed severe skin and stomach lesions. Table 8-5 summarizes the toxic effects of chronic exposure to 2,3,7,8-TCDD and provides Information on the exposure levels that result 1n 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 1n the litera ture search on the effects of chronic exposure to 1,2,3,7,8-PeCDD.
8 .1.3.1. STUDIES ON LABORATORY RODENTS -- In an early study, Van Miller et al. (1977a,b) defined the dietary level of 2,3,7,8-TCDD that adversely affected the longevity of rats following chronic exposure. Groups of 10 male Sprague-Dawley rats were maintained for 78 weeks on diets con taining 1, 5, 50, 500, 1000, 5000, 50,000, 500,000 or 1,000,000 ppt of 2.3.7.8-TCDD. Survival was monitored during the study or at termination 95 weeks after Initiation of treatment. No animals survived until the end of the study at the five highest exposure levels. The respective week after the start of treatment 1n which the first death occurred 1n these high-dose groups was 31, 31, 3, 2 and 2 weeks, with all animals 1n groups >50 ppb dead by week 4. The mortality rate 1n the 0.0, 1, 5, 50 and 500 ppt groups at 95 weeks was60, 20, 40, 40 and 50%. Although the small number of animals 1n each group makes 1t Impossible to precisely define a dose-response relation ship, 1t was apparent that exposure to >1 ppb curtailed survival.
8-51
Specles/Straln Sex/Ho.
Oose
TABLE 8-5
Effects of Chronic Exposure to 2,3,7,8-TCDD In Laboratory Rodents
Treatment Schedule
Ouratlon of Study
Parameters Monitored
Effects of Treatment
Reference
8-52
Rat/ Sprague-Oawley
H/10 H/10
H/10
H/10
H/10
H/10
H/10
0.0 ppt
NA 95 weeks
1 ppt 5 ppt
50 ppt 500 ppt 1000 and 5000 ppt 50,000, 500,000 and 1,000,000 ppt
continuous In
95 weeks
diet for 78 weeks
continuous In
95 weeks
diet for 78 weeks
continuous In
95 weeks
diet for 78 weeks
continuous In
95 weeks
diet for 78 weeks
continuous 1n
95 weeks
diet for 78 weeks
continuous In
95 weeks
diet for 78 weeks
Rats/
H&F/50&50
Sprague-Oawley
-2193 ppt (0.1 vg/kg/day)
continuous In diet for 2 years
2 years
survival survival survival survival survival survival survival
40X survived until 95
Van Hiller
weeks, the first death
et al., 1977a
occurred at week 68
BOX survived until 95
weeks, the first death
occurred at week 86
60X survived until 95
weeks, the first death
occurred at week 33
60X survived until 95
weeks, the first death
occurred at week 69
50X 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
extensive hlstopathology, hematology, urine analyses, and clinical chemistry
Cumulative mortality.
Koclba et al
Increased (F);
1978a, 1979
bw gain,
decreased (H,F);
Red blood cell count,
decreased (H.F);
Packed cell volume,
decreased (H,F);
Hemoglobin,
decreased (H.F);
Reticulocytes,
Increased (H.F);
White blood ceil count,
decreased (F);
Serum glutamic pyruvic
transaminase. Increased (F)
G-Glutamyl transferase,
Increased (F);
Alkaline phosphatase,
Increased (F );
iadlc o-3 i t u n i .j
8-53
Specles/Straln Sex/No.
Dose
Treatment Schedule
Duration of Study
Parameters Monitored
Effects of Treatment Reference
Rats/ Sprague-Dawley (cont.)
Rat/ n&F/so&so
Sprague-Dawley
-208 ppt (0.01 |ig/kg/day)
MF/50&50
-22 ppt (0.001 pg/kg/day)
Rat/ Osborne-Hendel
MF/7S&7S M F /50&50
0.0 pg/kg/week O.S pg/kg/week
N&F/50&50 0.0S pg/kg/week
M F / 5 0 & 5 0 0.01 |ig/kg/week
H1ce/B6C3F1
MF/7S&7S
0.0 pg/kg/week
H&F/50&50
O.S pg/kg/week (N) 2.0 pg/kg/week (F)
continuous In diet for 2 years
continuous In diet for 2 years
NA
administered by gavage biweekly for 104 weeks administered by gavage biweekly for 104 weeks administered by gavage biweekly for 104 weeks
NA
administered by gavage biweekly for 104 weeks
2 years
2 years 106 weeks 107 weeks
Urinary coproporphyrin. Increased (F);
Urinary uroporphyrin, Increased (F ); Urinary delta-amino levulinic acid, Increased hepatic degeneration, Increased (H,F)
Koclba et al., 1970a, 1979
extensive hlstopathology. hema tology, urine analyses and clinical chemistry
Urinary coproporphyrin. Increased (F); Urinary uroporphyrin,
Increased (F );
Hepatic degeneration, Increased (N,F)
Koclba et al., 1978a, 1979
extensive htstopathology, urine analyses and clinical chemistry
No differences In values obtained from control animals
extensive htstopathology
Toxic hepatitis; 0/74 (N). 0/75 (F)
NTP, 1980a
extensive hlstopathology
Toxic hepatitis; 14/50 (N), 32/50 (F)
107 weeks
extensive htstopathology
Toxic hepatitis; 0/50 (N). 1/50 (F)
107 weeks
extensive htstopathology
Toxic hepatitis; 1/50 (N), 0/50 (F)
105-106 weeks 107 weeks
extensive htstopathology
extensive htstopathology
Toxic hepatitis; 1/73 (N). 0/73 (F)
Toxic hepatitis; 44/50 (N), 34/47 (F)
NTP, 1980a
TABLE 8-5 (cont.)
8-54
Specles/Straln Sex/No.
Bose
Treatment Schedule
Duration of Study
H1ce/B6C3F1 (cont.)
Mtce/Swlss
M&F/50&50 H&F/50&50 M/38
0.05 pg/kg/week (N) 0.2 pg/kg/week (F)
0.01 pg/kg/week (N) 0.04 pg/kg/week (F)
0.0 pg/kg/week
administered by gavage biweekly for 104 weeks
administered by gavage biweekly for 104 weeks
NA
107 weeks 107 weeks 588 days
M/44 M/44 M/43
0.007 pg/kg/week 0.7 pg/kg/week 7.0 pg/kg/week
administered by gavage weekly for 1 year
administered by gavage weekly for 1 year
administered by gavage weekly for 1 year
649 days 633 days 424 days
NA >= Not applicable
Parameters Monitored
Effects of Treatment
Reference
extensive hlstopathology
Toxic hepatitis; 3/49 (M), 2/48 (F)
NTP, 1980a
extensive hlstopathology
Toxic hepatitis; 5/44 (H), 1/50 (F)
histology on all organs
histology on all organs
Dermatitis and amyloidosis; 0/38
Dermatitis and amyloidosis; 5/44
Toth et al., 1978, 1979
histology on all organs
Dermatitis and amyloidosis; 10/44
histology on all organs
Early mortality, dermatitis and amyloidosis; 17/43
Increased mortality was also observed 1n female Sprague-Dawley rats maintained for 2 years on a diet that provided a 2,3,7,8-TCDD dose of 0.1 pg/kg/day, while no Increased mortality was observed 1n male rats at this dose or In animals receiving doses of 0.01 or 0.001 pg/kg/day (Koclba et al., 1978a, 1979). The average dietary levels of 2,3,7,8-TCDD associated with these doses were 2193, 208 and 22 ppt. Interim hematologic, clinical chemistry and urine analyses revealed treatment-related changes 1n a number of parameters In the high-dose group, along with some of the same changes occurring 1n the mid-dose group, albeit to a lesser degree (see Table 8-6). At termination of the study, gross and histologic examination Indicated that the Uver was the most severely affected organ, with degenerative, necrotic and Inflammatory changes observed. Increases In urinary excretion rates of coproporphyrin and uroporphyrin 1n the high and middle dose females were consistent with the observed liver damage. Again, primary liver Injury was dose-related with the lowest dose representing a NOEL. Although the group sizes (50 males and 50 females 1n the treated groups, and 85 males and 86 females In the control groups) were reported, the description of the experi mental results did not enumerate the number of animals affected.
When 2,3,7,8-TCDD was administered by gavage 1n corn o1l-acetone (9:1) at dose levels of 0.0, 0.5, 0.05 or 0.01 pg/kg/week, "toxic hepatitis" was observed respectively 1n male Osborne-Mendel rats at Incidences of 0/74, 14/50, 0/50 and 1/50, and 1n female rats at Incidences of 0/75, 32/49, 1/50 and 0/50 (NTP, 1980a). Toxic hepatitis was defined as "lipidosis (lipoido sis) and hydropic degeneration of the cytoplasm of the hepatocytes" 1n the central, mldzonal and, at times, peripheral portions of the Uver. No other
8-55
non-neoplast1c lesions were observed even though extensive histologic exami nations were performed. The two preceding studies support a NOEL for rats of ~0.001 yg/kg/day, with a LOAEL of 0.05 yg/kg/day, and a FEL for Uver Injury and possibly decreased survival of 0.5 yg/kg/day.
Non-neoplastic effects of chronic exposure to 2,3,7,8-TCDD in mice have been briefly decMbed 1n studies Investigating the carcinogenic potential of 2.3.7.8- TCDD. In an NTP (1980a) bioassay, extensive histologic examinations were performed on B6C3F1 mice treated biweekly with 2,3,7,8-TCDD by gavage 1n corn oil-acetone (9:1) for 104 weeks followed by an additional 3-week observation period. The doses for male animals were 0.0, 0.01, 0.05 and 0.5 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 1n males at Incidence of 1/73, 5/49, 3/49 and 44/50, and 1n females at Incidences of 0/73, 1/50, 2/48 and 34/47, respectively, 1n the control, low-, medium- and high-dose groups. In a second study, weekly Intubation of 2.3.7.8- TCDD at doses of 0.0, 0.007, 0.7 or 7.0 yg/kg/week for 1 year resulted 1n amyloidosis of the kidney, spleen and Uver, and dermatitis at the time of death 1n male Swiss mice (Toth et al., 1978, 1979). The Inci dence of these lesions 1n the control, low-, medium- and high-dose groups, respectively, was 0/38, 5/44, 10/44 and 17/43. In the high-dose group, the amyloidosis was extensive and considered to be the cause of early mortal ity. The amyloidosis may have resulted from the chronic dermal Inflammation produced by the treatment. From the limited data presented 1n these studies, 1t appears that mice and rats were approximately equally sensitive to the toxic effects of 2,3,7,8-TCDD following chronic exposure. Severe toxic effects were observed at doses of 1 yg/kg/day (early mortality) and
8-56
0 .28-0 .07 y g / k g / d a y (toxic hepatitis), while a L O A E L for dermatitis a n d amyloidosis of 0.001 yg/kg/day was reported. A NOAEL for mice was not clearly defined by these studies.
The only information available on the effects of chronic exposure to HxCDD was provided by an NTP (1980c) bioassay of a 1:2 mixture of 1,2,3,6,7,8- and 1,2,3,7,8,9-HxCDD. Male and female Sprague-Dawley rats and B6C3F1 mice were exposed biweekly to this mixture for 104 weeks and followed for an additional 3-4 weeks before the terminal kill. Both male and female rats and male mice received doses of 0.0, 1.25, 2.5 and 5.0 yg/kg/week; female mice received doses of 2.5, 5.0 and 10 yg/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 reduced sur vival. No gross signs of toxicity were observed in mice of either sex. Al though extensive histologic examinations were performed, the only treatmentrelated effect was toxic hepatitis, which was defined as "degenerative hepatocytic changes and/or necrosis associated with mild fibrosis and infil tration." The incidence of this lesion in control-, low-, medium- and high dose groups, respectively, was as follows: male rats - 0/75, 28/48, 35/50 and 34/48; female rats - 0/73, 33/50, 37/50 and 44/50; male mice - 0/75, 28/50, 35/50 and 34/49; and female mice - 0/75, 33/50, 37/50 and 44/50. The severity of the toxic hepatitis was dose-related; however, it 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 FELs for liver toxicity.
8 .1.3.2. STUDIES IN NONHUMAN PRIMATES -- Initial studies indicating the effect of chronic exposure to PCDDs including 2,3,7,8-TCDD in nonhuman
\ 8-57
primates was conducted using "toxic fat," a contaminated poultry feed addi tive, which resulted 1n the death of a large number of chickens (Allen and Carstens, 1967). Groups of 4-5 monkeys, Hacaca mulatta. were fed diets con taining 0.0, 0.125, 0.25, 0.5, 1.0, 5.0 and 10% toxic fat until death. There was a dose-associated shortening of survival time: monkeys 1n the high-dose group survived only for an average of 91 days and animals 1n the low-dose group survived an average of 445 days (data for control animals were not provided). During the course of treatment the animals were moni tored for hematologic and gross clinical changes as well as histologic changes 1n the Uver 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 hypertrophic and 8 of the 27 animals treated with the "toxic fat" had small gastric ulcers. At the light microscopic level, the Uver had developed moderately distorted architecture with vacuolated cells containing neutral fat. The sternal bone marrow was nearly devoid of blood-forming elements, which was consistent with the observed decrease 1n packed blood cell volume and RBC counts. Also, electron micrographs revealed derangement of the rough endo plasmic reticulum and a loss of ribosomes, which the authors suggested may have resulted 1n the observed decrease 1n serum proteins. Skeletal muscle, lungs, GI tract, skin and heart had signs of edema as observed under the
8-58
light microscope; the electron mAcrographs of the heart revealed v a s c u l a r 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 TCDDs represented 64% by mass (Norback and Allen, 1973).
Allen et al. (1977) also assessed the toxicity 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 toxicity, monitored for hematologic changes and, following death or the termination of the study, were subjected to complete autopsies. Since no control animals were Included 1n this study, the data were compared with pre-exposure values where possible.
As observed 1n monkeys fed "toxic fat," the monkeys fed 2,3,7,8-TCDD lost hair and developed swollen eyelids and periorbital edema after 3 months of treatment. Blood parameters Including hemoglobin levels and hematocrit decreased; however, blood proteins (total serum protein and album1n/globul1n ratio) were not altered except 1n terminal animals. In the three animals that survived the 9-month exposure period, the toxic symptoms continued to develop during the 4 months of observation. The hematologic changes observed during the treatment period were consistent with the microscopic findings at autopsy of bone marrow degeneration. It was suggested that decreased platelet levels resulted 1n poor clotting and the widespread hemorrhage observed 1n many organs, which was particularly severe 1n the
8-59
stomach. Also, the decreased RBC count and resultant loss of oxygen-carry ing capacity resulted 1n an Increase 1n cardiac workload and hypertrophy of the heart. Cellular hypertrophy, hyperplasia and metaplasia of the epithe lium of the salivary gland, bile duct, lung and stomach were also observed microscopically. Although many effects of treatment were observed, 1t was concluded that the ultimate cause of death was related to the severe pan cytopenia.
The total dose of 2,3,7,8-TCDD used over 9 months 1n this study by Allen et al. (1977) was estimated to be between 2 and 3 yg/kg/day, which 1s approximately the same dose that resulted 1n severe toxic effects following chronic exposure 1n rats and mice. Schantz et al. (1979) reported 1n an abstract that similar, though less severe, effects were observed 1n female monkeys following chronic Ingestion of diets containing 50 ppt of 2,3,7,8TCDD. It was also noted that this exposure resulted 1n a decreased ability to successfully bear young (see Allen et al., 1977, 1n Section 9). It 1s apparent that the data available for nonhuman primates do not permit the determination of a NOAEL. 8.2. HUMAN 8.2.1. Acute Exposure. Symptoms of acute exposure to materials that con tained 2,3,7,8-TCDD are nausea and vomiting, headache and signs of Irrita tion to the eyes, skin and respiratory tract. Acute exposure to chemicals contaminated with 2,3,7,8-TCDD may also result 1n drenching and sweating with extensive dehydradtlon and weight loss, Increase 1n body temperature, severe respiratory distress, fatty degeneration of Uver, cyanosis, elevated blood urea nitrogen level, followed by fast deterioration of general condi tion and death from acute congestive heart failure (Regg1an1, 1982; Hay 1982). Initially a chemical burn-type cutaneous reaction will occur (pos sibly because of other chemicals), usually followed by chloracne after
8-60
several days to weeks (Taylor, 1979). Chloracne Is the most characterIstlc and frequently observed dermal lesion produced by 2,3,7,8-TCDD and other chlorinated aromatic hydrocarbons 1n humans (Crow, 1981; Taylor, 1979). This lesion consists of hyperplasia and hyperkeratosis of the InterfolUcular epidermis, hyperkeratosis of the hair follicle, especially at the Infundibulum, and squamous metaplasia of the sebaceous glands that form keratlnaceous comedones and cysts (Kimbrough, 1974). These cutaneous erup tions of comedones, cysts and possibly pustules 1n severe cases, usually occur on the face and shoulders (Crow, 1978a; Passl et al., 1981). The persistence of chloracne varies greatly, with severe cases lasting for up to 15 years, while mild cases may resolve 1n a matter of months. Similar epi dermal changes have been produced by 2,3,7,8- TCDD 1n rhesus monkeys (McConnell et al., 1978a; Allen et al., 1977), the ear of the rabbit (Polger and Schlatter, 1980), and hairless mice (Knutson and Poland, 1982). These changes have not generally been observed 1n other laboratory animals, such
as guinea p i g s , hamsters, r a t s and mice.
Chronic exposure to 2,3,7,8-TCDD has probably occurred most 1n chemical Industry workers exposed to low levels of this contaminant during the manu facture of 2,4,5-T on a dally basis. Chloracne 1s generally the first symptom noted 1n chronic exposure. Systemic symptoms, Including altered function of the neuromuscular system, Uver, kidneys, and pancreas, altered blood chemistry (serum bilirubin, GOT, GPT, lipid and cholesterol levels), porphyria cutanea tarda, hyperpigmentation and hyperkeratosis, have also been reported 1n Individuals that have had chronic 2,3,7,8-TCDD exposure (Crow, 1978b, 1981). A combination of acute, high-level exposure to 2,3,7,8-TCDD followed by chronic exposure for many years (or a lifetime) has been noted for residents of areas where PCDDs have been accidentally
8-61
released Into the environment (Taylor, 1979). Residents of Seveso, Italy, for example, where an explosion of a reactor vessel used to manufacture 2,4,5-T released PCDDs and other chemicals Into the atmosphere, were exposed acutely for a few days and are now exposed dally to diminishing levels of PCDDs 1n the soil.
The first cases of chloracne associated with exposure to PCDDs occurred after a 1949 explosion 1n a chemical factory producing 2,4,5-T 1n N1tro, WV (Holmstedt, 1980). A total of 228 workers were exposed. Symptoms Included nausea, headaches, fatigue, muscular aches and pains, and chloracne (Zack and Susklnd, 1980). Chemical tests revealed elevated lipid levels and prolonged prothrombin 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) 1n Ludwlgshafen, Germany. Most of the workers developed chloracne, while 21 workers developed nervous system and Internal organ damage 1n addition to severe chloracne. In 1963, an explosion at a 2,4,5-T producing factory 1n Amsterdam resulted 1n the exposure of 106 men to chlorinated 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) 1n 1968, which manufactured 2,4,5-tr1chlorophenol, at least 90 workers were exposed to dioxins. Clinical exami nations, Including Uver function tests, full blood counts and urinalysis, were conducted on 14 employees who were 1n the building at the time of the explosion (May, 1973; Hay, 1982). Eleven of these 14 men showed abnormal Uver function (zinc turbidity, thymol turbidity and serum transaminase) and altered hematological parameters or glucosurla. Later, after normal plant
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operations were resumed, additional workers apparently were e x p o s e d to 2,3,7,8-TCDD by contact and developed chloracne. Seventy-nine cases of chloracne developed by the end of 1968. The condition appeared on the face 1n all cases; however, other parts of the body were affected 1n more severe cases (May, 1973).
The most recent and extensively studied chemical plant explosion occurred on July 10, 1976, at the ICMESA (Industrie Ch1m1che-Meda-Soc1eta Az1onar1a) plant at Seveso, Italy. This accident, caused by the release of the reactor contents Into the atmosphere, exposed workers and residents (>8655 people) of the area to 2,3,7,8-TCDD 2,4,5-trlchlorophenol (Garatt1n1, 1982; Pocchlarl et al., 1983). A total of 447 patients developed chloracne and some complained of nausea, vomiting, headache, diarrhea, hyperhldrosls and Irritation of the eyes (Taylor, 1979). Serious cases of chloracne and dermal blistering occurred 1n children and appeared within several weeks of their exposure (G1anott1, 1977; Crow, 1981; Taylor, 1979). Pocchlarl et al.
( 1 9 79 ) c i t e d unpubl ished da t a r e p o r t e d t o the Lombardy Regional A u t h o r i t y
(Boerl, 1978; Ch1app1no et al., 1978; S1rch1a, 1978) on the health effects of 2,3,7,8-TCDD to children and adults at Seveso. Reduced peripheral nerve conduction velocities were noted 1n adults and children, with abnormalities being more frequent In people residing nearer the chemical plant. The Immunology of a group (n=45) of exposed children was compared with a similar unexposed group. No significant differences were noted; however, total serum complement activity, lymphocyte blastognie response and peripheral blood lymphocytes were elevated to some degree 1n the exposed children (Tognonl and Bonaccorsl, 1982). Exposure to 2,3,7,8-TCDD has been associ ated with Irtcreased serum glutamate-oxalacetate transaminase (GOT), serum GPT and gamma-glutamyl transferase (g-GT) levels 1n exposed children
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(Pocchlarl et al., 1979). Compared with normal values for "healthy" Individuals, lympho- cyte aberrations appeared more frequently; however, the findings were not statistically significant.
A comparison between children (under age 15) who developed chloracne and children of the same area who did not develop skin lesions was reported by
/ Caramaschl et al. (1981). A significant Increase 1n the frequency of head aches and eye Irritation (p=0 .01), 61 tract symptoms (nausea, vomiting, loss of appetite, abdominal pain or gastritis) (p=l.6xl0-4), and abnormal g-GT, serum GPT and aminolevulinic acid levels (p=2.3xl0-4, 0.035 and 1.2xl0"5, respectively) was noted 1n those children who had chloracne (Caramaschl et al., 1981). Ideo et al. (1982) measured urinary D-glucar1c acid levels to assess Uver microsomal enzyme activity 1n 67 children exposed to 2,3,7,8-TCDD at Seveso. A significant {p<0.05) Increase 1n the glucarlc acid levels, used to Indicate Increased microsomal 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 1n Hay, 1980, and the possible contamination of clean-up workers was closely monitored and safety measures were Implemented (Ghezzl et al., 1982).
j Laboratory tests on the blood (GOT, GPT, g-GT, alkaline phosphatase, bili rubin, hemoglobin, cell counts, thromboplastlc partial time, albumin, gamma globulin, cholesterol and triglycerides) and urine (porphyrin) of the workers were performed and compared with pre-employment values (of the same group of workers) and with a nonexposed group. No significant changes were noted, but exposure to 2,3,7,8-TCDD was believed to be minimal. A recent review of the Seveso Incident, Including Its history and human health effects, 1s reported by Tognonl and Bonaccorsl (1982).
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Three cases of accidental exposure to PCDDs (Isomer not specified) while scientists were attempting to prepare a pure standard in the laboratory were reported by Oliver (1975). All three laboratory scientists reported the same general symptoms: chloracne (within several weeks after exposure), GI pains, headaches, fatigability 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.
Since PCBs and PBBs can cause neurotoxic and behavioral effects (Safe, 1984; Agarwal et al., 1981; Anderson et al., 1978) and their toxic effects may be mediated by the same cytosolic receptor protein as 2,3,7,8-TCDD, it may be important to determine whether 2,3,7,8-TCDD has any neurotoxic activities (Silbergeld, 1984; Safe, 1985).
Additional reports of toxic effects as a result of acute 2,3,7,8-TCDD exposure in humans were noted by Kimbrough et al. (1977). Children were exposed to soil 1n horse arenas (In Eastern Missouri) sprayed with oil con ta mi nat ed w i t h 2 , 4 , 5 - t r i c h l o r o p h e n o l (5000 ppm i n the s o i l ) 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 in a contaminated horse arena. Three 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 toxicity (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-T. These workers, how ever, were also exposed to di- and trichlorophenols, PCDD contaminants and
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2,4-D. Thirteen employees developed moderate to severe chloracne; another 35 had minimal "active acne" (cysts, comedones or pustules). Other com plaints noted by the workers were eye Irritation, hyperpigmentation and hirsutism. Gastrointestinal symptoms (nausea, vomiting, diarrhea, abdominal pain or blood 1n the feces) were reported by 22 of the 73 workers. Findings as to cardiovascular, hepatic, pulmonary and neurological function were regarded as unremarkable and unrelated to occupational exposures. The authors noted that exposure was to several compounds and to assign a causa tive agent(s) would be conjecture (Poland et al., 1971).
In a brief report, Walker and Martin (1979) reported on some of the clinical findings of eight men who had contracted chloracne as a result of occupational exposure to 2,3,7,8-TCDD. Five men had elevated g-GT and tri glyceride levels. All eight men had decreased levels of high-density lipo protein (HDL) cholesterol and elevated total/HDL cholesterol ratios con sistent with higher than average risk of Ischaemic vascular disease. Abnor mal lipid levels, reported In 6 men, were attributed to enzyme Induction. May (1982), however, observed no differences 1n triglyceride, cholesterol, alkaline phosphatase D glucarlc acid or g-GT levels 1n 41 workers exposed to 2,3,7,8-TCDD. These determinations were made 10 years after the workers had developed 2,3,7,8-TCDD chloracne.
Blelberg et al. (1964) found 29 workers In a chemical plant manufactur ing 2,4-chlorophenol and 2,4,5-trlchlorophenol exhibiting features of chlor acne. These patients were tested for the presence of porphyria cutanea tarda (PCT). This Investigation revealed evidence of varying degrees of severity of PCT In 11/29 workers, but the authors could not determine any quantitative relationship between the chloracne and PCT. Urinary uropor phyrins were elevated 1n all 11 cases. A number of workers were noted to
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have hyperpigmentation, hirsutism, fragility of the skin and veslculobulbous
eruptions on exposed areas of the skin. In this paper the authors suggested PCT 1s perhaps an acquired disease occurring after various Insults to the liver (Blelberg et al., 1964).
In a survey of 204 employees engaged 1n the manufacture of 2,4,5-T for 1 month to 10 years, Ott et al. (1980) reported no cases of chloracne, por phyria cutanea tarda or other effects Indicative of dioxin exposure. Maxi mum allowable 2,3,7,8-TCDD levels 1n the final product were <1 mg/kg 1n 1966 and <0.1 mg/kg 1n 1972. Estimates of TWA exposure to 2,4,5-T ranged from 0.2-0.8 mg/m3, so that 2,3,7,8-TCDD levels would be exceedingly low. Cook et al. (1980) reported chloracne, from slight to severe cases, 1n 49 of 61 employees exposed to 2,3,7,8-TCDD during the manufacture of trlchlorophenol. Changes 1n Industrial and personal hygiene techniques decreased potential exposure to 2,3,7,8-TCDD and subsequent chloracne. Additional toxic effects were not reported. The National Institute for Occupational Safety and Health (NI0SH) 1n a survey of workers at a St. Louis, M0, trucking terminal contaminated with 2,3,7,8-TCDD (subsoil concentration of 2,3,7,8-TCDD was as high as 17 ppb) found one of the long-term former workers had developed porphyria cutanea tarda and angiosarcoma of the right 1l1um (Hope et al., 1984). Pazderova-Vejlupkova et al. (1981) reported that 80 workers developed chloracne, nausea, fatigue and weakness 1n the lower extremities while engaged 1n the production of 2,4,5-sodium trlchlorophenoxyacetate and trlchlorophenoxyacetate butylester. Prominent clinical symptoms among 55 of the 80 workers Included hypercholesterolemia, hyperlipemia and hyperphosphoUpemla, 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
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severe during a 3-4 year follow-up period. Long-term pathological symptoms (remaining evident 5 years after exposure) Include deviations 1n I1p1d metabolism, abnormal glucose tolerance and high urinary excretion of uropor phyrins (Pazderova-Vejlupkova et al., 1981). Polyneuropathy, usually of the lower extremities, occurred during the period of Illness and remained evi dent after 4 years. Singer et al. (1982) also Indicated a decrease 1n nerve conduction velocities of sural nerves 1n workers exposed to phenoxy acid herbicides (average exposure, 7 years) when compared with a similar group of nonexposed workers (40.3 m/sec 1n exposed vs. 42.8 m/sec 1n nonexposed, p=0.02). Although the causative agent 1s not known, PCDD contaminants are suggested.
The toxic effects attributed to 2,3,7,8-TCDD exposure were studied over a 10-month period 1n a group of 78 Vietnam veterans who claimed to have been exposed to Agent Orange (Bogen, 1979). Symptoms reported by the veterans Included gastrointestinal complaints (anorexia, nausea, diarrhea, constipa tion, abdominal pain), joint pain and stiffness, and neurological complaints (numbness, dizziness, headaches, depression and bouts of violent rage). These patients had previously been chronically 111 and had frequent Infec tions and allergies (Bogen, 1979). This study was apparently based on per sonal evaluations of health 1n a survey-type format. No control group was used for comparison and no clinical or medical evaluations of health were made. Host of these complaints are nonspecific, judgmental and occur com monly 1n the general public.
In an effort to evaluate the toxic effects attributed to 2,3,7,8-TCDD as a contaminant of Agent Orange, Stevens (1981) estimated a minimum toxic dose of 2,3,7,8-TCDD and determined the amount of this contaminant to which veterans may have been exposed during Agent Orange spraying. Based on
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studies 1n which rhesus monkeys were fed small amounts of dietary 2,3,7,8-
TCDD and analogy with human data on the minimum toxic dose of 2,3,7,8-tetra-
chlorod1benzo--furan (TCDF), the cumulative minimum toxic dose of 2,3,7,8-
TCDD 1n man was estimated to be 0.1 yg/kg (Stevens, 1981). Based on
application rates (4.1 g Agent Orange/m2) and 2,3,7,8-TCDD concentration
1n the herbicide (2 ppm), the average concentration of 2,3,7,8-TCDD on
sprayed surfaces of Vietnam was estimated to be ~8 yg/m2. Based on
accidental exposures to 2,3,7,8-TCDD 1n humans (Industrial accidents,
Eastern Missouri cases), Stevens (1981) estimated an average Intake transfer
factor (ratio of absorbed compound to environmentally available compound) of
1:2050 for 2,3,7,8-TCDD. Assuming this absorpt1on-to-exposure ratio and
even assuming that a soldier was directly sprayed (exposed to 8 yg/m2 )
for each day of h1s 1-year service 1n Vietnam, h1s cumulative Intake would
be only 1.4 yg or 0.02 yg/kg of 2,3,7,8-TCDD (Stevens, 1981). Based on
these calculations and assumptions, Stevens (1981) reported that 5 years of
direct dally contact with Agent Orange would be necessary to reach a toxic
level of 2,3,7,8-TCDD and felt that claims of Illness caused by 2,3,7,8-TCDD
1n Agent Orange were without merit. Exception 1s made, however, for certain
workers (forest Industries) who may have been exposed to 2,4,5-T and
2,3,7,8-TCDD for many years.
8.3. MECHANISM OF TOXICITY
A number of studies have attempted to determine the mechanism of toxic
ity of 2,3,7,8-TCDD. The ultimate purpose Is to provide a better estimate
of man's relative sensitivity to 2,3,7,8-TCDD and other compounds having a
similar mode of action. Specifically, these studies may be able to explain
the reason for the marked 1nterspec1es differences 1n 2,3,7,8-TCDD toxicity
and, thus, help determine 1f humans possess factors that are associated with
sensitivity to 2,3,7,8-TCDD toxicity.
i
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8.3.1. Receptor-Mediated Toxicity. Pharmacogenetlc studies have played an Important role 1n understanding the biologic and toxic effects of drugs and xenoblotlcs. Nebert and coworkers have shown that carcinogenic poly cyclic aromatic hydrocarbons (PAHs) Induce the cytochrome P-450-dependent monooxygenase AHH 1n certain responsive strains of mice (e.g., C57B1/6J, BALBc, C3HF/He), whereas this PAH Induction activity 1s minimal or nonexis tent 1n nonresponslve 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 that comprises regulatory, struc tural and possible temporal genes. Extensive studies on genetically Inbred responsive and nonresponslve mice (and their backcrosses) Indicate that these differences are related to the Aromatic Hydrocarbons (Ah) regulatory gene (termed "Ah complex" or "AH cluster") and Its gene product, the Ah cytosolic receptor protein. This receptor protein Interacts with PAH Ugands and the resultant PAH:Ah receptor complex translocates Into the nucleus and presumably Initiates the Induction of AHH by a process compar able to that proposed for the steroid hormones.
Since the carcinogenic and toxic effects of PAHs are dependent on their oxidative metabolism to reactive electrophilic forms, It 1s not surprising that the Ah receptor plays an Important role 1n mediating their toxicity and carcinogenicity (Kourl, 1976; Kourl et al., 1974; Benedict et al., 1973; Shum et al., 1979; Thomas et al., 1973; Legraverend et al., 1980; DuranReynolds et al., 1978; Robinson et al., 1975; Mattlson and Thorgelrsson, 1979). Responsive mice are more susceptible to the toxic (Inflammation, fetotoxlclty, primordial oocyte depletion) and carcinogenic effects of PAH at organs/tlssues 1n direct contact with the applied chemical; 1n contrast,
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nonresponslve mice are more susceptible to the tumorlgenlc effects of PAHs
at tlssue/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 detoxication.
2,3,7,8-TCDD can produce dermal lesions Including epidermal hyperplasia,
hyperkeratosis and squamous metaplasia of the sebaceous glands 1n hairless
mice (HRS/J), homozygous for hr/hr locus, but not 1n heterozygous (hr/+) or
normal haired wild type (+/+) mice. These effects on the skin seem to be
mediated through the Ah receptor (Poland, 1984).
8 .3.1.1.
2,3,7,8-TCDD: 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 microsomal AHH-1nduc1ng activity of
2.3.7.8- TCDD and 3-MC 1n several genetically Inbred mice strains were
similar. Like MC and related PAHs, 2,3,7,8-TCDD Induced AHH 1n several
responsive mouse strains (1.e., C57B1/6J). In contrast to 3-MC, 2,3,7,8-
TCDD Induced microsomal AHH 1n the DBA/2J nonresponslve mice; however, the
ED50 for this biologic response was significantly higher than values
reported for the responsive mice. In genetic crosses between responsive
C57B1/6 and nonresponslve DBA/2 mice 1t was also shown for both 3-MC and
2.3.7.8- TCDD that the trait of responsiveness 1s Inherited 1n a simple
autosomal dominant mode (Poland and Knutson, 1982). It has been suggested
that the observed differences 1n the activities of 3-MC and 2,3,7,8-TCDD are
related to their relative Ah receptor affinities (Poland and Knutson, 1982)
and the pharmacokinetic and metabolic factors that would more rapidly
diminish the "available" concentrations of 3-MC caused by metabolism and
excretion.
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Several studies with 2,3,7,8-TCDD 1n genetically Inbred mice support the receptor mediated hypothesis. The Induction of UDP-glucuranosyl transfer ase, DT dlaphorase, 6-am1nolevul1n1c acid, glutath1one-S~transferase B, T-aldehyde dehydrogenase and choline kinase 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; Kirsch et al., 1975; Dietrich et al., 1977; Ishldate et al., 1980; Poland and Glover, 1973a). Toxicology studies with genet1cally-1nbred mice confirm the role of the Ah locus 1n mediating several toxic effects Including porphyria, 1mmunotox1c1ty a wasting syn drome, thymic atrophy and cleft palate formation (Jones and Sweeney, 1980; Poland and Glover, 1980; Courtney and Moore, 1971; Vecchl et al., 1980, 1983). Poland et al. (1982) also linked the tumor-promoting activity of 2.3.7.8- TCDD 1n hairless mice to the cytosolic receptor. In vitro studies with XB cells 1n culture also support the role of receptor 1n mediating a dose-related cell kerat1n1zat1on by 2,3,7,8-TCDD that resembles some of the characteristics of chloracne (Knutson and Poland, 1980). This cell U n e Is also responsive to AHH Induction and contains a cytosolic receptor binding protein. Although the murine 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 phnobarbital, which 1s the only known agent at present that has been demonstrated to affect tissue concentrations of the receptor (Okey and
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Vella, 1984). Although the Ah receptor has not been detected 1n the cytosoT of DBA/2J mice, after the administration of radiolabeled 2,3,7,8-TCDD to these mice, some of the radlolabel 1s detected 1n the nuclei of the nonresponslve mice. Moreover, the sedimentation characteristics of the [3H]-2,3,7,8-TCDD:nuclear protein complex 1n DBA/2J mice are similar to those observed with the bound Ah cytosolic receptor protein 1n C57B1/6J mice using a sucrose density gradient centrifugation separation technique (Okey, 1983) . The cytosolic Ah receptor protein migrates Into the nucleus of the cell only after binding with 2,3,7,8-TCDD (Nebert and Jensen, 1979; Nebert, 1980; Greenlee and Poland, 1979; Okey et al., 1979, 1980; Tukey et al., 1982; Gonzalez et al., 1984), and this parallels the observations noted for the Interactions between steroids and their receptor proteins. The 2,3,7,8TCDD 1nducer-Ah receptor complex undergoes a temperature-dependent step before gaining high affinity for DNA (Okey et al., 1980; Klmura et al., 1984) . The 2,3,7,8-TCDD Ah-receptor complex thus binds to the nucleus and regulates the transcription of cytochrome P-j-450, which represents the gene product of Ah-structural loci, 1n mouse hepatoma cells 1n culture (Whitlock et al., 1984; Eisen, 1984) and 1n mice with various Ah genotypes (E1sen, 1984). This results 1n Induction of AHH activity which may remain elevated for a prolonged period. Such prolongation of activity may be because cytochrome P^-450 mRNA remains elevated even after 1 week follow ing single exposure to 2,3,7,8-TCDD (Eisen, 1984).
In elucidating the mechanisms of 2,3,7,8-TCDD Induced teratogenic effect 1n the formation of cleft palate 1n C57 mouse fetus, the presence of Ah-receptor 1n the palatal shelves of the embryo seems to be necessary for alterat1on/1nh1b1t1on of terminal differentiation of the medial epithelial cells 1n the palate (Denker and Pratt, 1981; Pratt, 1983; Pratt et al..
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1984a,b). Pratt and W1ll1s (1985) have even suggested utilizing growth
Inhibition of an established line of human embryonic palatal mesenchymal
cells for In vitro short-term screening for assessment of the teratogenic
potential of environmental agents.
The presence of Ah-receptor have been detected 1n normal lung, Uver,
kidney, spleen and Intestine from human fetus. In addition, normal lung
tissue from 10 of the 50 Individuals examined were found to have Ah-receptor
(Roberts et al., 1985). Ah-receptor has also been observed 1n cell lines of
human squamous cell carcinoma at a concentration of 5-10 fmol/mg (Hudson et
al., 1983; Roberts et al., 1985). Whether variation 1n Ah-receptor content
1n human 1s genetically determined and 1s a critical determinant of Individ
ual susceptibility to PCDDs 1s not known and warrants further Investigation.
8 .3.1.2.
2,3,7,8-TCDD AND RELATED TOXIC HALOGENATED ARYL HYDROCARBONS;
STRUCTURE-ACTIVITY CORRELATIONS -- The evidence for a receptor mediated
mechanism of action for 2,3,7,8-TCDD 1s supported by data reported for the
effects of other halogenated aryl hydrocarbons 1n genetically Inbred mice
i
and other diverse animal species. A number of reviews and comparative
studies (Allen et al., 1979; Kimbrough, 1974; Kimbrough 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 that Include 1) a
wasting syndrome which 1s manifested by a progressive weight loss and
decreased food consumption by the treated animals; 2) skin disorders Includ
ing acneform eruptions or chloracne, alopecia, edema, hyperkeratosis, and
hypertrophy of the Meibomian glands; 3) lymphoid Involution and atrophy;
4) porphyria (resembling porphyria cutanea tarda); 5) endocrine and repro
ductive disorders; 6) modulation of chemical carcinogenesis; and 7) the
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Induction of numerous enzymes Including the cytochrome P-448 (or P-450C) dependent monooxygenases. It is apparent that the effects of these com pounds are not manifested in all the animal species tested. McConnell and Moore (1979) summarized the pathologic findings observed in several animal species after pretreatment with PCDDs, PCDFs, PCBs and PBBs; these data illustrate the different species and organ/tlssue susceptibilities to these compounds. It is also evident that for most of these effects, all the toxic halogenated aromatics elicit similar effects in these species that also contain the cytosolic receptor protein (Carlstedt-Duke, 1979; Carlstedt-Duke et al., 1979, 1981; Okey, 1983; Okey and Vella, 1982; Mason and Okey, 1982). These observations support a common mechanism of action for all the toxic halogenated aryl hydrocarbons (Poland and Knutson, 1982; Safe et al., 1982; McConnell and Moore, 1979).
Several reports have demonstrated the effects of structure on the activ ity of PCDDs. The most active member of this group is substituted in the lateral 2, 3, 7 and 8 positions; activity is decreased with 1) decreasing lateral substituents, and 2) increasing Cl substitution. Moreover, for several PCDDs, there is an excellent correlation between the toxicity of individual PCDD congeners in guinea pigs and mice (McConnell et al., 1978b) and their AHH induction potencies in 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, 2,3,7,8TCDF, 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 correlation among the toxicity of several individual PCDFs (Yoshihara et
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a!., 1981), their AHH Induction potencies 1n rat H-4-II-E hepatoma cells and binding affinities to male Wlstar rat hepatic cytosolic receptor protein (Bandlera et al., 1983).
Correlations between structure-activities of PCDDs and Ah-receptor site binding, AHH Induction potencies and systemic toxicity have also been sug gested (Safe et al., 1984). 2,3,7,8-TCDD, the Isomer substituted with Cl 1n all four lateral positions 1s most active for all of the above three param eters. Increased or decreased substitution of 2,3,7,8-substltuted PCDDs tend to decrease receptor binding affinity and toxic action.
The most active PCB congeners, 3,4,4',5-tetra-, 3,3',4,4'-tetra-, 3,3',4,4',5-penta- and 3,3',4,4',5,5'-hexachlorob1phenyl, are substituted at both para and at two or more meta positions. The four coplanar PCBs Induce rat hepatic microsomal 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, 1980; 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 microsomal AHH, DMAP 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,-q ) In rat hepatoma H-4-II-E cells and their binding affinities (EDj-q ) for the 2,3,7,8-TCDD rat cytosolic recep tor protein (Sawyer and Safe, 1982; Bandlera et al., 1983). The results showed that there was an excellent correlation between AHH Induction potencies and receptor binding avidities of these compounds and the order or activity was coplanar PCBs (3,3',4,4'-tetra-, 3,3',4,4',5-penta- and
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3 ,3 ', 4 , 4 ', 5 , 5 '-hexachloroblphenyls) > 3,4,4',S-tetrachlorob1phenyl > mono
o r t h o coplanar PCBs > diortho coplanar PCBs. It was also apparent that the relative tox1c1t1es 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; Kurokl and Masuda, 1977).
The coplanar and monoortho coplanar PCBs also exhibit differential effects 1n the Inbred C57B1/6J and DBA/2J mice. These compounds Induce AHH and cause thymic atrophy 1n the former "responsive" mice whereas at compar able or higher dose* none of these effects are observed 1n the nonresponslve DBA/2J mice (Parkinson et al., 1982). The results obtained for structurally diverse PCDDs, PCBs and PCDFs clearly support the role of the receptor pro tein 1n Initiating the broad spectrum of biologic and toxic effects elicited by these chemicals. Bandlera et al. (1983) demonstrated that the 2,3,7,8TCDD receptor protein 1s not only susceptible to halogen substitution
p a tt e r n s but als o the s t r u c t u r e of the s u b s t i t u e n t . The cytosol receptor
binding avidities and AHH Induction potencies 1n rat hepatoma H-4-II-E cells for several 4'-X-2,3,4,5-tetrachlorob1phenyls were remarkably dependent on the structure of the X substituent. The binding data for 13 different sub stituents was subjected to multiparameter regression analysis to correlate binding avidities with the physical and chemical characteristics of the critical lateral X substituents. The equation
log (1) = 1.53<j + 1.47 1 + 1.09 HB + 4.08 EC50
showed that ligand binding was dependent on substituent electronegativity (cr), 11pophl 11c1ty (1) and hydrogen binding (HB) with a correlation coefficient (r) equal to 0.978 for 13 different substituents.
8-77
Dependency of 11gand-receptor complex and the biological activity of
PCDDs on their electronic and geometric structure Investigated by an n
vitro molecular fragment analysis has also been suggested (Cheney, 1982).
The receptor mediated hypothesis for the mechanism of action of 2,3,7,8-
TCDD still requires further confirmation and numerous problems must be
clarified. For example:
1. Several cell culture Unes that appear to have the Ah receptor are highly resistant to the toxicity of TCDD; the nonrespons1ve HTC and responsive H-4-II-E cell Unes (1.e., forAHH 1nduc1b1l1ty by TCDD) do not possess cytosolic receptor; how ever, the nonresponslve HTC cells possess more nuclear recep tor binding protein than the responsive H-4-II-E cells (Okey, 1983; Okey et al., 1980).
2. Hepatic cytosolic receptor levels 1n rats (Wlstar and SpragueDawley), C57B1/6J mice, hamsters and guinea pigs are compar able (Gas1ew1cz et al., 1983b); however, their susceptibility to the biologic and toxic effects of TCDD are highly variable: guinea pigs are highly susceptible to the lethal effects of TCDD (LD50 = 1-2 yg/kg) whereas thesusceptibility of the other species follows the order rat > C57B1/6J mice > DBA/2J mice > hamster (Neal et al., 1982).
3. "Responsiveness" of the mouse to 2,3,7,8-TCDD Induced toxicity seems to be highly dependent on the genetic conditions, as regards the Ahb allele gene, of the animal. However,cell Unes "nonresponslve" to P]-450 Induction by 2,3,7,8-TCDD have also been found to possess Ah-receptor protein (Guenthner and Nebert, 1977).
Ah receptor protein 1s also present 1n human tissue (Roberts et al., 1985). Whether variation 1n Ah-locus 1s critical for Individual susceptibility to toxicity by PCDDs remains to be demonstrated 1n human population.
8.3.2. Metabolism. The metabolism of 2,3,7,8-TCDD has been examined 1n
the guinea pig, rat, mouse and hamster. Urine and bile from 14C-TCDD-
treated animals were found to be free of unmetabollzed 2,3,7,8-TCDD, demon
strating that metabolism was required for elimination through these routes
(Olson et al., 1983). The direct Intestinal elimination of unchanged
2,3,7,8-TCDD 1n feces suggests, however, that some routes of excretion may
not be dependent on prior metabolism of the toxin (Olson et al., 1983).
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Thus, 1t 1s not possible to directly correlate the half-Hfe for elimination of 2,3,7,8-TCDD with Its in vivo rate of metabolism 1n a given species. The relative persistence of 2,3,7,8-TCDD 1n a given species may be related to the iji vivo rate of 2,3,7,8-TCDD metabolism, excretion of the toxin not dependent upon metabolism (direct Intestinal elimination, lactation, sebum), and the relative tissue distribution of 2,3,7,8-TCDD, particularly to adipose stores. Qualitative and quantitative differences 1n the metabolism and disposition of 2,3,7,8-TCDD have been observed between various species, and these may 1n part be related to the remarkable 1nterspec1es differences 1n sensitivity to 2,3,7,8-TCDD toxicity (Olson et a!., 1983).
Polger et al. (1982a) suggested that 2,3,7,8-TCDD metabolism represents detoxification, since they observed relatively little toxicity 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 uroporphyrinogen decarboxylase activity and lead to 2,3,7,8-TCDD-1nduced p o r p h y r i a (De V e r n e u l l e t al., 1983). C u r re n t data on the s t r u c t u r a l Identification of 2,3,7,8-TCDD metabolites suggest that reactive epoxide Intermediates 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 radio activity to hepatic DNA was 4 orders of magnitude less than the levels of binding observed with other chemical carcinogens. The study found much higher levels of 2,3,7,8-TCDD derived radioactivity bound to hepatic protein of the rat. No data 1s available, however, on the degree 2,3,7,8-TCDD derived radioactivity Is bound to tissues of various species of laboratory animals, which have demonstrated remarkable variability In sensitivity to 2,3,7,8-TCDD. While biliary excretion products may represent detoxified,
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polar metabolites of 2,3,7,8-TCDD, 1t remains to be shown whether unexcreted reactive metabolites initiate some of the toxic responses associated with exposure to this toxin. 8.3.3. Vitamin A Depletion. Many of the toxic effects of 2,3,7,8-TCDD resemble the effects of vitamin A deficiency, such as epithelial lesions, keratosis and Immunosuppression. 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 1n the hepatic storage of retinol 1n Sprague-Dawley rats (Thunberg, 1984; Thunburg et al., 1979, 1980). The authors suggested, but did not demon strate, that the low storage of retinol 1n the 2,3,7,8-TCDD-treated animals 1s the result of an Increased turnover of retinol.
Hakansson and Ahlborg (1985) pretreated male Sprague-Dawley rats with 2,3,7,8-TCDD at 10 yg/kg bw 4 days before the oral administration of 1200 IU/kg of1 retlnyl acetate. One hundred ninety-two hours postadm1n1strat1on of retlnyl acetate the 2,3,7,8-TCDD-pretreated rats excreted 41% of the retlnyl acetate compared to the control excreting only 30%. After 2,3,7,7TCDD treatment the decrease 1n vitamin A content was 39-53, 19-67 and 18-44% 1n the Uver, Intestine and epididymis, respectively. 2,3,7,8-TCDD treat ment also Influenced vitamin A content 1n the thymus, Initially increasing by 42% 1n 6 hours and then decreasing by 40% 1n 192 hours as compared to the controls. 2,3,7,8-TCDD pretreatment Increased the vitamin A content 1n the kidney 3-30 times that of the control. It 1s Important to note that the kidney becomes the primary vitamin A storage organ 1n vitamin A deficient animals (Johnson and Baumann, 1947; Moore and Sharman, 1950). In a similar study Thunberg and Hakansson (1983) has also found an Increase of vitamin A storage 1n the kidney after a single oral dose of 2,3,7,8-TCDD 1n male Sprague-Dawley ratsv Results from these observations suggest strongly that
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p r e t r e a t m e n t with a single oral dose of 2,3,7,8-TCDD can affect both storage
and excretion of retlnyl acetate as well as the vitamin A storage 1n several tissues.
These results suggest that an Induced vitamin A deficiency may be responsible for some, but not all, of the toxic effects produced by 2,3,7,8TCDD. At the highest dose of 2,3,7,8-TCDD, dietary retinol supplements could not fully compensate for the 2,3,7,8-TCDD-produced decrease 1n hepatic retinol content. 8.3.4. Lipid Peroxidation. Increased lipid peroxidation has been sug gested as a possible mechanism of 2,3,7,8-TCDD-1nduced toxicity (Sweeney and Jones, 1983). This hypothesis 1s based on the following limited pieces of evidence. First, Iron deficiency Inhibits in vitro Up l d peroxidation (Bus and Gibson, 1979; Sweeney et al., 1979) and reduces the hepatotoxlc effects of 2,3,7,8-TCDD (Sweeney et al., 1979). Secondly, Upofuscln pigments, by-products of 11p1d peroxidation, 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 antioxidant butylated hydroxyanlsole (BHA) at a level of 0.75% 1n the diet provided some protection from
r-
2,3,7,8-TCDD-1nduced prophyMa and neutral 11pid 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 antioxidant vitamin E (0.01%) was Included 1n the diet.
Recently, Stohs et al. (1983) obtained direct evidence that 2,3,7,8-TCDD accelerates lipid peroxidation 1n Sprague-Dawley rats. Groups of 4-8 female rats were treated for 3 days with 2,3,7,8-TCDD at doses of 0, 10, 20 or 40 vg/kg by gavage (1n a corn oil vehicle). At days 1, 6 and 11 after the
8-81
last treatment the animals were sacrificed and 11p1d peroxidation was deter mined 1n Isolated liver mlcrosomes by the reaction of formed malondlaldehyde with th1obarb1tur1c acid. At all sacrifice periods, Increased Upld peroxi dation was observed and the Increase was dose-related. The maximal Increase detected on day 6 after the last treatment was 5- to 6-fold greater than 1n the controls. In addition, these workers measured I1p1d peroxidation 1_n vivo by the determination of conjugated dienes 1n rats receiving 2,3,7,8TCDD at 40 yg/kg. Using this latter method, similar Increases In lipid peroxidation were detected, although the maximal Increase of 2.35-fold was observed at day 1 postexposure rather than day 6. The authors suggested that the In. vivo formation of reactive free radicals during lipid peroxida tion could account for the nonspecific nature of 2,3,7,8-TCDD toxicity.
Since 0-carotene can quench singlet oxygen (102 ) and vitamin E 1s an antloxldent, Hassan et al. (1985) studied the effects of vitamins A and E on 2,3,7,8-TCDD Induced 11p1d peroxidation. Vitamin A was found to Inhibit I1p1d peroxidation, elevated the activity of glutathione peroxldate and prevented a 2,3,7,8-TCDD-1nduced decrease 1n 6SH content 1n the liver. Vitamin E markedly Inhibited microsomal I1p1d per1ox1dat1on, but did not have any effect on glutathione peroxidase activity or glutathione content. 8.3.5. Endocrine Imbalance. Some of the toxic response to 2,3,7,8-TCDD, Including hirsutism and diminishing libido, Indicate that 2,3,7,8-TCDD may produce some of Its toxicity through endocrine disturbances (Oliver, 1975). Nlenstedt et al. (1979) reported that a single oral dose of 20 yg 2,3,7,8TCDD/kg bw significantly reduced testosterone catabolism. Catabolism of exogenous estrogen 1n ovarlectomlzed rats 1s also decreased by 2,3,7,8-TCDD pretreatment (Shlverlck and Muther, 1982). In this study, there was a 57% Increase 1n serum estrone concentrations following administration of 10 mg
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e s t r o n e / 1 0 0 g bw/day f o r 4 days t o e i t h e r c o n t r o l or 2,3,7,8-TCDD p r e t r e a t e d ovar1ectom1zed rats. No differences were observed 1n the Increase 1n uterine wet weight following estrone administration 1n control and 2,3,7,8TCDD pretreated rats. Thus, the uterotrophlc response was not altered by any 2,3,7,8-TCDD-med1ated change 1n estrone disposition.
Sh1ver1ck and Muther (1983) also measured estradiol metabolism 1n female Holtzman rats given 2,3,7,8-TCDD at a dose of 1 yg/kg bw on days 4-19 of gestation. At this fetal toxic dose, the catechol estrogen formation abil ity of Isolated liver mlcrosomes from the dams was decreased 50% when mea sured on day 20 of gestation. These mlcrosome preparations had a 4-fold Increase 1n the 7a-hydroxylat1on of testosterone, while there was no change 1n the 16a- or 68-hydroxylase activity. Although steroid metabo lism was altered 1n mlcrosomes Isolated from 2,3,7,8-TCDD-treated pregnant rats, similar exposure of pregnant rats on days 4-15 of gestation resultd 1n no change 1n circulating levels of serum 178-estradlol. The authors sug gested that other mechanisms besides Uver metabolism of steroids may be Involved 1n the fetotoxlc effect of 2,3,7,8-TCDD.
Gustafsson and Ingelman-Sundberg (1979) observed that 2,3,7,8-TCDD pro duced greater change 1n steroid metabolism 1n female Sprague-Dawley rats than 1n male rats of the same strain, resulting 1n a Uver enzyme pattern displaying less sex differentiation than 1n uninduced rats. Based on this result, they propose that some of the effects of 2,3,7,8-TCDD resulted from an Interaction with the hypothalamo-pltultary axis, rather than from a direct effect on steroid metabolism.
Since glucocorticoid hormones are known to have a catabolic effect on lymphoid 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
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the ability of 2,3,7,8-TCDD to either stimulate the production or mimic the effects of these hormones. In male Sprague-Dawley rats treated by gavage with 2,3,7,8-TCDD at a dose of 50 yg/kg (the ~LD5Q), there was a slight depression 1n blood glucocorticoids during post-treatment days 1-4, followed by an ~2.5-fold Increase on post-treatment days 7 and 14. While 1n competi tive binding assays between 2,3,7,8-TCDD and a synthetic hormone, dexamethasone, 2,3,7,8-TCDD had no affinity for the hormone receptor. Thus, 2,3,7,8TCDD may have stimulated glucocorticoid production but was not able to mimic the action of these hormones by binding to the glucocorticoid receptor. It was determined, however, that the Increase 1n glucocorticoids was likely not to participate 1n the toxicity of 2,3,7,8-TCDD through adrenal hyperfunc tion, since prior adrenalectomy did not provide any protection from the lethal effects ofj2,3,7,8-TCDD 1n rats. 8.4. SUMMARY 8.4.1. Experimental Animal Data. A wide range of lethal doses has been reported for 2,3,7,8-TCDD depending on the species tested. The male guinea pig was the most sensitive, with an LD5Q value of 0.6 yg/kg; the male hamster was the least sensitive, with an LDcn value of 5051 yg/kg
dU
(Schwetz et al., 1973; Henck et al., 1981). At least for acute exposure, the toxicity 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 thymic atrophy. Although Uver damage was not observed 1n the guinea pig, the most sensitive species
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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 LDrn for 9 congeners of PCDDs, 1t appeared
i)U
that biologic activity required chlorine 1n the 2,3,7,8-positions (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 toxicity 1n rats and mice. Single high doses, 200 yg/kg, of 2,3,7,8-TCDD produced Uver necrosis 1n rats (Jones and Butler, 1974); however, lower doses of 5 and 25 yg/kg produced fatty changes and proliferation of the ER (Fowler et al., 1973). Along with Increases 1n ER, there was an associated marked Increase 1n 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, there was also a decreased ability to excrete some xenoblotlcs Into the bile (Yang and Peterson, 1977; Hwang, 1973). Porphyria was also observed, with the mouse being more sensitive than the rat. In addition to effects on the Uver, 2,3,7,8-TCDD also affects Intestinal absorption by Increasing and decreasing the absorption of specific nutrients. In some species, the cellularlty 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 1s undlsputably an acute 1mmunotox1c substance 1n animal models, causing decreases 1n thymic 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-indepen dent antigen (type III pneumococcal polysaccharide) has been reported fol lowing 2,3,7,8-TCDD exposure (Vecchl et al., 1980). 2,3,7,8-TCDD presumably affects lymphocytes or thymic cells directly, since several studies have
8-85
negated Indirect routes of immunosuppression (hormonal controls). 2,3,7,8TCDD at 1mmunotox1c 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 1mmunoglob1n levels 1n mice at oral doses as low as 0.01 and 0.1 pg/kg/week when administered for up to 8 weeks (Sharma and Gehrlng, 1979). Thomas and HInsdl11 (1979) reported reduced hypersensitivity to DNFB, decreased Immune response to . coll LPS and decreased thymic weight 1n young mice exposed to 2.5 and 5 ppb 2,3,7,8TCDD (0.33 and 0.65 pg/kg) through maternal dosing. Thigpen et al. (1975) postulated a NOEL of 0.5 pg 2,3,7,8-TCDD/kg/week for 4 weeks, but more precise tests of 1mmunotox1c1ty suggest a lower NOEL would be appropriate, especially for neonatal and young animals.
The mechanism of 2,3,7,8-TCDD-1nduced 1mmunotox1c1ty 1s not yet known. 2,3,7,8-TCDD 1s not likely to decrease Immune responsiveness through an endocrine control. 2,3,7,8-TCDD may act as an antigenic agent causing Immunosuppression and thymic 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 subchronic toxicity studies 1n rats and mice, the Uver appeared to be a target organ. The Induction of Uver damage after repeated exposure to small doses of 2,3,7,8-TCDD was shown 1n rats. Histologic changes 1n the Uver of rats killed 2, 4, 8 , 16 and 28 weeks after exposure to weekly doses of 1 pg/kg bw revealed no fatty changes until week 28; however, 12 weeks after termination of the 28-week exposure, there was still evidence of fatty
8-86
changes in the liver (King and Roesler, 1974). A similar long induction period was observed by Goldstein et al. (1982b) for porphyrin accumulation in the liver of rats. Following 16 weeks of exposure to 2,3,7,8-TCDD and a 6-month postexposure period, porphyrin levels were still elevated. The only study in mice (NTP, 1980a) described toxic hepatitis as the only effect of subchronic exposure to low levels of 2,3,7,8-TCDD. In these and other sub chronic studies, NOELs of 0.01 yg/kg/day (Kociba et al., 1976), 0.5 yg/ 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 yg/kg/week was obtained in females, while males exposed to 1 yg/kg/week (the lowest dose tested) developed toxic hepatitis. Similar hepatic lesions were observed after exposure to a mixture of HxCDDs with NOELs of 2.5 and 1.25 yg/kg/week reported for rats and mice, respectively (NTP, 1980b).
In chronic toxicity studies in rats and mice, it was again the liver that appeared to be the most sensitive organ. Changes in the liver of rats included initially fatty infiltration, and at higher doses, necrosis. The studies in rats indicated that 0.001 yg/kg/day was a NOEL, while 0.05 and 0.1 yg/kg/day were the N0AEL and FEL for liver damage (Kociba et al., 1978b, 1979; NTP, 1980a). In mice, a NOEL was not determined, with the lowest doses tested, 0.0015 and 0.006 yg/kg/day, producing liver damage in male and female B6C3F1 mice (NTP, 1980a), while the lowest dose tested in Swiss mice, 0.001 yg/kg/day, produced amyloidosis of the kidney, spleen and liver (Toth et al., 1978, 1979). In nonhuman primates, chronic exposure to 2,3,7,8-TCDD in the diet at 50 or 500 ppt resulted in hair loss, edema and pancytopenia (Allen et al., 1977; Schantz et al., 1979). Data were not available to determine a NOEL for monkeys. Also, in the only study avail
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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 yg/kg/week for males and females, respectively, produced toxic hepatitis and represented a FEL (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 Uver function, hematological abnormalities, porphyria cutanea tarda, hyper pigmentation and hirsutism. Recently, Susklnd and Hertzberg (1984) have demonstrated an association between exposure to 2,4,5-T contaminated with 2.3.7.8- TCDD and the history of GI ulcer. No evidence of Increased risk for cardiovascular disease, hepatic disease, renal damage or central or peri pheral nervous system problems could be found 1n a group of workers exposed to 2,4,5-T following a run way reaction (Susklnd and Hertzberg, 1984). How ever, occupational or accidental exposure to 2,3,7,8-TCDD has been shown to produce neurological ailments 1n addition to the above ailments. The neuro logical problems Include peripheral polyneuropathies, Impairment of sensory functions Including sight disorders, loss of hearing, taste and sense of smell, central lassitude, weakness, Impotence and loss of libido (Regg1an1, 1982; Kimbrough et al., 1984). Only one estimate was available, which speculates a cumulative minimum toxic dose of 0.1 yg/kg for man (Stevens, 1981). The available follow-up reports and epidemiological studies, primar ily on populations exposed occupationally, accidentally or 1n Vietnam, Indi cate that toxic effects noted soon after exposure to 2,3,7,8- TCDD may sub side or may persist for many years. 8.4.3. Mechanisms of Toxicity. In the preceding sections, five possible mechanisms by which 2,3,7,8-TCDD may produce U s 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
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t h a n t h e p a r e n t compound, although Intermediate or minor metabolites of
2,3,7,8-TCDD may be Involved 1n toxicity. Vitamin A depletion, Increased lipid peroxidation and effects on the hypothalamo-pltultary axis have all been Implicated as possible mechanisms for 2,3,7,8-TCDD-1nduced toxic response. It seems probable that these mechanisms are responsible for some, but not all, of the toxic effects of 2,3,7,8-TCDD.
The major mechanism of 2,3,7,8-TCDD toxicity that has received Intense Investigation Involves effects mediated by specific cytosolic receptors pro duced by the Ah locus. The toxicity of various dioxins has been correlated with binding to the cytosolic 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 In several species, species differences 1n the toxic response to 2,3,7,8-TCDD do not correlate with species differences 1n receptor concentration or affinity, or with the degree of enzyme Induction. It thus appears that the toxicity of 2,3,7,8TCDD may be mediated by binding to the cytosolic 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 cytotoxic process.
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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 teratogenic 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 teratogenic and fetotoxlc to mice at >46.4 mg/kg. Rats were more sensitive, exhibiting fetotoxlc responses at 10 mg/kg for this 2,4,5-T/2,3,7,8-TCDD mixture. Since this Initial report, research has focused on determining the role of 2,3,7,8-TCDD contamination 1n eliciting the teratogenic response. These studies are summarized In Table 9-1.
Neubert and DUlmann (1972) conducted a detailed study to determine the significance of 2,3,7,8-TCDD contamination. These Investigators assayed three 2,4,5-T samples: a highly purified sample containing <0.02 ppm 2.3.7.8- TCDD (referred to as Sample A), a purified sample Identical to that used by Roll (1971) that contained 0.05+0.02 ppm 2,3,7,8-TCDD (Sample B), and a commercial sample containing an undetermined quantity of 2,3,7,8-TCDD (Sample C). All three samples Induced cleft palates at sufficiently high doses (30-90 mg/kg). In terms of the number of fetuses with cleft palate/ the total number of fetuses, the dose/response pattern observed by Neubert and DUlmann (1972) was similar to that observed by Roll (1971) using a similar grade of 2,4,5-T. In addition to the three 2,4,5-T samples, Neubert and DUlmann (1972) also assayed a sample of 2,3,7,8-TCDD alone and 1n various combinations with the highly purified sample of 2,4,5-T. This approach allows at least partial quantification of the significance of 2.3.7.8- TCDD contamination 1n 2,4,5-T-1nduced cleft palates. When the
9-1
TABLE 9-1
Studies on the Potential Teratogenic Effects of 2,3,7,8-TCBO Contaminated 2,4,5-T
9-2
Spec1es/Stra1n
Vehicle
Form of 2,4.5-T TCDD Level
Dally Oose
Treat Obser ment vation Haternal Response Days Day
Fetal Response
Reference
Hlce/NNRI
Hlce/NNRI Hlce/NNRI Hlce/NNRI Hlce/NNRI
N1ce/CD-1
Rape-seed oil
acid
<0.02 ppm (Sample A)
0. 15, 30, 45, 60, 90 and 120 mg/kg
6-15
Rape-seed oil
acid
0.05*0.02 ppm 30, 60 and
(Sample B)
90 mg/kg
6-15
Rape-seed oil
acid
NR (Sample C) 90 mg/kg
6-15
Rape-seed oil
butyl ester
NR
12 and 17 mg/kg
6-15
NR
acid
0.05*0.02 ppm 20, 35, 60,
6-15
90 and 130
mg/kg
Corn o11:acetone (9:1)
acid
<0.05 ppm
115 mg/kg
10-15
18 No toxic effects; Significant Increases In
decreased maternal the Incidence of cleft
weight at doses of palates at doses above
90 mg/kg and
30 mg/kg (see text for
greater
additional details).
Significantly decreased
(p<0.005) fetal weight
at all dose levels.
Neubert and Dlllmann, 1972
18 No toxic effects; Increases In the Incidence Neubert and decreased maternal of cleft palate at 60 and Dlllmann, 1972 weight at 90 mg/kg 90 mg/kg; significant (p<0.005) at all dose
levels
18 No toxic effects but decreased maternal weight
Increase 1n the Incidence of cleft palate; signifi cant (p<0.005) decrease
1n fetal weight
Neubert and Dlllmann, 1972
18 No toxic effects
Significant decrease 1n fetal weight but no effect on mortality; Increase In the frequency of cleft palate similar to that seen with acid (see text)
Neubert and Dlllmann, 1972
NR Toxic effects observed at 90
and 130 mg/kg
Increases In the percent age of resorptions and/or dead fetuses at 90 and 130 mg/kg; Increases In the
Incidence of cleft palate and retardation of skeletal development at 35 mg/kg and above
Roll, 1971
18 No significant
No effect on fetal mortal Courtney, 1977
effect on weight
ity or fetal weight but
gain or llver-to- an Increase In the Inci
bw ratios
dence of cleft palate
1ABIE 9-1 (cont.)
9-3
Spedes/Straln
Vehlcle
N1ce/C57Bl/6 Honey:water (1:1)
H1ce/AKR
Honey:water ( 1: 1)
Rats/SpragueOawley (groups of
25 rats)
Gavage/hydroxypropyl-methylcellulose
Rats/Ulstar
Gavage/aqueous gelatin or corn oil
Rats/Wtstar
Gavage/aqueous gelatin or
c o m oil
Form of 2,4,5-T TCOO Level
a d d 30 ppm
add
30 ppm
add
0.5 ppm
add
<0.5 mg/kg
butyl ester
<0.5 mg/kg
Dally Dose
Treat- Obserment vatlon Maternal Response Days Day
Fetal Response
Reference
46.4 and 113 mg/kg
6-14
113 mg/kg
6-15
1, 3, 6, 12 or 6-15 24 mg/kg/day
25, 50, 100 or 6-15 150 mg/kg/day
50 or 150 mg/kg/day
6-15
NR
Increase 1n Uverto-bw ratio
No effect on bw and no observable signs of toxicity
Some maternal mortality and decreased bw gain at 150 mg/kg; no signs of toxicity at 100 mg/kg or below NR '
Significant (p<0.01) Increases In the Incidence of cleft palate In the high dose group and cystic kidney In both dose groups; Increased fetal mortality also observed In the high dose group
Courtney et al., 1970a,b
Significant (p<0.05) Increases In the Incidence of cleft palate and fetal mortality
Courtney et al., 1970a,b
A slight but statistically significant (p<0.05) decrease In Implantations and litter size In lowest dose group only; no frank teratogenic effects based on a detailed examination of the control and 24 mg/kg dose group; the only effect noted was an Increase In the Incidence of 5th par tially ossified sternebrae
Emerson et al., 1970, 1971
At 100 or 150 mg/kg, decreased fetal weight.
Increased fetal mortality and an Increase In the Incidence of skeletal anomalies; no significant effect at the two lower dose levels
Khera and McKinley, 1972; Khera et al., 1971
No significant effect on fetal mortality, fetal weight or the Incidence
of anomalies
Khera and McKinley, 1972;
Khera et al., 1971
TABLE 9-1 (coni.)
9-4
Specles/Straln
Vehicle
Form of 2.A.5-T TCDD Level
Dally Dose
Treat Obser ment vation Maternal Response Days Day
Fetal Response
Reference
Rats/Holtzman Gavage/1:l solution of
honey and water
acid
Rats/CD
Gavage/15% sucrose solution
acid
Rats/straln Gavage/methocel not specified
acid
Rats/straln Gavage/methocel not specified
acid
Syrian hamsters/ Hesocrlcetus euratus
Gavage/acetone, corn oil, and
carboxymethyl cellulose In ratio of 1:5.8:10
acid
30 ppm
0.5 ppm 0.5 ppm 0.5 ppm <0.1-4.5ppm
4.6, 10.0 and 10-15 46.4 mg/kg/day
10.0, 21.5, 46.4 and 80.0 mg/kg/day
6-15
50 mg/kg
6-15
100 mg/kg
6-10
20, 40, 80
6-10
and 100 mg/kg
20 NR
Significant (p<0.01) Increases In fetal mor tality at the 2 higher
dose levels; dose-related Increases In the percent of abnormal fetuses ^er litter; a high Incidence
of cystic kidneys In treated groups
Courtney et al., 19T0a,b
20 Reduced maternal
Increase In the Incidence Courtney and
weight gain at the of kidney anomalies, but
Moore, 1971
2 higher dose
no Increase In cleft
levels (p<0.05)
palate
and Increased
llver-to-bw ratio
at the highest dose
level (p<0.05)
NS No effect on mor No significant effect on tality or bw gain fetal mortality or fetal weight; a significant
(p<0.05) Increase In the Incidence of delayed ossification
Sparschu et al.. 1971a
NS Increased mor
Increase In the Incidence Sparschu
tality and
of delayed ossification
et al., 1971a
decreased bw gain and poorly ossified or
malallgned sternebrae
(p<0.05)
14 NS
Dose-related Increases In fetal mortality, gastro intestinal hemorrhages, and fetal abnormalities; see text for discussion of effect TCDD level on development
Collins et al., 1971
NS = Not specified; NR = Not reported
l e t t e r 1s used as th e ba sic e x p e rim e n ta l u n i t , th e In c id e n c es o f c l e f t
palate (number of Utters with cleft palate/total numbers of Utters) versus the dose can be plotted on log dose/prob1t response paper, correcting for background response using Abbott's equation. According to this method, the EDscun (by eye-fit) for cleft palate Induction are as follows:
2,3,7,8-TCDD: 4.6 vg/kg bw 2.4.5-T (Sample A): 115 mg/kg bw 2.4.5-T (Sample B): 46 mg/kg bw If the assumption were made that all teratogenic activity 1n the 2,4,5-T samples were attributable to 2,3,7,8-TCDD contamination, the expected ED^q for samples A and B would be 230,000 mg/kg (0.0046 mg/kg x 0.02 ppm-1) and 92,000 mg/kg (0.0046 mg/kg x 0.05 ppm-1), respectively. Since the observed ED^q was lower by a factor of over 1000, this suggests that 2,3,7,8-TCDD 1s not the sole factor 1n 2,4,5-T-1nduced cleft palate. The nature of possible Interaction between 2,4,5-T and 2,3,7,8-TCDD 1s
more d i f f i c u l t to d e f i n e . Based on assays o f f i v e m ix tu re s o f 2 ,3 ,7 ,8 -T C D D
and the highly purified 2,4,5-T, Neubert and Dlllmann (1972) noted a greater than additive effect on the Induction of cleft palates. A similar conclu sion can be reached 1f one assumes that Sample A was a "totally pure" sample of 2,4,5-T. Using the assumptions of simple similar action (Finney, 1971) and treating Sample B as a mixture of 2,3,7,8-TCDD and 2,4,5-T, the expected EDg0 for Sample B would be 119.8 mg/kg. The observed value of 46 mg/kg again suggests a greater than additive effect. A more detailed statistical analysis of these data, however, would be required to support the assump tions of simple similar action or Independent joint action that are Implicit 1n these analyses. Furthermore, the Inability to define precisely the
9-5
levels of 2,3,7,8-TCDD 1n the 2,4,5-T samples and the possible significance of other contaminants would preclude an' unequivocal Interpretation of the results of the analysis.
Nevertheless, three of the studies summarized 1n Table 9-1 (Neubert and DUlmann, 1972; Roll, 1971; Courtney, 1977) have demonstrated the Induction of cleft palate 1n mice by using 2,4,5-T samples containing 2,3,7,8-TCDD levels of 0.05 + 0.02 ppm or less. Although 2,3,7,8-TCDD contamination Is undoubtedly a factor 1n the teratogenic 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 teratogenic 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 teratogenic potential. A summary of this study and others assessing the teratogenic potential of purified 2.3.7.8- TCDD are presented In Table 9-2. CD-I, DBA/2J and C57B1/6J mice were given subcutaneous Injections of 2,3,7,8-TCDD at 1 or 3 yg/kg/day on days 6-15 of gestation 1n the study by Courtney and Moore (1971). This dose regime did not result 1n maternal toxicity, although an Increase 1n the maternal llver/bw ratio was observed 1n DBA/2J and C57B1/6J mice. 2,3,7,8TCDD had no measurable effect on fetal mortality; however, anatomical abnormalities were observed 1n all strains and at all dose levels, with C57B1/6J being the most sensitive strain. The abnormalities observed were cleft palate and unspecified kidney anomalies.
Moore et al. (1973) treated pregnant C57B1/6 mice with an oral dose of 2.3.7.8- TCDD at 1 or 3 yg/kg/day on days 10-13 of gestation, or 1 yg/kg on day 10 of gestation. At the high dose level, the average Incidence of cleft palate was 55.4%. Kidney anomalies (hydronephrosis) were observed on
9-6
TABLE 9-2
Studies on the Potential Teratogenic Effect of 2,3,7,8-TCDO
Specles/Straln
Vehicle
Mouse/C57BI/6 Mouse/AKR
Nouse/CD-1 Nouse/DBA/2J Nouse/C57Bl/6J House/C57Bl/6
House/CD-1
DMSO or honey:water (1:1)
DMSO
acetone: corn oil (1:9)
DMSO or c o m oil
Nouse/CF-1 Nouse/NHRI
c o m oil/ acetone (98:2)
rape-seed oil
Rat/CD
DMSO
Rat/SpragueDawley
c o m oil/ acetone
Rat/Ulstar
corn oil/ anlsole
Dally Dose
Treatment Days
21.5, 4 6 . 4 , 113.0 mg/kg
6-14 or 9-17
0.5, 1, 3 ug/kg
6-15
1. 3 ug/kg
10-13 or 10
25, 50, 100, 200, 400 u9/k9
7-16
0.001, 0.01, 0.1, 1.0, 3.0 ug/kg
0.3, 3.0, 4.5, 9.0 u9/k9
6-15 6-15
0. 0.5, 2.0 ug/kg
0. 0.03, 0.125, 0.5, 2.0 and 8.0 ug/kg
6-15, 9 and 10, or 13 and 14
6-15
0.0, 0.125, 0.25, 1, 2, 4, 8, 16 ug/kg
6-15
Observation Day iga
17a or 18 18a 18b 18a 18 20a 20a
22
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 ug/kg
maternal toxicity observed at or above 1 u9/kg
fetal Response
Reference
fetoctdal, cleft palate, cystic kidney
Courtney et al., 1970b
cleft palate, kidney anomalies
Courtney and Moore, 1971
cleft palate, kidney anomalies
Moore et al., 1973
cleft palate, hydronephrotlc kidneys, hydrocephalus, open eyes, edema, petechlae
cleft palate, dilated renal pelvis
Courtney, 1976
Smith et al., 1976
fetoddal at the high dose, cleft palate at doses at or above 5 ug/kg
kidney malformations at both dose levels
Neubert and Dtllmann, 1972
Courtney and Moore, 1971
Intestinal hemorrhage at 0.125 and 0.5 u9/kg, fetal death at higher doses, subcutaneous edema
Increased fetal death observed at or above 1 ug/kg. subcutaneous edema and hemorrhages In the 0.25-2 ug/kg groups
Sparschu et al., 1971b
Khera and Ruddlck, 1973
TABLE 9-2 (cont.)
9-8
Specles/Stratn
Vehicle
Dally Dose
Treatment Days
Observation Day
Haternal Response
Fetal Response
Reference
Rat/SpragueDawley
Rat/SpragueDawley
corn oil/ acetone (9:1)
diet
Rabbit/ New Zealand
corn oil/ acetone (9:1)
0.1, 0.5, 2.0 pg/kg
1-3
0.001, 0.01 and 0.1 pg/kgc
throughout gestation
0.0, 0.1, 0.25, 0.5 and 1 pg/kg
6-15
21 post parturition
28
decrease In bw gain In the high dose group
low fertility at 0.01 and 0.1 pg/kg decreased bw at 0.01 and 0.1 pg/kg dilated renal pelvis
maternal toxicity at doses of 0.25 pg/kg and above
decreased fetal weight 1n the 0.5 and 2 pg/kg group
Glavlnl et al., 1982a
low survival at 0.01 and 0.1 pg/kg, decreased bw at 0.01 pg/kg, slight dilated renal pelvis at 0.001 pg/kg 1n the F^ but not succeeding generations**
Increases 1n extra ribs and total soft tissue anomalies
Hurray et al., 1979
Glavlnl et al., 1982b
First day of gestation designated day zero **F1rst day of gestation designated day one
cThe high dose level (O.T pg/kg/day) was discontinued due to very low fertility In adults
**H1sbet and Paxton (1982) re-evaluated the study by Hurray et al. (1979) using different statistical methods and considered the effects In the 0.001 pg/kg group to be statistically significant.
an average of 95.1% of the fetuses/lltter, with 83.1% having bilateral
kidney anomalies. When the dose was decreased to 1 yg/kg/day, the average Incidence of cleft palate dropped to 1.9%; however, the Incidence of kidney anomalies remained relatively high, with an average Incidence of 58.9%. On the average, bilateral kidney anomalies occurred 1n 36.3% of the fetuses/ Utter. A single dose of 1 yg/kg on day 10 of gestation produced kidney anomalies 1n 34.3% of the fetuses; however, no cleft palates were observed. When 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 2,3,7,8TCDD-treated mother. When pups from a 2,3,7,8-TCDD-treated mother nursed on control mice, kidney anomalies were found 1n only 1/14 Utters. In contrast, when pups from control mothers nursed on 2,3,7,8-TCDD-treated mice, kidney anomalies were observed 1n 4/14 Utters. In the pups exposed to 2,3,7,8-TCDD both in utero and during the postnatal period, kidney anomalies were observed In 5/7 litters. Kidney anomalies observed following In utero exposure or exposure through the milk were similar, and these kidney anomalies may not be considered a purely teratogenic response.
Neubert et al. (1973) reviewed what was known of the embryotoxlc effects of 2,3,7,8-TCDD 1n mammalian species. Also reported were their own studies and previous work (Neubert and Dlllmann, 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 2,3,7,8TCDD 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 yg/kg. At day 18 of gestation, extensive reabsorption was observed 1n the high-dose group with 6/9 Utters totally resorbed. In the few surviving fetuses, there was an 81% Incidence of cleft
9-9
i
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 1n 138 fetuses examined 1n the 0.3 yg/kg group. Fetal mortality was Increased at the 9.0 yg/kg dose 1f animals were treated only on days 9 through 13; however, the Incidence of cleft palate remained high at a frequency of 60%. In a series of experiments to determine the time of gestation at which 2,3,7,8-TCDD was effective 1n Inducing cleft palate, mice were treated for a single day between days 7 and 13 of gestation with 2,3,7,8-TCDD at a dose of 45 yg/kg. A maximum nutober of Induced cleft palates occurred when animals were treated on either day 8 or 11 of gestation; exposure to 2,3,7,8-TCDD after day 13 of gestation produced no cleft palates 1n the fetuses.
Courtney (1976) compared the teratogenic potential of 2,3,7,8-TCDD administered orally with 2,3,7,8-TCDD administered subcutaneously. CD-I mice were dosed with 2,3,7,8-TCDD on days 7 through 16 of gestation at levels of 25, 50, 100, 200 or 400 yg/kg/day; the 400 yg/kg dose was not used 1n animals treated by subcutaneous Injection. Doses of 200 or 400 yg/kg/day produced vaginal bleeding and high rates of abortion. A dose of 100 yg/kg/day was fetotoxlc, resulting 1n decreased fetal weight and survival. Anatomic abnormalities were observed at all dose levels, with cleft palate and hydronephrotlc kidneys being most common. Other abnormal ities observed Included hydrocephalus, open eye, edema and petechlae. Subcutaneous administration of 2,3,7,8-TCDD produced a greater 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-TCDD on the Incidence of fetal anomalies were also studied by Smith et al. (1976) 1n CF-1 mice. The mice were given
9-10
0 . 0 0 1 - 3 . 0 yg 2,3,7,8-TCDD/kg/day by gavage from day 6 through 15 of gesta
tion. The Incidence of cleft palate was found to be significantly Increased 1n 1.0 and 3.0 yg/kg/day dose groups, and the Incidence of kidney anomalies was significantly Increased at 3.0 yg/kg/day. There were no observable teratogenic effects 1n the study at 0.1 yg/kg/day; however, some were noted at lower dose levels, although not statistically signif icantly elevated.
Poland and Glover (1980) compared cleft palate formation by 2,3,7,8-TCDD 1n the responsive C57B1/6J, the nonresponslve DBA/2J and the hybrid B6D2F1/J strains of mice. Female mice were mated with male mice of the same genetic strain, and on day 10 of pregnancy the pregnant mice were given a single subcutaneous dose of 3.0, 10.0 or 30.0 yg/kg of 2,3,7,8-TCDD dissolved In p-d1oxane or the solvent (control) alone (0.4 ml/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 yg/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 yg/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 yg/kg of 2,3,7,8-TCDD, 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 teratogenic 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
9-11
between 0 and 3% at the 30 yg/kg dose level. In a reciprocal blastocyst transfer study between 2,3,7,8-TCDD "responsive" (NMRI) and "nonresponslve" (DBA) strains of mice 1t has been demonstrated that 2,3,7,8-TCDD exposure (30 yg/kg bw) on day 12 of gestation developed cleft palate 1n 75-100% of NHRI fetuses, Irrespective whether these embryo were kept 1n their own (NHRI) dams or transferred to DBA dams. However, none of the 2,3,7,8-TCDD exposed DBA fetuses transferred to NMRI dams or kept 1n their own (DBA) dams had cleft palate (D"Argy et al., 1984). These results suggest that the responsive mice, containing high levels of the Ah receptor, are highly sus ceptible to the effects of 2,3,7,8-TCDD 1n producing cleft palate, whereas the nonresponslve mice, which contain low (or 0) levels of the Ah receptor protein, are resistant to this teratogenic effect of 2,3,7,8-TCDD. These data and other results (Hassoun and Dencker, 1982) suggest that cleft palate formation elicited by 2,3,7,8-TCDD segregates with the Ah locus.
Dencker et al. (1981), Pratt (1983) and Pratt et al. (1984a,b) found an association between 2,3,7,8-TCDD-1nduced cleft palate and Ah activity 1n mice. Significant concentrations of TCDD have been detected In the placenta of pregnant TCDD-dosed mice, resulting 1n cleft palate Induction 1n the fetus without any apparent effect 1n the dams. Sensitivities to TCDD vary among strains. The AKR strain lack Ah receptors and remain Insensitive to cleft palate whereas the C57 strain possess Ah responsiveness and are sensi tive to TCDD-1nduced cleft palate. These observations further prove that the Ah locus Is the cause of strain differences 1n cleft palate production. It 1s thought that TCDD forms a complex with the Ah receptor and becomes Incorporated Into the chromatin. This alters the terminal differentiation of the medial epithelial cells 1n the palate.
9-12
9.1.3. 2,3,7,8-TCDD Studies 1n Rats. In an early study, Courtney a n d Moore (1971) tested the teratogenic potential of 2,3,7,8-TCDD 1n pregnant rats (CD) Injected subcutaneously on a dally basis with 2,3,7,8-TCDD (0.5 or 2 yg/kg) 1n 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 transplacentaily at a dose of 0.5 yg/kg, 4/6 Utters had fetuses with kidney mal formations (average number of kidney defects/Htter 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 hemorrhagic GI tracts were observed 1n 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 anlmals/group) of pregnant Sprague-Dawley rats at dose levels of 0, 0.03, 0.125, 0.5, 2.0 or 8.0 y g /k g /d a y on days 6 through 15 of g e s t a t io n (Sparschu e t a l . , 1971b). No adverse teratogenic effects were reported 1n fetuses exposed transplacentally at the 0.03 yg/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 given doses of 0.5 yg/kg, the number of viable fetuses was reduced, resorptions 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 resorptions were prevalent. No live fetuses, but many early resorptions, were reported 1n the group exposed to 8.0 yg 2,3,7,8-TCDD/kg/day. Subcutaneous edema appeared dose-related.
9-13
occurring 1n a considerable number of fetuses from the higher dose groups. Hale fetuses appeared to be more susceptible to 2,3,7,8-TCDD exposure; however, there was no significant difference 1n the sex ratio of live fetuses.
Khera and Ruddlck (1973) tested a wide range of 2,3,7,8-TCDD doses for teratogenic and fetotoxlc potential. Groups of 7-15 Wlstar rats were Intubated with 2,3,7,8-TCDD at doses of 0.125, 0.25, 1, 2, 4, 8 or 16 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 1n the 1 and 2 yg/kg group. Unspecified maternal toxic ity 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 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 1n 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 toxicity was reported 1n the 0.5 and 1 yg/kg group. At birth, there were fewer viable pups, and the pups had lower body weight 1n all but the 0.125 yg/kg
9-14
group. At weaning on day 21 after birth, there were no surviving pups In 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 dams exposed to 2,3,7,8-TCDD did not Increase pup mortality. These data suggest that poor pup survival was a result of delayed toxicity from 1_n utero exposure to 2,3,7,8-TCDD.
G1av1n1 et al. (T982a) assessed the effect of small doses of 2,3,7,8TCDD administered during the prelmplantatlon period 1n Sprague-Dawley rats. The animals, 1n 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 1n this paper Indicated that the low dose was 0.125 yg/kg.) At day 21 of gestation, no toxic effects were observed In the dams except for a decrease from 19.3-12.9 g 1n average maternal weight gain 1n the high dose animals as compared with controls. In the fetuses, weight was signifi cantly reduced (p<0.05) 1n the 0.5 and 2 yg/kg groups. Malformed Utters and malformatlon/fetuses examined were 2, 5, 5 and 6, and 2/270, 8/260, 5/255 and 8/253, respectively, 1n the control 0, 0.1, 0.5 and 2 yg/kg groups; however, these Increases 1n the treated animals were not statisti cally significant. The anomalies observed were restricted to cystic kidney. This exposure to 2,3,7,8-TCDD early 1n pregnancy did not affect Implantation frequency, and the decrease 1n fetal weight was considered a result of 2.3.7.8- TCDD delayed Implantation.
In a second study, G1av1n1 et al. (1983) administered the same doses of 2.3.7.8- TCDD (0.0, 0.125, 0.5 or 2 yg/kg) dally to 15 female CRCD rats per group by gavage 1n corn o1l:acetone (9:1) for 2 consecutive weeks before mating. Females that did not become pregnant during three estrous cycles
9-15
were necropsled to determine signs of toxicity, 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 yg/kg), there were no overt clinical signs of toxicity 1n the dams or adverse effects 1n any of the fetal parameters examined. At the 0.5 and 2 yg/kg levels, average maternal weight was decreased. Also, one animal 1n each of these groups did not become pregnant, although necropsy did not reveal any obvious dysfunc tions. The only other overt sign of toxicity was Ustlessness during the treatment period 1n the animals of the high-dose group. The only signif icant (p<0.01) fetal effect observed 1n the 0.5 yg/kg group was an Increase In 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 1n both pre- and postlmplantatlon losses of 11.7% for controls and 19.5% (p<0.05) 1n treated animals, and 2.9% 1n control and 30.3% {p<0.001) 1n treated animals, respectively. In addition to these signs of fetal toxic ity, 9/10 Utters 1n the high-dose group contained at least one malformed fetus as compared with 1/13, 2/13 and 2/13 1n the control, 0.125 and 0.5 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-TCDD was administered during gestation.
The reproductive effects of 2,3,7,8-TCDD were also studied 1n a 3-generatlon 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
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maintained for 90 days on the test diets before mating. The fQ rats were mated twice, producing the filial generations ( f ^ and f-|g)* Selected f1B and f^ rats were mated at -130 days of age to produce the f2 and fg Utters, respectively. In later generations, the high-dose group (0.1 yg 2,3,7,8-TCDD/kg/day) was discontinued because few offspring were pro duced 1n this group. At the Intermediate dose (0.01 yg/kg/day), 2,3,7,8TCDD caused lower body weight 1n exposed rats of both sexes (f^ and f2). At the low dose, no toxic effects were discerned.
Fertility was greatly reduced 1n the fQ generation exposed to 0.1 yg 2,3,7,8-TCDD/kg/day. At 0.01 yg 2,3,7,8-TCDD/kg/day, fertility was significantly (p<0.05) reduced 1n the f^ and f2 rats. Fertility 1n rats (of any generation) exposed to 0.001 yg 2,3,7,8-TCDD/kg/day was not different from that of control rats. Decreases 1n Utter size were noted 1n the f1A group exposed to 0.1 yg/kg/day and the f2 and f3 Utters exposed at 0.01 yg/kg/day. Statistically significant decreases 1n fetal survival throughout gestation were noted 1n f2 and f^ Utters of the 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 f2 Utters, but not for other generations. Decreased neonatalsurvival was noted among f1A and f2 pups exposed to 0.01 yg 2,3,7,8-TCDD/kg/day, but not among f1D or f,, pups. Postnatal body weights of the f,, and f_ Utters at 0.01 yg 2,3,7,8-TCDD/kg/day were significantly depressed. At the low dose (0.001 yg 2,3,7,8-TCDD/kg/day), necropsy of 21-day-old pups revealed a statistically significant (p<0.05) Increase 1n dilated renal pelvis 1n the f.j generation. Subsequent generations at this dose level or any at the Intermediate dose (0.01 yg 2,3,7,8-TCDD/kg/day) did not have a significant Increase 1n this abnormality. Significantly decreased thymus weight and
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Increased liver weight were reported 1n the fg generation, but not 1n the f.| generation (f? generation data not obtained) of the Intermediate dose group. Hurray et al. (1979) concluded that 2,3,7,8-TCDD Ingested at 0.01 or 0.1 pg/kg/day Impaired reproduction among rats, and NOAELs were associated with 0.001 pg 2,3,7,8-TCDD/kg/day.
Nlsbet and Paxton (1982) reevaluated the primary data of Hurray et al. (1979) using different statistical methods. From this rvaluation 1t was concluded that 2,3,7,8-TCDD significantly reduced the gestational Index, decreased fetal weight, and Increased Uver-to-body weight ratios and the Incidence of dilated renal pelvis 1n both lower-dose groups. Nlsbet and Paxton (1982) concluded that the dose of 0.001 pg/kg/day was not a NOAEL In this study. The FIFRA Scientific Advisory Panel has also reviewed the data from this 3-generat1on study and concluded that the effects observed at the 0.001 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-tr1chlorophenoxyacet1c acid (2,4,5-T) contaminated with 30 ppb of TCDD appears to have behavlorally teratogenic effect 1n Long-Evans rats at doses as low as 6 mg 2,4,5-T/kg bw administered to mother rats on day 8 of gestation. 9.1.4. 2,3,7,8-TCDD Studies 1n Rabbits and Ferrets. A report by G1av1n1 et al. (1982b) describes the effects of exposure to 2,3,7,8-TCDD on fetal development 1n rabbits. Groups of 10-15 New Zealand rabbits were adminis tered 2,3,7,8-TCDD by gavage at doses of 0.0, 0.1, 0.25, 0.5 and 1 pg/kg on days 6 through 15 of gestation. The dams were examined for Implantation sites, resorptions and live fetuses, and the fetuses were examined for
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malformations on day 28 of gestation. Decreased maternal weight gain and unspecified signs of maternal toxicity occurred 1n dams exposed to 2,3,7,8TCDD at doses of >0.25 yg/kg. At doses of 0.5 and 1 yg/kg, there were 2 and 4 deaths, respectively, among the dams. There were Increases 1n abor tions and resorptions at a dose of >0.25 yg/kg, and no live fetuses were detected 1n the high dose group. In the fetuses, the most common observa tion was a significant Increase 1n extra ribs from 33.3% 1n the controls to 82, 66.6 and 82% 1n the 0.1, 0.25 and 0.5 yg/kg dose groups. Although there was no significant Increase 1n specific soft-tissue anomalies, there was an Increase from 0/87 to 3/78, 2/33 (p<0.05) and 2/28 (p<0.05) 1n total soft-tissue anomalies 1n the control, 0.1, 0.25 and 0.5 yg/kg groups. The most prevalent soft-tissue anomaly was hydronephrosis, which the authors pointed out was a common finding 1n rat fetuses exposed to 2,3,7,8-TCDD 1n utero. These effects were considered to be signs of embryotoxicity rather than a teratogenic effect.
In addition to the fetotoxlc effects of prenatal exposure to 2,3,7,8TCDD, Norman et al. (1978b) demonstrated that 2,3,7,8-TCDD could Induce liver microsomal enzymes following in utero exposure. Pregnant New Zealand rabbits were given subcutaneous Injections of 2,3,7,8-TCDD at a dose of 30 nmol/kg (9.6 yg/kg) on day 24 of gestation, and the livers of newborns were examined for enzyme activity within 12 hours after birth. While this treatment Increased the Uver 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 electrophoresis 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 Uver. The Identity of form 6 was confirmed by Immunologic
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reaction and Its peptide 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 Uver microsomal enzymes has not been established.
Huscarella 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 1n fetal deaths and resorbed fetuses, along with growth retardation. Terata observed Included unilateral and bilateral patalosch1s1, open eyelids, anasarca and brachygnathla. The author concluded that 2.3.7.8- TCDD was a teratogen 1n ferrets. 9.1.5. 2,3,7,8-TCDD Studies 1n Nonhuman Primates. Dougherty et al. (1975) found no evidence of teratogenicity or embryotoxldty 1n rhesus monkeys that were given on days 22-38 of gestation dally oral doses (In gelatin capsules) of up to 10 mg/kg/day of 2,4,5-T containing 0.05 ppm 2.3.7.8- TCDD. The 2,3,7,8-TCDD dose at the highest dose level of 2,4,5-T administered (10 mg/kg/day) would correspond to 0.5 yg 2,3,7,8-TCDD/kg/ day. Palate closure 1n the monkey, however, occurs on gestational days 42-44 and the kidney 1s also a late developing organ.
Adverse effects of exposure to 2,3,7,8-TCDD on reproductive success 1n monkeys have also been described. Schantz et al. (1979) fed a diet contain ing 50 ppt 2,3,7,8-TCDD to rhesus monkeys for 20 months. Seven months Into
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the study the female monkeys were bred to control males. There were f o u r 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 yg/kg, corresponding to a calculated dally dose of 0.0015 v9 2.3.7.8- TCDD/kg/day.
Allen et al. (1979) and Barsottl et al. (1979) fed adult female rhesus monkeys for 6-7 months on diets containing 50 or 500 ppt of 2,3,7,8-TCDD. These exposure levels correspond to total doses per animal at the end of 7 months of 1.8 and 11.7 v9 2,3,7,8-TCDD. Although menstrual cycles were not affected 1n either treatment group, 5/8 animals 1n the high-dose group had either decreased serum estradiol 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 lowand 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 birthto "normal" offspring. The high dose resulted 1n the death of five animals between the 7th and 12th month of treatment.
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 t1mes/week during weeks 4, 5 and 6 (days 20 through 40) after conception. Group II, consisting of 12 animals, received single doses of 1yg/kg bw of
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2,3,7,8-TCDD on days 25, 39, 35 and 40 after conception. Three animals were exposed In each of these 4 days. The vehicle control group, consisting of 11 animals, was treated with corn o1l:acetone only, on the same schedule as Group I animals. Both of the females that received the highest dose (5 vg/kg) had fetal losses. In the next lower-dosed animals (1 v9/kg 1n both groups), 12 of 16 females had fetal losses; and In the lowest-dosed animals (0.2 v9/kg 1n Group I), one abortion occurred 1n four pregnancies. Maternal toxicity was observed 1n many of these treated females. The difference 1n frequency of fetal loss between all pregnant animals given 1 vg/kg and the rate of historical abortion 1n the author's breeding colony was found to be significant. The author concluded that short exposure to 1 vg/kg bw of 2,3,7,8-TCDD during early pregnancy results 1n fetal loss 1n rhesus monkeys. In a recent report, McNulty (1984) reveals that he failed to detect any malformations 1n the fetus but observed widespread maternal toxicity and fetoddal effects 1n monkeys as a result of 1ntragastr1c exposure to 2,3,7,8-TCDD. 9.1.6. Studies 1n Chickens. The effects of 2,3,7,8-TCDD on the develop ment of the heart 1n chicken embryos was studied by Cheung et al. (1981) as a consequence of the known Induction of hydropericardium by 2,3,7,8-TCDD 1n adult chickens and the relation between changes 1n hemodynamics and cardio vascular malformation. Groups of at least 20 White-Leghorn eggs were Injected with 2,3,7,8-TCDD 1n acetoneicorn oil (0.5:9.5 v/v) on day zero of embryo development. Administered doses ranged from 0.009-77.5 pmol/egg (0.00029-2.5xl0-2 v9/egg) 1n 5 v8>* The embryos were examined on day 14 of development. A dose-related Increase 1n cardiovascular malformations was observed with 1 pmol/egg resulting 1n malformations 1n 50% of the embryos. Increases In all types of malformations (ventricular septal
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d e f e c t , aortic arch anomaly, aortic arch anomaly and ventricular septal defect, and conotruncal malformations) occurred. Hydropericardium was observed in some embryos (not enumerated), but it could not be concluded that this was the cause of the cardiovascular malformations. Malformed legs and crossed beaks associated with micropthalmia was observed in treated embryos, however, the incidence, 7/284 and 2/284, respectively, was low. 9.1.7. Studies of the Teratogenic and Reproductive Effects of HxCDD. In addition to 2,3,7,8-TCDD, the teratogenic potential of a related chlorinated dibenzo-p-dioxin 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 yg HxCDD/kg/day on days 6-15 of gestation (Schwetz et al., 1973). Treatment with high levels of HxCDD (10 and 100 yg/kg) was highly lethal to fetuses during late gestation. There was a significant dose-related increase in late resorptions from 0% (at 0.1 yg/kg/day) to 79% (at 100 yg/kg/day). Decreases in the weight and length of surviving fetuses were due to HxCDD. The incidences of cleft palate, subcutaneous edema, malformed vertebrae and split sternebrae were significantly increased in fetuses of rats treated with 100 yg HxCDD/kg/day. No increase in fetal anomalies was noted in fetuses exposed to 0.1 yg HxCDD/kg, and only subcutaneous edema was more prevalent in groups exposed at 1 or 10 yg HxCDD/kg/day when compared with controls.
Pertinent information regarding the teratogenicity or reproductive effects of PeCDDs was not located in the available literature. 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 in human populations in Oregon (U.S. EPA, 1979c), New Zealand (Hanify et al., 1981), and Australia (Field and
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Kerr, 1979). A lack of any such association has been reported 1n human populations 1n Arkansas (Nelson et al., 1979), Hungary (Thomas, 1980b), New Zealand (Dept, of Health, New Zealand, 1980; McQueen et al., 1977), and Australia (Aldred, 1978). Almost all of the reports are geographic correla tion studies, and because of the uncertainties Inherent 1n this type of epidemiologic Investigation, as well as the difficulties 1n distinguishing the effects of 2,4,5-T from those of 2,3,7,8-TCDD contamination, none of the reportedly positive associations unequivocally Identify either 2,4,5-T or 2,3,7,8-TCDD as the causative agent. Similarly, the reportedly negative associations do not rule out 2,4,5-T or 2,3,7,8-TCDD as potential teratogens or abortlfaclents 1n humans.
Based on a report of a high Incidence of abortions 1n a small group of women living around Alsea, Oregon, who may have been exposed to the herbi cide 2,4,5-T from aerial spraying (Smith, 1979), the U.S. EPA (1979c) Initiated a study, often referred to as the "Alsea II study," to determine 1f spontaneous abortion rates differed between the exposed and unexposed populations, 1f spontaneous abortion rates evidenced seasonal variation 1n these two groups, and 1f such seasonal variations were associated with 2,4,5-T spray application.
The Spontaneous Abortion Rate Index, as defined by the U.S. EPA, 1s "basically the ratio of the number of hospitalized spontaneous abortions to the number of births corresponding to the spontaneous abortions, based on the residence zip code of the women contributing to each event." Upon completion of the study, the U.S. EPA concluded that (1) the 1972-1977 Spontaneous Abortion Rate Index for the study area was significantly higher than 1n the Rural Control Area or the Urban area; (2) there was a statisti cally significant seasonal cycle 1n the abortion Index 1n each of the areas with a period of ~4 months. In particular there was an outstanding peak 1n
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the study area In 3une; and (3) there was a statistically significant corre lation between the Spontaneous Abortion Rate Index and spray patterns 1n the study area when a lag-time of 2 or 3 months was Included. The U.S. EPA concluded, however, that "This analysis 1s a correlational analysis, and correlation does not necessarily mean causation."
Mllbyy et al. (1980), citing three critiques of the Alsea II study (not published 1n the open literature), state that the statistical method and basic design of the Alsea II study were sufficiently flawed to make this study of no use 1n human risk assessment. The Alsea II study has also been reviewed by a panel of scientists who, 1n a published report of their meeting, also concluded that the basic design of the study was Inadequate to demonstrate either an effect or absence of an effect of exposure to 2,4,5-T (Coulston and Olajos, 1980). The major Inadequacies of the study were that the data collection methods were likely to result 1n the underestimation of abortions, particularly 1n the urban area (the Incidence of abortions 1n all three groups was within the expected background rate of 8-15%); only a small part of the area from which the exposed subjects were selected was actually sprayed with 2,4,5-T, and the study was not controlled for other factors such as age, smoking habits and alcohol consumption, which may affect the spontaneous abortion rate. Based on a new report by Smith (1979), the U.S. EPA 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.
Nelson et al. (1979) noted a general Increase 1n the reported Incidence of facial cleft 1n both high and low exposure groups 1n Arkansas from 1948-1974. In this study, exposure estimates were based on average
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rice production 1n 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 1s 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 1f the spraying had been Implicated, a lack of Information on 2,3,7,8-TCDD levels 1n the spray and the absence of any monitoring data on 2,4,5-T or 2,3,7,8-TCDD would limit the usefulness of this report.
The study by McQueen et al. (1977) 1s not published 1n th^ open litera ture but 1s summarized by Mllby et al. (1980). According to the summary, McQueen et al. (1977) "...examined the epidemiology of neural-tube defects 1n three areas 1n New Zealand and concluded 'there 1s no evidence to Implicate 2,4,5-T as a causal factor 1n human birth defects.'" No addi tional details are provided.
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Vianlfy et al. (1981) performed an epidemiologic study 1n Northland, New Zealand, 1n areas where spraying of 2,4,5-T was done by various companies for a number of years. The rate of birth defects was obtained from an examination of hospital records in seven 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 epispadias, 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 statisti cally significant association between the occurrence of talipes and exposure when the fractional removal rate was assumed to be 0.25. There was, how ever, no statistically significant association where 1.0 was used as the fractional removal rate.
Smith et al. (1982a) Investigated the outcome of pregnancy 1n families of professional 2,4,5-T applicators and agricultural contractors 1n New Zealand. Agricultural contractors were chosen as the control population since both sprayers and contractors were of the same economic group with similar outdoor occupations. The. survey was conducted by mall with 89% of the chemical applicators responding and 83% of the agricultural contractors
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responding to questions asking whether they used 2,4,5-T and Its temporal relationship to reproductive histories regarding birth, miscarriages, still births and congenital defects. The relative risks of congenital defects and miscarriages were 1.19 (0.58-2.45% confidence limits) and 0.89 (0.61-1.30% confidence limits) for the wives of chemical sprayers as compared with the wives of agricultural contractors. These data Indicate that exposure of fathers and mothers (l.e., while cleaning clothes) had no effect on the outcome of pregnancy. Biases that may have affected the results, such as the age of the mother at childbirth, smoking habits and birth to Maori parents were Investigated and eliminated as possible confounders.
The two reports from Australia (Aldred, 1978; Field and Kerr, 1979) also present apparently conflicting results. The report by Aldred (1978) 1s not published In the open literature, but the following summary Is taken from M1lby et al. (1980): "The report concluded that birth defects 1n a group of babies born 1n the [Yarram] district 1n 1974 and 1976 could not be attrib uted to exposure to 2,4,5-T or 2,4-D." Additional details that might be useful 1n assessing the rationale for this statement are not provided 1n the summary. The report by Field and Kerr (1979) plotted the Incidence of neural-tube defects (anencephaly and meningomyelocele) 1n New South Wales, Australia, over the years 1965-1975, and the usage of 2,4,5-T 1n all of Australia during the previous years. The authors noted a decrease 1n the Incidence of 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 epidemiologi cal data on neural-tube defects to be "relatively complete," they do not comment on the Increasing Incidence of neural-tube defects during the time
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period of this study and whether or not an Increase 1nthe thoroughness of reporting neural-tube defects could have contributed to the apparent corre lation of 2,4,5-T exposure with these defects. A replotting 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 U p, cleft palate, spina bifida, anencephalus and cystic kidney disease 1n all ofHungary 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
g r e a t e s t , and because annual use o f 2 , 4 , 5 - T 1n Hungary had r i s e n from 4 6 ,0 0 0
kg In 1969 to 1,200,000 kg 1n 1975, Thomas (1980b) considered Hungary to be "...probably the best country 1n which to examine possible health effects of this herbicide." All Indices of birth defect rates decreased or remained stable over the period of study.
In addition to contamination of 2,4,5-T being a potential source of 2,3,7,8-TCDD exposure, 2,3,7,8-TCDD 1s also an Inadvertent contaminant of 2.4.5- trlchlorophenol (TCP). Chronic exposure to 2,3,7,8-TCDD may occur during the manufacture of TCP and high level acute exposure to 2,3,7,8-TCDD has occurred after an accident 1n July, 1976 at the ICMESA TCP chemical factory 1n Seveso, Italy (Bonaccorsl et al., 1978). In this accident, the reaction used to produce TCP became uncontrolled, producing conditions
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favorable for 2,3,7,8-TCDD formation before venting the contents of the chemical reactor Into the atmosphere. The resulting cloud of chemicals settled over a heavily populated area. Although the amount of 2,3,7,8-TCDD released was not known, the reported cases of chloracne, a symptom of acute exposure to 2,3,7,8-TCDD, Indicated that exposure to 2,3,7,8-TCDD had occurred. Some preliminary results are available from epidemiologic 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 2,3,7,8TCDD (Townsend et al., 1982).
Epidemiologic 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), Regg1an1 (1980) and B1sant1 et al. (1980). The spontaneous abortion rate 1n the contaminated area for the three trimesters following the acci dent was 13.1, 11.0 and 13.05%, which was similar to the worldwide 15-20% frequency of spontaneous abortion. Subdividing the contaminated area Into highly, moderately, and least contaminated, and examining the rates for each area Individually, also failed to demonstrate any change 1n the spontaneous abortion rate. The Incidence rates of malformations also were examined; however, the numbers were too few for meaningful assessment. There are several Inadequacies 1n these studies that might make them Insensitive 1n detecting reproductive effects. The authors noted that there are many difficulties 1n Interpreting these data. Adequate data on the Incidence
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rites of spontaneous abortions and birth defects were not adequately avail able for the region before the accident as a result of suspected under reporting. There was Inadequate reporting even after the accident because of political turmoil with regard to the management of health services. Also, an unknown number of pregnancies were surgically aborted for fear of 2,3,7,8-TCDD-lnduced birth defects. In a recent review of the progress of epidemiologic Investigations of the Seveso accident, Tognonl and Bonaccorsl (1982) Indicated that the data on spontaneous abortions and malformation rates still needed verification, and that these data were too preliminary to allow for conclusions.
Townsend et al. (1982) Investigated the reproductive history of wives of employees potentially exposed to 2,3,7,8-TCDD during chlorophenol production 1n Midland, MI. A total of 930 potentially exposed males were Identified who had worked for >1 month between January 1939, and December 1975, 1n a job with potential 2,3,7,8-TCDD exposure. Exposure estimates of low, moderate and high were made by an Industrial hygienist 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 control population was an equal number of male employees not Involved 1n any process that might Involve exposure to 2,3,7,8-TCDD and matched for date of hire. In these groups, 586 wives were Identified and 370 agreed to partici pate as the exposed group, while 345 wives of a potential control group of 559 agreed to participate. After Identification of the participants, a personal Interview was conducted with the wives to determine pregnancy outcome. Of the total of 737 conceptions 1n the exposed category and 1785 conceptions 1n the control category (conceptions that occurred In the
9-31
exposed group before work records Indicating potential exposure to 2,3,7,8TCDD were placed 1n the control group), there was no statistically signifi cant Increase 1n spontaneous abortions, stillbirths, Infant deaths or selected congenital malformations. Sample sizes were too small to provide meaningful data 1f the populations were subdivided by extent of exposure. The authors suggested that many confounding factors could account for these negative results, such as the Inappropriate selection of the populations, the use of "exposed11 persons 1n both exposed and control groups, unidenti fied covarlables 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-TCDD does not affect the conceptus.
Poole (1983), 1n testimony before the House Committee on Science and Technology, described a reanalysis of the primary data used by Townsend et al. (1982). In this reanalysis, the relative risk of cleft palate and cleft U p 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 U p with or without cleft palate for births 1n Midland County, Michigan (the site of Dow's chlorophenol production facility) during the years 1970-1981. The data Indicated an Increased rate for these defects of between 50 and 100% 1n the years 1971-1975, with the rate returning to normal from 1976-1981. The observed Increase was statistically signif icant If the rates for cleft palate alone and cleft lip with or without cleft palate were combined; however, 1t was the opinion of Houk (1983) that these defects should not be combined since the causal mechanism may be
9-32
d i f f e r e n t . The M ichigan Department o f P u b lic H e a lth (1983a) a ls o r e p o rte d these results and, in addition, demonstrated that the same results occurred if the comparison was made with other counties in Michigan as well as with the general population of the United States. It was noted in 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 if any common factors may exist, such as exposure to chemicals contaminated with 2,3,7,8-TCDD.
A similar but limited study of the reproductive history of the wives of employees of the Long Island Railroad was performed by Honchar for NIOSH (1982). The employees were concerned about the use of 2,4,5-T for mainte nance along the right-of-way. There were 170 live births as indicated by union files during the study period from 1975-1979. For each birth, insur ance claims were reviewed to determine any health problems during the first year of life. The incidence of major birth defects was underrepresented in the study population when compared with data from the Metropolitan Atlanta Congenital Defects Program (3 observed and 3.81 expected). Some minor health problems (1.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 in this study.
To test any possible association between birth defects and exposure to Agent Orange in Vietnam veterans, Erickson et al. (1984) conducted a casecontrol study on newborns with various types of congenital defects in the metropolitan Atlanta area during the years 1968 through 1980. Though most of the Vietnam veterans received from the Army Agent Orange Task Force an
9-33
estimated opportunity Index score regarding their exposure to Agent Orange, 25J4 of the Vietnam veterans Interviewed 1n this study felt that they were exposed and approximately an equal proportion did not know 1f they were exposed to Agent Orange. Increased estimated risks for fathering babies with 1) spina bifida, 2) cleft U p with or without cleft palate and 3) cer tain tumors were found 1n this study. However, the authors concluded that "Vietnam veterans who had greater estimated opportunity for Agent Orange exposure did not seem to be at a greater risk for fathering babies with all types of defects combined" (Erickson et al., 1984). 9.3. OTHER REPRODUCTIVE EFFECTS
The effects of a mixture of 2,4,5-T, 2,4-D and 2,3,7,8-TCDD (simulated Agent Orange; however, the free acids 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 yg of 2.3.7.8- TCDD (Group II);40 mg each of 2,4,5-T and 2,4-D and 0.16 yg of 2.3.7.8- TCDD (Group III);or 20 mg each of2,4,5-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 motility and sperm abnormalities were evaluated. In addition, the males were mated with virgin females (3 females/week for 8 post-treatment weeks) to assess mating frequency, average fertility, percent Implantations and resorptions, and percent fetal malfor mations. There was no significant decrease 1n any of the parameters used as a measure of fertility and reproductive capacity 1n any groups of treated
9-34
mice when compared with controls. Lamb et al. (1981b), in a further report of this work, Indicated that germ cell toxicity was not apparent and survival of offspring of exposed mice was unaffected. No external, visceral or skeletal terata were noted 1n offspring whose sires were exposed to the phenoxy ac1ds/2,3,7,8-TCDD mixture 1n this study. The only effects noted were dose-related decreases 1n body weight 1n the treated males, and these effects were reversed when treatment was terminated. 9.4. SUMMARY
2,3,7,8-TCDD has been demonstrated to be teratogenic 1n all strains of mice tested. The most common malformations observed are cleft palate and kidney anomalies; however, other malformations have been observed occasion ally. With an MED of 1 yg/kg/day for mice, 2,3,7,8-TCDD 1s the most potent teratogen known. At higher doses, 2,3,7,8-TCDD has a marked fetotoxlc effect, as measured by decreased fetal weight and Increased fetal toxicity. Hemorrhagic GI tract has been associated with 2,3,7,8-TCDD fetal toxicity.
In rats, 1t has also been observed that 2,3,7,8-TCDD produced terato genic 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 microsomal enzymes 1n the fetus exposed 1_n utero. and this Induction 1s accompanied by damage to the fine structure of the Uver cell; however, other reports Indicate that enzyme Induction occurs only after birth following exposure to 2,3,7,8-TCDD through the mother's milk. As 1n mice, hemorrhagic GI tracts have been observed 1n rat fetuses exposed in utero to 2,3,7,8-TCDD.
9-35
Rabbits and monkeys are also susceptible to the fetotoxlc effects of 2.3.7.8- TCDD; however, the studies of these species have been too limited to clearly evaluate a teratogenic response or define a threshold dose for fetotoxlclty.
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, other studies have not. In Investigations concerning potential exposure to 2.3.7.8- TCDD through the manufacture of TCP, there has been no positive substantiated association between exposure and reproductive difficulties. In these studies, exposure was always mixed, with 2,3,7,8-TCDD being only a minor component. Hence, 1t 1s not possible to attribute with certainty any positive finding to 2,3,7,8-TCDD. It 1s also possible, since levels of 2.3.7.8- TCDD contamination of 2,4,5-T and TCP were only estimated, that the negative results reflect the exposure was too low or the study designs too Insensitive to elicit a detectable response. From an extensive review of d1ox1n-1nduced animal and human reproductive toxicity data by Mattlson et al. (1984) and another review of 15 reports dealing with human exposure to dioxins and reproductive effects by Hatch (1984), 1t can be concluded that epidemiologic observations from well designed studies are warranted before deriving any conclusion on dioxin-induced reproductive toxicity In humans. Although the evidence from human studies 1s Insufficient to prove 2,3,7,8TCDD 1s teratogenic, the animal data clearly Indicate teratogenic or feto toxlc effects 1n all animal species tested.
9-36
10. MUTAGENICITY AND OTHER INDICATIONS OF GENOTOXICITY 10.1. RELEVANT STUDIES 10.1.1. Assays 1n Microorganisms. Short-term In. 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 xenoblotlcs, carcinogens and crude environmental extracts. The most widely used short-term test system, the Ames test for bacterial mutagenesis, employs several strains of Salmonella typh1mur1um that are highly susceptible to the effects of mutagenic chemicals. Despite the obvious utility of the Ames test and related short-term assays, their predictive capabilities (1.e., the correlation between bacterial mutagenicity and carcinogenicity) have not been fully assessed (Bartsch et al., 1982).
Mutagenicity 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 mutagenic effects (Table 10-1).
Hussain et al. (1972) exposed S. typh1mur1um histidine-dependent strains TA1530 and TA1532 1n liquid suspension to 2,3,7,8-TCDD followed by plating Into selective medium to observe reversion to prototypes. No Increase 1n the reversion rate was observed with strain TA1530 at exposure levels of 1 and 10 pg/ml. These exposures resulted 1n cell survivals of 90 and <1%, respectively. In strain TA1532, Increased reversion frequency was not observed at 2,3,7,8-TCDD concentrations of <2-3 yg/ml, which resulted 1n a 0-50% decrease 1n survival; however, at 2,3,7,8-TCDD levels that resulted 1n a 99% decrease 1n survival, there was an Increased number of revertant colon1es/surv1v1ng cells. This positive response 1s questionable because of the extremely high toxicity observed. The dose levels were not specified.
10-1
TABLE 10-1 The Results of Mutagenicity Assays for 2,3,7,8-TCDD In Salmonella tvohlmurlum
10-2
Type of Assay
__________________________________ Strains of Salnonella typhlmurlum__________________________________ S-9 TA98 TA1530 TA1535 TA1537 TA1538 TA1532 TA1950 TA1975 TA1978 G46 TA100 TA1531 TA1534
Reference
Spot test
/- NT
NT
00
0
0
NT NT NT NT NT NT NT McCann, 1978
Plate
/- NT
NT
00
0
0
NT NT NT NT NT NT NT McCann, 1978
Incorporation
Plate
/- 0
Incorporation*
0
0 0 0 0 0 0 0 0 0 NT NT Gilbert et al., 1980
fluctuation test
/- 0
0
0 0 0 0 0 0 0 0 0 NT NT Gilbert et al., 1980
Spot test
- NT
0
NT NT NT f
NT NT NT 0 NT QR QR Seller, 1973
Plate
f0
NT
00
0
NT NT NT NT NT 0
NT NT Geiger and Neal, 1981
Incorporation
Plate
- NT
NT
NT
0
Incorporation
NT NT NT NT NT NT NT NT NT Geiger and Neal, 1981
Suspension assay
- NT
0
NT NT NT QR NT
NT
NT NT NT NT NT Hussain et al., 1972
Suspension assay
0 NT
00
NT NT NT NT NT NT 0
NT NT Mortelmans et al., 1984
The assay was performed under both aerobic and anaerobic conditions. NT = Not tested; QR = Questionable response; 0 = Negative response; <- = Positive response
The source o f the 2,3,7,8-TCDD sample s tu d ie d 1n t h i s paper was the F o o d a n d Drug Administration, and Its reported purity was 99%. Also, Seller (1973) observed a positive mutagenic response 1n a spot test of 2,3,7,8-TCDD per formed In the absence of a metabolic activation system. However, the purity of the sample studied was not provided. In tester strains G46 and TA1530, the ratio of revertants/108 cells 1n the treated plates divided by spon taneous revertants/103 cells was <1. In strains TA1531 and TA1534, the ratio was between 1 and 2, which was considered a "doubtful" mutagenic response, while 1n strain TA1532, the ratio was >10. There was no mention of the 2,3,7,8-TCDD levels tested 1n this assay. The positive controls, dlethylsulfate, 2-am1nopur1ne and 2-am1nofluorene, produced ratios of 2 to 5, <1 and 5 to 10, respectively, 1n strain TA1532. In both the study by Hussain et al. (1972) and the study by Seller (1973), 2,3,7,8-TCDD produced a positive mutagenic response only 1n the S. typh1mur1um strain TA1532, which 1s sensitive to frameshlft mutagens.
Hussain e t a l . (1972) a ls o performed a m u ta g e n ic ity t e s t o f 2,3,7,8-TCDD 1n two other microbial test systems. A positive response was observed 1n Escherichia 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 yg/ma.. The greatest mutation frequency (256 mutants x 10-8, as compared with the control frequency of 2.2 mutants x 10-8) occurred at a dose level of 2 yg/ma.. The absolute number of colonles/plate was 7 for the control and 46 for the treated plate. The dose of 2 yg/ma. caused an 89% decrease In cell survival. A duplicate sample resulted 1n an 82% decrease 1n survival and a mutation frequency of 34xl0-8. These results Indicate that the reproducibility of the assay may not have been perfect, but both results are well above the control value of
10-3
2.2xl0-8. A dose-response relationship was not observed, Indicating that the results at 2 yg/ma, are only suggestive of a positive response. In addition, the positive results were obtained at a concentration of 2,3,7,8TCDD (2 yg/ma) that was well above solubility 1n water (0.2 yg/a), which also casts doubt on the significance of the positive result. In the second test system, the ability of 2,3,7,8-TCDD to Increase prophage Induc tion 1n . coll K-39 cells was examined. The vehicle control, DMSO, Inhib ited prophage Induction as compared with the untreated controls, while the most effective dose level of 2,3,7,8-TCDD (0.5 yg/ma) resulted 1n an Increased prophage Induction as compared with the vehicle control but not as compared with the untreated controls. Hussain et al. (1972) concluded that 2,3,7,8-TCDD was capable of causing Increases 1n the reverse mutation rate 1n . coll Sd-4 and that 2,3,7,8-TCDD had a weak ability to Induce prophage 1n . coll K-39 cells.
The studies that followed these two early reports of Hussain et al. (1972) and Seller (1973) failed to detect mutagenic activity of 2,3,7,8-TCDD 1n S. typh1mur1um. Wassom et al. (1978) cited a personal communication from McCann (1978), ^h1ch reported that 2,3,7,8-TCDD was Inactive 1n both the spot test and plate Incorporation assay with S. typh1mur1um 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. typh1mur1um strains TA98, TA100, TA1530, TA1535, TA1537, TA1538, G46, TA1532, TA1950, TA1975 and TA1978. Both the standard plate Incorporation assay and the bacterial fluctuation test were used, and both were performed with and without S-9 prepared from the livers of Aroclor 1254 pretreated rats. In the plate Incorporation assay, the test compound was tested at 1-2000 yg/plate under both aerobic
10-4
a n d a n a e r o b i c 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 mutagenicity assays, since information on experimental conditions was limited in the negative studies.
In an attempt to resolve the conflicting results and observe a mutagenic response, Geiger 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 maximize the amount of compound tested, dioxane, a better solvent for 2.3.7.8- TCDD than the commonly employed DMSO, was used. Even with the use of dioxane, the limited solubility of 2,3,7,8-TCDD allowed only 20 yg/ plate to be tested, a dose that was shown to be nontoxic to the cells. The S-9 used 1n 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-TCDD concentrations of 0.2, 2, 5 or 20 yg/plate, and regardless of the source of the S-9, there was no observed mutagenic response. In further attempts to duplicate the previous positive results, Geiger and Neal (1981) tested the same concentrations of 2,3,7,8-TCDD in strain TA1537, a more sensitive direct descendent of strain TA1532, for mutagenic activity in the absence of S-9. Again, no increase in the number of revertants was observed. In assays either with or without S-9, positive controls had predictable increases in 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 polychlorinated aromatic compounds are not mutagenic in the Ames test, even though there is positive evidence of carcinogenicity.
10-5
The National Toxicology Program (NTP) provided data on 2,3,7,8-TCDD from
four assay systems: the S. typh1mur1um (strains TA98, TA100, TA1535 and
TA1537) histidine reversion assay, the sex-Hnked recessive lethal test In
Drosophila, and cytogenetic studies (sister chromatid exchange and chromo
some aberrations) 1n Chinese hamster ovary cells. Negative results were
obtained In all of these assays (Mortelmans et al., 1984; Z1mmer1ng et al.,
1985; NTP, 1985).
Mutagenic effects of 2,3,7,8-TCDD 1n yeast were observed by Bronzettl et
al. (1983). Positive results for reversion and gene conversion were
obtained In. vitro and 1n the host-mediated assay. The In. vitro experiments
yielded small dose-related Increases 1n trp+ convertants and 1lv+ rever-
tants. An S10 metabolic activation system was required. Exposure of the
yeast to 2,3,7,8-TCDD at the highest level tested (10 yg/ml) resulted 1n
16% survival and yielded 4-fold Increases 1n reversion and gene conversion.
In the host-mediated assay, male mice were exposed to 25 y g of
2.3.7.8- TCDD/kg (Bronzettl et al., 1983). After 5, 10, 20 or 30 days, 0.2
ms. of a yeast culture (4 x 10 cells) was Instilled retroorbltally.
Four hours later, the Uver and kidneys were removed and the yeast cells In
these organs were assayed for mutagenic responses. Increases (4- to 6-fold)
1n reversion and gene conversion were observed 1n yeast cells obtained from
the livers and kidneys. The toxic response of the animals to an exposure of
25 yg/kg was not described 1n this report. The positive results described
1n this paper suggest that 2,3,7,8-TCDD 1s mutagenic In yeast, but more
definitive studies are needed before a firm conclusion can be drawn.
Hay (1982) has found that 2,3,7,8-TCDD dissolved 1n DMS0 transformed
baby hamster kidney cells (BHK) n vitro. The dioxin Isomers
2.8-
d1chloroand 1 ,3,7-tr1chlorod1benzo-j)-d1ox1n also transformed BHK cells,
10-6
b u t t h e r e s p o n s e was weak. The unchlorlnated d1benzo--d1ox1n and the t u n y
chlorinated octachlorod1benzo--d1ox1n were both negative 1n the BHK assay (1.e., there was no cell transformation).
Abernethy et al. (1985) failed to transform C3H/10T^2 cells 1n cul ture by single treatments with 0.06 mM to 5 y dosage of 2.3.7.8-TCDD or Initiate transformation 1n these treated cells by subsequent exposure with tumor promoter 12-0-tetradecanoylpharbol-13-acetetate (TPA). However, these authors could transform C3H/10 T . ^ cells In vitro by N-methyl-N'-n1tro-Nnltrosoguanldlne (MNNG) and this transformation could be enhanced by subse quent treatment with low concentration (> 4 pM) of 2,3,7,8-TCDD. Maximum enhancement was observed at a concentration of 40 pM of 2.3.7.8-TCDD. This study Indicates that 2,3,7,8-TCDD Induced promotional activities can be observed 1n C3H/10 T,1/2 cells 1n cultured.
Rogers et al. (1982) reported that 2,3,7,8-TCDD Induced mutations 1n the excess thymidine, thloguanlne and methotrexate selective systems 1n L5178Y mouse lymphoma cells 1n culture. However, no significant mutation was noted 1n ouabln or cytosine arablnoslde selective systems. 10.1.2. Interactions with Nucleic Acids. In vitro reactions of 2,3,7,8TCDD with bacteriophage QB RNA were evaluated by Kondorosl et al. (1973). Active RNA was purified from QB phage followed by Incubation for 1 hour at 37C with 0.0, 0.2, 2.0 or 4.0 yg/mJL of 2,3,7,8-TCDD. At all concentra tions tested, 2,3,7,8-TCDD had no effect on the transfectlvlty of QB RNA. Other compounds tested Included the alkylating agents methyl, ethyl and Isopropyl methane-sulfonate, and diethyl pyrocarbonate, all of whlbh 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
10-7
an Intercalating agent, and hence would require double stranded DNA 1n order to Interact. The data presented 1n this study, however, were Insufficient to support this conjecture.
In vivo binding of radiolabeled 2,3,7,8-TCDD to Uver macromolecules was studied 1n Sprague-Dawley rats by Poland and Glover (1979). Both male and female animals were administered [1,6-sH]2,3,7,8-TCDD 1.p. at a dose of 7.5 yig/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 phno barbital or unlabeled 2,3,7,8-TCDD. Following sacrifice, Isolation of macromolecules, and removal of free labeled 2,3,7,8- TCDD, the amount of label bound to protein, RNA and DNA was determined. The greatest nonex tractable binding of labeled 2,3,7,8-TCDD occurred to protein; however, the amount of label bound was small and only amounted to 0.03-0.1% of the total radioactivity administered. The total amount of label associated with RNA and DNA was, respectively, only 50 and 4 cpm above background. Time after exposure, sex or prior enzyme Induction had no significant effect on 2,3,7,8-TCDD binding. As a result of the extremely low levels of radioacti vity associated with RNA and DNA, 1t 1s uncertain whether 2,3,7,8-TCDD truly binds covalently to these macromolecules and, 1f so, whether there 1s any biological significance to this low level of apparent binding. 10.1.3. Cytogenetic Effects of 2,3,7,8-TCDD. The effects of 2,3,7,8-TCDD exposure on the extent of chromosomal aberrations 1n the bone marrow of male rats were reported 1n an abstract by Green and Moreland (1975). In the Initial experiment, no Increase 1n 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 Intraperitoneal
10-8
Injection of 2,3,7,8-TCDD at 5, 10 or 15 pg/kg or a single gavage treat ment at 20 pg/kg. The animals at the two highest exposure levels were killed 24 hours post-treatment, while the remaining animals were killed 29 days post-treatment. Again, no Increase 1n chromosomal aberrations was observed, except 1n the positive control group exposed to trlethylenemelam1ne.
In a later report, a small but significant Increase 1n chromosomal aber rations was observed 1n the bone marrow cells of male and female OsborneMendel rats (Green et al., 1977). Bone marrow cells for cytogenetic analysis were obtained from Osborne-Mendel rats used 1n a range-finding study preliminary to a chronic bioassay (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 pg/kg for 13 weeks. Because 1t was not required for the range-finding study, a control group was not Included. Bone marrow cells were analyzed for abnormalities and cells 1n mitosis 1n the animals that survived to the end of the study (4-8 anlmals/group). The only significant Increases 1n chromosomal aberrations 1n comparison with the low dose group were 1n males at 2 and 4 pg/kg and females at 4 pg/kg. The greatest Incidence observed was 4.65% of the cells with chromosomal breaks 1n the high-dose males; this was considered only weakly positive. The weak response, as well as the lack of data from control animals and the reported difficulty of obtaining cells from the high-dose animals as a result of 2.3.7.8- TCDD toxicity, makes the conclusion from this study that 2,3,7,8TCDD produced chromosomal breaks tenuous.
10-9
A similar weak response was observed by Loprleno et al. (1982) 1n male and female CD-I mice that received an 1ntraper1toneal Injection of 2,3,7,8TCDD at a dose of 10 pg/kg. At 96 hours post-treatment, there was a significant (p<0.01) Increase 1n bone marrow cells with gaps and chromatid 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 pg/kg for 45 weeks. Analysis of bone marrow cells for chromosomal aberrations 24 hours after the last treatment failed to detect significant Increases.
Czelzel and Klraly (1976) reported an Increased Incidence (p<0.001) of chromatid-type and unstable chromosome aberrations 1n the peripheral lympho cytes of workers exposed to the herbicides 2,4,5-trIchlorophenoxyethanol (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 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 chromatid exchange nor structural aberrations Including both gaps and breaks were Increased. The authors noted that the long time between exposure and analysis may have accounted for the negative results.
10-10
In addAA.Aon, ReggAanA (1980) and M ottura e t al. (1981) studied the 2,3,7,8-TCDD exposed inhabitants in Seveso. Reggiani (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 in these 17 individuals indicated chemical exposure, no increase in chromosomal aber rations was detected. The methods of performing the analyses and the actual number of aberrations detected were not described. Similar negative results were reported in an abstract by Mottura et al. (1981). In this study, sub jects were chosen from the area of heavy contamination following the acci dent (acute high level exposure), from the working population of the plant (chronic low level exposure) and a nonexposed control population. The num ber of subjects in each group was not provided. The specimens were examined by three independent laboratories and no laboratory reported an increase in chromosomal aberrations, although there was a significant difference in the
r e p o r te d scores between l a b o r a t o r i e s . There was no in fo r m a tio n in t h i s
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.
Tenchini et al. (1979) also conducted a cytogenetic study of the exposed individuals at Seveso, Italy and of the aborted fetal tissue from exposed mothers. No significant chromosomal aberrations could be observed in the peripheral lymphocytes of the exposed idividuals. But aborted fetuses showed a nonsignificant increase in chromosomal abnormalities compared to the spontaneously aborted fetuses as observed in the general population. In a subsequent study, Tenchini et al. (1983) observed a significant increase
10-11
1n the frequencies of aberrant cells and in the average number of aberra tions per damaged cell 1n fetal tissues from exposed pregnancies. This 1s a potentially Interesting observation, but the study has the following pit falls. First, the controls were nonconcurrent. This Is a major problem In the Interpretation of the results from pregnancies before and after expo sure. Second, cells carrying the chromosomal aberrations described are not expected to survive more than one cell cycle, but 1n this study cells were examined that had undergone several cell divisions. This casts doubt on the validity of a positive result.
D1Lern1a et al. (1982) conducted additional studies on lymphocytes pre pared 1n 1976 and 1979 from eight persons considered acutely exposed to 2,3,7,8-TCDD In 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 (Satellite Associations; evidence for functional rlbosomal genes), both on a cell basis and for the large acrocentric chromosomes (D group chromosomes). There was no change In the frequency of SAs on a per cell basis 1n any of the groups as compared to control values, nor 1n D group chromosomes from acutely exposed subjects examined Immediately after the accident. There was, however, a decrease 1n the average frequency of SAs 1n group D chromosomes of acutely exposed subjects examined In 1977 and 1n ICMESA workers at both the 1976 and 1979 examinations. Although the bio logic relevance of these observations has not yet been confirmed, DILernla et al. (1982) observed a similar decrease 1n SAs after exposure of lympho cytes to x-1rrad1at1on. It was concluded that the decrease 1n SAs may have resulted from mutagenic damage to functional nucleolar organizing regions.
10-12
1 0 . 2 . SUMMARY
A limited number of Initial studies on the mutagenicity of 2,3,7,8-TCDD In bacteria reported positive results 1n S. typh1mur1um strain TA1532 1n the absence of a mammalian metabolic activation system (Hussain et al., 1972; Seller, 1973). More recent attempts to repeat these results with strain TA1532 or related strains have failed (Geiger and Neal, 1981; Nebert et al., 1976; Gilbert et al., 1980; McCann, 1978). These authors have also reported no Increase in mutation rate when 2,3,7,8-TCDD was tested 1n the presence of a mammalian metabolic activation system. In other in vitro assays, 2,3,7,8TCDD has produced a positive response 1n reversion to streptomycin Indepen dence 1n E. coll Sd-4 cells and questionable positive response with prophage Induction 1n E. coll K-39 cells (Hussain et al., 1972). Also, 2,3,7,8-TCDD has been reported to be mutagenic 1n the yeast S. cerevlslae in both the in vitro assay with S-10 and the host-mediated assay (Bronzettl et al., 1983). Rogers et al. (1982) also reported positive mutagenicity results in the mouse lymphoma assay system. In the E. coll studies, the poor survival of the cells or the Interference of the vehicle solvent, DMSO, with the assay makes the evaluation of the studies difficult. With the data available, 1t Is not possible to resolve the conflicting reports on the mutagenic poten tial of 2,3,7,8-TCDD.
Overall, the data Indicate little potential for the Interaction of 2,3,7,8-TCDD with nucleic acids or the ability of 2,3,7,8-TCDD to produce chromosomal aberrations. Kondorosl et al. (1973) demonstrated that 2,3,7,8TCDD did not react with RNA 1_n vitro 1n the absence of a metabolic activa tion system. In vivo studies using radiolabeled 2,3,7,8-TCDD Indicated some association of nonextractable label with RNA and DNA (Poland and Glover, 1979); however, the level of bound label was very low. Similar marginal
1 0 -1 3
data were available on the clastogenlc effect of 2,3,7,8-TCDD. Although two 1n vivo studies 1n rats (Green and Moreland, 1975; Loprleno et al., 1982) failed to demonstrate treatment-related chromosomal aberration, a second study by the same authors (Green et al., 1977) using a longer exposure period reported a small Increase 1n the number of aberrations. A similar small Increase was observed by Loprleno et al. (1982) following a single 1ntraper1toneal Injection of 2,3,7,8-TCDD 1n mice. In humans exposed to 2,3,7,8-TCDD during the manufacture of 2,4,5-TCPE and Bumlnol, Czelzel and Klraly (1976) reported an Increase 1n the number of chromosomal aberrations; however, no Increase was detected 1n Individuals exposed to 2,3,7,8-TCDD following an Industrial accident 1n Seveso, Italy (Regg1an1, 1980; Mottura et al., 1981; Tench1n1 et al., 1979). In contrast, Tench1n1 et al. (1983) reported positive results 1n a Seveso study, but this study has problems. The studies of the clastogenlc effect of 2,3,7,8-TCDD were presented with little or no experimental detail to assist 1n evaluating the merits of the reports. The data available are too limited to Indicate whether 2,3,7,8TCDD 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 toxicity of 2,3,7,8-TCDD. This chemical may be a weak mutagen, but because 1t Is 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 bac terial studies and the sex-Unked recessive lethal test In Drosophila.
Pertinent Information regarding the mutagenicity of PeCDDs and HxCDDs were not located In the available literature.
10-14
11. CARCINOGENICITY
The purpose of this section 1s to provide an evaluation of the likeli hood that 2,3,7,8-tetrachlorod1benzo-j)-d1ox1n (TCDD), and a mixture of 1.2.3.7.8.9- and 1,2,3,6,7,8-hexachlorod1benzo--d1ox1n (HxCDD), are human carcinogens and, on the assumption that they are human carcinogens, to provide a basis for estimating their public health Impact, Including a potency evaluation, 1n relation to other carcinogens. The evaluation of carcinogenicity depends heavily on animal bioassays and epidemiologic evidence. However, Information on mutagenicity and metabolism, particularly 1n relation to Interaction with DNA, as well as to pharmacokinetic behavior, has an Important bearing on both the qualitative and quantitative assessment of carcinogenicity. The available Information on these subjects Is reviewed In other sections of this document. This chapter presents an evaluation of the animal bioassays, the human epidemiologic evidence, the quantitative aspects of assessment, and finally, a summary and conclusions section dealing with all of the relevant aspects of carcinogenicity. 11.1. ANIMAL STUDIES 11.1.1. Studies Using 2,3,7,8-TCDD. The polychlorinated d1benzo-j)-d1ox1ns (PCDDS), 2,3,7,8-TCDD and a mixture of 1,2,3,7,8,9- and 1 ,2,3,6,7,8-HxCDD, have been tested for carcinogenicity 1n rats and mice by administering the compound 1n the diet and by gavage. Also, the tumor Incidence In native mice Inhabiting an area with heavy exposure to the herbicide Agent Orange has been assessed and compared with mice from an uncontaminated habitat. The results of these bioassays are discussed 1n this section. Along with studies using the oral route, both 2,3,7,8-TCDD and a mixture of 1.2.3.7.8.9- and 1,2,3,6,7,8-HxCDD have been tested for tumor1gen1city by
11-1
dermal application. Using the skin two-stage tumor1gen1c1ty model, 2,3,7,8-
TCDD has been tested for promoting and Initiating activity as well as ant1-
carclnogenlc activity. Other model systems have been used to a more limited
extent 1n studies of the effect of 2,3,7,8-TCDD on the carcinogenic poten
tial of chemical carcinogens.
11.1.1.1.
VAN MILLER ET AL. (ORAL) RAT STUDY (1977a,b) -- In a limited
study, Van Miller et al. (1977a,b) maintained small groups of male Sprague-
Dawley rats on diets containing 2,3,7,8-TCDD. The animals, 1n groups of 10,
were fed diets containing 0.0, 0.001, 0.005, 0.05, 0.5, 1.0, 5.0, 50, 500 or
1000 ppb of 2,3,7,8-TCDD for 78 weeks. As determined from the food consump
tion of two animals from each group, these exposure levels corresponded to
doses of 0.0, 0.0003, 0.001 , 0.01, 0.1, 0.4, 2.0, 2.4, 240 and 500 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 1n Table 11-1.
All animals 1n groups maintained on diets containing 1-1000 ppb of
2,3,7,8-TCDD were dead by week 90 of treatment; the first deaths 1n groups
at the 1000 and 1 ppb levels were observed at 2 weeks and 31 weeks of
treatment, respectively. Animals exposed to 0.001-0.5 ppb of 2,3,7,8-TCDD
had similar food consumption and survival as control animals; however, all
treated animals had histopathologic degenerative changes 1n the kidneys.
11-2
TABLE 11-1 2,3,7,8-TCDD Intake and Mortality 1n Male Sprague-Dawley Rats3
Doseb (ppb)
Weekly Dose/Rat (yg/kg bw)
Week of First Death
Number of Rats Dead at 95th Week
0.0 0.001 0.005 0.05 0.5
1 5
0.0003 0.001 0.01 0.1
0.4 2.0
68 6/10 (60%) 86 2/10 (20%) 33 4/10 (40%) 69 4/10 (40%) 17 5/10 (50%)
31 10/10 (100%) 31 10/10 (100%)
aSource: Van Miller et al., 1977a,b bRats at 50, 500 and 1000 ppb dose levels were all dead within 4 weeks.
11-3
Complete necropsies were done and samples of tissues were taken for
microscopic examination from the control groups and each treatment group
(Laboratory audit* and personal communication with author).
Special staining methods were used as an aid 1n the diagnosis of neo
plasms. Various benign and malignant tumors were found 1n each treatment
group. No tumors were observed 1n the controls (Table 11-2).
Statistically significant Increases of squamous cell tumors of the lungs
and neoplastic nodules of the Uver were observed 1n rats Ingesting 5 ppb
TCDD (Table 11-3). In addition, two animals 1n the 5 ppb dose group and one
animal 1n the 1 ppb dose group had Uver cholanglocardnomas, which are rare
1n Sprague-Dawley rats. These results provide evidence of a carcinogenic
effect.
The observation of no tumors of any kind 1n the controls 1s unusual for
Sprague-Dawley rats. In addition, the reporting of the study was not exten
sive. These factors may tend to lessen the reliance that can be placed on
the positive results of this study. However, this study 1s suggestive of a
carcinogenic response upon exposure to TCDD 1n rats.
11.1.1.2.
KOCIBA ET AL. (ORAL) RAT STUDY (1978a) -- Although this
study was published as Koclba et al., 1978a, a fuller version was submitted
1n an unpublished report (Koclba 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 pg/kg/day 2,3,7,8-TCDD. Vehicle control groups consisted of 86
animals of each sex. The test was appropriately conducted with the
*The audit of this study brought out the fact that 1t was Intended to be only a range-finding study. Therefore, only small numbers of animals were used. This may have made the study relatively Insensitive for detecting carcinogenic effects at doses <1 ppb.
11-4
TABLE 11-2 Benign and Malignant Tumors 1n Rats Ingesting 2,3,7,8-TCDDa
Dose^
Benign
Malignant
Number of Tumors
Number of Rats With Tumors
0 1 ppt
5 ppt
50 ppt 500 ppt
0 0
1
2 2
0 0
5 1
2
0 0/10 (0%)c 0 0/10 (0%) 6d 5/10 (5094)e
3f 3/10 130%) 49 4/10 (40%)"
1 ppb
0
4
51 4/10 (40%)
5 ppb
8
2
103 7/10 (70%)
aSource: Van Miller et al., 1977a,b
bRats 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 lymphocytic leukemia 1 adenocarcinoma (kidney) 1 malignant histiocytoma (retroperitoneal)
1 angiosarcoma (s k in )
1 Leydlg cell adenoma (testis)
e3 rats died with aplastic anemia
^1 fibrosarcoma (muscle) 1 squamous cell tumor (skin) 1 astrocytoma (brain)
9l fibroma (striated muscle) 1 carcinoma (skin) 1 adenocarcinoma (kidney) 1 sclerosing seminoma (testis)
bl rat had a severe Uver Infarction
11 rat cholanglocardnoma and malignant histiocytomas (retroperitoneal) 1 anglosarcoma (skin) 1 glioblastoma (brain) 1 malignant histiocytoma (retroperitoneal)
3l rat had squamous cell tumor (lung) and neoplastic nodule (Uver) 2 cholanglocardnomas and neoplastic nodules (Uver) 3 squamous cell tumors (lung) 1 neoplastic nodule (Uver)
11-5
TABLE 11-3 Liver Tumors 1n Rats Ingesting 2,3,7,8-TCDDa
Dose (ppb)
Neoplastic Nodules
Cholanglocard nomas
Squamous Cell Tumors of the Lungs
0 0/10 (0%) 0/10 (054)
1 0/10 (034) 1/10 (1054)
5
4/10 (4034)
2/10 (2054)b
p=0.043c
0/10
0/10
4/10 (40%) p=0.043c
aSource: Van Hiller et al., 1977a,b
bThe two animals had both neoplastic nodules of the Uver and cholanglocardnomas.
cp-va1ues calculated using the Fisher Exact Test.
11-6
high-dose group given a dose which induced signs of tissue toxicity, reduced weight increments in both sexes, and shortened lifespans in female rats. Clinical tests performed at intervals during the study monitored organ specific toxicity, particularly of the liver. Pathologic examinations in cluded histopathologic evaluation of all major tissues in both the high-dose and control animals, but only of selected tissues identified as possible target organs and suspect tumors in lower-dose group:;. This approach is suitable for the identification of a carcinogenic effect, but does not determine actual tumor incidences in all groups except in those organs iden tified as target organs. It, therefore, is adequate to define dose-response relationships only in these target organs. Tissues examined from most animals in all dose groups included liver, lungs, kidneys, urinary bladder, tongue, brain, testes/ovaries and prostate/uterus. For these tissues, a quantitative analysis can be performed using the actual number of tissues examined histopathologically for animals at risk. For other tissues
(e x c lu d in g s k i n , mammary glands and nasal t u r b i n a t e s / h a r d p a l a t e ) , a c t u a l
tumor incidence cannot be evaluated for the two lower doses. For skin, mammary glands and nasal turbinates/hard palate, the number of animals necropsied is the appropriate denominator to determine incidence, because detection of these tumors is based on observation of the tumor at necropsy.
A laboratory audit of this study by H. Spencer and W.S. Woodrow, Hazard Evaluation Division, Office of Pesticide Programs, U.S. EPA, did not reveal significant new information. Reviewers concluded that the study was properly conducted, adhering to the accepted procedures (Spencer and Woodrow, 1979).
Based on data reported for food consumption, body weight and dietary level of TCDD, the daily doses were reasonably constant for most of the
11-7
study, although somewhat below the value expected 1n most groups during the third month.
High early mortality was observed 1n all groups 1n this study but was only statistically significant 1n the high-dose group. The survival curves show progressive mortality beginning as early as the 12th month and leading to 50% mortality by 21 months.* The effects of this early mortality are a reduction 1n expected tumor Incidence because of a truncated latency period, and a reduction 1n sensitivity of the study because of a reduction 1n number of animals at risk during the time of expected tumor manifestation. Cumula tive mortality and Interval mortality rates are given 1n Tables A-l to A-4 of Appendix A (Clement Associates, 1979).
The results of this study provide substantial evidence that 2,3,7,8-TCDD 1s carcinogenic In rats. 2,3,7,8-TCDD Induced a highly statistically significant Increase of both hepatocellular carcinomas and hepatocellular neoplastic 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, 1n the high-dose females, was also highly significant. In addition, at the highest dose level, 2,3,7,8-TCDD Induced a statistically significant Increase 1n stratified squamous cell carcinomas of the hard palate and/or nasal turbinates 1n both males and females, squamous cell carcinomas of the tongue 1n males, and highly significant keratinizing squa mous cell carcinomas of the lungs In females (Tables 11-4, 11-5 and 11-6).
*In the 0.001 group of males, 44% of the animals had died by 18 months. The mortality patterns were analyzed by the Wh1tney-W1lcoxon test and the Kolmogorov-SImonov test. These tests showed that mortality was signifi cantly higher 1n the high-dose females than 1n controls, and while Indica tions of Increased mortality were found 1n other groups, they were not part of a consistent pattern.
11-8
TABLE 11-4
Hepatocellular Carcinomas and Hepatocellular Hyperplastic Nodules 1n Female Sprague-Oawley Rats Maintained on Diets Containing 2,3,7,8-TCDDa
Dose Level (vg/kg/day)
Hepatocellular Hyperplastic
Nodules
Hepatocellular Carcinomas11
Total Number With Both
Types of Tumors15
0
0.001 (22 ppt)
0.01 (210 ppt)
0.1 (2200 ppt)
8/86 (9%) 3/50 (6%)
18/50 (36%) 23/49 (48%)
1/86 (1%) 0/50 (0%)
2/50 (4%)
11/49 (22%) (p=5.6 x 10"s)
9/86 (10%)
3/50 (6%)
18/50 (36%)c (p=4.36 x 10") 34/50 (71%) (p=4.56 x 10~13)
aSource: Koclba et al., 1977 bp-values calculated using the Fisher Exact Test (one-ta1led). cTwo rats had both hepatocellular carcinomas and hyperplastic nodules.
11-9
TABLE 11-5 Tumor Incidence 1n Female Rats Fed Diets Containing 2,3,7,8-TCDDa
Dose Level (pg/kg/day)
Stratified Squamous Cell Carcinomas of Hard Palate
or Nasal Turbinates
Keratinizing Squamous Cell Carcinomas of Lungs
0
0.001 (22 ppt)
0.01 (210 ppt)
0.1 (2200 ppt)
1/54 (2%) 0/30 (0%)
1/27 (4%)
5/24 (2154) (p=0.01)b
0/86 (0%) 0/50 (054)
0/50 (0%)
7/49 (14%) (p=0.0006)0
aSource: Kodba et al ., 1977 bp-values calculated using the Fisher Exact Test (one-tailed).
11-10
TABLE 11-6 Tumor Incidence 1n Male Rats Fed Diets Containing 2,3,7,8-TCDDa
Dose Level (vg/kg/day)
Stratified Squamous Cell
Hard Palate/Nasal Turbinates
Carcinomas of the Tongue Stratified Squamous Cell Carc1nomab
0
0.001 (22 ppt)
0.01 (210 ppt)
0.1 (2200 ppt)
0/76 (0%) 1/49 (2%) NS 1/50 (2%) NS 3/42 (7%) (p=4.3 x 10"2 )c
0/51 (0%) 1/34 (3%) NS 0/27 (0%) NS 4/30 (13%) (p=0.016)c
aSource: Koclba et al.t 1977 bIncludes examinations from both original and updated report (5/20/79). cp-values calculated using the Fisher Exact Test. NS = Not significant at p=0.05.
11-11
Dr. Robert Squ4r-e, pathologist at the Johns Hopkins University Medical
School and consultant to the CAG, evaluated the histopathologic 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 h1s associates examined all Uver, lungs,
tongues, hard palates and nasal turbinates available from the 2,3,7,8-TCDD
study. Their hlstopathologlcal findings, as well as Dr. Koclba'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. Koclba and Squire, the conclusions about the target organ for cancer
Induction and the dose levels at which Induction occurred are the same.
11.1.1.3.
NATIONAL TOXICOLOGY BIOASSAY PROGRAM (ORAL) RAT STUDY
(1980a,b) -- A cancer bioassay for the possible carcinogenicity of 2,3,7,8-
TCDD was tested by the Illinois Institute of Technology In 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 o1l-acetone by gavage 2
days/week for 104 weeks at doses of 0.01, 0.05 or 0.5 yg/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 pentachlorodlbenzo-pdloxln. The presence of 0.1-0.2% hexachlorodlbenzo-p-dloxln was also detected by gas chromatography and mass spectrometry.
11-12
TABLE 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 Substrain (2 years)a *b
11-13
Tissues and Diagnoses
0 (control 1 SK
S
0.001 K
Dose levels luo/kg/dav)
0.01
s
K
0. 1 SK
Lung Squamous cell carcinomas
0/86 0/86 0/50
0/50
Nasal turblnate/hard palate squamous cell carcinomas
0/54 1/54
0/30
0/30
Liver Neoplastic nodules/ hepatocellular carcinomas
16/86
9/86
8/50
3/50 (p=4.37 x 10")
0/49 1/27
0/49 1/27
27/50 (p=2.42 x 10"*)
18/50 (p=4.37 x 10")
8/47 (17%) (p=1.61 x 10")
7/49 (14%) (p-6.21 x 10")
5/22 (23%) (p-1.43 x 10" )
5/24 (21%) (p-9.46 x 10")
33/47 (70%) (p=4.92 x 10"*)
34/48 (71%) (p=9.53 x 10" *
Total combined (each animal had at least one tumor above)
16/86 9/86 19% 10%
8/50 16%
3/50 6% (p = 4.37 x 10')
27/50 54%
(p=2.42 x 10"*)
18/50 34%
(p=4.37 x 10")
34/47 72%
(p=l.20 x 10")
34/49 69%
(p=2.13 x 10" 1*
aSource: Koclba et al., 1977; Squire, 1980 bp-va1ues calculated using the Fisher Exact Test. S Dr. Squire's histopathologic analysis; K = Dr. Koclba's histopathologic analysis
TABLE 11-8
Dow 2,3,7,8-TCDD Oral Rat Study by Dr. Koclba, With Dr. Squire's Review (8/15/80) Sprague-Dawley Hale Rats - Spartan Substrain (2 years)*
n-14
Tissues and Diagnoses
0 (control) SK
Dose Levels (uq/kq/day)
0.001
0.01
SK
SK
S
0.1
K
Nasal turblnate/hard palate squamous cell carcinomas
Tongue squamous cell carcinomas
0/55 0/51
1/34 1/34
0/26 0/27
6/30 (20%)
4/30 (13%)
(p=l.36 x 10"3) (p=l.6 x 10~2)
0/77 0/76
2/44 1/49
1/49 1/49
3/44 m
3/42 (7%)
(p=4.60 x 10"2) (p=4.34 x 10"2)
Total - 1 or 2 above (each rat had at least
one tumor above)
0/77
2/44 5%
1/49 2%
9/44 20% (p=6.28 x 10"s)
*p-values calculated using the Fisher Exact Test. S = Dr. Squire's histopathologic analysis K = Dr. Koclba's histopathologic analysis
In rats, a dose-related depression 1n mean body weight gain became
evident 1n the males after week 55 of the bioassay and In the females after
week 45.
The results of histopathologic diagnosis of primary tumors caused by the
oral administration of 2,3,7,8-TCDD are presented 1n Table 11-9. In male
rats an Increased Incidence of follicular-cell adenomas or carcinomas of the
thyroid was dose-related and was statistically significantly higher In the
low-, mid- and high-dose groups than In the vehicle controls. In addition,
a statistically significant Increase 1n subcutaneous tissue 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 1n Table 11-10.
These results confirm the carcinogenic effect observed 1n the Koclba et
al. (1978a) study using Sprague-Oawley (S p a rta n s u b s t r a in ) r a t s .
11.1.1.4.
TOTH ET AL. (ORAL) HOUSE STUDY (1979) -- This study Investi
gated the carcinogenicity of 2,3,7,8-TCDD 1n Swiss mice. Ten-week-old
outbred Sw1ss/H/R1op mice were used. 2,3,7,8-TCDD was administered 1n a
sunflower oil vehicle by gavage to groups of 45 male mice once a week at
doses of 7.0, 0.7 and 0.007 yg/kg bw for a year (groups 9, 10, 11, respec
tively, In Table 11-11). Matched male vehicle controls were administered
sunflower oil once a week. Matched controls to a companion study Investi
gating the carcinogenicity of (2,3,5-tr1chlorophenoxy)ethanol (TCPE) contam
inated with low levels of 2,3,7,8-TCDD, were administered carboxymethyl
cellulose (the vehicle used 1n that study) once a week. Two untreated
controls were also maintained.
11-15
TABLE 11-9 Incidence of Primary Tumors 1n Hale Rats Administered 2,3,7,8-TCDD by Gavage3
9L-LL
Type of Tumor
Vehicle Control
Low Doseb 0.01
Subcutaneous tissue Fibroma
Liver Neoplastic nodule or hepatocellular carcinoma
Adrenal Cortical adenoma
Thyroid Follicular cell adenoma
Thyroid Follicular cell adenoma or carcinoma
3/75 (4%)
0/74 (OX) 6/72 (8X) 1/69 (IX)
1/69 (IX)
1/50 (2X)
0/50 (OX) 9/50 (18X)
5/48 (10X) p=0.042
5/48 (10X) p=0.042
aSource: NTP, 1980a bp-values calculated using the Fisher Exact Test.
tiq/kq/week Hid Doseb
0.05 3/50 (6X)
0/50 (OX) 12/49 (24X)
6/50 (16X) p=0.021
8/50 (16%) p=0.004
High Doseb 0.5
7/50 (14X) p=0.048
3/50 (6X) 9/49 (18X)
10/50 (20X) p=0.001
11/50 (22X) p<0.001
TABLE 11-10
Incidence of Primary Tumors 1n Female Rats Administered 2,3,7,8-TCDD by Savage3
Type of Tumor
Vehicle Control
uo/ka/week
Low Doseb 0.01
Mid Dose 0.,05
High Doseb 0.5
Subcutaneous tissue Fibrosarcoma
Liver Neoplastic nodule
0/75 (0%) 5/74 (7%)
Liver Neoplastic nodule or hepatocellular carcinoma
5/75 (7%)
Pituitary Adenoma
1/66 (2%)
Adrenal Cortical adenoma
11/73 (15%)
2/50 (4%)
3/50 (6%) 4/49 (8%) p=0.023
1/49 (2%)
3/50 (6%) 12/49 (24%) p=0.006
1/49 (2%)
3/50 (6%) 14/49 (29%) p=0.001
5/47 (11%) 2/44 (5%) p=0.044
3/43 (7%)
8/49 (16%) 4/49 (8%) 14/46 (30%) p=0.039
aSource: NTP, 1980a bp-values calculated using the Fisher Exact Test.
11-17
TABLE 11-11 emulative Data on Tumor Incidence9
Group
TCPEb (mg/kg)
Treatment
Number
Effective
of Tumor
TCDD
Vehicle:
Sex
Number of
Bearing
(vg/kg)
(mg/kg)
Nice
Nice
1 67.0
0 .112
50 H 88
(1 . 6 ppm)
F 83
2 70.0
0.007
50 H 98
(0 . 1 ppm)
F 96
3 control H 93
F 84
4 7.0 0.07
50 H 93
( 1 0 ppm)
F 96
5 7.0
0.0007
50
H
94
(0 . 1 ppm)
F 93
6 0.7
0.00007
50
H
97
(0 . 1 ppm)
F 94
7 __ 50 H 96
F 84
8 control
H 96
F 91
9 10 11 12 --
7.0 0.7 0.007
--
10 H 43 10 H 44 10 N 44
1 0 n 38
69 61
78 59 63 57
79 60 77 71 78 64
74 55 78 57
27 36 39 27
aSource: Toth et al., 1979 bTCPE = Trlchlorophenoxy ethanol cCarboxymethy1 cellulose In groups 1-8, sunflower oil In groups 9-12. dp<1X ep<0.1X
Liver (X)
Number of Animals with Tumors of:
Lung
Lymphomas
Other Organs
42d (18) 7 (8 )
57e (58) 9 (9)
24 (26) 4 (5)
25 (27) 10 (1 0 ) 23 (24)
8 (9) 24 (25)
5 (5)
32 (33) 4 (5)
32 (33) 4 ()
13 (30) 2 1 (48) 13 (29)
7 (18)
50 52
18 39 44 41
38 38 50 42 51 38
44 38 38 31
11 18 27 15
7 15
11 15
8 23
18 19 23 36 20 22
14 18 22 24
6 12 10
6
16 25
16 23 17 13
22 19 17 21 17 21
22 17 15 19
7 4 6 7
595 652
571 582 577 639
641 589 660 590 643 566
615 565 651 549
424 633 649 588
This study appears to have been generally well conducted. However, the administration of 2,3,7,8-TCDD over a period of only 1 year, which 1s far short of the life expectancy of the mice used, made the study relatively Insensitive. Animals were followed for their entire lifetimes. Autopsies were performed after spontaneous death or when the mice were moribund, and all organs were examined histologically. Sections were stained with hematoxylin and eosln for light microscopy. Pathological findings were evaluated and analyzed statistically. The findings of the 2,3,7,8-TCDD study and the comparison study on TCPE are given 1n Table 11-11.
Analysis of the results of this study focused on the Incidence of Uver tumors 1n the groups treated with 2,3,7,8-TCDD and the Incidence of these tumors 1n the matched controls (group 12) and 1n the males 1n the three other control groups. Males 1n groups 3 and 8, the two untreated control groups, had 26% and 33% Uver tumors, respectively (p<0.20). The carboxymethyl cellulose male controls (group 7) had 33% (32/96) Uver tumors. No significant differences 1n Uver tumors were observed when males 1n all four control groups were compared with each other (p<0.05). Nevertheless, there was evidence that the Incidence of Uver tumors 1n the control groups was associated with the average lifespan 1n the respective groups. The two groups that had <600 days average survival (groups 3 and 12) had the fewest Uver 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% Uver 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 Uver tumors (21/44 = 48%).
11-19
This Incidence was significantly higher than the Incidence of liver tumors
1n either the sunflower oil controls (p<0.01) or the pooled controls (all
four control groups combined) (p<0.025).
The h1ghest-dose group (7.0 jjg/kg) had an Increased Incidence of Uver
tumors compared with the matched sunflower oil controls (13/43 = 30%), but
this Increase was not statistically significant (p=0.11). The Incidence of
Uver tumors 1n the high-dose group was comparable with that of the pooled
controls. The h1ghest-dose group, however, had a much reduced average sur
vival 1n comparison with any of the control groups (only 424 days compared
with 577, 588, 615 and 651 days 1n the four control groups). This poor
survival may have accounted for the lack of a statistically significant
Increase 1n Uver tumors 1n the high-dose group. Furthermore, If t1me-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 Uver tumors that was observed In the
high-dose group In comparison with the matched control group, although not
statistically significant, 1s considered to be consistent with an oncogenic
effect.
In conclusion, the results of this study provide suggestive evidence of
an oncogenic effect.
11.1.1.5.
NATIONAL TOXICOLOGY BIOASSAY PROGRAM (ORAL) MOUSE STUDY
(1980a,b) -- A cancer bioassay for the possible carcinogenicity of 2,3,7,8-
TCOO was tested by the Illinois Institute of Technology 1n mice under a
contract sponsored by the NCI.
In the mouse study, groups of 50 B6C3F1 mice of each sex were admini
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 g/kg/week for male mice
1 1 -2 0
and 0.04, 0.2 and 2.0 yg/kg/week for female mice. Seventy-five mice of each sex were used as vehicle controls. One untreated control group of 25 mice of each sex was present In the 2,3,7,8-TCDD treatment room. One untreated control group of 25 mice of each sex was present In the vehicle control room. In mice, the mean body weight gain In the treated groups was comparable with that of the vehicle control groups. However, the mean body weight of the treated mice was lower when 1t was compared with untreated controls.
The results of the histopathologic diagnosis of primary tumors are pre sented In Table 11-12. The results Indicate that, 1n male mice, 2,3,7,8TCDD Induced a statistically significant Incidence of hepatocellular carci nomas (p=0.002) and both hepatocellular carcinomas and neoplastic 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=O.OQ2) 1n the high-dose group. In addition, a statistically significant Increase 1n tumor Incidences of fibrosarcoma, histiocytic lymphoma, thyroid folUcular-cell adenoma and cortical adenoma or carcinoma were also observed In the high-dose group (Table 11-13).
The Incidence of Uver tumors observed In this study confirms the earlier observations of an Increase In Uver tumors 1n the male mouse study performed by Toth et al. (1979).
11.1.1.6. OTHER RELATED STUDIES -- 11.1.1.6.1. Pitot et al. Promotion Study 1n Rats (1980) -- Pitot et al. (1980) Investigated a two-stage model of hepatocardnogenesls. Twentyfour hours after a partial hepatectomy (to enhance cell proliferation), female Sprague-Dawley rats were divided Into seven groups (Table 11-14).
11-21
TABLE 11-12
Incidence of Primary Tumors 1n Male Mice Administered 2,3,7,8-TCDD by Gavage3
Type of Tumor
Vehicle Control
Low Dose 0.01
ug/kq/week
M1d Dose 0.05
High Dose*5 0.5
Liver Hepatocellular adenoma
Liver Hepatocellular carcinoma
Liver Hepatocellular adenoma and carcinoma
7/73 (10%) 8/73 (11%)
15/73 (21%)
3/49 (6%)
5/49 (10%) 10/50 (20%)
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.
1 1 -2 2
TABLE 1 1 -1 3
Incidence of Primary Tumors 1n Female Mice Administered 2,3,7,8-TCDD by Gavage3
Type of Tumor
Vehicle Control
Low Dose 0.04
ua/kq/week
M1d Dose 0.2
High Doseb 2.0
Subcutaneous tissue Fibrosarcoma
Hematopoietic system
Histiocytic lymphoma
Hematopoietic system
All lymphoma
Hematopoietic system
Lymphoma or leukemia
Liver Hepatocellular carcinoma
Liver Hepatocellular adenoma or carcinoma
Thyroid Follicular-cell adenoma
1/74 (1%) 9/74 (12%) 18/74 (24%) 18/74 (24%) 1/73 (1%) 3/73 (4%) 0/69 (0%)
1/50 (2%)
1/48 (2%) 5/47 (11%) p=0.032
4/50 (8%)
4/48 (17%) 14/47 (30%) p=0.016
11/50 (22%) 13/48 (27%) 20/47 (43%) p=0.029
12/50 (24%) 13/48 (27%) 20/47 (43%) p=0.029
2/50 (4%) 2/48 (4%) 6/47 (13%)
6/50 (12%) 6/48 (13%) 11/47 (23%) p=0.002
3/50 (6%)
1/47 (2%) 5/46 (11%) p=0.009
3Source: NTP, 1980a bp-values calculated using the Fisher Exact Test.
11-23
Group No.
TABLE 11-14
Promoting Effect of 2,3,7,8-TCDD on Hepatocarc1nogenes1s by a Single Dose of D1ethyln1trosam1ne (DEN) and Partial Hepatectomy {PH)a
Treatment
Nc No. of Enzyme-Altered Percent Liver Volume Which Number of Rats
fod per cm3 of Liver
Is Enzume-Altered foci
with Carcinoma
1 PH + DEN
4
2 PH 4- TCDD (low dose)
5
3 PH 4- TCDD (h1gh dose)
5
4 PH 4- Phnobarbital
6
5 PH 4- DEN 4- TCDD (low dose)
5
6 PH 4- DEN 4- TCDD (hlgh dose)
7
7 PH 4- DEN 4- Phnobarbital 4
346 i 65 46 15 76 + 20 138 t 40
1582 300
1280 i 40
1510 i 185
5.0
0 .1 0 .1 0 .1
7.8
35.0
5.0
0 0 0 0
0<*
5/7e (p=0.0075)f
2
n-a
aSource: Pitot et al., 1980
^Female rats (200 g) were Intubated where shown with DEN. Seven days later TCDD (Injected subcutaneously) or phnobarbital (0.05% In the diet) administration was begun and continued for 28 weeks at which time the animals were sacrificed and the livers examined. The low and high doses of TCDD were 0.14 and 1.4 yg/kg/ 2 weeks, respectively, administered subcutaneously. DEN was given at a dose of 10 mg/kg. See text for further details.
cDenotes the number of animals used In each group. ^Three rats showed "neoplastic nodules." e0ne rat showed a "neoplastic nodule."
^p-value calculated using the Fisher Exact Test.
The animals 1n groups 1, 5, 6 and 7 received d1ethyln1trosam1ne (DEN). The rats 1n group 1 were then maintained on a standard laboratory diet for 32 weeks. The rats 1n groups 2 and 3 received no DEN, but starting 1 week after hepatectomy received biweekly subcutaneous Injections of 0.14 or 1.4 yg/kg of 2,3,7,8-TCDD 1n corn oil for a period of 28 weeks (2,3,7,8-TCDD was 98.6% pure and provided by Dow Chemical Co.). Groups 5 and 6 received DEN, and 1 week later were Initiated on a regimen of 14 biweekly Injections of 0.14 and 1.4 yg/kg of 2,3,7,8-TCDD. The animals In group 4 received 0.05% sodium phnobarbital In 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 phnobarbital 1n the diet for the duration of the experiment. At the end of the experiment, rats were killed and sections of the Uver were removed and frozen on solid CO^. Serial sections of the frozen blocks of Uver were cut and stained consecutively for glucose-6-phosphatase (G6Pase), canalicular ATPase, glutamyl transpepti dase (GGTase) with hematoxylin and eosln. The number of enzyme-altered foci were determined from photographs of hlstochemlcally stained sections. Hepatocarclnomas were diagnosed by standard hlstopathologlcal criteria.
The results presented 1n Table 11-14 showed that the number of foci with single enzyme changes, the number of foci with multiple enzyme changes, and the total Uver 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 in addition to DEN had hepatocellular carcinomas, and six of seven rats had hepatocellular carcinomas or hepatocellular neoplastic 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
11-25
neoplastic 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 neoplasia.
The results of this study provide evidence that 2,3,7,8-TCDD acts as a potent promoter In this two-stage model of hepatocarclnogenesls, causing Increased neoplasia and Increases 1n enzyme-altered foci at exceedingly low levels.
11.1.1.6.2. National Toxicology Bioassay Program Skin Painting Study In Mice (1980b) -- This cancer bioassay of 2,3,7,8-TCDD for possible carcino genicity 1n Swlss-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 Swlss-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-TCDD per application, and the female mice received 0.005 yg 2,3,7,8-TCDD per application.
In another experiment, the same number of animals were pretreated with one application of 50 yg 7,l2-d1methylbenz(l)anthracene (DMBA*) 1n 0.1 mi acetone 1 week before 2,3,7,8-TCDD application was Initiated. Forty-five mice of each sex received 0.1 ms. acetone 3 t1mes/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%.
11-26
treated and vehicle control groups of females were lower than those of
untreated controls. Mean body weights of males were less than that of
untreated controls.
The results of histopathologic 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.
11.1.1.6.3.
Berry et al. Skin Painting Study 1n 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-
TCDD, no papillomas were observed on the DMBA-1n1t1ated mice. In the posi
tive controls, DMBA-1n1t1ated mice were treated with 12-0-tetradecanoyl-
phorbol-13-acetate (TPA) for 30 weeks; 92% of these mice developed tumors.
Berry et al. (1979) also studied the effects of treatment with 2,3,7,8-
TCDD and 7,l2-d1methylbenz(a)anthracene (DMBA) 1n a two-stage tumorlgenesls
bioassay 1n mouse skin. In this study, tumors on the shaved skin of female
CD-I mice were Initiated by topical application of DMBA and were promoted
with TPA. Pretreatment with 2,3,7,8-TCDD markedly Inhibited the Initiation
of tumors by DMBA. The effects were greatest when 2,3,7,8-TCDD was applied
3-5 days before Initiation and were negligible when 1t was applied only 5
minutes before Initiation. The Inhibition was almost complete (94-96%) when
a single dose of 1 yg 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.
11-27
TABLE 11-15
Incidence of Primary Tumors 1n Mice Administered 2,3,7,8-TCDD or 2,3,7,8-TCDD Following DMBA by Dermal Application3
Type of Tumors Vehicle Control
TCDD
Dose Levels*5
DMBA (50 yg) plus TCDD
Integumentary system
Fibrosarcoma
3/42 (7%)
MALE
0.001 yg x 3/weeks
6/28 (21%) p=0.08
0.001 yg x 3/weeks
6/30 (20%) p=0.10
Fibrosarcoma
2/41 (5%)
FEMALE
0.005 yg x 3/weeks
8/27 (30%) p=0.007
0.005 yg x 3/weeks
8/29 (28%) p-0.010
aSourceT~NTP, 19805 bp-value calculated using the Fisher Exact Test.
11-28
The time course of the Inhibitory effects was closely parallel to the time course of Induction of arylhydrocarbon hydroxylase 1n the skin of the mice. It was also associated with substantial reduction 1n the covalent binding of the DMBA metabolite to DNA and RNA, but with no change 1n their binding to protein.
The same authors also reported Inhibitory effects of 2,3,7,8-TCDD on the Initiation of mouse skin tumors by benzo(a)pyrene (BaP), although the effect was not as great (maximum 65%) with BaP as with DMBA.
11.1.1.6.4. Cohen et al. Skin Painting Study 1n Mice (1979) -- Cohen et al. (1979) showed that pretreatment of mice with dermally applied 2,3,7,8-TCDD resulted 1n the Inhibition of skin tumor Induction by subse quent treatment with DMBA and BaP. The Inhibition of skin carcinogenesis by BaP 1n mice after pretreatment with 2,3,7,8-TCDD was associated with an Increase 1n covalent binding of BaP metabolites to DNA, RNA and protein (In contrast to the results with DMBA, which showed a reduction In binding to DNA and RNA). However, the BaP metabolites that were bound to DNA and RNA 1n mice pretreated with 2,3,7,8-TCDD differed from those In untreated mice. In particular, pretreatment with 2,3,7,8-TCDD markedly reduced the formation of the presumptive ultimate carcinogenic metabolite of BaP, 7,8-d1ol-9,l0epoxy-BaP and Its covalent binding with guanoslne 1n DNA.
11.1.1.6.5. Kourl et al. Mouse Study (1978) -- This study was designed as an Investigation of the cocarclnogenlc activity of 2,3,7,8-TCDD adminis tered to mice 1n conjunction with subcutaneous administration of 3-methylcholanthrene (3-MC). Two Inbred strains 1n mice, C57BL/6Cum (abbreviated B6) and DBA/2Cum (abbreviated D2), were used. These strains are responsive and nonresponslve, respectively, to the Induction of aryl hydrocarbon hydroxylase (AHH) by 3-MC.
11-29
Groups of mice of both sexes were Injected subcutaneously at 4-6 weeks of age with either 150 yg of 3-MC dissolved 1n trloctanoln or with trloctanoln alone. Some groups were also Injected with 2,3,7,8-TCDD dissolved 1n ja-dloxane, either simultaneously with the administration of 3-MC or 2 days earlier. Two doses of 2,3,7,8-TCDD (1 yg/kg and 100 yg/kg) were used, and the effects of both Intraperitoneal and subcutaneous Injections were Investigated.' Two sets of experiments Involving 29 groups of mice were conducted ~1 year apart (Tables 11-16 and 11-17).
After treatment, the mice were observed for 36 weeks, during which time they were palpated weekly for the presence of tumors; latency was calculated when the subcutaneous tumors became 1 cm 1n diameter. Only tumors charac terized histologically 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 IT-1-7.
No subcutaneous tumors were observed 1n controls or 1n mice treated with 2.3.7.8- TCDD alone. In B6 (responsive) mice, the administration of 2.3.7.8- TCDD did not significantly enhance the Induction of tumors by 3-MC. However, 1n 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 1n groups treated with 3-MC alone to 14 1n the group treated subcutaneously
11-30
TABIf It 16
E ffe c ts o f I n tr a p e r t to n e a l A d m in is tra tio n o f 2 ,3 ,7 ,8 -T C D O on 3 - M C - In iH a te d Su bcuta neous Tum ors3
LE-LL
In b re d S tra in
-2 Oays
Treatm ent
0 Days
No. o f H1ce D ying 8ecause o f T r e a t m e n t 15
No. o f H lce a t R isk fo r
Tumorsc
No. of H lce w ith
Tumors**
X o f H lce w ith Tumors
A ve ra g e L a te n c y (days)
C a rc in o g e n ic In d e x e
B6 l . p . p - d l o x l n
s .c . trlo c ta n o ln
1 .p . TC00 (100 p g /k g )
s .c . trlo c ta n o ln
None
s . c . 3-HC
None
l . p . TCDD ( 1 0 0 p g / k g )
None
1 . p . TCDD ( 1 0 0 p g / k g ) s . c . 3-HC
None
l . p . TCDD (1 p g / k g )
None
1 . p . TCDD (1 p g / k g ) t s . c . 3-HC
1 . p . TCDD ( 1 0 0 p g / k g )
s . c . 3-HC
l . p . TCDD (1 p g / k g )
s . c . 3-HC
1 20
1 20 30
4 6
20 6
39 0 27 0 36 29 30 0 43 33
0 0 81 125 65 0 71 12 3 6 3
46 0
0
27 27 100 132 76
25 21 23 16
84 129 65 70 140 50
02 l . p . p -d 1 o xa n e
s .c . trlo c ta n o ln
l . p . TCDD ( 1 0 0 p g / k g )
s .c . trlo c ta n o ln
None
s . c . 3-HC
None
1 . p . TCDD ( 1 0 0 p g / k g )
None
l . p . TCDD ( 1 0 0 p g / k g ) t s . c . 3-HC
None
l . p . TCDD (1 p g / k g )
None
1 . p . TCDD (1 p g / k g ) s . c . 3-HC
l . p . TCDD ( 1 0 0 p g / k g )
s . c . 3-HC
l . p . TCDD (1 p g / k g )
s . c . 3-HC
6 24
3 30 43
5 5
20 6
22 0 25 0 34 1 38 0 43 10
48 0 34 5
28 0 31 0
0
0
3 217
1
0
23 178 13f
0 15 199
7
0 0
a S o urce : K o u rl e t a l . , 1978
bDur1ng th e f i r s t 28 days f o llo w in g tre a tm e n t.
c D ef1ned as th e number o f m ice s u r v iv in g th e 36-w eek o b s e rv a tio n p e r io d .
dA t th e end o f th e 36-week e x p e rim e n t.
P e r c e n t a g e o f I n c id e n c e o f tu m o r s , d i v i d e d by t h e a v e r a g e la t e n c y 1n d a y s , m u l t i p l i e d b y 100 ( 8 ) .
^Th1s c a rc in o g e n ic Index v a lu e lie s o u ts id e (g re a te r th a n ) the 9 9 * c o n fid e n c e I n te r v a l ( 1 . e ., p < 0 .0 1 ) c o n s tru c te d from seven s tu d ie s o v e r th e p a s t S y e a rs d u r in g w h ic h 150 pg o f 3-HC was g iv e n s . c . to 0? m ic e . These s t u d ie s In c lu d e d 295 02 I c e , th e mean a l l seven s tu d ie s was a c a rc in o g e n ic Index o f 5 .4 3 * 2 .7 0 .
d iffe re n t - 5 0 fo r
TABI E 11-17
E f fe c t o f I n t r a p e r it o n e a l o r S ubcutaneous A d m in is tr a tio n o f 2 ,3 ,7 ,8 -T C D D G iven 2 Days B e fo re o r S im u lta n e o u s W i t h S u b c u ta n e o u s A d m i n i s t r a t i o n o f 3-MC o n T u m o r lg e n e s ls I n D7 M ic e 3
11-32
-2 Days
Treatm ent
0 Days
No. o f M ice D ying Because o f Treatm ent
No. o f M ice a t R isk fo r
T uraors
No. o f M ice w ith
Tumors
% o f M ice w i t h Tumors
Average L a te n c y (d a y s )
C a rcin o g e n ic Index
None l. p . p -d lo x a n e 1 . p . TCDD ( 1 0 0 p g / k g ) None None
None
None None None
None None
s . c . 3 -MC
s . c . 3-HC
s . c . 3 -MC
l . p . p -d lo x a n e s . c . 3-HC
l . p . TCDD ( 1 0 0 p g / k g ) t s . c . 3 -MC
l . p . TCDD (1 p g / k g ) * s . c . 3-MC
s . c . p - d lo x a n e f s . c . 3-MC
s . c . TCDD ( 1 0 0 p g / k g )
s . c . TCDD ( 1 0 0 p g / k g ) t s . c . 3-MC
s . c . TCDD (1 p g / k g )
s . c . TCDD (1 p g / k g ) s . c . 3-MC
0 10 35
5 38
22
2 8 18
2 2
30 3 10 177 6
40 3 10 194 5
65
9
14
145
10
5
5
11
176
6
6?
17
27
183
15b
78 8 10 162 6
68 B 12 180 6
4? 0 0
B2
46
55
145
38b
48 0 0
98
21
21
154
14b
a S o u rce : K o u rl e t a l . , 1978 b lh e s e c a r c in o g e n ic In d e x v a lu e s l i e o u t s id e th e 99% c o n fid e n c e I n t e r v a l .
with 2,3,7,8-TCDD at 1 yg/kg, and 13-15 1n the groups treated 1ntraper1-
toneally with 2,3,7,8-TCDD at 100 yg/kg. The authors concluded that
2.3.7.8- TCDD acts as a cocarcinogen, 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 yg/kg). The reported results at the lower dose (1 yg/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
r e p o r t tumor In c id e n c e by s e x ), the r e s u l t s p ro v id e evidence t h a t 2 , 3 , 7 , 8 -
TCDD acts as a cocarcinogen. The failure of 2,3,7,8-TCDD to Induce tumors
when administered alone was not unexpected since only a single dose was
administered and the duration of the study was very short (36 weeks).
11.1.1.6.6.
Poland et al. Study (1982) -- Poland et al. (1982)
described studies which Indicate that genetic differences 1n mice affect the
tumor-promoting capacity of 2,3,7,8-TCDD 1n the mouse skin two-stage tumorl-
genesls model. Both 2,3,7,8-TCDD and TPA were compared for tumor-promoting
activity 1n DMBA-1n1t1ated HRS/J mice that were either heterozygous (hour/+)
or homozygous (hour/hour) for the recessive "hairless" trait. Promotion
with biweekly applications of 2 yg of TPA for 25 weeks resulted 1n
papilloma Incidences of 100 and 70% 1n (hour/+) and (hour/hour) mice,
1 1 -3 3
TABLE 11-18
Incidence of Tumors 1n Mice Treated With 3-MC and With 3-MC and 2,3,7,8-TCDDa
Experiment
Dose of TCDD
(vg/kg)
Route of Administration
Tumor Incidence TCDD and 3-MC 3-MC
p-Valueb
1
100 Intraperitoneal
10/43
1/34 p=0.01
2
100 Intraperitoneal
17/62
5/45 p=0.03
2
100 subcutaneous
46/82
5/42 p=3.0 X 10"7
2
1 subcutaneous
21/98
5/45 p=0.1
aSource: Kourl et al., 1978 bp-value calculated using the Fisher Exact Test (one-ta1led).
11-34
respectively. Promotion of DHBA-initiated (hour/*) mice with 2,3,7,8-TCDD
(50 ng/application for 8 weeks followed by 20 ng/appl1cat1on) did not result
in the formation of tumors, while promotion of (hour/hour) mice resulted in
both the same incidence and multiplicity of tumors as observed in TPA-pro-
moted mice. With either DMBA or methyl-N-nitrosoguanidine (MNNG)-initiated
(hour/hour) mice, the effective dose of 2,3,7,8-TCDD was ~100-fold less than
TPA on a molar basis. Histologic examination of the skin showed that TPA
produced both acute inflammation and hyperplasia in (hour/*) and (hour/hour)
mice, while 2,3,7,8-TCDD produced hyperplasia and hyperkeratosis only in
(hour/hour) mice with no inflammatory response. The lack of a 2,3,7,8-TCDD-
induced inflammatory response suggested to the authors that 2,3,7,8-TCDD
promoted skin papillomas in (hour/hour) mice by a mechanism different from
TPA.
11.1.1.6.7.
DiGiovanni et al. Study (1977, 1980) -- Investigations
have also been conducted on the effects of prior or simultaneous treatment
with 2,3,7,8-TCDD on the subsequent development of skin tumors by chemical
carcinogens. When 2,3,7,8-TCDD (0.1 yg) was administered simultaneously
with DMBA (200 nmol) to the backs of CD-I mice in a single initiation dose,
the skin 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-initiated tumors when CD-I mice were pretreated 3
days before DMBA initiation with 1 yg/mouse of 2,3,7,8-TCDD. The time of
treatment with 2,3,7,8-TCDD in relation to initiation was shown to be
critical in the antitumorigenic effects of 2,3,7,8-TCDD (Berry et al., 1979;
11-35
D1G1ovann1 et al., 1979a, 1980), as shown 1n Figure 11-1. Maximum tumor Inhibition of between 86 and 94% occurred when pretreatment was between 1 and 5 days before Initiation. If pretreatment was 10 days before 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 1n 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 Senear mice pretreated with 1 yg of 2,3,7,8-TCDD 3 days before Initia tion by DMBA.
D1G1ovann1 et al. (1980) Investigated the ant1tumor1gen1c 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 In a decrease 1n tumor yield as compared with acetone-pretreated animals; however] pre treatment with 2,3,7,8-TCDD 5 minutes before or 1 day after Initiations was Ineffective 1n changing the tumor yield. The maximum decrease 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-lTCDD to Inhibit tumor formation by BaP-d1ol-epox1de as compared with \the previously studied polyaromatic hydrocarbons (PAH). When 2,3,7,8-TCDD was applied 3 days or 5 minutes before, or 1 day after Initiation with BaP-diol epoxide, there was an 81.5 and 49% decrease 1n tumor yield. Examination of PAH metabolism 1n the skin of mice treated with 2,3,7,8-TCDD showed la
11-36
FIGURE 11-1
Time-Dependent Inhibition by 2,3,7,8-TCDD of Tumor Initiation
Summary of the time-dependent Inhibitory effect of 2,3,7,8-TCDD on tumor Initiation by DMBA (a), BaP (o), 3-MC (a) and BaP-d1ol-epox1de (o). Animals were Initiated with 10 nmol DMBA, 100 nmol BaP, 100 nmol 3-MC and 200 nmol BaP-d1ol-epox1de and promoted 1 week later with twice weekly appli cation of TPA.
11-37
21-fold Increase 1n aryl hydrocarbon hydroxylase (AHH) activity 72 hours
after treatment (D1G1ovann1 et al., 1980). The in vitro metabolism of DMBA
by dermal homogenates from 2,3,7,8-TCDD-treated mice Indicated both qualita
tive and quantitative changes 1n metabolism (Cohen et al., 1979; D1G1ovann1
et al., 1979a; Berry et al., 1979). The similarity 1n the time frame of AHH
Induction and the ant1tumor1gen1c effect of pretreatment with 2,3,7,8-TCDD
suggested that the ant1tumor1gen1c properties of 2,3,7,8-TCDD resulted from
2,3,7,8-TCDD Induced alteration 1n the metabolism of the Initiating chemi
cal. Although metabolic change was a possible mechanism for the Inhibition
of 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 ant1carc1nogen1c effect of 2,3,7,8-TCDD.
11.1.1.6.8.
Cockerham et al. 1980 Field Study on Beach Mice -- Cocker-
ham et al. (1980) performed a field study on beach mice, Peramyscus polle-
notus. that Inhabited an area which was heavily treated with the herbicide
2,4,5-T, of which 2,3,7,8-TCDD was a contaminant. Analysis of the soil 1n
the contaminated area revealed average 2,3,7,8-TCDD levels of 150 ppt at the
surface. Measured levels of 2,3,7,8-TCDD 1n the liver of beach mice from
the contaminated area were determined to be 1300 ppt 1n males and 960 ppt 1n
females. Detection of 2,3,7,8-TCDD 1n the Uver 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 histopathologic differences, Including neo
plastic lesions, observed 1n the Uver as compared with 9 male and 6 female
mice captured 1n a noncontamlnated area. The only observed difference 1n
11-38
the two groups of mice was a statistically significant (9556 confidence) increase in Uver-to-body weight ratios. The authors back-calculated from the 2,3,7,8-TCDD levels of the liver and estimated a daily 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 in laboratory studies to produce tumors. 11.1.2. Studies Using HxCDD.
11.1.2.1. NATIONAL TOXICOLOGY BIOASSAY PROGRAM (ORAL) STUDY IN RATS AND MICE (NTP, 1980d) -- Although 1,2,3,6,7,8-HxCDD and 1,2,3,7,8,9-HxCDD have not been tested individually for carcinogenicity, the NTP has performed a chronic bioassay in both Osborne-Mendel rats and B6C3F1 mice to determine the carcinogenicity 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-HxCDD congener, with a total HxCDD purity of 98%. The following impurities were detected in HxCDD used for this bioassay: PeCDD, 0.04%; TCDD, 0.09%0.03%; TriCDD, 0.004%; DCDD, 0.004% and Bromo
PeCDD, <0.004%. The s p e c i f i c isomers o f these i m p u r i t i e s were not i d e n t i
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 in 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 yg/kg/week, and the female mice received doses of 0.0, 2.5, 5.0 and 10 yg/kg/week. Treatment was con tinued for 104 weeks followed by a 3- to 4-week observation period. Complete necropsies, including extensive histologic examinations, were performed on animals at the time of natural death, when moribund or at the termination of the study.
11-39
A decrease 1n body weight gain was seen at the two higher exposure levels. A dose-related "toxic hepatitis" that was noninflammatory and consisted of degenerative changes 1n the Uver, eosinophilic foci of cellu lar alteration, mild fibrosis and bile duct hyperplasia was also observed. Cytomegaly and lipidosis were Included 1n these degenerative changes. The only neoplastic lesions that appeared to be treatment-related were neoplas tic nodules of the Uver 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 Uver tumors 1n male rats showed a positive dose-related trend by the Cochran-Armltage test; the Incidence 1n the high-dose male rat group was statistically different from the control group by the Fisher exact test (p=0.022) but the requirements by NTP for overall significance were not met based on the Bonferronl Inequality. The NTP thus concluded that the evidence for the carcinogenicity of HxCDD 1n male rats was Inconclusive. In female rats, the Cochran-Armltage test was significant at p<0.001, and the Uver tumor Incidence of the high-dose animals was significantly {p<0.001) different from that of the control group, as well as with the m1d-dose group (p=0.006).
Subsequent to the release of the NTP gavage study of HxCDD 1n rats and mice (NTP, 1980d), several pathologists reevaluated the microscopic slide material of the female rats. These reviews resulted from a report by Squire (1983) which stated that many of the entitles diagnosed as tumors by NTP were actually nonneoplastlc regenerative nodules; but his report concluded that the HxCDD bioassay still provided evidence of a weak hepatocarclnogenlc
11-40
TABLE 11-19
Liver Tumor Incidences 1n Male and Female Osborne-Mendel Rats Administered HxCDD for 104 Weeks3
Diagnoses
Untreated Control
Treatment Group
Vehicle Low Dose Control
M1d Dose
High Dose
Neoplastic nodule (NN)
Hepatocellular carcinoma (HC)
Combined NN + HC
2/75b
0/75 2/75
MALE
0/74
0/49
0/74 0/74
0/49 0/49
1/50
0/50 1/50
3/48
1/48 4/48 p=0.002c
Neoplastic nodule (NN)
Hepatocellular carcinoma (HC)
Combined NN + HC
1/73 0/74 1/73
FEMALE
5/75
10/50 p=0.026
0/75
0/50
5/75
10/50 p=0.026
12/50 p=0.006
0/50
12/50 p=0.006
30/50 p=6.94xl0-11
4/50 p=0.024
30/50 p=6.94x10 11
aSource: Adapted from NTP, 1980c D,I_nci*d,en__ce = -------N-o.---o-f--r-a-t-s--w-i-t-h--l-e-s-i-o-n-------
No. of rats examined microscopically cp-values calculated using the Fisher Exact Test.
11-41
effect 1n rats and mice. Drs. R. Schueler and B. Haberman also reported discrepancies 1n the diagnoses of Uver tumors from the NTP gavage study. Their findings were reported 1n an Internal U.S. EPA memorandum from CAG to J. BelUn (U.S. EPA 1983b) with an attached report prepared by Dr. R. Schueler, Research Pathology Associates, Inc. (Schueler, 1983). Finally, Dr. E. HcConnel of NTP requested that Dr. P. Hlldebrandt of Tracor-J1tco, Inc., review the microscopic slides of the HxCDD bioassay (gavage) 1n the female rat; h1s 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 findings, still confirmed that the HxCDD mixture administered by gavage produced an Increased Incidence of Uver tumors 1n treated female rats as compared with control animals, as well as an Increase In "toxic hepatitis."
In mice there were no gross signs of HxCDD toxicity; however, as observed 1n rats, there was a dose-related Incidence of "toxic hepatitis" consisting of degenerative Uver changes and/or necrosis associated with cellular Infiltration and mild fibrosis. The only neoplastic changes that were treatment-related were Increases 1n hepatocellular adenomas and carci nomas (Table 11-21). The adenomas were characterized as groups of cells with a uniform cell type that did not conform to the lobular architecture and which caused compression of the surrounding normal Uver, while the carcinomas contained cells with greater histologic deviations, disorganized growth and more cells In mitosis. A few Uver tumors 1n control and dosed groups metastasized 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 Uver tumor
11-42
TABLE 11-20
Liver Tumor Incidences 1n Female Osborne-Mendel Rats Administered HxCDD by Gavage for 104 Weeks3
Diagnoses
Untreated Control
Vehicle Control
Low Dose 1.25
uq/kq/week
M1d Dose 2.5
High Dose 5
Neoplastic nodule (NN)
Hepatocellular carcinoma (HC)
Combined NN + HC
1/7 3b
0/73 1/73
2/75
0/75 2/75
5/50
0/50 5/50
7/50 p=0.02c
0/50 7/50 p=0.02
16/50 p=6.0xl0 6
2/50 18/50 p=7.3xl0-7
aSource: Adapted from HUdebrandt, 1983
Incidence =
No. of rats with lesion
No. of rats examined microscopically
cp-values calculated using the Fisher Exact Test.
11-43
TABLE 11-21
Liver Tumor Incidences 1n Male and Female B6C3F1 Mice Administered HxCDD by Gavage for 104 Weeks3
Diagnoses
Untreated Control
Hepatocellular adenoma (HA)
15/75b
Hepatocellular carcinoma (HC)
Combined HA + HC
12/75 27/75
Treatment Group
Vehicle Control
Low Dose M1d Dose
MALE
i
7/73
5/50
9/49
8/73 15/73
9/50 14/50
5/49 14/49
High Dose
15/48 p=0.003c
9/48 24/48 p=7.33xl0~4
Hepatocellular adenoma (HA)
Hepatocellular carcinoma (HC)
Combined HA + HC
2/74
0/74 2/74
FEMALE
2/73
4/48
1/73 3/73
0/48 4/48
4/47
2/47 6/47
9/47 p=0.003
2/47
10/47 p=0.004
aSource: Adapted from NTP, 1980c
Incidence________No. of rats with lesion------No. of rats examined microscopically
cp-values calculated using the Fisher Exact Test.
11-44
^cAdence showed a significant dose-related trend by the Cochran-Armltage test, and the Incidence of tumors 1n the high-dose group was significantly higher than the Incidence 1n the control group by the Fisher exact test.
The obvious question was raised concerning the presence of tetrachlorod1benzo-p-d1ox1n as an Impurity (0.09%) 1n the test material, which may have contributed to the observed Uver tumor Incidence. The analysis presented 1n Table 11-22 shows that the calculated 95% upper-Umlt Uver cancer response due to 0.09% TCDD Impurity 1s so low as compared with the observed Uver cancer response due to HxCDD 1n this cancer bioassay study, 1t 1s reasonable to conclude that the Impurity 1n the test material did not
contribute significantly to the observed carcinogenic response for HxCDD. McGaughy and R1sp1n (1985) made the following comments regarding the
three documents listed below, which evaluated Issues that have been raised with respect to the NCI/NTP HxCDD carcinogenicity bioassay on rats and mice:
1. The responses outlined by the Office of Health and Environ mental Assessment (OHEA) were prepared f o r p r e s e n t a t io n to EPA's Science Advisory Board on November 28, 1984.
2. The document entitled "Response to Comments" was prepared by Agency staff and a consultant pathologist.
3. Amemorandum from Dr. John Doull, member of EPA's Science Advisory Board, concerning the HxCDD audit by Dr. G. Schoenlg. The above documents respond to questions that were raised
concerning many aspects of the bioassay study. These questions relate, for example, to allegations of problems 1n :
test procedures, such as problems 1n preparation of the test material; flaws 1n methods of administration; flaws 1n recordkeeping procedures and practices.
pathology practices, such as non-un1form and substandard tissue harvesting practices; non-un1form histologic pro cedures; bias 1n histology review; and deficiencies 1n correlation between gross and microscopic observations.
11-45
TABLE 11-22
Liver Tumor Response for HxCDD (Observed) and TCDD Contaminant (Calculated)
Animal
HxCDD Dose (vg/kg/week)
Liver Cancer
Response Observed
0.09% TCDD
Contaminant Dose3
(vg/kg/week)
Liver Cancer
Response Calculated 95%
Upper Limit
Rat (OH) Hale
Female
5
4/48b
0.0045
TCDD has shown
no effect 1n
NCI study
5
18/50c
0.0045
0.02/50d
House (B6C3F1) Hale
Female
5 10
24/48e 10/47f
0.0045 0.009
0.20/48d 0.22/47e
aIt 1s assumed that all of the contaminant 1s 2,3,7,8-TCDD. bNTP reviewed cRe-evaluat1on by Hlldebrandt (see Table 11-35) dBased on response 1n NCI 2,3,7,8-TCDD study; see Table B-10. eBased on response 1n NCI 2,3,7,8-TCDD study; see Table B-l 1. ^Based on response 1n NCI 2,3,7,8-TCDD study; see Table B-l 2.
11-46
alleg ed bias 1n the above practices with respect lo treated and control animals.
pathology Interpretation: disagreement 1n conclusions reached by different pathologists.
From our review of these documents we conclude that there~were Indeed some procedural flaws during the 1n-11fe portion of the study, and there were minor recordkeeping problems. The management of a two-year rodent study Is a very complex undertaking. It "1s therefore not surprising that the procedural and recordkeeping deficiencies highlighted by the two audits occurred. They do not Invalidate the study.
The detailed review by Agency staff and Dynamac Corporation of Dr. Schoenlg's findings concerning room bias did not substantiate h1s allegations. Similarly, review of Dr. Schoenlg's criticism of the histologic practices did not reveal meaningful deficiencies 1n tissue harvesting, preparation of microscopic slides, and histo logic diagnoses.
Differences 1n Interpretation among pathologists have pre viously been addressed by the Agency (see the OHEA document attached hereto). The slight differences 1n Interpretation among the different pathologists do not alter the conclusion as to the carcinogenic potential of HxCDD.
We conclude that the HxCDD bioassay 1s valid, and that 1t can appropriately be used for the assessment of the carcinogenic poten tial of HxCDD.
Under the test conditions of this bioassay, the 1:2 mixture of
1.2.3.7.8- and 1,2,3,7,8,9-HxCDD was carcinogenic, as Indicated by a statis
tically significant Increased incidence In tumors of the Uver 1n female
rats and In both male and female mice, and by a borderline Uver tumor
response 1n male rats.
11.1.2.2. NATIONAL TOXICOLOGY BIOASSAY PROGRAM SKIN-PAINTING STUDY IN
MICE (NTP, 1980b,c) -- Both 2,3,7,8-TCDD (NTP, 1980b) and a 2:1 mixture of
1.2.3.6.7.8- and 1 ,2,3,7,8,9-HxCDD (NTP, 1980c) have been tested 1n mice for
tumorlgenlc potential by dermal application. These studies were conducted
under the NTP and the description of the chemicals used was the same as
previously presented 1n the discussion of NTP (1980a,d). There was no
11-47
Information found 1n the literature searched on the tumorlgenlc effect of 1,2,3,7,8-PeCDD following dermal exposure. The tumorlgenlc response after chronic dermal exposure to HxCDD was presented 1n Table 11-23.
In both NTP bioassays (1980b,c), groups of 30 male and 30 female SwlssWebster mice were treated with 100 yi of a solution of the test compound 1n acetone 3 t1mes/week for 104 weeks. Groups of 45 animals were employed as vehicle controls, and 2 groups of 15 animals were used as untreated controls. The concentration of 2,3,7,8-TCDD used resulted 1n a dose of 0.01 vg/appl1cat1on 1n male mice and 0.005 yg/appUcatlon 1n female mice; the concentration of HxCDD used resulted 1n a dos-e of 0.005 yg/appHcation for the Initial 16 weeks of the study, followed by a subsequent Increase to 0.01 vg/appl1cat1on for the remainder of the study. Subchronic toxicity studies used to define the dose levels for the chronic bioassay Indicated that all the doses used resulted 1n some Uver damage but no Increase 1n mortality. In the chronic study, animals were killed when moribund at the termination of the study and examined for gross tumors. Microscopic exami nations were also made of all major organs.
In mice exposed to 2,3,7,8-TCDD (NTP, 1980b), there was no treatmentrelated difference 1n body weight of either sex between exposed animals and control groups; however, male mice treated with 2,3,7,8-TCDD had a signifi cant shortening of lifespan. Nontumorlgenic hepatic lesions were observed 1n treated female mice; no mention was made of these lesions occurring 1n male mice. The only tumors that were treatment-related were Integumentary system fibrosarcomas, with tumors developing on or near the site of applica tion. The Incidence of these tumors 1n male mice was 3/42 and 6/28, and 1n female mice the Incidences were 2/41 and 8/27, respectively, for the vehicle control groups and the treated animals. Only the tumor Incidence 1n female
11-48
lABLt II-23 Carcinogenicity Bioassays of 2,3,7,8-TCDD and HxCDD by Dermal Application to M1cea
11-49
Compound
Sex
2,3,7,8-TCDD
M
M
H
2,3,7,8-TCDD
F
F
F
HxCDD
H
H
Dose*1
0.01 yg/appllcatlon
0.0 yg/appl1cat1on (vehicle control) 0.0 yg/appl1cat1on (untreated control) 0.005 yg/appllcatlon
0.0 yg/app11cation (vehicle control) 0.0 yg/applicat1on (untreated control) 0.01 yg/appl1cat1onc
0.0 yg/appllcatlon (vehicle control)
Duration of
Exposure 104 weeks 104 weeks NA 104 weeks 104 weeks NA 104 weeks
104 weeks
Target Organ
Integumentary system Integumentary system Integumentary system Integumentary system Integumentary system Integumentary system lung
lung
Tumor Type
fibrosarcoma
fibrosarcoma
fibrosarcoma
fibrosarcoma
fibrosarcoma
fibrosarcoma
alveolar/ broncholar carcinoma alveolar/ broncholar carcinoma
Tumor Incidence
6/28 3/42 0/28 7/28 2/41 1/27 5/30
1/41
TABLE 11-23 (cont.)
0 9 " LL
Compound
Sex
Doseb
Duration of
Exposure
Target Organ
HxCDD (cont.) M
0.0 yg/appl1cat1on (untreated control)
NA
lung
HxCDD
F
0.01 yg/appl1cat1onc
104 weeks
skin
F 0.0 yg/appllcatlon (vehicle control)
104 weeks
skin
F 0.0 yg/appllcatlon
NA
(untreated control)
skin
aSource: NTP, 1980b,c ^The compound was applied 3 times/week 1n 100 yil of acetone. cFor the Initial 16 weeks of the study, the dose was 0.005 yg/appl1cation. NA = Not applicable
Tumor Type
alveolar/ bronchlolar carcinoma fibrosarcoma fibrosarcoma
fibrosarcoma
Tumor Incidence
4/28
4/27 2/41
0/30
mice was statistically (p=0.007) greater than control values; however, life table analyses indicated that the time to tumor was shorter 1n both male and female treated mice. The Incidence of tumors 1n untreated and vehicle control groups was similar.
In the bioassay of HxCDD (NTP, 1980c), no gross or nonneoplastlc histo logic effects associated with treatment were observed. Although there was a slight Increase 1n the Incidence of skin fibrosarcomas 1n female mice, this Increase was significant 1n comparison with the vehicle control group, but not significantly different from the untreated control group. The opposite occurred with the Incidence of alveolar/bronchlolar carcinomas of the lung In male mice, which was significantly elevated 1n comparison with untreated but not vehicle-treated controls. It was concluded that although dermal exposure to 2,3,7,8-TCDD resulted 1n a carcinogenic response 1n both male and female Swlss-Webster mice, dermal exposure to a mixture of 1,2,3,7,8TCDD and 1,2,3,7,8,9-HxCDD did not result 1n a carcinogenic response under
th e c o n d itio n s o f t h i s b io a s s a y . A summary of the c a r c i n o g e n i c i t y bioassays
1s given 1n Table 11-24. 11.1.3. Summary of Animal Carcinogenicity. In a preliminary study by Van Miller (1977a,b), 2,3,7,8-TCDD was tested for carcinogenicity following oral administration to rats. At the five highest dietary levels, 0.005, 0.05, 0.5, 1.0 and 5.0 ppb, which allowed long-term survival of the animals, an Increased Incidence of total tumors was observed. In animals at an exposure level of 0.001 ppb and 1n the control animals there were no tumors. This study, however, provides only suggestive evidence of a carcinogenic response since no Increase 1n s1te-spec1f1c tumors was detected and the group sizes, ~10 anlmals/group, were too small for an assessment of a treatment-related response. In a second, more extensive study by Koclba et al. (1978a) a positive carcinogenic response was detected. In this study the estimated
11-51
TABLE 11-24
Carcinogenicity Bloassays of PCDD Administration by the Oral and Dermal Route
11-52
Exposure Route/
Compound
Specles/Straln Sex Dose or Exposure
Gavage/
rats/
H 0.0 yg/kg/week
2,3,7,8-TCDD Osborne-Hendel
0.1 yg/kg/week
0.05 yg/kg/week
0.5 yg/kg/week
Gavage/
rats/
F 0.0 yg/kg/week
2,3,7,8-TCDD Osborne-Hendel
0.1 yg/kg/week
0.05 yg/kg/week
0.5 yg/kg/week
Gavage/ 2,3,7,8-TCDD
mtce/B6C3F1
H 0.0 yg/kg/week 0.1 yg/kg/week
Duration of
Treatment
Duration of Study
Vehicle
104 weeks 104 weeks 104 weeks 104 weeks 104 weeks 104 weeks 104 weeks 104 weeks 104 weeks 104 weeks
105 weeks 107 weeks 107 weeks 105 weeks 105 weeks 107 weeks 107 weeks 107 weeks 105 weeks 107 weeks
corn oilacetone (9:1)
corn ollacetone (9:1)
corn oilacetone (9:1)
corn oliacetone (9:1)
corn ollacetone (9:1)
corn ollacetone (9:1)
corn oliacetone (9:1)
corn ollacetone (9:1)
corn oilacetone (9:1)
corn ollacetone (9:1)
Tumor Type
folltcular-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
neoplastic nodule or hepatocellular carcinoma of the liver
neoplastic nodule or hepatocellular carcinoma of the liver
neoplastic nodule or hepatocellular carcinoma of the liver
neoplastic nodule or hepatocellular carcinoma of the liver
hepatocellular carcinoma
hepatocellular carcinoma
Tumor Incidence
Reference
1/69
NTP, 1980a
5/48
8/50
11/50
5/75
1/49
3/50
14/49
8/73
NTP, 1980a
9/49
TABLE 11-24 (cont.)
11-53
Exposure Route/
Compound
Spectes/Straln Sex Dose or Exposure
Gavage/ 2,3,7,8-TCDO (cont.)
m1ce/B6C3Fl
0.05 yg/kg/week 0.5 yg/kg/week
Gavage/ 2,3.7,8-TCOD
m1ce/B6C3Fl
F 0.0 yg/kg/week 0.04 yg/kg/week
0.2 yg/kg/week
2.0 yg/kg/week
Oral/
rat/
h 0.0 ppb
2,3,7,8-TCDD Sprague-Dawley
0.001 ppb
0.005 ppb
0.05 ppb
0.5 ppb
1.0 ppb
5.0 ppb
Duration of
Treatment
Duration of Study
Vehicle
104 weeks 104 weeks 104 weeks 104 weeks 104 weeks 104 weeks 78 weeks
107 weeks 107 weeks 105 weeks 107 weeks 107 weeks 107 weeks 95 weeks
corn oilacetone (9:1)
corn oilacetone (9:1)
corn oilacetone (9:1)
corn oilacetone (9:1)
corn oilacetone (9:1)
corn oilacetone (9:1)
In diet
78 weeks 78 weeks 78 weeks 78 weeks 78 weeks 78 weeks
95 weeks 95 weeks 95 weeks 95 weeks 95 weeks 95 weeks
In diet In diet In diet In diet In diet In diet
Tumor Type hepatocellular carcinoma
Tumor Incidence
Reference
8/49
NTP, 1980a
hepatocellular carcinoma
17/50
hepatocellular carcinoma, follicular-cell adenomas of the thyroid
hepatocellular carcinoma, follicular-cell adenomas of the thyroid
hepatocellular carcinoma, follicular-cell adenomas of the thyroid
hepatocellular carcinoma, follicular-cell adenomas of the thyroid
all tumors
all tumors
all tumors
all tumors
all tumors
all tumors
all tumors
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
NTP, 1980a
Van Hiller et al.. 1977a
TABLE 11-24 (coni.)
frS-LL
Exposure Route/
Compound
Specles/Straln Sex Dose or Exposure
Duration of
Treatment
Duration of Study
Vehicle
Oral/
rat/
H 0.0 yg/kg/day
2,3,7,8-TCDO Sprague-Dauley
105 weeks 105 weeks In diet
0.001 yg/kg/day
105 weeks 105 weeks In diet
Oral/
rat/
H 0.01 yg/kg/day
2,3,7,8-TCDD Sprague-Dawley
105 weeks 105 weeks In diet
0.1 yg/kg/day
105 weeks 105 weeks In diet
Oral/ 2,3,7,8-TC00
rat/ SpragueDauley
f 0.0 yg/kg/day
105 weeks 105 weeks In diet
' 0.001 yg/kg/day
105 weeks 105 weeks 1n diet
0.01 yg/kg/day
105 weeks 105 weeks In diet
Tumor Type
Tumor Incidence
Reference
squamous cell carcinoma of the hard palate, squamous cell carcinoma of the tongue, adenoma of the adrenal cortex
squamous cell carcinoma of the hard palate, squamous cell carcinoma of the tongue. adenoma of the adrenal cortex
squamous cell carcinoma of the hard palate, squamous cell carcinoma of the tongue, adenoma of the adrenal cortex
squamous cell carcinoma of the hard palate, squamous cell carcinoma of the tongue, adenoma of the adrenal cortex
hepatocellular carcinoma. squamous cell carcinoma of the tongue, squamous cell carcinoma of the lung
hepatocellular carcinoma. squamous cell carcinoma of the tongue, squamous cell carcinoma of the lung
hepatocellular carcinoma. squamous cell carcinoma of the tongue. squamous cell carcinoma of the lung
0/85 0/85 0/85
0/50 1/50 0/50
0/50 1/50 2/50
4/50 3/50 5/50
0/86 0/86 0/86 0/50 0/50 0/50 2/50 1/50 0/50
Koclba et al., 1978a
Koclba et al., 1978a
Koclba et al., 1978a
TABLE 11-24 (cont.)
11-55
Exposure Route/
Compound
Specles/Straln Sex Dose or Exposure
Duration of
Treatment
Duration oF Study
Vehicle
Tumor Type
Tumor Incidence
Reference
Oral/ 2,3,7.8-TCDD
rat/ Sprague-Dawley
F 0.1 pg/kg/day
105 weeks
105 weeks
In diet
hepatocellular carcinoma, squamous cell carcinoma of the tongue, squamous cell carcinoma of the lung
Gavage/ 2,3.7,8-TCOO
mice/Swiss/ H/Rlop
H 0.0 pg/kg/week
365 days
588 days
sunflower oil
liver tumors
0.007 pg/kg/week
365 days
649 days
sunflower oil
liver tumors
0.7 pg/kg/week
365 days
633 days
sunflower oil
liver tumors
7.0 pg/kg/week
365 days
424 days
sunflower oil
liver tumors
Oral/ 2,3,7,8-TCOO
mice/ Peramyscus pollenotus
H&F 0.0012 pg/kg/day
NA
NA
contaml-
liver
nated soli
0.0 pg/kg/day
NA
NA
contami
liver
nated soil
Gavage/HxCDD
rats/ Osbome-Hendel
H 0.0 pg/kg/week
104 weeks 105 weeks corn oil-
(vehicle control)
acetone
(9:1)
liver neoplastic nodules or hepatocellular carcinoma
Gavage/HxCDO rats/
H 1.25 pg/kg/week
Osbome-Hendel
104 weeks
106 weeks
corn oilacetone (9:1)
liver neoplastic nodules or hepatocellular carcinoma
2.5 pg/kg/week
104 weeks
107 weeks
corn oilacetone
liver neoplastic nodules or hepatocellular carcinoma
5.0 pg/kg/week
104 weeks
107 weeks
corn oilacetone
liver neoplastic nodules or hepatocellular carcinoma
11/49 4/49 7/49 7/38
13/44 21/44 13/43
0/15
0/15 0/74
0/49
1/50
4/48
Koctba et al., 1978/
Toth et al., 1979
Cockerham et al.. 1980
NTP, 1980d NTP, 1980d
TABLE 11-24 (cont.)
9S-LL
Exposure Route/
Compound
Specles/Straln Sex Dose or Exposure
Gavage/HxCDD rats/
F 0.0 yg/kg/week
Osborne-Hendel
1.25 yg/kg/week
2.5 yg/kg/week
5.0 yg/kg/week
Gavage/HxCDD mlce/B6C3Fl
H 0.0 yg/kg/week
1.25 yg/kg/week
2.5 yg/kg/week
5.0 yg/kg/week
Gavage/HxCDD mlce/B6C3Fl
F 0.0 yg/kg/week
2.5 yg/kg/week
5.0 yg/kg/week
10.0 yg/kg/week
NA = Not available
Duration of
Treatment
Duration of Study
Vehlcle
104 weeks 104 weeks 104 weeks 104 weeks 104 weeks 104 weeks 104 weeks 104 weeks 104 weeks 104 weeks 104 weeks 104 weeks
105 weeks corn oliacetone
--- (9:1)
107 weeks
corn oliacetone
(9:1)
107 weeks
corn oliacetone (9:1)
107 weeks
corn oliacetone
(9:1)
105 weeks
corn oliacetone (9:1)
10B weeks
corn oliacetone (9:1)
107 weeks
corn oliacetone (9:1)
108 weeks
corn oliacetone (9:1)
106 weeks
corn oliacetone (9:1)
108 weeks
corn oliacetone (9:1)
10B weeks corn oliacetone
' (9:1)
107 weeks
corn oliacetone (9:1)
Tumor Type
liver neoplastic nodules or hepatocellular carcinoma
liver neoplastic nodules or hepatocellular carcinoma
liver neoplastic nodules or hepatocellular carcinoma
liver neoplastic 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 Incidence
Reference
5/75
HIP, 1980d
10/50
12/50
30/50
15/73
NIP. 1980d
14/50
14/49
24/48
3/73
NTP, 19B0d
4/48
6/47
10/47
Intake o f 2 ,3 ,7 ,8 -T C D D fro m th e d i e t w as 0 .0 , 0 .0 0 1 , 0 .0 1 an d 0 .1 y g / k g /
day.
In th e h ig h -d o s e g ro u p , b o th m ale and fe m a le a n im a ls had s i g n i f i c a n t
I n c r e a s e s In s l t e - s p e c l f 1 c tu m o rs.
The t a r g e t o r g a n s an d tum or t y p e s 1n
m ale a n im a ls w ere squam ous c e l l ca rc in o m a s o f th e to n g u e , squam ous c e l l
carcin o m as o f th e hard p a la te and n a sa l tu r b in a te s , and adenom as o f th e
a d r e n a l c o r t e x ; In fe m a le a n im a ls th e t a r g e t o r g a n s and tum or ty p e s w ere
h e p a t o c e llu la r c a rc in o m a s , squam ous c e l l carcin o m as o f th e to n gu e and n a sa l
t u r b in a t e s , and squam ous c e l l carcin o m as o f th e lu n g .
The d ata dem on strate
th a t d ie t a r y e x p o su re to 2 , 3 ,7,8 -T C D D a t le v e ls th a t p ro d u ce a d a lly d o se o f
0 .1 y g / k g r e s u l t s 1n I n c r e a s e d tum or I n c id e n c e s 1n b o th m ale and fe m a le
ra ts.
Under the National Toxicology Program, 2,3,7,8-TCDD was tested for carcinogenicity In 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 follicular cell adenomas or carcinomas of the thyroid In male animals, and neoplastic nodules or hepatocellular carcinomas of the liver 1n female animals. The Incidence of these tumors was significantly greater than control In the high-dose groups, and the Incidence of both tumors showed a positive dose-related trend. Under the conditions of this assay, 2,3,7,8TCDD was concluded to be carcinogenic 1n both male and female rats.
Further studies 1n mice exposed by gavage have provided support for the carcinogenicity of 2,3,7,8-TCDD. Toth et al. (1979) exposed male mice to 2,3,7,8-TCDD at doses of 0.0, 0.007, 0.7 and 7.0 yg/kg/week 1n a study to determine whether 2,4,5-TCPE, Its contaminant 2,3,7,8-TCDD or both were carcinogens. At the 0.7 yg/kg/week level there was a significantly Increased Incidence of Uver tumors. Liver tumors were not significantly Increased 1n the high-dose group; however, early mortality 1n this group may
11-57
have precluded observing late-developing tumors. Similar Increased Inci dences of Uver tumors were observed 1n the NTP (1980a) study 1n the high dose male mice exposed to 0.5 ug/kg/week and In the high-dose female mice exposed to 2 vQ/kg/week of 2,3,7,8-TCDD by gavage. Female mice also had an Increased Incidence of follicular-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 tumor1gen1c1ty model has also been used to test the carcinogenic potential of 2,3,7,8-TCDD. Following long-term dermal application 3 tlmes/week of 2,3,7,8-TCDD at levels of 0.01 and 0.005 yg/app11cation to male and female mice, respectively, there was an Increased Incidence of skin tumors only In 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 promotiononly 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 In mice heterozygous for this recessive trait. Pitot et al. (1980) also reported that 2,3,7,8-TCDD was a promoter for DEN-1n1t1ated hepatocarclnogenesls 1n rats following parenteral administration of the compounds. On mouse skin, 2,3,7,8-TCDD was a complete carcinogen and possibly a tumor Initiator, while no tumor-promoting activity could be attributed to 2,3,7,8TCDD 1n the assays. In rat Uver Initiated with DEN, 2,3,7,8-TCDD was a tumor promoter.
11-58
In studies of the Interaction of 2,3,7,8-TCDD with other chemical carcinogens, Kourl et al. (1978) reported that 2,3,7,8-TCDD was a cocar cinogen with 3-MC when administered by subcutaneous Injection. In the mouse skin bioassay, Initiation with simultaneous administration of 2,3,7,8-TCDD and DMBA, however, did not affect tumor yield (DIGIovannl et al., 1977). Similarly, no effect was observed when 2,3,7,8-TCDD was administered either Immediately before (5 minutes) or 1 day after DMBA Initiation (Berry et al., 1979; DIGIovannl et al., 1977, 1979b; Cohen et al., 1979). When treatment with 2,3,7,8-TCDD occurred 1-10 days before DMBA Initiation, 2,3,7,8-TCDD demonstrated a potent ant1carc1nogen1c action. Although 1-5 days prior exposure to 2,3,7,8-TCDD Inhibited tumor Initiation by BaP, 3-MC and BaPd1ol-epox1de, the tumor Initiating ability of the latter compound was also Inhibited when 2,3,7,8-TCDD exposure occurred either 5 minutes before or 1 day after Initiation (DIGIovannl et al., 1980). The Increased AHH activity resulting from 2,3,7,8-TCDD exposure may account for the ant1carc1nogen1c activity by altering the metabolism of the Initiating compound; however, DIGIovannl 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 ant1carc1nogen1c activity of 2,3,7,8-TCDD.
HxCDD has also been tested for carcinogenicity In rats and mice treated by gavage and by dermal application to mice (NTP, 1980c,d). In these studies, a 1:2 mixture of 1,2,3,6,7,8- and 1,2,3,7,8,9-HxCDD was tested. In the oral study, animals received HxCDD at doses of 0.0, 1.25, 2.5 or 5.0 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 Uver occurred at a significantly greater Incidence than controls. In male rats and male and female mice, the Uver tumor Incidence was significantly
11-59
Increased over control values only In the high-dose groups, while 1n female rats the Incidence was significantly greater at both the medium- and high dose levels. In the study of HxCDD carcinogenicity 1n mouse skin conducted by NTP (1980c), there were no treatment-related tumors 1n either the car cinogenicity bioassay or the tumor promotion assay using DMBA as an Initi ator. It was concluded that this mixture of HxCDD was carcinogenic to rats and mice following administration by gavage; however, there was no tumorigenie activity when HxCDD was applied to mouse skin.
No chronic animal bioassays were found 1n the literature searched on the carcinogenicity of 1,2,3,7,8-PeCDD. 11.2. CASE REPORTS AND EPIDEMIOLOGICAL STUDIES* 11.2.1. Case Reports. Observations of an unusual occurrence of relatively rare soft-tissue sarcomas were first made by 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 acids 10-20 years earlier. The tumors were 2 leiomyosarcomas, 1 llposarcoma, 1 rhabdo myosarcoma, 1 myxofibrosarcoma and 2 additional sarcomas of which the hlstopathology 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 epidemiological studies be done to determine 1f exposure to phenoxy acids and the Impurities they contain are related to the occurrence of soft-tissue sarcomas.
Portions of this section were taken from U.S. EPA (1980c).
11-60
Zack and Susklnd (1 9 80 ) re p o rte d a soft-tissue sarcoma death 1n a cohort study of workers exposed to 2,3,7,8-TCDD 1n a trlchlorophenol process accident 1n N1tro, West Virginia. This tumor, a fibrous histiocytoma, was noted by the author as a rare event. This study, referred to as the N1tro study, 1s discussed later.
Cook et al. (1980) 1n 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.
Ott et al. (1980) 1n a cohort mortality study of 204 employees exposed to 2,4,5-T during Its manufacture from 1950 to 1971, found no soft-tissue sarcomas among 11 deaths that had occurred by 1976. One of these 11 deaths was due to a malignant neoplasm.
In a review of the studies of Zack and Susklnd (1980), Cook (1980), an unpublished study by Zack (1n which a Uposarcoma was found), a study by Ott et al. (1980) and Honchar and Halperln (1981) noted 3 (2.9%) soft-tissue sarcomas 1n a total of 105 deaths. Among U.S. males aged 20-84, 0.07% of the deaths were reported as soft tissue sarcomas (ICO 171, 8th Revision, 1975)* Indicating an unusual excess of such tumors. This may be an under estimate because of the possibilities that some soft-tissue sarcomas may have been coded to categories other than ICO 171. Individually, none of the reported case studies reported a significant excess of soft-tissue sarcomas.
Department of Health, Education, and Welfare. U.S. Public Health Service. National Center for Health Statistics of the United States, 1974. Vol. II. Mortality, Part A.
11-61
Cook (1981a) found an additional malignant fibrous histiocytoma after a later review of the medical records from h1s 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 histiocytoma. A third person diagnosed as having a fibrosarcoma worked 1n a trlchlorophenol (TCP) process area contaminated with 2,3,7,8TCDD. This Individual exhibited facial dermatitis but there was no diagnosis of chloracne. The fourth case (diagnosed as a llposarcoma) was an Individual who had been employed earlier 1n a plant producing 2,4,5-T. Cook (1980) noted that although chloracne was not reported, 1t could not be discounted. He also noted that all four were cigarette smokers and suggested that smokers with chloracne caused by 2,3,7,8-TCDD exposure may be subject to an Increased risk of fibrous soft-tissue sarcomas, although no prior reports have shown soft tissue sarcomas associated with cigarette smoking.
Hardell and Eriksson (1981) discounted this hypothesis by citing that only one of 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 h1s cases and controls.
Hoses and Sellkoff (1981) reported a fifth soft-tissue sarcoma 1n a worker employed at the Monsanto Chemical Company at a time when trlchloro phenol and 2,4,5-T were being produced. The worker died of a retroperito neal neurogenic sarcoma (malignant schwanoma) In 1980 at the age of 58. The employee, before h1s death, In a detailed occupational history said that he believed he was exposed to these chemicals while he was a truck driver, hauler and maintenance worker, but that he did not work 1n the production of either chemical. He was a nonsmoker and had no history of chloracne.
11-62
Johnson et al. (T98T) treated a father and son with soft-tissue sarcomas (the 33-year-old son was diagnosed as having a fIbrosarcomatous mesothe lioma, while the 53-year-old father had a Uposarcoma). 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 h1s disease. The son supposedly had a shorter latency, accord ing to the author. In neither case was the follow-up time given.
Sarma and Jacops (1981) reported three cases of thoracic soft-tissue sarcoma In Individuals who were presumably exposed to Agent Orange while serving 1n Vietnam. The diagnoses were fibrous histiocytoma, mediastinal fibrosarcoma, and a pleural/dlaphragmatlc leiomyosarcoma. 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 pentachlorophenol manufacturing plant in Wales. Homologues of 2,3,7,8-TCDD occurred as contaminants at up to 300 ppm at Intermediate manufacturing stages and 5 ppm 1n the final products. Mild, moderate and severe cases of chloracne were seen In many employees, Including the two men who subsequently developed lymphomas. Both men worked 1n processes where exposure to other chemicals occurred, Including exposure to aromatic hydrocarbons. The authors reported that only 0.28 tumors of this type could be expected to occur In 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 In Sweden with a recent diagnosis of non-Hodgkin's lymphoma (NHL), 5 had cutaneous lesions as the only clinically detectable manifestation of
11-63
NHL. Four of the five were reported to have repeatedly sprayed large areas with phenoxy acid herbicides. In the remaining 118 NHL patients, only seven had a similar occupational exposure to phenoxy acids. The authors reported this to be significant at p<0.001. Olsson and Brandt suggested that a relationship exists between cutaneous presentation of NHL and occupational exposure to phenoxy acids, and believed their observations were similar to those of Bishop and Jones (1981).
The total number of workers with these Illnesses who were exposed to phenoxy acids and/or chlorophenols 1s small, but considering the rarity of this cancer, 1t Is unusual that so many cases of soft-tissue sarcomas have occurred. A Lancet editorial (Anonymous, 1982) calls this phenomenon "disturbing." 11.2.2. Epidemiologic Studies.
11.2.2.1. SOFT-TISSUE SARCOMAS -- Soft-tissue sarcomas (STS) consti tute a collection of heterologous lesions that Include both malignant and nonmallgnant tumors. Not all of them have their origin In primordial mesen chymal cells. Some exceptions are tumors of peripheral nerves, and neuro ectodermal tumors that are classified as STS but are derived from nonmesen chymal cells. Classification, grading and staging of STSs Is difficult because of the capacity of such cells to differentiate Into many different tissues. Fairly precise hlstogenetlc classification of such tumors 1s accomplished through consideration of growth patterns and cell morphology and evaluation of 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.
11-64
STSs can be induced in any of these tissue types (Hajdu, 1983). The classi fication of STSs for cause of death coding 1n the ninth and latest revision of the International Classification of Diseases (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 STSs of the bone are coded elsewhere. In some Instances, 1f site 1s mentioned, 1t 1s coded to the site [1.e., leio myosarcoma of the stomach (ICD 151.9), neurofibroma of the chest wall (215.4)].
Questions have been raised concerning the appropriateness of lumping together malignant tumors of different sites and tumor types 1n order to derive risk estimates. It may not be scientifically appropriate to do so because an elevated risk cannot readily be ascribed to a particular site or type as 1s usual with most carcinogenic chemicals and substances. Unfortu nately, with respect to STSs, tallies of deaths from STSs of particular sites and types are not maintained separately by the vital statistics offices because of their rarity; therefore, 1t 1s Impossible to derive risk estimates for particular types at given sites. Altogether, -2000 deaths/ year can be attributed to STSs 1n the United States, most of which are coded to ICD category 171 for purposes of developing Incidence and mortality rates for this composite cause. Within ICD 171, Individual types that may be correlated with exposure cannot be Identified.
A separate problem that potentially could arise from assigning STSs to multiple ICD codes 1s that Incidence and death rates from STSs may be under estimated. Furthermore, risk estimates derived from dividing observed cases (or deaths) by expected cases (or deaths) could be biased upward. This could happen when observed STSs classified to ICD codes other than ICD 171
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are lumped together 1n ICD 171 while expected STSs are based upon STSs classifiable to ICD 171 only. Thus, action of this sort, especially with respect to cohort studies of Individuals exposed to d1ox1n-conta1n1ng herbi cides and/or chlorophenols, could lead to risk estimates that may be biased upward by the Inclusion of STSs 1n the observed category for risk estimation that should be coded to categories other than 171.
Prompted by clinical observations over a 7-year period of malignant sarcomas 1n seven men with previous occupational exposure to phenoxyacetlc acid herbicides (Harden, 1977), researchers at the Department of Oncology, University Hospital, Umea, Sweden, Initiated case-control epidemiologic studies to test the hypothesis of an etlologlc association (Harden and Sandstrom, 1979). Cases were defined as male patients with sarcomas of soft connective tissue, such as smooth muscle (leiomyosarcoma) and fat (Uposarcoma). The distribution of tumor types 1n the two studies 1s shown 1n Table 11-25. Sarcomas of tissues, such as bone and cartilage, were excluded as cases. According to the authors, these tumors may have a different etiology and there occurred a different age-d1str1but1on 1n patients with these tumors as compared with that of STS (Harden, 1983).
Two case-control studies were conducted: the first 1n northern Sweden (referred to below as Study A) and the second 1n the southern part of the country (Study B). The exposures to the substances of primary Interest are shown 1n Table 11-26. In the north (Study A), occupational exposure to phenoxyacetlc acids took place 1n both forestry and agricultural work. In the south (Study B), these exposures were predominantly agricultural. The phenoxyacetlc acids to which exposure occurred consisted predominantly of 2,4,5-T and 2,4-D 1n both studies. Exposure to 2,4,5-T 1n the absence of 2,4-D was rarely reported 1n either study. Exposure to chlorophenols, which
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TABLE 11-25
Distribution of Tumor Types 1n Two Case-Controls Studies of Soft-Tissue Sarcoma
Diagnosis
Tissue of Origin
Percent of Cases
Study Aa (n=52)
Study Bb (n=110)
Leiomyosarcoma Fibrous histiocytoma
Llposarcoma Neurogenic 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
aUnpubl1shed Information supplied by Hardell to EPA (Harden and Sandstrom, 1979)
bEr1ksson et al., 1979, 1981
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TABLE 11-26 Exposure Frequencies 1n Two Case-Control Studies of Soft-Tissue Sarcoma
Substance(s)
Percent Exposed
Studv A
Study B
Cases (n=52)
Controls (n=206)
Cases (n=110)
Controls (n=219)
Phenoxyacetlc acids only Chlorophenols only Both
Total
23.1 11.5
1.9
36.5
6.3 12.7 2.3
2.4 10.0 3.6
0.5 0
0
9.2 22.7 5.9
Sources: Study A, Hardell and Sandstrom, 1979; Study B, Eriksson et al.t 1979, 1981
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c o n t a i n c h l o r i n a t e d dibenzodloxin Impurities (Levin et al., 1976), occurred mostly 1n sawmill work and paper pulp production. Very few persons reported exposure both to phenoxyacetlc acid and chlorophenols 1n these studies. Of the two predominant phenoxyacetlc acids, only 2,4,5-T 1s known to be contam inated with 2,3,7,8-TCDD. In Study B, a relative risk of 4.9 (90% confi dence Intervals 1.6-11.1) was found 1n relation to exposure to phenoxyacetlc acid herbicide other than 2,4,5-T (2,4-D, MCPA, mecoprop, dlchloroprop).
Relative risks 1n relation to the three major categories of exposure are shown 1n Table 11-27.* Studies A and B Indicate a risk of developing STSs among workers exposed to phenoxyacetlc acids only, chlorophenols only, or phenoxyacetlc acids and/or chlorophenols several times higher than among persons not exposed to these chemicals. In each comparison, the relative risk 1s high and was thus unlikely to have resulted by chance alone.
Since little 1s known of the etiology of STSs, the consideration of confounding 1n these studies was largely a hypothetical matter. The authors presented the effects of age, sex, and place of residence as possible confounding factors 1n the selection of controls.+ 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 1n these occupations was responsible for the elevated relative risks.
*In the analyses considering phenoxyacetlc acids only and chlorophenols only, persons exposed to the other categories of substances were excluded. In Study A, the three persons exposed to both chlorophenols and phenoxy acetlc acids were Included 1n all comparisons.
fControls were matched Individually to cases on the basis of these factors. Unmatched analyses are presented 1n 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) 1n Study A and 5.1 (p<0.001) 1n Study B.
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TABLE 11-27
Relative Risks of Soft-Tissue Sarcoma In Relation to Exposure to Phenoxyacetlc Acids and Chlorophenols In Two Case-Control Studies3
n-70
Phenoxyacetlc Acids Onlv
Study A
Study B
Chlorophenols Onlv
Study A
Study B
Phenoxyacetlc Acids and/or
Chlorophenols
Study A
Study B
Relative risk** 90% Confidence 1ntervalc Significance level^
5.3 2.7-10.2 <0.001
6.8 3.1--H.9 <0.001
6.6 2.8-15.6 <0.001
3.3 1.6-7.0 <0.005
5.7 3.2-10.2 <0.001
4.7 2 .7-8.3 <0.001
aSource: Study A, Harden and Sandstrom, 1979; Study B, Eriksson et al., 1979, 1981 ^Unmatched odds ratio cTest-based method of Mlettlnen, 1976 ^Ch1 square statistic, no continuity correction, one-ta1led test
To test this hypothesis, 1t 1s possible to calculate the relative risk 1n relation to the phenoxyacetlc acid exposure 1n Study B, restricting the analysis to workers within agriculture and forestry. The result 1s a rela tive risk of 6.1 (90% confidence Interval 2.4-15.4). This finding suggests that a confounding risk factor for STS distributed throughout agriculture and forestry work was not responsible for the overall Increase 1n risk found 1n relation to phenoxyacetlc acid exposure.
Because exposure histories were obtained by means of questionnaires and Interviews, the major potential source of bias In 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 acids or
c h l o r o p h e n o l s and c o m p a r i n g the m w i t h unexposed persons 1n other occupations
was 0.9 (90% confidence Interval 0.3-2.4) 1n Study B. This suggests that little recall bias was present (Axelson, 1980).
In an update of their earlier study, Eriksson et al. (1981) obtained Information on the effects of phenoxy acids 1n the absence of the Impuri ties -- polychlorinated d1benzod1ox1ns and dlbenzofurans. The risk ratio given exposure to phenoxy acids free of polychlorinated d1benzod1ox1ns and dlbenzofurans equaled 4.2 based upon 7 of 14 respondents who Indicated exposure to phenoxy acid herbicides. When consideration was given to persons exposed only to phenoxy acids that contain such Impurities, the relative risk was 17.0. A description of the basis for the determination of exposure or nonexposure to dioxins 1s not well presented 1n this study.
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The author concluded that exposure to phenoxy acids and chlorophenols "might constitute a risk factor 1n the development of soft-tissue sarcomas." This risk relates not only to 2,4,5-trlchlorophenoxy acids containing dioxin Impurities but to other phenoxy acids as well. Some doubt was raised concerning the possible mlsclasslf1cat1on of Individuals who were exposed to phenoxy acids free of polychlorinated d1benzod1ox1ns [1.e., 1n particular, "dlchloroprop" 1n the Eriksson et al. (1981) study]. In a recent communica tion from Hardell (1983), Eriksson recalculated h1s risk estimates after reclassifying h1s dlchloroprop-exposed cases and controls Into the category of probable exposure to phenoxy acids contaminated with polychlorinated d1benzod1ox1ns and removing them from the nonexposed category. H1s new estimates were 4.0 based upon 5 of 8 respondents who were exposed to phenoxy acids allegedly free of contamination and 10.9 for those exposed to contami nated phenoxy acid. 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 dioxin.
In a cohort mortality Investigation Cook et al. (1980) studied 61 males Involved 1n a 1964 exposure Incident who had absorbed 2,3,7,8-TCDD through the skin and developed chloracne. The skin lesions characterizing chloracne ranged from a few comedones on the back of one employee (predating his entry Into the process area where exposure could occur) to severe cysts and comedones over the faces, scalps, ears, necks and backs of the remaining employees of the group. Since the main route of exposure was not through the respiratory tract, no measurements of dioxin 1n the air were provided by the author. On the other hand, the author divided the cohort of 61 males Into potentially "high" vs. "low" exposure by place of work based upon
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dermal exposure, although not stated. Vital status was traced from the data
of the Incident through 1978. Altogether only 4 deaths were observed by the end of the follow-up, vs. 7.8 expected. Of these, 3 were cancer deaths vs. 1.6 expected. The remaining death was hypersensitive heart disease vs. 3.8 expected. The histopathologic causes of death of the three cancer victims Were 1) fibrosarcoma, 2) glioma with metastases, and 3) adenocarcinoma. The authors report that all three victims smoked a minimum of one pack of cigarettes a day for "many years." Not enough Information 1s provided by the authors to conclude that any of these four deaths were smoking related. Site of tumor 1s not mentioned 1n the cancer deaths.
Cancer mortality 1s slightly elevated 1n this cohort. The study has low sensitivity and lacks a sufficient latent period. This Increased mortality was not attributable to any particular cause and no deaths were attributable to Uver cancer. Additionally, the authors state that only one of the cancer deaths possessed "documented" evidence of chloracne, although this
appears to be at variance with the definition of the cohort, which was
reported by the authors to consist of males who reported to the medical department with skin conditions subsequently "diagnosed as chloracne." The authors concluded that the latency period was sufficient to "allow the Identification of a potent human carcinogen," since 1t "exceeded 14 years." Orris (1981) noted that 1n the 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.
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Smith et al. (1982b) conducted an Initial case-control study of 102 males Identified from the New Zealand Cancer Registry as having STSs (ICD 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 histological types 1n the cases 1s given 1n Table 11-28. An Interview to elicit occupational history Information was accomplished by telephone either with the next of kin to the patient or the patient himself 1f he was well enough, although the Informa tion was not used 1n this preliminary analysis.
Comparisons between cases and controls were accomplished by use of occupational groupings according to the Standard Classification System of New Zealand focusing on those occupational groups with a potential for exposure to phenoxy herbicides and chlorophenols. Expected cases for each major occupational classification were derived based upon the occupational distribution of the controls. The authors found no unusual excess of cases of STS 1n any major occupational category. In agriculture, forestry and fishing, 14 cases were observed vs. 14.0 expected. In laborers, production and transport workers, 35 cases were observed vs. 37.0 expected. A further breakdown of these two broad categories Into finer subcategories 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 acids 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
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TABLE 11-28 Distribution of Histological Types of Soft-Tissue Sarcomas*
Cell Type
Fibrosarcoma Llposarcoma Rhabdomyosarcoma Leiomyosarcoma Malignant Histiocytoma Other Unspecified Total
Source: Smith et al., 1982b
Number of Cases
25 20
9 7 6 22 13 102
Percent
24 20
9 7 6 21 13 100
11-75
manifest themselves could Introduce a negative bias Into any estimates of relative risks. The latency for STS was suggested to be a minimum of 15 years (Hueper and Conway, 1964).
The finding of no switching from one occupational category to another that was noted 1n the "first 20 Interviews" 1n which a change could be noted 1s not necessarily Indicative of fidelity to the same Job over long periods 1n all 408 cases and controls. Information Identifying a change may be lacking 1n those cases and controls 1f 1n fact one did occur possibly because of several reasons, for example, separation of the earlier work history from the latter and purging of earlier employment records. Besides the "first 20 Interviews" where a change could be noted 1s not necessarily representative of the entire cohort 1n any case.
Furthermore, the authors do not know absolutely that any of their cases and controls were exposed to phenoxy acids or chlorophenols or to both since apparently no effort was made to confirm "potential" exposures. Only dif ferences 1n occupational classification were noted where "potentially" cases or controls could have had exposure to the d1ox1n-conta1n1ng herbicides. It was pointed out that the risk estimates noted do not "preclude" the possi bility that an association may be found 1n this study when the cases and controls (or surviving kin) are Interviewed for chemical spraying at a later time. The authors themselves concluded that the preliminary study results "should not be taken as substantial evidence against the hypothesis that phenoxy herbicides and chlorophenols may cause human cancer."
The distribution of tumor types differed considerably from the Hardell and Eriksson study to the Smith study. Leiomyosarcomas, malignant hlstocytomas, neurogenic sarcomas and myxosarcoma seem to predominate 1n the Hardell and Eriksson study, whereas fibrosarcomas and llposarcomas appear
11-76
p r o m i n e n t l y In 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 date of birth + 2 years. Inquiries were made by the authors with the hos pital consultant, family doctor, and finally the next-of-k1n or patient 1f alive. Telephone Interviews were conducted by only one Interviewer, who had no knowledge of the patient's cancer history, and were completed on 80 cases and 92 controls. Because some 32 potential cases (14 Ineligible) and 20 controls were excluded or lost from the study for various reasons, 1t raises a question whether control of confounding by age and year of registration was maintained In the final group of 172 cases and control Included 1n the analysis. Presumably the corresponding "matched" case or control to each of the 52 lost members of the total study group were not excluded. However, since the span of registration was only 5 years, not much age confounding could occur.
Patients were classified as having had potential exposure to phenoxyacetlc acids 1f they had definite, probable or possible exposure to phenoxyacetlc acid through spraying or hand contact. The actual chemical was Identified only 1n some Instances. The authors concluded 1n all remaining
11-77
situations that 1f the member sprayed "gorse" and/or "blackberries" this was tantamount to potential exposure to phenoxyacetlc acid. Smith (1983) calcu lated elevated but nonsignificant relative risks of exposure to phenoxy acetlc acid ranging from 1.3 1n those Individuals who were "probably exposed" for a minimum of 5 days not 1n the previous 10 years before cancer registration to 1.6 1n Individuals "probably exposed" for a minimum of 1 day not 1n the previous 5 years before cancer registration. When risk ratios were calculated after stratifying by year of birth and whether or not the patient or a relative was Interviewed, the rates Increased to 1.7 (from 1.6) 1n the latter and 1.4 (from 1.3) In the former calculation, although still nonsignificant. If the numbers would allow, 1t would be of Interest to repeat the above calculations excluding only those with potential exposure occurring only within the 15-year period just before cancer registration. The small numbers that remain following the 15-year lapse probably precludes such an analysis. Furthermore, the categories of exposure "probably or definitely" exposed for >1 day or even 5 days raises a question whether any of the cases or controls could really be said to have ever come In contact with enough phenoxyacetlc acid to justify such a designation. It could be that, In fact, potentially exposed Individuals In 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 In Individuals with >1 day exposure not In the last 5 years cannot be entirely discounted. But then the authors stated that 1f length of exposure was >5 days prior to 10 years before cancer registration, they would expect an Increase, and since they do not see an Increase, there 1s no evidence of a "real causal link." One might ask whether this 1s a suitable criterion for providing evidence of a causal association. Perhaps a more valid group for
11-78
study would be one where the potential exposure was considerably longer than "5 days" and >15 years before Initial cancer registration. As kind of a subtle justification for the finding of no significant risk 1n workers exposed In phenoxy acids, the author alluded to the fact that there were 500 full-time workers registered In New Zealand who did full time ground spray ing and altogether some 2000 workers who were at some time professionally Involved 1n phenoxyacetlc acid herbicide spraying from the air or ground with exposure "very much greater" than that of patients In this study. This kind of argument has appeal 1f these workers could be shown to have had their exposure sufficiently far In the past that latency considerations could be adequately addressed. However, the real question again remains; how much real exposure did those patients In the study really have 10-15 years earlier, and 1n what numbers. The author remarked that 1t was surprising that he found no STS victims who had ever worked full-time In phenoxyacetlc acid herbicide spraying. Perhaps they have not yet been
observed f o r a long enough p e r io d . The time I n t e r v a l of 10 years and/or 5
years from exposure to registration may not have been long enough to allow latent effects to become evident. However, as was pointed out by the author, the findings do not support the hypothesis that exposure to phenoxy acetlc acid herbicides causes STS. But neither do they support a negative finding without better documentation regarding actual exposure and time of actual exposure. Smith (1983), however, noted that h1s documentation of exposure to 2,4,5-T (and 2,4-D) was at least as good as that 1n the Hardell and Sandstrom (1979) study, and that although Hardell and Sandstrom (1979) noted higher relative risks of <30 days exposure, Smith (1983) did not. Hence the paradox. Smith (1983) admitted the possibility that 2,3,7,8-TCDD contaminations might be lower In New Zealand as opposed to 2,3,7,8-TCDD contamination In the Swedish studies, although there 1s no evidence for 1t.
11-79
He still maintains that his study showed that exposure to phenoxyacetlc acids may not be associated with STS.
Pazderova-Vejlupkova et al. (1981) studied 80 workers Involved In the production of 2,4,5-sodium trlchlorophenoxyacetate and butylester of tr1chlorophenoxyacetlc acid who subsequently became 111 from exposure to 2,3,7,8-TCDD during the period 1965-1968. Only 55 members of this group were followed for 10 years. The remaining 25 either refused participation or moved leaving no forwarding address. Most patients developed chloracne while 11 developed porphyria cutanea tarda. Chief chemical signs were metabolic disturbances, pathologically elevated 11plds with abnormalities 1n the lipoprotein spectrum, and "pathological" changes In glucose tolerance. Other symptoms noted were biochemical deviations consistent with "a mild Uver lesion," light steatosis, periportal fibrosis or activation of Kupffer cells, or nervous system focal damage (peripheral neuron lesion In lower extremities). Altogether six patients were reported to be deceased during this 10-year period, 2 from bronchogenic carcinoma, 1 from cirrhosis, 1 atherosclerosis preclpue cerebl and 2 In auto accidents. No STSs or lympho mas were found. Since there was no comparison population with which to estimate relative risk for cancer, the study must be classified at best as clinical with respect to cancer. The 6 deaths (of 55) that occurred during the 10-year observation period cannot be construed to be associated with exposure to the 2,4,5-T. Because of the small number of cases and the short follow-up period, nothing can be said concerning the association of exposure with cancer, especially specific types of cancer such as STS or nonHodgkin's lymphoma.
R11h1mak1 et al. (1982, 1983) studied a cohort of 1926 herbicide appli cators formed In 1972 from personnel records of four Finnish employers
11-80
( e .g ., the F o restry A u th o rity , Highway A u th o rity , S tate Railways and a state-owned electric power company). Chlorinated phenoxyaclds had been used since the 1950s 1n Finland for spraying. They constituted 2:1 mixtures of emulsified esters of 2,4-D and 2,4,5-T dissolved 1n water. Analyses from old herbicide formulations dating back to the 1960s revealed that these mixtures contained 0.1-0.9 mg/kg of 2,3,7,8-TCDD.
This cohort of male workers was exposed a minimum of 2 weeks during at least one growing season from 1955-1971. Follow-up continued 9 years through 1980 for mortality but only until 1978 for morbidity. Fifteen Individuals could not be traced by 1980. Expected deaths were generated based upon cause- and age-spec1f1c national Finnish death rates for 1975. Expected cases were similarly calculated based upon national Incidence rates of 1975.
By 1980, 144 deaths had occurred vs. 184.0 expected, a deficit of 2 2 % 1n observed mortality. Only 26 cancer deaths had occurred vs. 36.5 expected, a 29% deficit. The authors separated out "natural" deaths from the total. The observed residual deaths equaled 39 while the expected deaths equaled 28.7. This excess was of borderline significance. The authors also con sidered 10-year and 15-year latent periods. Even after 15 years, the defi cit of deaths continued to manifest Itself both 1n categories of all causes and total cancers; 35 observed vs. 53.6 expected and 5 observed vs. 11.3 expected, respectively. Similarly, the 7-year follow-up of cancer morbidity revealed 26 cases of cancer vs. 37.2 expected. After a 10-year latent period, 16 cancer cases were observed vs. 20.1 expected. None of the 26 cancer deaths or 26 cancer cases were of the STS or lymphoma type. (How ever, only 0.1 STS and 0.5 lymphomas were expected.) In no Instance was cancer of any site significantly elevated.
11-81
The authors noted that this unusual deficit of mortality and morbidity of between 70 and 82% (even after 15 years from Initial exposure) was prob ably a consequence of the "healthy worker effect" In that only able-bodied and healthy Individuals were selected Into the Industry. The fact that the cohort was assembled 1n 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.
The authors correctly noted that, because of limitations In the study material, only powerful carcinogenic effects could be detected. Risk ratios higher than 1.5 for all cancers, 4.0 for lymphomas and 10.0 for STS could be excluded based on this data set from the authors' own calculations. More follow-up 1s needed 1n order to provide a stable assessment of the relation ship between exposure and cancer. The authors concluded that this study will allow no assessment of STS because "the number of persons having a suf ficiently long latency period 1s too small." It was suggested that more valid conclusions could be made only with the passage of time (R11hlmakl et al., 1983).
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
1 1 -8 2
estimates are based upon 5 deaths and 7 deaths, respectively, and are listed 1n Table 11-29. No excess risk was reported among white males, however. The Michigan Department of Health concluded that because of the occurrence of these two successive elevated rates, 1t 1s unlikely to be a chance happening. At the same time the age-adjusted male and female cancer mortal ity rates for Midland were below that of the State of Michigan for the period 1970-1979. Midland County 1s the home of a major chemical company that produced phenoxyacetlc acid herbicides until recently. The authors stated that a detailed review of death certificates, hospital records, resi dency and occupational histories of the 20 male and female cases revealed no "commonalities" suggesting a "single causative agent," although a majority or their spouses had worked at this chemical facility. They recommend that a case-control study should be employed to evaluate possible Influences, such as lifestyle, occupation or location of residence on the risk of STS.
In a series of reports prepared under the auspices of the U.S. A1r
For c e, C o l . W i l l i a m H. Wolfe and h i s a s s o c i a t e s j u s t completed the f i r s t
phase of a study of A1r Force personnel Involved 1n the aerial dissemination of TCDD-conta1n1ng herbicides 1n the Republic of Vietnam (RVN). During the period of time beginning 1n 1962 and ending 1n 1971, -1278 male A1r Force personnel (Ranch Handers) were Identified as having been Involved 1n the effort to 1) defoliate vegetation 1n Vietnam 1n order to decrease the risk of ambush and 2) destroy enemy crops (Wolfe et al., 1985). Based on an 1984 report of baseline mortality study results (Wolfe et al., 1984), the cohort Involved 1n the mortality study was smaller at 1256 because of the
1 1 -8 3
TABLE 11-29 Midland County Soft and Connective Tissue Cancer Deaths 1960-1981*
11-84
Identification
Year of Sex Age Death
1961 F 24
1963 1964 1968 1969
F 75 F 51 F 37 F 45
1970 1970
F 59 F 56
1974 1976
F1 F 77
1978
F 64
Type, Site and Progression of Malignancy
Type
Primary Site
Metastases
Month and Year Diagnosed
Hemanglosarcoma
LIposarcoma Leiomyosarcoma Llposarcoma Fibrosarcoma Leiomyosarcoma Kaposi sarcoma Fibrosarcoma Leiomyosarcoma Rhabdomyosarcoma Llposarcoma
Leiomyosarcoma
Face
Right gluteal Uterus Spine Right thigh Uterus Right leg Right thigh Abdominal wall Inguinal area Right thigh
Left knee
Skull and upper lobe of lung
Unknown
Widespread
Lungs, pelvis
Lung, liver Adrenal gland and skin
Lymph nodes
Spine Lung
Unknown
Buttock, lung, rib, lymph nodes
Liver, lymph nodes, lung, bone
5-58
Unknown 11-63
1-66 10-68
8-68 1960 1967 8-73 12-74
7-70
TABLE 11-29 (cont.)
11-85
Identification
Year of Sex Age Death
Tvoe. Site and Progression of Malignancy
Type
Primary Site
Mtastass
1978 1978 1979 1962 1967 1967 1969 1971 1972
1976
F
26
Rhabdomyosarcoma
Rectum
Lung, neck, Inguinal region
F 88 Fibrosarcoma
Right cheek
Facial area
F 27 Leiomyosarcoma
Left thigh
Lung
H
63
Rhabdomyosarcoma
Left lower leg Lung and right outer
chest wall
M 77 Mesothelioma
Lung
Lung, peritoneum and diaphragm
M
20
Rhabdomyosarcoma
Pharynx
Periorbital area and liver
M 32 Llposarcoma
Left arm
Perineum and buttock
M 76 Leiomyosarcoma
Small Intestine
Liver
M 89 Leiomyosarcoma
Retroperl tonal region
Hepatic system
M 53 Fibrosarcoma
Perltloneum
Lung, Uver
Source: Adapted from Michigan Department of Public Health, 1983b
Month and Year Diagnosed
6-76 6-78 3-78 8-61 6-67 1-67 6-64 10-69 7-72
3-75
exclusion of 22 killed 1n action and was divided Into three main occupa tional categories as follows:
1 . Officers (pilots, navigators and others) 2. Enlisted (flight engineers) 3. Enlisted (others)
466 206 584
TOTAL
1256
The authors categorized the Ranch Handers as having had "exposure" to the TCDD-conta1n1ng herbicides 1f they were Involved 1n the aerial spraying of the herbicides. They were matched to 6171 cargo mission air crew members and support personnel generally on a 5 to 1 basis according to similarity of training and military background experiences, occupation and race. The comparison population presumably had no exposure to TCDD. In an earlier 1983 report (Lathrop et al., 1983), 50 deaths were Identified 1n the study group versus 250 1n the comparison population. Of these 50 deaths, 23 were due to external causes, 4 were malignant neoplasms, 16 were circulatory causes, 5 were digestive disorders and 1 was an endocrine disorder.
In the later December 1984 update, Wolfe et al. (1984) added 4 more deaths to the study population for a total of 54 deaths occurring to Ranch Hands while adding 15 to the 250 that had already occurred 1n the comparison group through December 31, 1983. Altogether this update produced a total of 6 cancer deaths 1n the Ranch Hands versus 43 cancer deaths 1n the comparison population. The greatest cause of death 1n both Ranch Handers and the comparison population were accidents with 19 and 94, respectively. None of the 6 cancer deaths and 1 of the 43 deaths In the comparison group were STSs. Comparison of overall mortality 1n the Ranch Handers with other A1r Force military personnel was nearly Identical (~4.354). Ranch Hand ground
11-86
enlisted personnel su ffere d somewhat greater (although not s ig n ific a n t) mortality than did Ranch Hand officers. Comparison of mortality 1n the Ranch Handers with other groups such as U.S. white males, Department of Defense retired enlisted men, U.S. civil servants, active duty A1r Force and West Point officers from the class of 1956, were similar except for A1r Force active duty officers who exhibited significantly less mortality. The authors attribute this to higher health qualification standards.
There were few biological markers that might tend to support the assump tion that Ranch Hands were exposed to 2,3,7,8-TCDD. In the Banbury report, Lathrop et al. (1984) reported that the dermatologic evaluation revealed no cases of chloracne through clinical diagnosis or bioassay. A questionnaire analysis of acne 1n Ranch Handers and comparison groups showed no unusually different Incidence, severity, duration or distribution of anatomical locations 1n either group. Lathrop et al. (1984) said 1n fact that the "historical occurrence of chloracne was highly unlikely 1n the Ranch Handers".
This study suffers from several deficiencies that limit Its usefulness 1n a determination of human health effects, notably cancer, and especially STS from exposure to 2,3,7,8-TCDD-contam1nated phenoxy herbicides. First, 1t 1s mainly a study of basically young men who were Involved 1n the A1r Force aerial spraying missions. This 1s evidenced by the exceptionally large number of accidents attributable to members of the cohort. It 1s the largest single cause of death 1n these men. Because this 1s a young group 1t 1s unlikely that substantial mortality will occur to the cohort until many more years of follow-up have passed. In fact, even after 15 years following Initial exposure <5% of the cohort have died. Since most cancers
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have a latency of >15 years following Initial exposure 1t 1s not likely that a cancer risk from 2,3,7,8-TCDD, 1f any, will manifest Itself for some time.
Furthermore, the relatively rare STS, which 1s thought to have an even longer latency period, may not appear as a risk 1n this cohort until well after the 20th year. Additionally, this cohort exhibits little evidence of actual exposure to the herbicide 1n question, thus raising the possibility of m1sclass1f1cat1on. In other small cohort studies (Cook et al., 1980; Ott et al., 1980; Zack and Susklnd, 1980) substantial numbers of the study cohorts exhibited evidence of exposure to 2,3,7,8-TCDD as Indicated by the presence of chloracne, a clear biological marker. Few of the Ranch Handers exhibited evidence of this condition (Lathrop et al., 1984). In fact, as was suggested by the authors, the historical, occurrence of chloracne was considered highly unlikely 1n the Ranch Hands. Neither do they present convincing evidence of other conditions suggestive of an association with exposure to the d1ox1n-conta1n1ng herbicide that cannot be explained by confounders, according to the authors. In fact Ranch Handers, who were heavily populated with officers, pilots, navigators and flight engineers, may not have been as heavily exposed to the phenoxy herbicides as other U.S. mili tary personnel 1n Southeast Asia. Perhaps Army combat foot soldiers or the non-Ranch Hand personnel who did the spraying on the ground around the mili tary bases would constitute a more appropriate cohort for study. Lathrop et al. (1984) concluded that the absence of any association of "clinical end points" with herbicide exposure must be viewed as Insufficient evidence supporting a cause-and-effect relationship. But this absence of any "clini cal endpoints" might also Indicate evidence of a lack of exposure to the
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p h enoxy herbicides In question by the Ranch Handers. This study must b e viewed as Inadequate 1n assessing the risk of cancer from exposure to 2,3,7,8-TCDD-conta1n1ng phenoxy herbicides.
In a separate review of the epidemiological 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 acid herbicides and related wood preservatives as "presently cleared" are safe and may continue to be used. This report readily discounts the posi tive studies of Harden and Eriksson (1D79) as being biased, and 1t makes no reference to the later validity study by Harden (1981) of h1s own work utilizing colon cancer controls (see Section 11.2.2.2.). In this report Harden answered these early criticisms that were reiterated by the British 1n their report. At the same time, the British report appears to put undue emphasis on nonposltlve studies that do not demonstrate a risk, although most of them have methodological limitations (e.g., low power, Insufficient latency and Inappropriate study methods). In short, the British review appears to be overly optimistic about the safety of 2,4,5-T herbicides.
Flngerhut et al. (1984) recently completed a review of medical and available exposure records of seven U.S. chemical workers that have been diagnosed as having STS and who were reported to have had possible exposure to dioxin. These cases collectively produced a clustering effect of the relatively rare STSs among former employees of a portion of the U.S. chemi cal Industry where exposure to compounds contaminated with 2,3,7,8-TCDD 1s most likely to have occurred. Flngerhut et al. (1984) reported that a subsequent review of the Armed Forces Institute of Pathology and a review of one of the authors of the Flngerhut paper confirmed the diagnosis of 5 of the 7 U.S. chemical workers as STSs.
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In terms of occupational exposure, Flngerhut et al. (1984) proposed a strict definition of exposure as follows: a record must exist somewhere that shows an assignment to either a 2,4,5-T department or to a trlchlorophenol department at some time 1n the past. If such a record did not exist, then the Individual would not have been considered to have had a confirmed exposure. Four of the seven who had a confirmed exposure 1n this manner were also members of cohorts that had been studied previously, while the remaining three could not be confirmed as having been assigned to any 2.4.5- T department or trlchlorophenol department. The latter three were not Identified as having been part of any earlier study but were case reports of Johnson et al. (1981) and Hoses and Sellkoff (1981). Individuals who were members of study cohorts of "exposed Individuals" might be expected to have better documentation of exposure, based upon employment records, than would cases turning up 1n a medical practice.
However, Flngerhut et al. (1984) pointed out that of these three cases, one worked 32 years 1n production, clerical, truck driving and maintenance jobs 1n a chemical manufacturing site that produced trlchlorophenol and 2.4.5- T; the second worked 2.5 years as a production worker 1n a plant that made 2,4,5-T; and the third was a production and maintenance worker for 29 years at the same facility as the second worker. It would seem that the opportunity for exposure to 2,3,7,8-TCDD containing 2,4,5-T or trlchloro phenol must be considered a distinct possibility 1n the first two cases, especially since both were Involved with maintenance for many years.
Johnson et al. (1981) pointed out that the second case could not have satisfied a minimum latency requirement for exposure to TCDD since his 2.5 years as a production worker occurred just before his diagnosis and death.
11-90
However, this man's father was employed with this same plant almost as long as h1s son was alive and 1t seems plausible that because of this connection the son may have been exposed.
One must have reservations about the usefulness of a classification scheme that relies on documentation of an assignment to a specific area of a plant as proof of exposure to dioxin without real evidence substantiating that exposure (1.e., either biological or physical measurements), while at the same time assignment to all other areas of the same plant 1s considered Insufficient evidence of exposure although nothing Is offered to substan tiate the presence or lack of exposure to 2,3,7,8-TCDD 1n either case. In most occupational prospective cohort epidemiologic studies, employment at a plant where the suspect agent 1s produced or found has been considered sufficient enough to call such a person "exposed" and thus Included 1n a cohort for study. On the other hand, 1f the Flngerhut et al. (1984) defini tion were retrospectively applied to the already small occupational cohorts from which the first four STSs came, even two of these relatively rare STSs might probably constitute an excessive risk 1n the much smaller cohorts circumscribed by their definition. Flngerhut et al. (1984) agreed that an excess risk of STS would remain even with just two confirmed cases, and hence the possibility of a causal relationship between exposure to 2,3,7,8TCDD and the development of STSs cannot yet be ruled out.
In summary, the associations reported In the two Swedish soft-tissue sarcoma studies are strong enough to make 1t unlikely that they have result ed entirely from random variation bias or confounding, even though the possibility cannot be excluded. These studies provide a strong suggestion that phenoxyacetlc acid herbicides, chlorophenols or their Impurities are carcinogenic 1n humans.
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n . 2 .2.2. MALIGNANT LYMPHOMAS -- A separate series of clinical obser
vations at the Department of Oncology 1n Umea, Sweden (Hardell, 1979), led the researchers to conduct a case-control study of malignant lymphoma 1n relation to phenoxyacetlc acid, chlorophenols, and other organic compounds (Hardell et al., 1980, 1981). Approximately 33% of the cases 1n this study were patients with Hodgkin's disease; the remainder of the cases were nonHodgkin's lymphomas.
This study employed essentially the same methods and produced results comparable with those of the STS studies: statistically significant 5-fold to 6-fold relative risks 1n relation to phenoxyacetlc acids and chloro phenols were confirmed. In addition, an elevated relative risk was found 1n connection with exposure to organic solvents, such as benzene, trichloro ethylene, and styrene. In the published report, the methods and results were Incompletely documented, especially the possibility of confounding by exposure to the organic solvents.
In the update of the earlier 1980 study, Hardell et al. (1981), utiliz ing the same basic data source, found that 36.1% of the cases had been exposed to phenoxy herbicides or chlorophenols, while only 9.6% of their controls were so exposed. The estimated relative risk was 6.0 when matching was considered and 5.3 when matching was eliminated. When cases and con trols that were exposed to chlorophenols only were excluded, the relative risk of lymphoma from phenoxy acids alone was 4.8 (95% C.I. 2.9-8.1). On the other hand, 1f exposures to phenoxy acids are excluded and consideration 1s given to just chlorophenols (which Includes combined exposure to phenoxy acids 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
1 1 -9 2
month was classified as low-grade. The relative risk for high-grade expo sure was 8.4 (95% C.I. 4.2-16.9), while that for low-grade exposure equaled 9.2 (95% C.I. 1.6-5.2). If exposure to organic solvents 1s examined, given that cases and controls exposed to only phenoxy 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 noted that expo sure to phenoxy acids and high-grade organic solvents (exposure to chloro phenols excluded) produced a relative risk of 11.2 (95% C.I. 3.2-39.7) based upon a few cases and controls with exposure to both. The authors concluded that "exposure to organic solvents, chlorophenols and/or phenoxy acids constitutes a risk factor for malignant lymphoma."
The Harden et al. (1981) study 1s still subject to the same methodo logical criticisms to which the earlier study was subjected. Chief among those 1s the possibility of observational and/or recall bias creeping Into
t he responses t h a t a r e e l i c i t e d from s e l f - a d m i n i s t e r e d q u e s t i o n n a i r e s on
kind and length of exposure. Secondly, confounding by exposure to poten tially carcinogenic organic solvents and other agents could have had an effect on the risk estimate, although Hardell (1981) Insists that they did not.
Other research has tentatively suggested that lumberjacks may be at
\
Increased risk of lymphoma (Edllng and Granstam, 1979). The N1tro study found three deaths from cancers of the lymphatic and hematopoietic system, against only 0.88 expected (p=0.06, one-ta1led Poisson 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.
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The two Swedish case control studies on STSs and a later case control study of malignant lymphoma (Hardell et al., 1981) were subjected to a validity analysis with respect to the assessment of exposure by Hardell and Eriksson (1981). To answer the question raised regarding the recall of occupation 1n a forestry/agrlculture job, secondary to the recall of expo sure to phenoxy acids and/or chlorophenols, the cases and controls were divided Into three groups: those who worked their entire time since 1950 1n an agrlculture/forestry job; those who worked some time 1n an agriculture/ forestry job but not exclusively; and the remainder who never worked 1n a forestry/agrlculture job. The study found that the risk ratio was still 8.2 for STS 1n exclusively agrlculture/forestry workers who were exposed to phenoxy acids compared with workers found 1n other occupations having no apparent exposure to phenoxy acids or chlorophenols. Even when comparing phenoxy acid- and/or chlorophenol-exposed agrlcultural/forestry workers exclusively with nonexposed agrlcultural/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 agrlculture/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 acids and/or chlorophenols (Table 11-30).
When woodworkers are separated out (possible exposure to chlorophenols 1n treatment of wood) the risk ratio becomes 9.7 (Table 11-31). These data suggest the presence of some recall bias.
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TABLE 11-30 Other Occupations (Minus Forestry/Agriculture)*
Group
Phenoxy Acids/Chlorophenols
Cases Referents
11 5
RR = 5.4
*Source: Hardell and Erikkson, 1981 RR = Relative risk
Non-exposed
68 167 X2 = 11.01 (P<0.01)
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TABLE 11-31 Other Occupations (Minus Forestry/Agriculture/Woodworkers)*
Group
Phenoxy Aclds/Chlorophenols
Cases Referents
4 1 RR = 9.7
Source: Hardell and Erikkson, 1981 RR = Relative risk
Non-exposed
66 160 X2 = 5.98 (P<0.05)
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Another focus of t he H a r d e l l and Er lkkson ( 1 9 81 ) study was t o d e t e r m i n e
1f observational bias on the part of the Investigators could explain the significantly high risk estimates. To answer the question, the study com pared the exposure data derived from the Interviewee's returned question naires only with the combined Information from both the phone Interviews and questionnaires. The study found no substantial differences 1n the frequency of reporting exposure.
Still a third consideration of possible bias Involves recall of exposure to phenoxy acids and/or chlorophenols because of subject knowledge of having cancer 1n the cases versus no knowledge of cancer 1n the referent popula tion. The study chose as a referent group for the 52 STS cases (Hardell and Sandstrom, 1979) and the 169 malignant lymphomas (Hardell et al., 1981) a group of 154 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.
U t i l i z i n g a Mantel-Haenszel ra te r a t i o , the study found the r is k of
exposure to phenoxy a d d s remaining significantly high at 5 . 5 and to chloro phenols 5 . 4 1n the STS cases compared with the colon cancer controls. Simi larly, with the malignant lymphomas, the Identically derived risk ratios remain significantly high at 4 . 5 with respect to phenoxy acids and/or chlorophenol exposure 1n the cases, hence, the study concludes, no "sub stantial observational bias" exists. If 1t 1s assumed 1n this study that recall bias was and 1s the same as observational bias, then such a conclu sion may not be entirely warranted from the comparison. Certainly, 1t appears that no recall bias existed because of subject "knowledge of having cancer" based on the authors' analysis. But 1t does not rule out the possi bility that recall bias can still be present 1n their data for other
11-97
reasons. Hardell et al. (1981) refers to an Intense "debate about phenoxy acids and their presumptive risk" 1n Sweden at the time the colon cancer study was conducted. But, there 1s no reason to think that colon cancer victims would assume their disease was brought about from exposure to dioxin containing chemicals 1f no connection was suggested.
It seems plausible that STS and non-Hodgkin's lymphoma patients would either learn at the time of their diagnosis that exposure to dioxin-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 dioxin could cause this rare form of cancer whereas colon cancer victims (a rather common form of cancer) would not necessarily be led to believe that exposure to the same d1ox1n-conta1n1ng chemicals caused their disease. Hence, 1t 1s not difficult to Imagine that such unusual victims of cancer could better "remember" exposure to such chemicals than could colon cancer patients.
Therefore, although the Harden (1981) study may explain any biases Introduced from secondary recall of occupation, observational bias Intro duced from the telephone Interviewer and recall bias based on subject knowledge of cancer, 1t does not adequately answer questions of recall bias Introduced through the acquired awareness on the part of the victim of STS or non-Hodgkin's lymphoma that h1s condition may have been caused by exposure to d1ox1n-conta1n1ng herbicides.
11.2.2.3. STOMACH CANCER -- Studies of two of the oldest cohorts of workers known to have been exposed to 2,3,7,8-tcdd containing phenoxyacetlc acid herbicides report stomach cancer mortality rates significantly higher than expected. The results 1n each study were based on small numbers of deaths. In one study (Axelson et al., 1980), 348 Swedish railroad workers
11-98
with at least 46 d a y s of herbicide exposure between 1955 and 1972 were followed through October 1978. The workers were grouped on the basis of their primary herbicide exposures: those primarily exposed to phenoxyacetic acids (2,4-0 and 2.4,5-T) only, to amitrole (aminotriazole) only, and to both types of herbicides. After a 10-year latency was achieved, 3 stomach cancer deaths were observed vs. 0.71 expected (p<0.05). None were attribut able to amitrol alone, but two were assigned to phenoxy acids alone while the remaining stomach cancer death occurred in a worker exposed to both amitrol and phenoxy acids. The excess was more pronounced (3 observed vs. 0.57 expected, p<0.05) among those with early exposure (1957-1961) to phenoxy acids and/or amitrol. If persons who were exposed to just amitrol alone are excluded, thus leaving individuals exposed to phenoxy acid alone and amitrol in combination, the excess is enhanced further (3 observed vs. 0.41 expected, p<0.01).
Axelson et al. (1980) also noted an excess in total "tumors" after 10 years latency as well (15 observed vs. 6.87 expected, p<0.005). This is pronounced in those exposed early to phenoxy acids alone (6 observed vs. 2.60 expected, p<0.01) and phenoxy acids in combination with amitrol (5 observed vs. 1.34 expected, p<0.05). Presumably, "tumors" in Sweden are analogous to malignant neoplasms in the United States. The author states that no specific type of tumor predominates and no breakdown by tumor type is provided.
The other study showing increased stomach cancer mortality is the follow-up of 75 workers exposed to 2,3,7,8-TCDD during and after a 1953 run away reaction at a trichlorophenol manufacturing facility in Ludwigshafen, Federal Republic of Germany (Thiess and Frentzel-Beyme, 1977). Two sources
IT-99
were used to calculate expected deaths: national mortality rates for the period 1971-1974, and .1972-1975 rates for Rh1nehessen-Palat1nate, the region 1n which Ludwlgshafen 1s located.*
The results, shown 1n Table 11-32, Indicate an Increased rate of stomach cancer mortality that also Is 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 1970s 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 1n West Germany during the late 1950s and 1960s would likely make these expected figures too large.
The researchers also used an Internal control group that does not raise the second concern discussed above. This group consisted of 75 men, each matched to study group members by age and date of entry Into employment, and selected at random from a 11st of over 10,000 persons who had been Included 1n previous cohort studies by the same Investigators. No stomach cancer deaths occurred 1n this control group during the follow-up period. Thus, use of the Internal control groups also Indicates an excess of stomach can cers 1n the exposed workers.
*The report originally Included expected deaths using rates for the city of Ludwlgshafen, which were later shown to be Inaccurate.
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TABLE 11-32
Analysis 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
Federal Republic
of Germany
1971-1974
3 0.559
RhlnehessenPalatlnate 1972-1975
3 0.495
Source: Thless and Frentzel-Beyme, 1977
Relative Risk
5.4 6.1
Significance Level
0.02 0.01
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In an update of this earlier study, Thless et al. (1982) continued the follow-up of h1s cohort through 1979 by adding 2 additional years of follow up and apparently reducing the size of h1s cohort from 75 to 74. Altogether 21 deaths (4 more than from the earlier study) occurred vs. 18 and 19 deaths 1n the 2 matched (1 to 1) Internal comparison groups. With respect to can cer deaths, the numbers were respectively 7, 5 and 5. The first control group was manually matched from the total number of persons (5500 Included 1n the cohort until the end of 1976) and the second, at random, by computer for some 8000 employees. In addition, 19 expected total deaths were esti mated based on 1970-1975 mortality statistics of Rh1nehess1n-Palat1nate, 18 expected deaths based on 1970-1975 mortality statistics of Ludwlgshafen, and 20 expected deaths based upon 1971-1974 mortality statistics of the Federal Republic of Germany. Just as 1n the earlier study, the three stomach car cinomas noted earlier appear to be significantly elevated regardless of which external comparison group 1s used (Table 11-33).
On the other hand, one stomach cancer appeared 1n the randomized Inter nal control group. None appeared 1n the manually matched Internal control. No other elevated risks for any other cause were evident and no STSs appeared. When latency was considered only, the risk of stomach cancer remained significantly elevated after a lapse of 10 years (3 observed, 0.52 expected, p<0.016) and then after a lapse of 15 years (2 observed, 0.23 ex pected, p<0.02) based upon death rates of Rh1nehess1n-Palat1nate, 1970-1975.
Again, these study conclusions are limited by the small size of the study group and the very few cancer deaths noted at any particular site. Thus, 1t 1s` Insensitive to the detection of a significantly elevated risk for most causes of cancer, especially STS and lymphomas. Although, stomach cancer 1s elevated significantly, 1t 1s based only upon three deaths and
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TABLE 11-33
Reanalysis 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
Federal Republic of Germany 1971-1974
RhlnehesslnPalatinate 1970-1975
Ludwlgs-Shafen 1970-1975
3 3 3
0.7 0.64 0.61
*Source: Thless et al., 1982
Relative Risk
4.3
4.7 4.9
Significance Level
0.034
0.027 0.024
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since one stomach cancer death has been noted 1n 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 1950s, 1960s and 1970s could make the comparison of stomach cancer mortality with expected deaths less valid based upon 1970-1975 rates.
In summary, the evidence that phenoxyacetlc acids and/or 2,3,7,8-TCDD might Increase the risk of stomach cancer consists of two studies, each of which reports a statistically significant excess that 1s based on only three stomach cancer deaths. Further follow-up of these and similar cohorts 1s warranted, but firm conclusions cannot yet be made.
Four additional cohort studies have reported results that do not show Increased stomach cancer mortality rates 1n groups of workers exposed to phenoxyacetlc acids 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 acid herbicide applicators (R11h1mak1 et al., 1978), the N1tro study 1n which workers were exposed to 2,3,7,8-TCDD (Zack and Susklnd, 1980) and trlchlorophenol manufacturing workers (Cook et al., 1980).
As previously mentioned, the N1tro study Included a single death from STS and a weakly suggestive Increase 1n lymphatic and hematopoietic system cancer mortality. The Midland study of 204 workers Included only one cancer death, a tumor 1n the respiratory system. In the Finnish study, histologic Information on tumor types was not provided; however, there were no deaths from lymphoma.
The results pertinent to stomach cancer mortality 1n the three studies are shown 1n Table 11-34. Results of neither the Midland study nor the
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TABLE 11-34
Stomach Cancer Mortality 1n Three Studies of Workers Exposed to Phenoxyacetlc Acid Herbicides and/or 2,3,7,8-TCDD
Stomach Cancer Deaths
Observed
Expected
Relative Risk
9554 Confidence Interval
Reference
0 0.14a
0
5 6.9a *b 0.7
0 0.5b
0
0-26.3 0 .2-1.7
0-7.4
Ott et al., 1980
RUhlmakl et al., 1978
Zack and Susklnd, 1980
aEst1mated from total cancer expected deaths (see footnote 1n text).
bEnt1re follow-up period without regard for minimum time for cancer Induc tion (Ott et a!., 1980 used a 10-year minimum Induction period).
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N1tro study contradict the findings of the Swedish and West German Investi gations previously discussed. This can be shown 1n two ways. First, the upper 95% confidence limits for the relative risk estimates from these two "negative" studies exceed even the highest point estimates of relative risk (6.1) from the two "positive" studies (see Table 11-31).
This Indicates that the relative risk estimates from the Midland and N1tro studies, even though equal to zero, are nevertheless not significantly different from the estimates of 6.1, given the sample sizes, follow-up per iods, age distribution and comparison group rates.
In addition, the smallest detectable relative risk In the Midland study (a = 0.05, <p = 0.2 one-tailed Poisson test) was 21.4 (3 observed deaths, 0.14 expected).* Similarly, the smallest detectable relative risk 1n the N1tro study (a = 0.05, <p = 0.2, one-ta1led Poisson test) was 10.0 (5 observed deaths, 0.5 expected). This calculation 1s based on results for the entire follow-up period. If, as 1n the Midland study, a minimum period of cancer Induction had been employed, the expected deaths would have been fewer and the smallest reasonably detectable relative risk would have been greater. This analysis of statistical power Indicates that the N1tro and Midland studies had very low probabilities of detecting the ~6-fold Increases In risk suggested by the Swedish and West German Investigations.
*0tt et al. (1980) did not report expected deaths from stomach cancers. The figure 0.14 was obtained by multiplying the numbers of expected deaths from all cancers (2.6, allowing a 10-year minimum Induction period) by the per centage of stomach cancers among the expected deaths 1n the N1tro study (0.5/9.04 = 5.5%). The two studies used United States white male mortality rates and covered similar calendar years In follow-up (1949-1978 1n N1tro and 1950-1976 In Midland), but a similarity In age distributions cannot be established from the published reports.
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Statistically, the study of Finnish herbicide applicators is inconsis tent with the results of the Swedish and West German cohort studies. The smallest reasonably detectable relative risk (a = 0.05, <p = 0.2, one tailed Poisson test) was only 3.1 (11 observed deaths, 3.6 expected).* The study, therefore, appears powerful enough to detect relative risks even smaller than those seen in the Swedish and West German studies. A partial explanation for this apparent Inconsistency could H e 1n the fact that the Finnish study set the minimum period of herbicide exposure for membership 1n the cohort at 10 days (2 working weeks) and noted that the "total strength of exposure has, 1n most cases, been a few weeks only." The Swedish study of herbicide applicators set the minimum exposure at 46 days (>1 spraying season).
There are also certain Inconsistencies 1n the data from the Finnish study that the authors note but find difficult to explain. In particular, no cancer deaths occurred during the latter part of the study period among Forestry Authority workers (1 of 4 groups Included 1n the cohort), even though 9.0 deaths were expected. This finding strongly suggests some defi ciency 1n follow-up or 1n 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-TCDD do not report Increased risks of stomach can cer. Only one of these, however, was statistically powerful enough to be Inconsistent with the two studies that tentatively suggest an Increase 1n stomach cancer risk. The available report of this study of Finnish herbi cide applicators contains methodologlc questions that require clarification.
*The expected stomach cancer deaths were estimated 1n the same manner as for the Midland study. A proportion of 2 0 % of all cancer deaths was applied because Finnish male mortality rates are known to be very high.
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11.2.3. Summary of Case Reports and Epidemiologic Studies. By adding together the number of workers exposed to phenoxy acids and/or chlorophenols from all case studies, an unusually high number of STSs 1s shown, consider ing the rarity of the disease. This excess 1s suggestive of an association of cancer with exposure to phenoxy acids and/or chlorophenols, and conse quently, with the Impurities found In these herbicides, Including 2,3,7,8TCDD.
Two Swedish case-control studies report highly significant association of STS with exposure to phenoxy acid and/or chlorophenols. They do not pin point the risk to the dioxin contaminants, however. In fact, In one study, the risk was found to extend to phenoxy acids free of dioxin Impurities. In that study, the risk Increases to 17 when phenoxy acids known to contain dioxin Impurities (polychlorinated 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 In the studies.
Later studies did not reveal a significant excess risk of STS. However, methodology problems make these latter studies limited with respect to evaluating the risk of STSs from exposure to phenoxy acids and/or chloro phenols and, consequently, 2,3,7,8-TCDD.
The Swedish case-control studies provide limited evidence for the carcinogenicity of phenoxy acids and/or chlorophenols In humans. However, with respect to the dioxin Impurities contained therein, the evidence for the human carcinogenicity for 2,3,7,8-TCDD based on the epidemiologic studies 1s only suggestive because of the difficulty of evaluating the risk of 2,3,7,8-TCDD exposure In the presence of the confounding effects of phenoxy acids and/or chlorophenol.
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There 1s less evidence Incriminating 2,4,5-T and/or 2,3,7,8-TCDD as the
cause of malignant lymphoma and stomach cancer 1n humans.
11.3. QUANTITATIVE ESTIMATION OF RISKS OF EXPOSURE TO 2,3,7,8-TCDD AND HxCDDs
11.3.1. Introduction. This quantitative section deals with the Incre
mental unit risk from exposure to 2,3,7,8-TCDD and HxCDDs by Inhalation and
oral routes, and their potencies relative to other carcinogens that the CAG
has evaluated. The Incremental unit risk estimate for
an air pollutant
present 1n such small quantities as the dioxins 1s defined asthe Increased
lifetime cancer risk occurring to an Individual exposedcontinuously from
birth throughout lifetime to an air concentration of 1 pg/m3 of the agent.
The unit risk from oral exposure 1s similarly defined 1n terms of either
iig/kg bw/day or 1n terms of ng/i water. These calculations are done to
estimate 1n quantitative terms the Impact of the agent as a carcinogen.
Unit risk estimates are used for two purposes: 1) to compare the carcino
genic potency of several agents with each other and 2) to give a crude
Indication of the population risk that might be associated with known (or
anticipated) air or water exposure to these agents.
The Incremental unit risks for both the Inhalation and oral routes will
be estimated from animal oral bioassays, since there are no animal Inhala
tion studies, and none of the epidemiology studies provides sufficient expo
sure Information for extrapolation purposes. The an1mal-to-man extrapola
tions for the oral route will assume equivalent absorption 1n both species.
However, the unit risk for the ambient air concentration of 2,3,7,8-TCDD
must be considered In terms of both Its 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, trlchlorophenol, sllvex and hexachloro-
phene; the application of 2,3,7,8-TCDD-contam1nated herbicides or wood
11-109
preservatives; the burning of municipal waste, wood and PCBs; and, possibly, dust from 2,3,7,8-TCDD-contam1nated soil.
Physically, 2,3,7,8-TCDD has a very low vapor pressure and 1s not normally airborne. At room temperature 1t 1s a crystalline solid, melting at 305C. When 2,3,7,8-TCDD 1s present 1n air, 1t 1s likely to be attached to particulates, to which 1t strongly binds. It has been measured 1n air only In the vicinity of burning processes and In dust from contaminated soil, and has not been found In the general air environment. 11.3.2. Procedures for the Determination of Incremental Unit Risk from Animal Data and Description of the Low-Dose Animal Extrapolation Model. Following 1s an abbreviated description of the procedures used 1n animal-to man extrapolation. A more complete description 1s given 1n Anderson et al. (1983).
In the development of quantitative estimates of carcinogenic risk from lifetime animal studies 1t Is assumed, unless evidence exists to the contrary, that If a carcinogenic response occurs at the dose levels used 1n the study, then responses will also occur at all lower doses with an Incidence determined by the dose as Indicated by the extrapolation model. While both TCDD and HxCDD cause cancer In animals at lower doses than any other known or suspect carcinogen, environmental levels are also extremely low. Thus, an extrapolation methodology must be employed.
There 1s no solid scientific basis for any mathematical extrapolation model that relates carcinogen exposure to cancer risks at the extremely low concentrations that must be dealt with 1n evaluating environmental hazards. Such low levels of risk cannot be measured directly either by animal experi ments or by epidemiologic studies.
11-110
I n th e absence of any s t r o n g ly s u g g estive evidence to the c o n t r a r y for TCDD or HxCDD, the linear nonthreshold model has been adopted as the primary basis for risk extrapolation 1n the low-dose region of the dose-response
relationship. The risk estimates made with this model should be regarded as
conservative, representing the most plausible upper limit for the risk; 1.e., the true risk 1s not likely to be higher than the estimate, but 1t could be lower.
The mathematical formulation chosen to describe the linear nonthreshold
dose-response relationship at low doses 1s the linearized multistage model.
It 1s called the linearized model because the procedure determines a linear
function, q^*, consistent with the observed data 1n a statistical sense. Thus, the multistage model procedure employs enough arbitrary constants to
be able to fit almost any monotonlcally Increasing dose-response data, and
then 1t Incorporates a procedure for estimating the largest possible linear
slope (1n the 95% upper confidence limit sense) at low extrapolated doses
t h a t 1s c o n s is te n t w ith the d a ta a t a l l dose l e v e l s o f th e e x p e rim e n t. The
multistage model has the form
where
P(d) = 1 - exp [-(qQ + q-|d + q2d2 + ... + q(<dk )]
q^ > 0, 1 = 0, 1, 2, ..., k and P(d) = the lifetime risk (probability) of cancer at dose d. Equivalently,
where
Pt(d) = 1 - exp [(q-,d + q2d2 + ... + qkdk )]
Pt(d) = P(d) - P(0) 1 - P(0)
1s the extra risk over background rate at dose d. The estimate q^* 1s the
95% upper-11m1t on q^ at lower doses. A more complete description of the
model 1s given 1n Appendix B.
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11.3.3. Selection of Data. For some chemicals, several studies 1n
different animal species, strains and sexes, each run at several doses and
different routes of exposure may be available. A choice must be made as to
which of the data sets from several studies to use 1n the model. The proce
dures used 1n evaluating these data are consistent with the approach of
making a maximum-likely risk estimate. They are listed below as follows:
1. The tumor Incidence data are separated according to organ sites or tumor types. The set of data (1.e ., dose and tumor Inci dence) used 1n the model Is the set where the Incidence 1s statistically significantly higher than the control for at least one test dose level and/or where the tumor Incidence rate shows a statistically significant trend with respect to dose level. The data set that gives the highest estimate of the lifetime carcinogenic risk, q-|*, 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 doseresponse relationship, and one has a very small sample size, the set of data having the larger sample size 1s selected for calcu lating the carcinogenic potency.
2. If there are two or more data sets of comparable size that are Identical with respect to species, strain, sex and tumor sites, the geometric mean of q-j*, estimated from each of these data sets, 1s used for risk assessment.
In some cases one or more of these studies may be negative, but the 9554 upper limit q-|* will still be greater than zero.
3. If two or more significantly Increased tumor sites are observed in the same study, and If 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.
11.3.4. Calculation of Human Equivalent Dosages for An1mal-to-Man Extrapo
lation. It 1s appropriate to correct for metabolism differences between
species and absorption factors through different routes of administration.
Following the suggestion of Mantel and Schnelderman (1977), 1t 1s
assumed that mg/surface area/day provides an equivalent dose between
species. To a close approximation, since the surface area Is proportional
11-112
to the 2 / 3 power of the weight, as would be the case for a perfect sphere, the exposure 1n mg/day per 2/3 power of the weight 1s also considered to be equivalent exposure. In an animal experiment, this equivalent dose 1s computed In the following manner. Let
Le = duration of experiment le = duration of exposure m = average dose/day 1n mg during administration of the agent
(1.e., during le ) and W = average weight of the experimental animal Then, the lifetime average exposure 1s
d = 1e x m Le x W2/3
A more expanded discussion 1s given 1n Anderson et al. (1983).
11.3.5. Alternative Methodological Approaches. The methods used by the
CA6 for quantitative assessment are consistently conservative, 1.e., tending
toward high estimates of risk. The most Important part of the methodology
contributing to this conservatism 1n 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 CA6 1n the following analysis, but
three are Included for comparison 1n the appendix. The models presented
there are the one-hit, probit and Welbull models. The CA6 feels that with
the limited data available from these animal bioassays, most of which are
conducted at high dosage levels, almost nothing 1s known about the true
shape of the dose response curve at low environmental levels. The position
1s taken by the CA6 that the risk estimates obtained by use of the linear
nonthreshold model are upper limits, and the true risk could be lower. 11-113
/
Another modification of the method described here Involves the choice of the specific animal bioassay as the basis for extrapolation. The present approach 1s to use the most sensitive responder. Alternatively, the average responses of all of the adequately tested 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 1n human pharmacological responses; 1t 1s not clear which of the two approaches Is more appropriate for carcinogens. In the absence of Information on this point, 1t seems appropriate to use the most generally accepted method, which also 1s more conservative. In the case of 2,3,7,8TCDD and HxCDD gavage studies, the use of extrapolation based on surface area rather than weights Increases the Incremental unit risk estimates by a factor of 5.8 for rats and about 13 for mice. 11.3.6. Interpretation of Quantitative Estimates. The Incremental unit risk estimate based on animal bioassays 1s an approximation to the excess risk 1n populations exposed to known carcinogen concentrations. This 1s because there may be Important species differences 1n uptake, metabolism and organ distribution of carcinogens, as well as species differences 1n target site susceptibility, Immunological responses, hormone function, and dietary factors and other diseases. The concept of equivalent doses for humans compared with animals on a mg/surface area basis has little experimental verification regarding carcinogenic response. Human populations are more variable than laboratory animals with respect to genetic constitution and diet, living environment, activity patterns and other cultural factors.
11-114
The unit risk estimate can give an Indication of the relative r e s p o n s e
per unit dose ("potency") of a given agent compared with other carcinogens.
The comparative potency of different agents should be more reliable when the
comparison 1s based on studies 1n the same test species, strain and sex, and
by the same route of exposure.
The quantitative aspect of the carcinogen risk assessment 1s Included
here because 1t may be of use 1n the regulatory decision-making process, for
example, setting regulatory priorities and evaluating the adequacy of
technology-based controls. However, the estimation of cancer risks to
humans at low levels of exposure 1s uncertain. At best, the linear extrapo
lation model used here provides a rough but plausible estimate of the upper
limit of risk; 1.e., 1t Is 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 1n subsequent sections should not be regarded as an
accurate representation of the true cancer risks even when the exposures are
a c c u r a t e l y d e f i n e d . The e s tim a te s presented may be fa c to r e d I n t o r e g u l a t o r y
decisions to the extent that the concept of upper risk limits 1s found to be
useful.
11.3.7. Incremental Unit Risk Estimates for 2,3,7,8-TCDD via the Oral and
Inhalation Routes. The positive animal cancer data available for calculat
ing an Incremental unit risk estimate for 2,3,7,8-TCDD are presented 1n
Appendix B 1n Tables B-l through B-5. These are as follows:
1. The Dow (1978) diet study on Sprague-Dawley rats, Spartan substrain. Significantly Increased cancers 1n the males Includ ed stratified squamous cell carcinomas of the tongue and squa mous cell carcinomas of the nasal turbinates and hard palate. Both the original pathological analysis (Koclba) and that of an Independent reviewer (Squire) are presented (Table B-l). Significant cancers 1n the females Included lung, nasal turbi nate and hard palate cancers, and Uver tumors (Table B-2). As with the males, the total number of animals with at least one of these significant tumors was recorded.
11-115
2. The NCI gavage study 1n Osborne-Mendel rats and B6C3F1 mice.
a. 2,3,7,8-TCDD 1n male rats caused an Increase 1n follicular 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 neoplastic 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 fibrosarcomas, lymphomas or leukemias of the hematopoietic system, Uver hepatocellu lar carcinomas and adenomas, and thyroid follicular cell adenomas (Table B-5).
The above data have been fit by the linearized multistage model
described 1n Section 11.3.2. These results are presented 1n Appendix B 1n
some detail "1n Tables B-6 through B-12, and summarized In Table 8-13. The
results of all estimates are within an order of magnitude, with the upper
limit estimates lowest for the Dow male rats, higher for the NCI study, both
rats and mice, and highest for the combined tumor sites of the female rats
1n the Dow study. The data from which the steepest slope factor (q-j*)
(1.e., greatest potency) was calculated were from the Squire review of the
slides. A summary of Squire's review 1s presented 1n Table B-2 and the
results of the linearized multistage model extrapolation procedure are
presented 1n Table B-9. An examination of Table B-9 shows that the high
dose group 1n the study was eliminated because Its Inclusion resulted 1n a
poor fit of the model (p<0.01). A second analysis of the female rat data
adjusted for early Increased mortality 1n the high-dose group by eliminating
all animals that died during the first year, so that the first tumors con
sidered were those detected during the 13th month of the study. The results
of the analysis from this adjustment are presented 1n Tables B-8A and B-9A.
11-116
The results yield acceptable fits of the data without dropping the responses at the highest dose levels, and these results were chosen for the final Incremental unit risk estimates. The slope estimates for the Kociba (Table B-8A) and Squire (Table B-9A) analyses, 1.51x10s and 1.61x10s (mg/kg/ day)-1, were averaged by taking the geometric mean, and the final estimate thus becomes
q.|* = [(1.51 x 10s) x (1.61 x 10s)]1/2 = 1.56 x 10s (mg/kg/ day)-1. This estimate is about one-third that derived from the Squire review in Table B-8.
This upper-limit estimate represents a range of uncertainty that is 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 estimatebased 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"cufve^ at low dose levels. It could also be argued on the basis of a saturation effect of either dose or response; the data can partially support either hypothesis. An adjustment of the multi stage modelneeded to incorporate such an effect or effects, however, is felt to be unwarranted by the sparsity of the supporting evidence. As an alternative, to incorporate this uncertainty, a range of 95% upper-limit estimates of q^* = 9.0xl04 to 4.25x10s (mg/kg/day)-1 has been chosen to accommodate this unusual data set.
In order to estimate an incremental unit risk for a 1 ng/8. concentra tion in drinking water, the following conversion is used:
1 yg/kg/day x 70 kg x 103 ng/yg x 1 day/2 a = 3.5 x 104 ng/l
11-117
based on human consumption of 2 a water/day for a lifetime. Therefore, the Incremental unit risk corresponding to 1 ng 2,3,7,8-TCDD/i water 1s
qi* = 1.56xl02 (yg/kg/day)-1 x 1 yg/kg/day = 4 5xl0-3 (ng/^-i 3.5xl04 ng/i
Similarly, the lower and upper limits of the range vary from q^* = 2.6xl0-a to 1 ,2xl0"2 (ng/a)-1.
This Incremental unit risk estimate from an oral study must be trans formed before an estimate can be made from exposure to 2,3,7,8-TCDD 1n the ambient air. Exposure will be assumed to occur only through respiration of 2,3,7,8-TCDD-contam1nated particulates. The amount of exposure depends on the particulate size distribution. Based on the report of the International Commission on Radiological Protection (ICRP, 1959), 1t can be assumed that 100% of particulates of <0.1 micron 1n size pass the nasopharyngeal (upper respiratory tract) barrier and are deposited on the tracheobronchial and alveolar passages. For the larger-sized particles, the percentage deposi tion of 5-m1cron particles In the lower respiratory tract 1s not more than 30%. Even those larger particles retained by the upper respiratory tract, however, may be swallowed and eventually absorbed by Ingestion. In the absence of specific data on the size distribution and eventual fate of the particles, the Information developed by the ICRP, Committee 2, will be used. The Committee developed the following estimates for retention of particulate matter 1n the lungs. For compounds not readily soluble, 25% will be exhaled, 50% will be deposited 1n the upper respiratory passages and subse quently swallowed, and the final 25% will be deposited 1n the lungs (lower respiratory passages). Of this final 25%, half 1s eliminated from the lungs and swallowed 1n the first 24 hours, making a total of 62.5% swallowed; the remaining 12.5% remains In the lung alveoli for long periods of time; eventually some are transferred to pulmonary lymph nodes.
11-118
If we take a worst-case estimate and assume that all of the swallowed
material 1s eventually absorbed Into the body, then 75% of the Inhaled mate rial will be absorbed. We further assume a breathing rate of 20 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-j*( resp.) = 1.56 x 10s (mg/kg/day)-1 x 1 x 10"9 mg/pg x .75 x 20 m3/70 kg or q-|*(resp.) = 3.3 x 10"5 (pg/m3)-1.
S1m1lary, the range of estimates 1s 1.9 x 10- 5 to 9.1 x 10"5 (pg/m3)"1 .
11.3.8. Incremental Unit Risk Estimate for HxCODs (1,2,3,6,7,8 and 1,2,3,7,8,9) Via the Oral and Inhalation Routes. The results of the National Toxicology Program (NTP) gavage study on a mixture of 1,2,3,6,7,8and 1 ,2,3,7,8,9-HxCDD showed positive results for male and female rats (com
bined liv e r neoplastic nodules or h e p ato ce llu lar carcinomas), the greater
response being In the females. In the females, carcinomas appeared only In the high-dose group. In the male rats, there was also a definite trend 1n neoplastic nodules and carcinomas combined, but this was only marginally significant. These results are presented 1n Table 11-35, which Includes the recent NTP rvaluation of the female rat Uver slides. The review shows responses 1n the range of 50% less than that of the original analysis. The responses for neoplastic nodules and combined nodules and carcinomas are still statistically significant. These results have been detailed 1n the qualitative section of this document.
11-119
TABLE 1 1 -3 5
NTF HxCDD (Gavage) Bioassay (NTP, 1980d) Osbome-Hendel Rats (2 years)
Incidences of Neoplastic Nodules and Hepatocellular Carcinomas
Tumor
Vehicle Control
Untreated Control
Low-Dose 1.25
uq/kq/week
Mid-Dose 2.5
High-Dose 5
Estimates3 of q-|*
(wg/kg/day)_1
Number of animals examined
Hepatocellular carcinoma (HC)
Neoplastic nodule (NN)
HC + NN combined
Human equivalent dose wg/kg/day
74 0 0 0 0
HALE (700 g)b
75 49
00
2(3%)
0
2(354)
0
0 0.04
50 0 1(2%) 1(254) 0.08
48 1(254) 3(654) 4(854)c 0.15
-- -- 5.6xl0-1 5.9xl0_1 --
11-120
TABLE 11-35 (cont.)
L2L" LL
Tumor
Vehicle Control
Untreated Control
Low-Dose 1.25
uq/kq/week
M1d-Dose 2.5
H1gh-Dose 5
Estimates3 of q-j*
(wg/kg/day)-1
Number of animals examined
Hepatocellular carcinoma (HC)
Neoplastic nodule (NN) HC 4- NN combined
Human equivalent dose vg/kg/day
75 0
2(3%)
2(3%) 0
FEMALE (450 g)d
73 50
00
1(1%) 1(1%)
0
5(10%) 5(10%)
0.03
50 0
7(14%)c 7(14%)c
0.06
50 2(4%) 16(32%)e 18(36%)e 0.12
-- 3.2xl0_1 3.3 3.5
--
a95% upper-Umlt estimate of linear term 1n the multistage model based on human equivalent dosages using surface area correction.
^Analysis by NTP (1980d)
cp<0.05 versus vehicle-control
^Rvaluation by Hlldebrandt (1983)
ep<0.001 versus vehicle-control
In female mice, there was a dose-related trend In hepatocellular carci nomas, but only the combined adenomas and carcinomas were significant. In male mice, there was a minor trend 1n hepatocellular adenomas, but no In crease, statistical or otherwise, 1n hepatocellular carcinomas (Table 11-36).
Although no statistically significant Increase In carcinomas occurred In mice or rats of either sex, when neoplastic nodules 1n the rats and hepato cellular adenomas In fhe mice were Included 1n the data, the results became significant for all groups. These combined results were then fitted to the multistage model for all four groups. As shown In Tables 11-35 and 11-36, the 9554 upper-limit unit risk estimates are as follows:
Rat - male q-|* = 0.59 (vg/kg/day)-1 female q-|* = 3.5 (vg/kg/day)-1
House - male q-|* = 11.0 (vg/kg/day)-1 female q^* = 2.9 (vg/kg/day)-1
The usual CA6 procedure Is to use the most sensitive sex-species for estimating the 95% upper-limit unit risk. Under that procedure, which Is based on the linearized multistage model with surface area correction for an1mal-to-man extrapolation, the male mouse data base yielding a q ^ = 11.0 (vg/kg/day)-1 would be selected to provide the upper limit estimate of potency. However, as examination of Tables 11-35 and 11-36 show, there are several reasons to give weight to the female rat data base also. These are as follows: 1) low spontaneous (control) rates In the rat vs. the male mouse Uver; 2) statistically significant Increases 1n both the mid and high level dose groups vs. control for the female rat; the male mouse response was significant only at the high dose; 3) a more distinct dose response trend 1n the female rat vs. the male mouse; and 4) the only hepatocellular carcinomas 1n the female rat were In the high dose group. There were none 1n 148 control animals. By comparison, the male mouse showed no clear trend In carcinomas.
11-122
IHULL I I - J U
NTP HxCDD (Gavage) Bioassay (NTP, 1980d) B6C3F1 Mice (104 weeks)
Incidences of Adenomas and Hepatocellular Carcinomas
11-123
Tumor
Vehicle Control
Untreated Control
Low-Dose 1.25
uQ/kq/week
M1d-Dose 2.5
H1gh-Dose 5
Estimates of q-|*a (vg/kg/day)"1
Number of animals examined
Hepatocellular carcinoma (HC)
Hepatocellular adenoma (HA)
Combined HA and HC
Human equivalent dally dose (vg/kg/day)
73 8(11%)
7(1054)
15(2154) 0
75 12(1654)
15(2054)
27(3654) 0
HALES 50
9(1854)
5(1054)
14(2954) 0.014
49 5(1054)
9(1854)
14(2954) 0.027
48 9(1954)
15(3l54)b
24 (5054)c 0.054
-- 3.71
6.99
11.00 --
TABLE 11-36 (cont.)
Tumor
Vehicle Control
Untreated Control
Low-Dose 2.5
uq/kq/week
Mid-Dose 5.0
High-Dose 10.0
Estimates of q-|*a (vg/kg/day)_1
Number of animals examined
Hepatocellular carcinoma (HC)
Hepatocellular adenoma (HA)
Combined HA and HC
Human equivalent dally dose Ug/kg/day)
73 1(1%)
2(3%)
3(4%) 0
FEMALES 74 48
00
2(3%)
4(8%)
2(3%) 0
4(8%) 0.027
47 2(4%)
4(9%)
6(13%) 0.054
47 2(4%)
9(19%)b
10(23%)b 0.107
--
9.5xl0_1
2.61
2.94
--
a95% upper-limit estimate of linear term In the multistage model based on human equivalent dosages using surface area correction.
bp<0.01 versus vehicle-control
cp<0.001
11-124
In addition to the above reasoning, we point to the uncertainty of the surface area correction. Nearly all the quantitative Increase 1n the estimate of the 95% upper limit risk of the male mouse vs. the female rat (11.0/3.5 = 3.1) can be attributed to the surface area correction 1n the extrapolation procedure, which 1s greater for mice than for rats by a 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 Its procedure slightly and to take the geometric mean of the 95% upper-Umlt estimates from the male mouse and the female rat. The final estimate 1s
q-j* = (3.5x11.0)1/2 = 6.2 (vg/kg/day)_1 In terms of exposure to 1 yg/i. of HxCC contaminate and 2 i/day for a lifetime, we use the same assumptions as with 2,3,7,8-TCDD:
1 vg/kg/day = 3.5x10* ng/l. Thus, for 1 ng/l 1n the drinking water the estimate of Incremental risk 1s
nP = .1-e-6.2/3.5x10"* 1.8x10"* In terms of continuous lifetime exposure to ambient air containing 1 pg/ni3 HxCDD, the transformation as was done before with 2,3,7,8-TCDD, 1s q.|*(HxCDD) (resp.) = 6.2xl03 (mg/kg/day)-1 x 1x10" mg/pg x 0.75x20 m3/70 kg
q1*(HxCDD) (resp.) = 1.3 x 10"* (pg/m3)'1 . 11.3.9. Relative Potency. One of the uses of unit risk 1s to compare the relative potencies of carcinogens. Potency 1s defined for this purpose as the linear portion of the dose-response curve, which was used to calculate the unit risk factors. To estimate the relative potency on a per-mole
11-125
basis, the unit risk slope factor 1s multiplied by the molecular weight, and the resulting number 1s expressed 1n terms of (mMol/kg/day)-1. This Is called the "relative potency Index."
Figure 11-2 Is a histogram representing the frequency distribution of potency Indices of 55 chemicals evaluated by the CAG as suspect carcinogens. The actual data summarized by the histogram are presented 1n Table 11-37. Where human data are available for a compound, they have been used to calcu late the Index. When no human data are available, animal oral studies have been used 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 Uver, lung and nasal turbi nate and hard palate tumors 1n the female rat 1n the Dow 2,3,7,8-TCDD feed ing study (Koclba et al. (1978a) 1s 5xl07 (mMol/kg/day)-1. This number 1s derived by multiplying as follows: the 95% upper-Umlt slope estimate from the Dow study using the geometric mean of the Squire and Koclba anal yses, q^* = 1.56x10s (mg/kg/day)-1, by the molecular weight of 322. Rounding off to the nearest order of magnitude gives a log 10 value of 8, which 1s the scale presented on the horizontal axis of Figure 11-2. The Index of 5xl07 1s the most potent of 55 chemicals that 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 HxCDD, based on combined hepatocellu lar adenomas and carcinomas In male mice 1n the NTP gavage study (NTP, 1980d), and combined nodules and hepatocellular carcinomas 1n female rats by gavage (NTP, 1980d) 1s 2.4x10*^ (mMol/kg/day)-1. This 1s derived by
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4th QUARTILE ,
3rd QUARTILE
2nd QUARTILE
1st QUARTILE
1 * IO"1
4 * 10*2
2 * 10*3
FREQUENCY
FIGURE 11-2 Histogram Representing the Frequency Distribution of the Potency Indices
of 55 Suspect Carcinogens Evaluated by the Carcinogen Assessment Group
11-127
TABLE 11-37
Relative Carcinogenic Potencies Among 55 Chemicals Evaluated by the Carcinogen Assessment Group as Suspect Human Carcinogens
8ZI-LI
Compounds
CAS Number
Acrylonitrile
Aflatoxln B-|
Aldrln Ally! chloride
Arsenic B[a]P
Benzene Benzldene Beryllium 1,3-Butadiene
Cadmium Carbon tetrachloride Chlordane Chlorinated ethanes
1,2-Dlchloroethane Hexachloroethane 1,1,2,2-Tetrachloroethane 1,1,2-lrlchloroethane Chloroform Chromium VI
DDT
Dlchlorobenzldlne
107-13-1 1162-65-8
309-00-2 107-05-1 7440-38-2
50-32-8 71-43-2 92-87-5 7440-41-7 106-99-0 7440-43-9 56-23-5 57-74-9
107-06-2 67-72-1 79-34-5 79-00-5 67-66-3
7440-47-3 50-29-3 91-94-1
Level of Evidence3
Humans
Animals
Ls Ls
IL
SI IS SS
sS
LS IS LS IS IL
IS IL IL IL IS SS
IS IS
Grouping
Based on IARC
Criteria
Sloped (mg/kg/day)"
2A 0.24 (H) 2A 2900 3 11.4
1.19x10' 1 15 (H) 2B 11.5 1 2.9x10" (U) 1 234 (U) 2A 2.6 (W) 2B I.OxIO'i (I) 2A 6.1 (W) 2B 1.30x10' 3 1.61
2B 9.2x10' 3 1.42x10' 3 0.20 3 5.73x10'
2B 8.1x10"
1 41 (U)
2B 0.34
2B 1.69
Molecular Weight
53.1 312.3 369.4
76.5 149.8 252.3
78 184.2
9 54.1 112.4 153.8 409.8
98.9 236.7 167.9 133.4 119.4 100 354.5 253.1
Potency Indexc
1x10*1 9x10*5 4x10*3 9x10' 2x10*3 3x10*3 2x10 4x10** 2x10*1 5x10 7x10*2 2x10*1 7x10*2
9x10 3x10 3x10*1 8x10 1x10 4x10*3 1x10*2 4x10*2
Order of Magnitude (logic Inde
*1 6 4
0 3
0 5 1 *1 3 1 3
*1 0
*1 1 1 4
2
3
lAtfLt 11-4/ iconi.j
11-129
Compounds
1,1-Dichloroethylene (VinylIdene chloride)
Dlchloromethane (Methylene chloride)
Dleldrln
2,4-Dlnltrotoluene
Dlphenylhydrazlne
Eplchlorohydrln
B1s(2-chloroethy1)ether
B1s(chloromethyl)ether
Ethylene dibromide (EDB)
Ethylene oxide
Heptachlor
Hexachlorobenzene
Hexachlorobutadldne
Hexachlorocyclohexane technical grade alpha Isomer beta Isomer gamma Isomer
Hexachlorodlbenzodloxln 1.2.3.6.7.8- and 1.2.3.7.8.9-
Nickel refinery dust Nickel subsulftde
CAS Number
Level of Evidence3
Humans
Animals
Grouping
Based on IARC
Criteria
Slopeb (mg/kg/day)"1
75-35-4
I
L
3 1.16 (I)
75-09-2
1
S
2B 1.4x10" (I)
60-57-1
I
S
2B 30.4
121-14-2
I
s
2B 0.31
122-66-7
I
s
2B 0.77
106-89-8
I
s
2B 9.9x10"
111-44-4
I
s
2B 1.14
542-88-1
S
s
1 9300 (I)
106-93-4
I
s
2B 41
75-21-8
L
s
2A 3.5x10" (I)
76-44-8
I
s
2B 3.37
118-74-1
I
s
2B 1.67
87-68-3
I
L
3 7.75x10"
319-84-6 319-85-7
58-89-9
34465-46-8
I I I
I
S L L
S
4.75 2B 11.12 3 1.84 3 1.33
2B 6.2x10*3
0120-35-722
S
s
S S
1 1.05 (H) 1 2.1 (M)
Molecular Height
97
84.9
380.9 182 180
92.5 143 115 187.9
44.1 373.3 284.4 261
290.9 290.9 290.9 290.9 391
Potency Indexc
1x10*2
lxl 0
1x10** 6x10*1 1x10*2 9x10" 2x10*2 lxlO*6 8xl0*3 2x10*1 1x10*3 5x10*2 2x10*1
lxl0*J 3x10*3 5x10*2 4x10*2 2x10*b
Order of Magnitude (loQio Inde)
2
0
4 2 2
0
J
2 6 4 1 3 3 1
3 3 3 3 >6
240.2 240.2
2.5x10*2 5.0x10*2
2
*3
TABLE 11-37 (cont.)
oei-ii
Compounds
CAS Number
Level of Evidence3
Humans
Animals
Grouping
Based on IARC
Criteria
Slopeb (mg/kg/day)'a
Molecular Height
Potency Indexc
Order of Magnitude (logio Index)
Nltrosamlnes Dlmethylnltrosamlne Dlethylnltrosamlne Dlbutylnltrosamlne N-nltrosopyrrolldlne N-nltroso-N-ethylurea N-nltroso-N-methylurea N-nltroso-dlphenylamlne
PCBs
Phenols 2,4,6-Trlchlorophenol
2,3,7,8-Tetrachlorodlbenzo-pdioxin (1CDD)
Tetrachloroethylene
Toxaphene
Trichloroethylene
Vinyl chloride
62-75-9 55-18-5 924-16-3 930-55-2 759-73-9 684-93-5 86-30-6
1336-36-3
88-06-2
1746-01-6
127-18-4
8001-35-2
79-01-6
75-01-4
I I I I I I I
I
I
I
I
I
I
S
S
28 25.9 (not by qj*)
74.1
2xl0*3
S
28 43.5 (not by q-|*)
102.1
4xl0*3
S 2B 5.43
158.2
9x10*2
S 2B 2.13
100.2
2xl0*2
S 2B 32.9
117.1
4x10*3
S 2B 302.6
103.1
3x10**
S 2B 4.92x10'
198 lxlOG
S 28 4.34
324 1x10*3
S 2B 1.99x10' S 2B 1.56X10*5
197.4 322
4x10 5x10*7
L 3 5.1x10'
S 2B 1.13
L/S
3/2B
1.1x10'
S 1 1.75x10' (I)
165.8 414 131.4
62.5
8x10 5x10*2 1x10 1x10
3 *4 *3 *2 *4 4 0
3
1, 8
*1
*3
0
0
aS = Sufficient evidence; L * Limited evidence; 1 = Inadequate evidence
bAn1mal slopes are 95% upper-bound slopes based on the linearized multistage model. They are calculated based on animal oral studies, except for those Indicated by I (animal Inhalation), W (human occupational exposure) and H (human drinking water exposure). Human slopes are point estimates based on the linear nonthreshold model. Not all of the carcinogenic potencies presented In this table represent the same degree of certainty. All are subject to change as new evidence becomes available. The slope value Is an upper bound In the sense that the true value (which Is unknown) Is not likely to exceed the upper bound and may be much lower, with a lower bound approaching zero. Thus, the use of the slope estimate In risk evaluations requires an appreciation for the Implication of the upper bound concept as well as the "weight of evidence" for the likelihood that the substance Is a human carcinogen.
cThe potency Index Is a rounded-off slope In (mmol/kg/day)"1 and Is calculated by multiplying the slopes In (mg/kg/day)' by the molecu lar weight of the compound.
mu7t1p7ying the mean 95% upper-limit slope factor
= 6.2x10 (mg/kg/
day)-1 by the molecular weight, 391. This potency is about one-twentieth
that of 2,3,7,8-TCDD, making it the second most potent of 55 chemicals that
the CAG has evaluated as suspect carcinogens.
The ranking of relative potency indices is subject to the uncertainties
involved in comparing a number of potency estimates for different chemicals
based on varying routes of exposure in different species, using data from
studies whose quality varies widely. Furthermore, all the indices are based
on estimates of low-dose risk using linear extrapolation from the observa
tional range. These indices are, therefore, not valid for the comparison of
potencies in the experimental or observation range if linearity does not
exist there. Nevertheless, the potency rankings of one and two for these
dioxins cannot be easily dismissed.
11.4. SUMMARY AND CONCLUSIONS
11.4.1. Summary.
11.4.1.1.
QUALITATIVE ASSESSMENT OF 2,3,7,8-TCDD -- There are several
chronic animal cancer bioassay studies of 2,3,7,8-TCDD: 1) a Dow Chemical
Company (Kociba et al., 1977, 1978a) study in male and female Sprague-Dawley
(Spartan substrain) rats; 2) the Van Miller et al. (1977a,b) study in male
Sprague-Dawley rats; 3) the Toth et al. (1979) study in Swiss mice; 4) the
National Toxicology Program (1980a,b) studies in rats and mice; 5) the Pitot
et al. (1980) promotion study in rats; and 6) the Kouri et al. (1978)
cocarcinogenicity study in mice.
The 1978 study by the Dow Chemical Company of male and female Sprague-
Dawley rats fed 2,3,7,8-TCDD in doses of 22, 210 and 2200 ppt showed a
highly statistically significant excess of hepatocellular carcinomas in
female rats at the highest dose level and hepatocellular carcinomas and
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hepatocellular hyperplastic nodules In female rats at both the middle and high dose levels, as compared with the controls. In addition, at the high dose there were significant Increases In carcinomas of the hard palate/nasal turbinates 1n both males and females, of the tongue 1n males, and of the lungs 1n females. The Van Miller et al. (1977a,b) study also showed some evidence of a carcinogenic response 1n the Uver and lungs of male SpragueDawley 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 Uver tumors 1n male mice (females were not tested) receiving 0.7 yg/kg/week by gavage.
In the National Cancer Institute rat study (NTP, 1980a), male and female Osborne-Mendel rats were administered 2,3,7,8-TCDD by gavage at three dose levels: 0.01, 0.05 and 0.5 yg/kg/week. 2,3,7,8-TCDD Induced statistic ally significant Increases of hepatocellular carcinomas, subcutaneous fibro sarcomas and adrenal cortical adenomas In high-dose female rats. 2,3,7,8TCDD also Induced significant Increases of thyroid tumors In male rats at all dose levels.
In a companion mouse study by the National Cancer Institute (NTP, 1980a), male and female B6C3F1 mice were given 2,3,7,8-TCDD by gavage at dose levels of 0.01, 0.05 and 0.5 yg/kg/week for males and 0.04, 0.2 and 2.0 yg/kg/week for females. 2,3,7,8-TCDD Induced statistically slgnlfl-
/' cant Increases of hepatocellular carcinomas In the high-dose males and females, and thyroid tumors, subcutaneous fibrosarcomas and histiocytic lymphomas 1n females.
In the study by Pitot et al. (1980), 2,3,7,8-TCDD has been shown to be a potent Uver cancer promoter after Initiation with dlethylnltrosamlne.
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S e v e r a l 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 strain. In the study by Kourl et al. (1978), 2,3,7,8-TCDD has been shown to be a potent cocarcinogen with 3-methyl chloranthrene.
2,3,7,8-TCDD 1s a potent Inducer of arylhydrocarbon hydroxylase (AHH) 1n mammals. The AHH contains enzyme epoxldase that 1s known to mediate the formation of epoxides, that are potentially active carcinogenic metabolites. 2.3.7.8- TCDD may be metabolized In mammalian species by the reactive epoxide Intermediate to dlhydrodlol and further conjugated. 2,3,7,8-TCDD was found 1n Uver and fat at the end of the 2-year rat feeding study. Significant covalent binding of 2,3,7,8-TCDD (14C or tritium) derived radioactivity with protein has been demonstrated. Covalent binding of 2,3,7,8-TCDD (14C or tritium) derived radioactivity with DNA 1s not significant In liver cells.
Currently available studies on the mutagenicity of 2,3,7,8-TCDD are inconclusive. Two bacterial systems, Escherichia coll and S. typhlmuHum (without metabolic activation), exhibited positive mutagenic activity. However, In another study of S. typhlmuHum (with and without metabolic activation), the results were negative.
Several epidemiological studies have been conducted that are relevant to the carcinogenicity assessment of 2,3,7,8-TCDD. Two Swedish epidemiologic case-control studies (Hardell and Sandstrom, 1979; Eriksson et al., 1979, 1981) reported a significant association between STSs and occupational exposure to phenoxyacetlc acid herbicides and/or chlorophenols that contain 2.3.7.8- TCDD as an Impurity. These studies Indicated ~5-fold to 7-fold Increases In the risk of developing soft-tissue sarcomas among people exposed only to phenoxyacetlc acids and/or chlorophenols In comparison with people not exposed to these chemicals. The associations are high enough to
11-133
make 1t unlikely that they have resulted entirely from random variation bias or confounding, although the possibility exists that recall bias may account for a small part of the excess; but not enough to account for the exces sively high risks. When an attempt was made to separate exposures Into two categories based on expected presence or absence of polychlorinated dlbenzoj)-d1ox1n Impurities, the relative risks were 17 and 4.2, respectively. This Indicates that agents themselves, without the dioxin Impurities, may be con tributing to the risk of STSs as well. The nonpositive studies that seem ingly do not support the finding of an elevated risk of cancer, specifically STS, suffer from a variety of methodological problems that will make such a risk Impossible to detect In some and difficult to detect In others. Sev eral of these require many more years of follow-up before a significant elevated risk of the relatively rare STS Is found. Within this group of nonpositive studies are several where evidence of exposure to 2,3,7,8-TCDD Is questionable at best and as such no elevated risk of STS will ever be found. On the other hand, several small-scale cohort studies with proven evidence of exposure to chemicals containing 2,3,7,8-TCDD have produced a small number of the relatively rare STS that certainly would not have been expected at the time. However, several epidemiologic studies are now In progress, the results of which are not yet available, that will provide additional epidemiologic evidence that may Influence our conclusions at a later time. 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 acids.
Two cohort studies, one by Axelson et al. (1980) and the other by Thless and Frentzel-Beyme (1978) provide suggestive evidence that phenoxyacetlc acids and/or 2,3,7,8-TCDD Increase the risk of stomach cancer 1n humans.
11-134
Four other cohort studies by Ott et al. (1980), R11h1mak1 et al. (1978),
Cbok et al. (1980) and Zack and Susklnd (1980) Indicated no significantly
Increased risk of stomach cancer 1n people exposed to phenoxyacetlc acids
and/or chlorophenols, but two of these studies were of relatively low
statistical power, and another study has certain Inconsistencies requiring
clarification.
11.4.1.2.
QUALITATIVE ASSESSMENT OF HxCDD -- Hexachlorod1benzo--
dloxln has been tested for carcinogenicity 1n rats and mice by gavage (NTP,
1980d) and by dermal application to mice (NTP, 1980b,c). In these studies,
a 1:2 mixture of 1,2,3,6,7,8- and 1 ,2,3,7,8,9-HxCDD was tested. In the oral
study, animals received HxCDD at doses of 0.0, 1.25, 2.5 or 5.0 yg/kg/
week, except for female mice, which received 0.0, 2.5, 5.0 and 10.0 yg/kg/
week. In both species and both sexes, only tumors of the Uver occurred at
a significantly greater Incidence than 1n controls. In male rats and male
and female mice, the liver tumor Incidence was significantly Increased over
control values only in the high-dose groups, while in female rats the In c i
dence was significantly greater at both the medium- and high-dose levels.
At the request of EPA this study was audited during May-August 1985 by
several scientists as to the pathologic evaluation and conduct of the study.
The scientists have reconfirmed the NTP conclusions that the study provides
carcinogenic evidence 1n both rats and mice. In the study of HxCDD carcino
genicity 1n mouse skin conducted by NTP (1980c), there were no treatment-
related tumors In either the carcinogenicity bioassay or the tumor promotion
assay using DMBA as an Initiator. There are no available epidemiologic
carcinogenicity studies 1n the published literature for HxCDD as the sole
compound of concern. The mutagenic potential for HxCDD 1s unknown since no
tests are reported 1n the available literature.
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11.4.1.3.
QUANTITATIVE ASSESSMENT OF 2,3,7,8-TCDD AND HxCDD -- Quanti
tative estimates of the potential carcinogenic Impact on humans, due to both
oral and Inhalation exposure to both 2,3,7,8-TCDD and HxCDD, have been
calculated. These estimates are all based on an1mal-to-human extrapolation
procedures. The animal gavage and feeding studies provide the only data
base for estimating the carcinogenic potency (unit risk) for 2,3,7,8-TCDD
and HxCDD. While the epidemiology studies provide positive, although
limited, evidence for carcinogenicity, the population exposures are unknown
and the findings cannot be attributed to exposure to 2,3,7,8-TCDD alone.
Thus the Ingestion unit risks as well as the estimates for Inhalation unit
risk are derived from the gavage and feeding studies.
There 1s Insufficient metabolism and pharmacokinetic Information to
alter the typically used assumptions regarding dose extrapolation. The
reported 1ntragastr1c absorption for 2,3,7,8-TCDD 1n rats varies from
52-86%; there are no absorption data for HxCDD. The assumptions used 1n
both the TCDD and HxCDD unit risk estimates assume that human absorption by
oral exposure 1s equal to that of the rat. Information regarding absorption
by Inhalation 1s totally lacking and 1s assumed to be 75% based on an ICRP
(1959) lung uptake model. The upper limit unit risks were calculated using
a multistage extrapolation model that 1s linear at low doses as programmed
1n GLOBAL 79.
For cancer risk due to oral exposures, the upper-Umlt quantitative
Incremental unit risk estimate Is q^* = 1.56xl0_1 (ng/kg/day)_1,
derived from the Koclba et al. (1977, 1978a) 2,3,7,8-TCDD feeding study 1n
female rats that Induced a statistically significant Increased Incidence of
tumors 1n the Uver, lungs, hard palate and nasal turbinates. Based on
continuous lifetime exposure to 1 ng/a 2,3,7,8-TCDD In drinking water, the
11-136
9 5 % upper limit estimate of Individual Incre m e n ta l cancer risk 1s 4.5x70"8 with a range of upper limit values of 2.6xl0-3 to 1.2xl0'2, depending upon pathological Interpretation and mortality correction. Based on contin uous lifetime exposure to 1 pg/m3 2,3,7,8-TCDD 1n ambient air, the 95% Upper-Umlt estimate of Individual Incremental cancer risk 1s 3.3xl0-5, with a range of upper-Umlt estimates of 1.9x10~5 to 9.1x10-s depending upon pathologic Interpretation and mortality correction. Since the Inhala tion unit risk values are based upon the observed Incidence 1n the feeding study, an Implicit assumption 1s made that 2,3,7,8-TCDD 1s as potent by Inhalation as by Ingestion exposure.
An upper-Umlt Incremental unit risk estimate for a mixture of HxCDDs has been calculated from the NCI gavage study (NTP, 1980d). Based on com bined Uver heptacellular carcinomas and neoplastic nodules 1n female rats, and hepatocellular adenomas and carcinomas 1n male mice, q^* = 6.2xl0"3 (ng/kg/day)_1. A continuous lifetime exposure to 1 ng/n of HxCDD 1n drinking water Is estimated to result 1n an upper limit Incremental unit risk of 1.8xl0-4. Similarly, for ambient air, a continuous lifetime exposure to 1 pg/m3 of HxCDD 1s estimated to yield an upper-Umlt unit risk of 1.3x10".
The cancer potency of 2,3,7,8-TCDD as represented by a potency Index 1s also estimated relative to 54 other chemicals which the CA6 has evaluated as carcinogens. The relative potency Index 1s 5xl07 (mMol/kg/day)-1, making 2,3,7,8-TCDD the most potent animal carcinogen evaluated by the CA6. 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.
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Tl.4.2. Conclusions. There 1s evidence from chronic animal cancer bio assay studies that 2,3,7,8-TCDD and HxCDD are probable human carcinogens. There are no chronic animal cancer bioassay studies available that evaluate the carcinogenic potential for other polychlorinated d1benzo-p-d1ox1n com pounds. The available data for 2,3,7,8-TCDD and HxCDD come from gavage and feeding studies, there being no studies available for Inhalation exposure. The epidemiologic evidence for the carcinogenicity of 2,3,7,8-TCDD alone 1s Inadequate, and there have been no epidemiologic studies, as yet, for HxCDD as the sole compound of concern.
2,3,7,8-TCDD has Induced hepatocellular carcinomas 1n two strains of female rats and both sexes of one mouse strain, along with the Induction of thyroid tumors, subcutaneous fibrosarcomas and tumors of the lung, nasal turb1nates/hard palate 1n male rats, and tongue tumors 1n female rats. These effects notably occur at extremely low doses. There 1s evidence that 2.3.7.8- TCDD 1s also a promoter and a cocarcinogen. The evidence of carcinogenicity for 2,3,7,8-TCDD 1n animals 1s regarded as "sufficient" using the ERA Interim we1ght-of-ev1dence classification system for carcino gens (U.S. EPA, 1984).
The human evidence for the carcinogenicity of 2,3,7,8-TCDD alone 1s regarded as "Inadequate" using the EPA classification criteria, because of the difficulty of attributing the observed effects solely to the presence of 2.3.7.8- TCDD that occurs as an Impurity 1n the phenoxyacetlc acids and chlorophenols. However, the human evidence for the carcinogenicity of chlorinated phenoxy acetic herbicides and/or chlorophenols with chlorinated d1benzod1ox1n Impurities 1s judged to be "limited" according to the EPA criteria.
11-138
The overall evidence for carcinogenicity, considering both animal and human studies, would place 2,3,7,8-TCDD alone In the B2 category of EPA's classification scheme, and 2,3,7,8-TCDD 1n association with the phenoxy herbicides and/or chlorophenols 1n the B1 category. Chemicals 1n category B are regarded as being "probably" carcinogenic 1n humans.
The EPA has, 1n the past, used an IARC we1ght-of-ev1dence classification scheme for evaluating carcinogenicity data. Using IARC classification criteria, the positive evidence 1n the rat and mouse studies, together with Inadequate evidence 1n humans for 2,3,7,8-TCDD alone, 1s equivalent to an IARC 2B category, meaning that 2,3,7,8-TCDD 1s "probably" carcinogenic 1n humans. However, the overall we1ght-of-ev1dence for 2,3,7,8-TCDD 1n combi nation with chlorinated phenoxyacetlc acid herbicides and/or chlorophenols would be classified as IARC 2A, meaning that chlorophenoxyacetlc acid and/or chlorophenols containing 2,3,7,8-TCDD are "probably" carcinogenic 1n humans.
Hepatocellular tumors have been Induced 1n mice and rats of both sexes following administration of a 1:2 mixture of 1,2,3,6,7,8- and 1,2,3,7,8,9HxCDD. This level of carcinogenic evidence In animals would be regarded as "sufficient" according to the EPA classification scheme. Based on animal evidence and the lack of epidemiologic data, HxCDD would be placed 1n EPA's B2 category, which characterizes HxCDD as "probably" carcinogenic 1n humans. Using the IARC classification scheme, based on animal evidence and no epidemiology data, HxCDD would be considered to be 1n a 2B category meaning that HxCDD 1s "probably" carcinogenic 1n humans.
Assuming that 2,3,7,8-TCDD and HxCDD are carcinogenic 1n humans, upper bound Incremental unit cancer risks have been estimated for both Ingestion and Inhalation exposure. The development of these unit risk estimates 1s for the purpose of evaluating the magnitude of the possible health Impact
11-139
from exposure to these compounds. The upper bound nature of these risk estimates 1s such that the true risk 1s not likely to be exceeded and may be lower.
Using the data from a feeding study with female rats, the cancer potency (unit risk per mg/kg/day) for 2,3,7,8-TCDD 1s 1.56xl0_1 (ng/kg/day)"1 . The upper limit estimate of Incremental cancer risk 1s 4.5xl0"3 for a continuous lifetime exposure to 1 ng/Jt of 2,3,7,8-TCDD 1n drinking water. The upper limit estimate of Incremental cancer risk 1s 3.3xl0"s for a continuous lifetime exposure to 1 pg/m3 of 2,3,7,8-TCDD 1n ambient air.
Using data from a gavage study with female rats and male mice the cancer potency for HxCDD 1s 6.2xl0"3 (ng/kg/day)"1 . The upper limit estimate of Incremental cancer risk Is 1.8xl0"4 for a lifetime exposure to 1 ng/i. of HxCDD 1n drinking water. For ambient air a lifetime exposure to 1 pg/m3 of HxCDD 1s estimated to have an upper limit risk of 1.3x10".
In terms of low dose response, 2,3,7,8-TCDD and the 1:2 mixture of 1,2,3,6,7,8- and 1,2,3,7,8,9-HxCDD rank as the most potent and second most potent, respectively, carcinogens evaluated by EPA's CA6.
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12. 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 1n either potentiation or Inhibition of their biological effects. 12.1. CHEMICAL CARCINOGENS Synergistic and antagonistic activities of 2,3,7,8-TCDD with chemical carcinogens have been discussed 1n depth 1n Chapter 11 of this document. 12.2. NONCARCINOGENIC CHEMICALS 2,3,7,8-TCDD pretreatment has been observed to modify the effects of anesthetics (Grelg, 1972). Adult male Porten rats were given a single oral dose of 200 yg 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 h e x a b a r b lto n e . A r e c e n t r e p o r t compares the 1mmunotox1c1ty o f 2.3.7.8- TCDD, 2,3,7,8-TCDF and 2,3,7,8-TCDF plus 2,3,7,8-TCDD (coadmin istered) (R1zzard1n1 et al., 1983). Seven days after administration of 1.2 yg/kg of 2,3,7,8-TCDD to C57B1/6J mice, sheep red blood cells were Injected 1ntraper1toneally and plaque-forming cells (PFC) 1n the spleen were counted 5 days later. 2,3,7,8-TCDD Inhibited antibody production by 80%. In a parallel study, a dose of 2,3,7,8-TCDF was administered (10 yg/kg) and no significant 1mmunotox1c effects were observed. Coadm1n1strat1on of 2.3.7.8-TCDD (1.2 yg/kg) plus 2,3,7,8-TCDF (10 yg/kg) resulted 1n 50% reduction 1n antibody production and demonstrates a significant antagonistic effect by 2,3,7,8-TCDF. Coadm1n1strat1on of these two Isostereomers resulted In antagonistic effects with respect to the Induction of hepatic
12-1
microsomal cytochrome P-450 and 7-ethoxycoumar1n O-deethylase. Sweeney et al. (1979) found that Iron deficiency protected mice against the development of hepatocellular damage (Including porphyria) normally caused by 2,3,7,8TCDD exposure. 12.3. SUMMARY
Exposure to 2,3,7,8-TCDD has been observed to alter the biological response of many species to some compounds. This altered response 1s pre sumed to be the result of altered enzyme activities 1n tissue 1n which 2,3,7,8-TCDD exerts an Inductive effect (vide ante, see Section 8.1.1.5.), although other mechanisms are possible (see Section 8.3.).
2,3,7,8-TCDD pretreatment Increases the conversion of some chemical car cinogens to mutagens by hepatic S-9 preparations 1n in vitro test systems; however, exposure to 2,3,7,8-TCDD often has an ant1carc1nogen1c effect in vivo (see Section 11.1.1.1.). This ant1carc1nogen1c 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 carcinogens. This modification may reduce the effectiveness, as 1n the case of zoxazolamlne, or Increase the effectiveness, as 1n the case of hexabarbltone (Grelg, 1972). The direction and extent of the alteration depends both on the effect of 2,3,7,8-TCDD on the particular enzyme system Involved and on whether metabolism 1s an activating or deactivating process.
12-2
13. REGULATIONS AND STANDARDS 13.1. WATER
Previous release of PCDD-conta1n1ng herbicides has been one mechanism by which these agents enter the environment. Their high environmental stabil ity and low water solubility (0.2 ppb) make the 2,3,7,8- TCDD tend to settle 1n the bottom sludge of waterways. The major risk to humans comes from eating bottom-feeding fish 1n which 2,3,7,8-TCDD has bloaccumulated. The U.S. EPA has set criteria of 1.3xl0-7, 1.3xl0-8 or 1.3xl0"9 yg 2.3.7.8-TCDD/i. 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 Jt of contaminated drinking water (U.S. EPA, 1984). No Information 1s available regarding concentration limits of 1.2.3.7.8- PeCDD, 1,2,3,7,8,9-HxCDD or 1,2,3,6,7,8- HxCDD 1n ambient water. 13.2. AIR
Many normal combustion processes are suspected of releasing dioxins to the atmosphere. However, the effect on human health from this source 1s unknown, and no criteria exist regarding concentration limits. 13.3. FOOD
According to the FDA (Cordle, 1981, 1983; FDA, 1981, 1983) and the Code of Federal Regulations (41 CFR 321), fish with a 2,3,7,8-TCDD content averaging <25 ppt pose no serious health concern. Federal legal limits for Great Lakes fish distributed 1n Interstate commerce are deemed unnecessary because most of the samples analyzed by the FDA contained <25 ppt. Canada has established a 20 ppt concentration limit for 2,3,7,8-TCDD 1n Lake Ontario commercial fish Imported Into the United States to comply with the levels believed by the FDA to be safe (NRCC, 1981a).
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A tolerance for hexachlorophene methylenebls (2,3,6-trlchlorophenol) 1n or on feedstock cottenseeds has been established at 0.05 ppm, with the con dition that 1t not contain >0.1 ppm of 2,3,7,8-TCDD (U.S. ERA, 1982c).
No Information regarding concentration limits of other dioxin Isomers 1s available. 13.4. SUMMARY
The regulation of dioxin by-products 1n substances such as chlorophenols and 2,4,5-tr1chlorophenoxyacet1c acid 1s apparently expected to eliminate dioxin releases to the environment. The Canadian concentration limit for 2,3,7,8-TCDD 1n fish 1s the only known criterion, and 1t agrees with levels regarded by the FDA as being protective of human health. In the absence of specific guidelines and standards regarding concentration limits of 2,3,7,8TCDD, the FDA examines Individual contamination situations separately, and gives only general guidance regarding relative risk to humans (Delgado, 1983). No Information 1s available regarding concentration limits for other PCDDs.
13-2
74. EFFECTS OF MAJOR CONCERN AND HEALTH HAZARD ASSESSMENT Of the four congeners of PCDDs discussed 1n this report (2,3,7,8-TCDD, 1.2.3.7.8- PeCDD, 1,2,3,7,8,9- and 1,2,3,6,7,8-HxCDD), the majority of toxi cologic data are on 2,3,7,8-TCDD. The limited data on the other congeners Indicate that they are qualitatively similar 1n their toxic action to 2.3.7.8- TCDD when comparisons are made 1n a single species; however, they are less toxic than the 2,3,7,8-TCDD congener. This 1s Illustrated 1n mice, 1n which 2,3,7,8-TCDD has an LD5Q value of 0.88 pmol/kg and 1,2,3,7,8PeCDD; 1,2,3,6,7,8- and 1,2,3,7,8,9-HxCDD have LDcn values of 0.94, 3.19
O il
and 3.67 pmol/kg, respectively (McConnell et al., 1978b). This suggests that either the position or the number of chlorine effects the toxicity of the PCDDs.
In more recent studies using biochemical endpoints, Poland et al. (1979), Bradlaw and Casterllne (1979) and Bradlaw et al. (1980) supported the contention that the position and number of chlorines on TCDD, PeCDD and HxCDD are critical for the biologic activity of the compound. In this study, the E D ^ for the Induction of AHH activity 1n hepatoma cells 1n culture was used to establish a range of potency for congeners of PCDDs. Although acute toxicity and Induction of AHH activity have been used to quantify the difference 1n the biologic activity of the congeners 2,3,7,8TCDD, 1,2,3,7,8-PeCDD and 1,2,3,7,8,9-HxCDD, the extrapolation of this data to estimate quantitative dose-response relationships for the chronic tox icity of Individual congeners 1s not sufficiently supported at th^ present time. From the following data described, 1t 1s clear that sufficient Infor mation for quantitative hazard assessment 1s available only for 2,3,7,8-TCDD and a mixture of the two HxCDD congeners.
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14.1. PRINCIPAL EFFECTS 14.1.1. Toxicity. The principal effect observed 1n all species after acute exposure to 2,3,7,8-TCDD 1s weight loss and thymic atrophy (see Table 8-1). The decrease 1n weight proceeds over a protracted length of time even after a single exposure to a lethal dose. By the time of death, an almost complete absence of body fat stores was often observed. At death, severe deterioration of the animal was observed; however, there was no specific lesion to associate with the cause of death. This was particularly evident 1n the guinea pig, the most sensitive species to 2,3,7,8-TCDD toxicity. Necropsy revealed no remarkable alteration 1n any Internal organ except for thymic atrophy (Gupta et al., 1973). Although Uver damage was observed 1n rats, rabbits and mice (Schwetz et al., 1973), there are Insufficient data to Indicate that this effect 1s the underlying cause of mortality after acute exposure to 2,3,7,8-TCDD. Also, 1n the guinea pig and monkey, which have the same general progression of gross signs of toxicity as do rats, rabbits and mice, there 1s only mild Uver damage (see Section 8.1.). In addition, 2,3,7,8-TCDD 1s an Immunosuppressant 1n mice (see Section
8 .1 .1 .4 .).
As a result of the long time necessary for the development of toxic symptoms 1n 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 1n Table 14-1 for orally administered 2,3,7,8-TCDD. The NOEL for subchronic exposure 1s ~10 times higher than that observed for chronic exposures, suggesting that the cumulative dose might be an Important factor 1n 2,3,7,8-TCDD toxicity. There are only limited data on the NOEL and LOEL for HxCDD
14-2
TABLE 14-1
No-Observed-Effect Levels and Low-Observed-Effect Levels Obtained from Subchronic and Chronic Oral Toxicity Studies of 2,3,7,8-TCDD
14-3
Spectes/Stratn
ua/ka/dav
NOEL
LOEL
Duration of
Exposure
Duration of Study
Reported Effect
Reference
Rat/Sprague-Dawley Rat/Osborne-Mendel Rat/Sprague-Dawley
0.01 0.07 0.0014
0.1 0.14 0.014
Rat/Sprague-Dawley ND
0.014
M1ce/B6C3F1 Monkey/Rhesus Rat/Sprague-Dawley
ND ND 0.001
0.014 <0.02
0.01
Rat/Osborne-Mendel M1ce/Sw1ss
0.0014 ND
0.007 0.001
13 weeks 13 weeks 16 weeks
28 weeks
13 weeks 36 weeks 104 weeks
104 weeks 62 weeks
26 weeks 13 weeks 40 weeks
40 weeks
13 weeks 52 weeks 104 weeks
107 weeks life
decreased bw
toxic hepatitis
elevated porphyrin levels
fatty changes In the liver, decreased bw
toxic hepatitis
pancytopenia
degenerative and necrotic changes 1n the liver
toxic hepatitis
dermatitis and amyloidosis
Koclba et al., 1976 NTP, 1980a Goldstein et al., 1982b King and Roesler, 1974
NTP, 1980a Allen et al., 1977 Koclba et al., 1978a, 1979
NTP, 1980a Toth et al., 1979
ND = Not determined
(Table 14-2) and these were obtained from studies using a 1:2 mixture of 1,2,3,6,7,8- and 1 ,2,3,7,8,9-HxCDD. As observed with 2,3,7,8-TCDD, there 1s a suggestion that the cumulative dose of this mixture 1s an Important consideration 1n defining a NOEL. For both 2,3,7,8-TCDD and the mixture of HxCDD, the Uver 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, 2,3,7,8TCDD 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 teratogenicity of 2,3,7,8-TCDD 1n rabbits reported by G1av1n1 et al. (1982b) 1n which Increases 1n extra ribs and total soft-tissue anomalies were observed. In mice, 1 yg/kg/day given for 9-10 days during the middle of gestation was the minimum dose necessary to elicit a teratogenic response (Smith et al., 1976; Moore et al., 1973), while dilated renal pelvis and decreased fetal weight were observed 1n the rat fetuses of dams receiving doses of 2,3,7,8-TCDD as low as 0.001 yg/kg/day throughout gestation. The statistical and biological signifi cance of effects at this later dose, however, 1s argued (Murray et al., 1979; Nlsbet and Paxton, 1982; U.S. EPA, 1979c). The fetuses of rats appear to be very sensitive to the effects of 2,3,7,8-TCDD, with adverse effects occurring at maternal exposures that were similar to the NOEL observed 1n chronic studies (see Table 14-1). Also, Schwetz et al. (1973) demonstrated that HxCDD (Isomers not specified) was both fetotoxlc and teratogenic when administered to pregnant rats at 100 yg/kg on days 6-15 of gestation.
Some epidemiology studies have shown a positive association between exposure to 2,4,5-T, of which 2,3,7,8-TCDD 1s a known contaminant, and birth
14-4
TABLE 14-2
No-Observed-Effect Levels and Low-Observed-Effect Levels Obtained from Subchronic and Chronic Oral Toxicity Studies of HxCDDa *b
Spec1es/Stra1n
ng/kq/day NOEL LOEL
Duration of
Exposure
Duration of Study
Reported Effects
Rat/Osborne-Mendel M1ce/B6C3F1 Rat/Osborne-Mendel M1ce/B6C3F1
0.35 0.7 0.7 1.4 ND 0.18 ND 0.18
13 weeks 13 weeks 104 weeks 104 weeks
13 weeks 13 weeks 107 weeks 107 weeks
hepatotoxldty hepatotoxldty toxic hepatitis toxic hepatitis
aSource: NTP, 1980b bThe HxCDD was a 1:2 mixture of 1,2,3,6,7,8- and 1,2,3,7,8,9--HxCDD. ND = Not determined
14-5
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 teratogenicity data, since among many other difficulties 1n Interpreting human data the exposures were always mixed, and there were Inadequate data concerning the levels of 2,3,7,8-TCDD to which the popula tions were exposed.
Animal studies also demonstrate that 2,3,7,8-TCDD 1s a carcinogen (see Table 11-1). The limited studies by Van Miller et al. (1977a,b) and Toth et al. (1978, 1979) Indicated that 2,3,7,8-TCDD caused a variety of tumors 1n rats and mice, and the more Intensive studies by Koclba et al. (1978a) and NTP (1980a) support these early findings. Also, papillomas have been re ported 1n female mice after dermal application of 2,3,7,8-TCDD (NTP, 1980b), and using the skin tumorlgenesls model, 1t has been shown that 2,3,7,8-TCDD may affect the carcinogenic potential of other chemical carcinogens (see Section 11.1.1.2.). Human exposure to 2,3,7,8-TCDD has resulted from contamination of other polychlorinated compounds with 2,3,7,8-TCDD (see Section 11.1.3.).
A 1:2 mixture of 1,2,3,6,7,8- and 1,2,3,7,8,9-HxCDD also has been tested for carcinogenicity 1n rats and mice treated by gavage and by dermal appli cation 1n mice (NTP, 1980c,d). In both species, this mixture produced Uver tumors when administered by gavage, while 1n 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-trlchlorophenoxyacetlc acid and 2,4,5-tr1chlorophenol are consistent with the position that 2,3,7,8-TCDD 1s probably carcinogenic for humans; the available evidence Indicates an excess Inci dence of soft tissue sarcoma. Because 2,3,7,8-TCDD 1s almost always found
14-6
in a s s o c i a t i o n w i t h the materials (chlorophenols, combustion products, etc.)
It may never be possible to evaluate the carcinogenicity of 2,3,7,8-TCDD by Itself In humans. 14.1.2. Mutagenicity. There have been many studies of the mutagenic potential of 2,3,7,8-TCDD (see Chapter 10). Tn vitro assays using bacteria and yeast have generally Indicated that 2,3,7,8-TCDD Is not a mutagen. These negative results were obtained both 1n the presence and absence^of a mammalian metabolic activation system. A few studies have reported positive results (Hussain 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, It 1s Impossible to assert whether or not 2,3,7,8-TCDD 1s devoid of mutagenic potential. There are also some conflicting data from humans and animal studies that Indicate that 2,3,7,8-TCDD causes chromosomal aberrations. Because the human data are derived from populations 1n which exposure to other biologically active compounds Is possible, and because the Increases observed 1n animal studies were small, 1t 1s still not substantiated that 2,3,7,8-TCDD produces clastogenlc changes.
Pertinent data regarding the mutagenic potential of 1,2,3,7,8-PeCDD, 1,2,3,7,8,9-HxCDD or 1 ,2,3,6,7,8-HxCDD could not be found 1n the available literature. 14.2. SENSITIVE POPULATIONS
Although there are no data from human studies to Indicate the presence of sensitive populations, the data from animal studies suggest that the fetus and newborn may be at greater risk. Studies 1n chickens, rats, mice,
14-7
rabbits, ferrets and monkeys have shown that in utero exposure to 2,3,7,8TCDD can result 1n malformations, fetal toxicity and abortions (see Table 9-2). The lowest dose reported to adversely affect the fetus In. utero was 0.001 yg/kg/day administered to the dams throughout gestation (from Hurray et al., 1979, according to Nlsbet and Paxton, 1982); this dose 1s similar to the NOEL reported for chronic exposure of adult rats (see Table 14-1). Hoore et al. (1973) observed that the nursing of pups on mothers exposed to 2,3,7,8-TCDD could also result 1n kidney anomalies detected at the time of weaning. These data suggest that both the fetus and the newborn may be more sensitive than the adult to the adverse effects of exposure to 2,3,7,8-TCDD.
In addition, 2,3,7,8-TCDD 1s known to be a powerful Inducer of the MFO system. There 1s Information to Indicate that MFO Induction by 2,3,7,8-TCDD can affect the biologic activity of other xenoblotlcs that require metabolic activation (see Chapter 12). ScarpelU et al. (1980), for example, demon strated that pretreatment of hamsters with 2,3,7,8-TCDD resulted 1n greater activation of mutagenic nltrosamlnes when assayed in vitro with Isolated mlcrosomes. Individuals exposed to chemicals that are activated by the MFO may experience a synergistic 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 1n the environment, and that human exposure may occur through Ingestion of contaminated food and water, by Inhalation of the compound absorbed to respirable particulates, or through dermal contact. Although potential exposure may occur by all routes, most of the toxicologic Informa tion 1s from studies of oral exposure. The limited observation of toxic
14-8
effects 1n humans and animals after dermal contact with 2,3,7,8-TCDD In organic solvents Indicates that dermal absorption occurs. Polger and Schlatter (1980) have shown In rats that both dermal and GI absorption Is dependent on the vehicle. Greatest absorption after oral exposure occurred when 2,3,7,8-TCDD was administered 1n organic solvent followed by aqueous suspension, with little absorption occurring 1f the 2,3,7,8-TCDD was adsorbed onto activated carbon. In a similar manner, dermal absorption was poor 1f the 2,3,7,8-TCDD was applied 1n a soil and water paste. Inhalation exposure 1s likely to occur through airborne particulate matter containing absorbed 2,3,7,8-TCDD; however, 1t 1s not possible with the available data to predict how efficiently absorption will occur through the respiratory tract. The use of standard respiratory absorption assumptions 1n risk assessment are most likely to provide conservative criteria levels. 14.4. QUALITATIVE HEALTH HAZARD ASSESSHENT
The data available from animal studies are sufficient to provide some assessment of the human health hazards associated with exposure to 2,3,7,8TCDD 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 LDOcUo value and studies of Induetlon of AHH activity. Although both types of data Indicate that 1,2,3,7,8PeCDD 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 toxicity, teratogenicity or carcinogenicity.
14-9
Although there have been human epidemiology studies Investigating the toxic, reproductive and carcinogenic effect of exposure to 2,3,7,8-TCDD, these studies have major deficiencies for use 1n health assessment. 2,3,7,8-TCDD 1s a contaminant of the chemicals 2,4,5-T and TCP, and all human data are derived from populations exposed to mixtures. In these studies, 1t 1s not possible to attribute with certainty any observed effect to exposure to 2,3,7,8-TCDD. Also, exposure data of sufficient quality are not available to define a dose-response relationship 1n human population. Without adequate exposure data, health assessments cannot be made. 14.4.1. Animal Toxicity 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 toxicity 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 1n rats (Koclba et al., 1978a, 1979; NTP, 1980a) are 0.001 and 0.0014 yg/kg/day; however, 1n the mouse (NTP, 1980a), the dose of 0.07 yg/kg/day was a FEL, as Indicated by fatty changes 1n the Uver, and 0.007 was a NOEL.
In addition, 1t may be Inappropriate to derive a tox1c1ty-based hazard assessment for 2,3,7,8-TCDD from these chronic studies, since a 3-generat1on study by Hurray et al. (1979) Indicates that exposure of pregnant rats to this dose of 2,3,7,8-TCDD (0.001 yg/kg/day) throughout gestation resulted 1n 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 1t occurred 1n only one generation at this dose and not at higher
14-10
doses. Hence, 0.001 yg/kg/day represented a NOAEL. H o w e v e r , a r v a l u a t i o n of these data by different statistical methods (Nlsbet and Paxton, 1982) Indicated a statistically significant Increase of dilated renal pelvis at higher doses, as well as the lowest one, and lower fetal weight 1n the 0.001 yg/kg group. With these data, 0.001 yg/kg could be considered a L0AEL. No other studies are available regarding the effects of 2,3,7,8-TCDD at even lower doses.
A toxldty-based hazard assessment 1s also possible for the mixture of HxCDO tested by NTP (1980b). As 1s shown 1n Table 14-2, however, the description of the histologic observations was not sufficiently detailed to determine whether the low dose represented a NOAEL or a L0AEL. These data could be used for hazard assessment 1n either case with an additional uncertainty factor for a L0AEL (Federal Register, 1980b). 14.4.2. Animal Carcinogenicity. In addition to the Inadequate data base for a tox1c1ty-based hazard assessment, the strong evidence of carcinogeni city 1n animals for 2,3,7,8-TCDD would justify a carcinogenicity-based assessment. That two adequate cancer bioassays used sufficiently large groups of animals exposed for an appreciable portion of their lifespan Indicates that 2,3,7,8-TCDD Is an animal carcinogen (NTP, 1980a; Kodba et al., 1978a) (Table 14-3). In the NTP (1980a) study, male rats developed folUcular-cell adenomas or carcinomas of the thyroid. Female rats and mice of both sexes had Increased Incidences of folUcular-cell adenomas of the thyroid. In the study by Kodba 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 Uver, tongue and lungs 1n females of the high-dose group. The evidence 1s suffi cient to Indicate that 2,3,7,8-TCDD 1s an animal carcinogen.
14-11
TABLE 14-3 Carcinogenicity Bioassays of 2,3,7,8-TCDD
14-12
Exposure Route
Specles/Straln
Sex
Dose or Exposure
Duration of
Treatment
Duration of Study
Vehicle
Tumor Type
Tumor Incidence
Reference
Gavage Gavage
rats/ .
H 0.0 pg/kg/week
Osborne-Hendel
104 weeks
105 weeks
corn oilacetone (9:1)
follicular-cell adenomas or carcinoma of the thyroid
0.01 pg/kg/week
104 weeks
107 weeks
corn oilacetone (9:1)
follicular-cell adenomas or carcinoma of the thyroid
0.05 pg/kg/week
104 weeks
107 weeks
corn oilacetone (9:1)
follicular-cell adenomas or carcinoma of the thyroid
0.5 pg/kg/week
104 weeks
107 weeks
corn oilacetone
(9:1)
follicular-cell adenomas or carcinoma of the thyroid
rats/
F 0.0 pg/kg/week
Osborne-Hendel
104 weeks
105 weeks
corn oilacetone
(9:1)
neoplastic nodule or hepatocellular carcinoma of the U v e r
0.1 pg/kg/week
104 weeks
107 weeks
corn oilacetone (9:1)
neoplastic nodule or hepatocellular carcinoma of the U v e r
0.05 pg/kg/week
104 weeks
107 weeks
corn oilacetone (9:1)
neoplastic nodule or hepatocellular carcinoma of the U v e r
0.5 pg/kg/week
104 weeks
107 weeks
corn oilacetone (9:1)
neoplastic nodule or hepatocellular carcinoma of the liver
1/69 5/48 8/50 11/50 5/75 1/49 3/50 14/49
NTP, 1980a NTR, 1980a
TABLE 14-3 (cont.)
14-13
Exposure Route
Specles/Straln
Sex
Oose or Exposure
Duration of
Treatment
Duration of Study
Vehicle
Tumor Type
Tumor Incidence
Reference
Gavage 6avage Oral
m1ce/B6C3F1
H 0.0 vg/kg/week
104 weeks
105 weeks
corn oilacetone (9:1)
hepatocellular carcinoma
0.01 vg/kg/week
104 weeks
107 weeks
corn oilacetone (9:1)
hepatocellular carcinoma
0.05 v9/kg/week
104 weeks
107 weeks
corn oilacetone (9:1)
hepatocellular carcinoma
0.5 v9/kg/week
104 weeks
107 weeks
corn oilacetone (9:1)
hepatocellular carcinoma
n>1ce/B6C3fl
F 0.0 v9/k9 /week
104 weeks
105 weeks
corn o 1 1acetone (9:1)
hepatocellular carcinoma. follicular-cell adenomas of the thyroid
0.04 vg/kg/week
104 weeks
107 weeks
c o m oilacetone (9:1)
hepatocellular carcinoma. follicular-cell adenomas of the thyroid
0.2 vg/kg/week
104 weeks
107 weeks
corn oilacetone (9:1)
hepatocellular carcinoma. follicular-cell adenomas of the thyroid
2.0 v9/kg/week
104 weeks
107 weeks
c o m oilacetone (9:1)
hepatocellular carcinoma. follicular-cell adenomas of the thyroid
rat/
N 0.0 v9/kg/day
Sprague-Dawley
105 weeks
105 weeks
In diet
squamous cell carcinoma of the hard palate, squamous cell carcinoma of the tongue. adenoma of the adrenal cortex
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
NTP, 1980a
NTP. 1980a
Koclba et al.. 1978a
TABLE 14-3 (cont.)
14-14
Exposure Specles/Straln Sex Route
Dose or Exposure
Duration of
Treatment
Duration of Study
Vehicle
Oral (cont.)
rat/ Sprague-Dawley
0.001 tig/kg/day 105 weeks
105 weeks
In diet
0.01 tig/kg/day
105 weeks
105 weeks
In diet
0.1 ug/kg/day
105 weeks
105 weeks
1n diet
Oral
rat/
F 0.0 ug/kg/day
Sprague-Dawley
105 weeks
105 weeks
In diet
0.001 pg/kg/day
105 weeks
105 weeks
1n diet
0.01 pg/kg/day
105 weeks
105 weeks
In diet
0.1 ug/kg/day
105 weeks
105 weeks
1n diet
Tumor Type
Tumor Incidence
Reference
squamous cell carcinoma of the hard palate, squamous cell carcinoma of the tongue. adenoma of the adrenal cortex
squamous cell carcinoma of the hard palate, squamous cell carcinoma of the tongue. adenoma of the adrenal cortex
squamous cell carcinoma of the hard palate, squamous cell carcinoma of the tongue. adenoma of the adrenal cortex
hepatocellular carcinoma, squamous cell carcinoma of the hard palate, squamous cell carcinoma of the lung
hepatocellular carcinoma, squamous cell carcinoma of the hard palate. squamous cell carcinoma of the lung
hepatocellular carcinoma, squamous cell carcinoma of the hard palate, squamous cell carcinoma of the lung
hepatocellular carcinoma, squamous cell carcinoma of the hard palate. squamous cell carcinoma of the lung
0/50 1/50 0/50
0/50 1/50 2/50
4/50 3/50 5/50
1/86 0/86 0/86 0/50 0/50 0/50 2/50 1/50 0/50 11/49 4/49 7/49
Koclba et al., 1978a
Koclba et al., 1978a
A single bioassay tested a mixture of the two congeners of HxCDD for carcinogenicity (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 neoplastic nodules or carcinomas of the Uver. Increased Incidence of tumors 1n two species 1s sufficient to Indicate that this mixture was carcinogenic to animals; however, caution 1s required 1n Interpreting these data for hazard evaluation since the NTP (1980a) study used a mixture containing two Isomers, 1,2,3,6,7,8- and 1,2,3,7,8,9-, of HxCDD and the HxCDD mixture used for this bioassay was found to be contami nated with other PCDDs Including 0.09% (+0.03%) of TCDD. The specific Isomer of PCDDs was not Identified. There 1s Insufficient evidence to confirm whether both Isomers are Independently carcinogenic or whether only one Isomer or this specific mixture 1s needed to elicit a carcinogenic response. Since the position of the chlorines may be extremely Important for the tox1c/carc1nogen1c properties of HxCDD, Information obtained from this combined exposure may not be applicable to the Individual congeners.
14-15
TABLE 14-4 Carcinogenicity Bioassays of a 1:2 Mixture of 1,2,3,6,7,8- and 1,2,3,7,8,9-HxCOO
9L-H
Exposure Spedes/Straln Route
Duration
Sex Dose or Exposure
of
Duration
Treatment of Study
Vehicle
Tumor Type
Tumor Incidence
Reference
Gavage
rats/ Osborne-Mendel
Gavage
rats/ Osbome-Mendel
H 0.0 pg/kg/week
104 weeks
105 weeks
c o m oilacetone (9:1)
liver neoplastic nodules or hepatocellular carcinoma
M 1.25 pg/kg/week 104 weeks 107 weeks corn oil-
U v e r neoplastic nodules
acetone (9:1)
or hepatocellular
carcinoma
2.5 pg/kg/week
104 weeks
107 weeks
c o m oilacetone (9:1)
liver neoplastic nodules or hepatocellular carcinoma
5.0 pg/kg/week
104 weeks
107 weeks
corn oilacetone (9:1)
U v e r neoplastic nodules or hepatocellular carcinoma
0/74
NTP, 19801
0/49
NTP, 1980
1/50
4/48
Gavage
rats/ Osbome-Mendel
r 0.0 pg/kg/week
104 weeks 105 weeks c o m oilacetone (9:1)
liver neoplastic nodules or hepatocellular carcinoma
1.25 pg/kg/week
104 weeks
107 weeks
corn oilacetone (9:1)
U v e r neoplastic nodules or hepatocellular carcinoma
2.5 pg/kg/week
104 weeks
107 weeks
corn oilacetone (9:1)
U v e r neoplastic nodules or hepatocellular carcinoma
5.0 pg/kg/week
104 weeks
107 weeks
c o m oilacetone (9:1)
U v e r neoplastic nodules or hepatocellular carcinoma
5/75
NTP, 1980c
10/50
12/50
30/50
TABLE 14-4 (cont.)
14-17
Exposure Specles/Straln Route
Duration
Sex Oose or Exposure
of
Duration
Treatment of Study
Vehicle
Tumor Type
Tumor Incidence
Reference
Gavage
rats/ Osborne-Hendel
F 0.0 pg/kg/week
104 weeks
105 weeks
corn oilacetone (9:1 )
hepatocellular adenomas or carcinomas
15/73
NTP, 1980d
1.25 pg/kg/week
104 weeks
108 weeks
corn oilacetone (9:1)
'hepatocellular adenomas or carcinomas
14/50
2.5 pg/kg/week
104 weeks
107 weeks
corn oilacetone (9:1)
hepatocellular adenomas or carcinomas
14/49
Gavage m1ce/B6C3F1
S.O pg/kg/week
104 weeks 108 weeks corn oilacetone (9:1 )
hepatocellular adenomas or carcinomas
F 0.0 pg/kg/week
104 weeks
106 weeks
corn oilacetone (9:1)
hepatocellular adenomas or carcinomas
2.5 pg/kg/week
104 weeks
108 weeks
corn oilacetone (9:1)
hepatocellular adenomas or carcinomas
5.0 pg/kg/week
104 weeks
108 weeks
corn oilacetone (9:1)
hepatocellular adenomas or carcinomas
10.0 pg/kg/week
104 weeks
107 weeks
corn o11acetone (9:1 )
hepatocellular adenomas or carcinomas
24/48
3/75
NTP, 1980d
4/48
6/47
10/47
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15-9
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15-10
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15-11
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APPENDIX A A-l
TABLE A-l Cumulative Mortality of Male Rats3
Time
(end of 30-day period) N=
Controls (86)
uq/kq/dav 2.3.7.8-TCDD
0.1 0.01 0.001 (50) (50) (50)
1-7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
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
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 74.0b 78.0 84.0 90.0
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 76.0b 84.0 88.0 92.0
2.0 2.0 2.0 2.0 2.0 2.0 2.0 4.0 14.0 14.0 24.0 44.0b 50.0 56.0 60.0 68.0 74.0 76. 78.0
aSource: Koclba et al., 1977
^Interval of greatest difference, D, 1n cumulative mortality curves of controls and treatment group. None of the differences were statistically significant (Kolmogorov-Smlrnov test, p>0.05).
A-2
TABLE A-2 Cumulative Mortality of Female Rats3
Time (end of 30-day period)
N=
Controls (86)
ug/kq/dav 2,3.7.8-TCDD
0.1 (50)
0.01 (50)
0.001 (50)
0-5 6-8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
0.0 1.2 1.2 1.2 1.2 1.2 3.5 3.5 7.0 12.8 15.1 18.6 25.6 34.9 40.7 58.1 64.0 70.9 70.9
0.0 0.0 2.0 4.0 8.0 16.0 20.0 26.0 28.0 32.0 38.0 44.0 56.0b 60.0 66.0 82.0 86.0 88.0 92.0
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 46.0b 60.0 66.0 72.0 72.0
0.0 0.0 0.0 0.0 0.0 4.0 4.0 6.0 10.0 12.0 18.0 22.0 34.0b 36.0 44.0 52.0 58.0 66.0 68.0
aSource: Koclba et al., 1977
^Interval of greatest difference, D, 1n cumulative mortality curves of controls and treatment group. The mortality curve for the rats fed 0.1 vg/kg/day differed significantly from that for controls (D = 30.4, p<0.01, Kolmogorov-Smlrov test). The other two groups did not differ significantly from, controls (p>0.05).
A-3
TABLE A-3 Hales: Interval Mortality Rates
Days
Control d/1 Rate
0.1 uq/kq/dav d/1 Rate
0.01 uq/kq/dav d/1 Rate
0.001 uq/kq/dav d/1 Rate
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
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
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
0/50
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
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
0/50 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
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
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
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
Terminal Kill
15
5
4
11
Corrected for continuity for combined Interval:
421-510
7/77 vs. 5/41(X2=0.04, n.s.) 12/48(X2= 4.63, p<0.05) 10/48 (Xa=2.54, n.s.
451-540
10/72 vs. 8/41(X2=0.37, n.s.) 10/43(X2=1.27, n.s.) 15/43 (X2=6.37, p<0.025)
481-570
13/72 vs. 12/40(X2=l.48, n.s.) 12/39(X2=1.67, n.s.) 18/43 (Xa=6.59, p<0.025)
511-600
20/70 vs. 9/36(X2=0.03, n.s.) 13/36(X2=0.32, n.s.) 16/38 (X2= 1/47, n.s.)
A-4
TABLE A-4 Females: Interval Mortality Rates
Days
Control d/1 Rate
0.1 uq/kq/dav d/1 Rate
0.01 uq/kq/dav d/1 Rate
0.001 uq/kq/dav d/1 Rate
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
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
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
0/50
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
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
0/50
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
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
0/50
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
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
Terminal
Kill
25
4
14
16
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=l.34, n.s.)
510-600
17/73 vs. 11/31(X2=l.08, n.s.) 9/41(X2=0.0, n.s.) 9/41 (X2=0.0, n.s.)
A-5
APPENDIX B Tables for 2,3,7,8-TCDD Quantitative Incremental
Unit Cancer Risk Estimates
B-l
Tables for 2,3,7,8-TCDD Quantitative Incremental Unit Cancer Risk Estimates
Tables B-l through B-5 are the 2,3,7,8-TCDD bioassay results judged suitable for quantitative estimates of Incremental unit risk. Tables B-l and B-2 show the results of the Dow rat feeding study for both males and females. The results Include both the original (Koclba) analysis and the (Squire) review. Individual organ sites where significantly Increased tumors occurred are tabulated separately, then the total number of animals with at least one of these tumors 1s compiled. Tables B-3, B-4 and B-5 compile similar data for the NCI bioassay. Table B-6 uses the data from Table B-l to estimate the parameters of the linearized multistage model. The X2 test for goodness-of-f1t of the model to the data determines whether or not the highest dose group 1s retained 1n the fit. The 95% upper-Umlt on the linear term a * 1s then adjusted by the surface area
aI
constant (70/Wa,)1/3 to derive the final extrapolated animal-to-human 95% upper-Umlt Incremental unit cancer risk estimate. Tables B-7 through B-l2 present the extrapolation procedure for the remaining data sets, with Tables B-8A and B-9A adjusting for high early mortality 1n the female rat high-dose group. Table B--13 summarizes the estimates derived 1n Tables B-6 through B-l2. The q.j* estimates from the female rat data of the Dow feeding study using both the Koclba and Squire readings are averaged to derive the final estimate q^* = 1.56x10s (mg/kg/day)5. Description of the An1mal-to-Human Extrapolation Procedure Using the Linearized Multistage Model
Let P(d) represent the lifetime risk (probability) of cancer at dose d. The multistage model has the form
It
P(d) = 1 - exp [--(qQ + q-jd + q2d2 + ... + qfcd )]
B-2
where
> O, 1 = O, 1, 2, ..., k
Equivalently,
Pt(d) = 1 - exp [(q-jd + q2d2 + ... + qkdk)]
where
Pt(d) = Pfdl - PO) 1 - P(O)
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, Pj.(d), at any given dose d, 1s
calculated by maximizing the likelihood function of the data.
The point estimate and the 95% upper confidence limit of the extra
risk, P^(d), are calculated by using the computer program GLOBAL 79
developed by Crump and Watson (1979). At low doses, upper 95% confidence
limits on the extra risk and lower 95% confidence limits on the dose
producing a given risk are determined from a 95% upper confidence limit,
q^* on parameter q^. Whenever q^>0, at low doses the extra risk
Pt(d ) has approximately the form Pt(d) = q^d. Therefore, q ^ x d
1s a 95% upper confidence limit on the extra risk and P^/Q-j* 1s a 95%
lower confidence limit on the dose producing an extra risk of P^. Let
LQ be the maximum value of the log-Hkel1hood function. The upper limit,
q.j*, 1s calculated by Increasing q1 to a value q^* such that when the
log-Hkel1hood 1s remax1m1zed subject to this fixed value q^* for the
linear coefficient, the resulting maximum value of the log-Hkel1hood L^
satisfies the equation
2 (LQ - Lj) = 2.70554
B-3
where 2.70554 1s the cumulative 90% point of the ch1-square distribution with one degree of freedom, which corresponds to a 95% upper limit (one sided). This approach of computing the upper confidence limit for the extra risk, Pt(d), 1s an Improvement on the Crump et al. (1977) model. The upper confidence limit for the extra risk calculated at low doses 1s always linear. This 1s conceptually consistent with the linear nonthreshold. The slope, q-|*, 1s taken as a plausible upper bound of the potency of the chemical 1n Inducing cancer at low doses. (In the section calculating the risk estimates, P^.(d ) 1s abbreviated as P.)
In fitting the dose-response model, the number of terms 1n the poly nomial Is chosen equal to (h-1), were h 1s the number of dose groups 1n the experiment, Including the control group.
Whenever the multistage model does not fit the data sufficiently well, data at the highest dose are deleted and the model 1s refit to the rest of the data. This 1s continued until an acceptable fit to the data 1s ob tained. To determine whether or not a fit 1s acceptable, the chi-square
h X2 = (R^ - N ^ ) 2
1 = 1 N ^d-P!)
statistic 1s calculated where
1s the number of animals 1n the 1th
dose group, R^ 1s the number of animals 1n the 1th dose group with a
tumor response,
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 fit Is determined to be unacceptable when
ever X2 1s larger than thecumulative 99% point of the ch1-square dis
tribution with f degrees of freedom, where f equals the number of dose
groups minus the number of nonzero multistage coefficients.
B -4
TABLE B-l
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)*
Tissue and Diagnosis
0 (control)
Dose Levels (ua/kq/dav)
0.001
0.01
0.1
Dow (Koclba) Analysis 1. Tongue
Stratified squamous cell carcinoma
2. Nasal turblnates/hard palate Squamous cell carcinoma
0/76 (0%) 0/51 (0%)
1/49 (2X) 1/34 (3X)
1/49 (2X) 0/27 (OX)
3/42 (7X) (p=0.043)
4/30 (13X) (p=0.016)
B-5
Total
0/76 (0%)
2/49 (4X)
1/49 (4X)
7/42 (17X) (p=5.12xl0_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 (3X)
1/49 (2X) 0/26 (OX)
3/44 (7X) (p=4.60xl0_2)
6/30 (20X) (p=l.36x10")
Total (1 or 2 above)
(each rat had at least one tumor above)
0/77 (OX)
Average body weight of male rat = 600 g
2/44 (5%)
1/49 (2X)
9/44 (20X) (p=6.28xl0~5)
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 Substrain (2 yrs)*
B-6
Tissue and Diagnosis
Dow (Koclba) Analysis
1. Lung Keratinizing squamous cell carcinoma
015
2* Nasal turb1nates/hard palate Stratified squamous cell
carcinoma (revised diagnoses
2/19/79)
3. Liver Hepatocellular hyperplastic nodules/hepatocellular
carcinoma
Total (1, 2, or 3 above) (each rat had at least one
tumor above)
0 (control)
Dose Levels (uq/kq/day)
0.001
0.01
0.1
0/86 (OX) 1/54 (2%)
0/50 (OX) 0/30 (OX)
9/86 (10%)
3/50 (6%)
9/86 (10%)
3/50 (6%)
0/49 (OX) 1/27 (4%)
7/49 (14X) (p=6.21xl0~4 )
5/24 (21X) (p=9.46xl0-3)
18/50 (36X) (2 had both) (p=4.37xl0-4)
34/48 (71X) (p=9.53xl0~13)
18/50 (36X) (p=4.37xl0~4 )
34/49 (69X) (p=2.13xl0_12)
TABLE B-2 (cont.)
Tissue and Diagnosis
_____________________ Dose Levels (ug/kq/day)______________
0 (control)
0.001
0.01
0.1
R. Squire's Review
1 . Lung Squamous cell carcinoma
2. Nasal turb1nate/hard palate Squamous cell carcinoma
OD
3. Liver Neoplastic nodules/hepatocellular carcinoma
0/86 (054) 0/54 (054) 16/86 (054)
0/50 (054) 0/30 (054) 8/50 (1654)
0/49 (054)
1/27 (454)
27/50 (5454) (p=2.42xl0~5 )
8/47 (1754) (poi. 61x10")'
5/22 (2354) (p=1.43xl0-3)
33/47 (7054) (p=4.92x10"*)
Total combined (1, 2 or 3 above) (each animal had at least
one tumor above)
16/86 (1954)
8/50 (1654)
27/50 (5454) (p=2.42xl0"s )
34/47 (7254) (p=l.20x10"*)
Average body weight of female rat = 450 g
B-7
TABLE B-3
NCI 2,3,7,8-TCDD (Gavage) Bioassay (No. 80-1765) Osbome-Hendel Female Rats (2 years; weight = 450 g)
B-8
Tissue and Diagnosis
Vehicle Control 0
Dose Levels (iiq/kq/week)
Low Medium 0.01 0.05
High 0.5
1. Liver Neoplastic nodule or hepatocellular carcinoma
2. Adrenal* Cortical adenoma, or carcinoma
5/75 (7%) 11/73 (15%)
1/49 (2%) 9/49 (18%)
3/50 (6%) 5/49 (10%)
14/49 (28%) (p=0.001)
14/46 (30%) (p=0.038)
*The biological significance of this tumor 1n old rats Is questionable, since It Is commonly observed In control rats and associated with the aging process.
TABLE B-4
NCI 2,3,7,8-TCDD (Gavage) Bioassay (No. 80-1765) B6C3F1 Male Mice (2 years; weight = 48 g)
B-9
Tissue and Diagnosis
Vehicle Control 0
Liver Hepatocellular adenoma or carcinoma
Hepatocellular carcinoma**
/ 1^/73 (21%) (p<0.001)a /
8/73 (11%) (p<0.001)a
aCochran-Arm1tage test for linear trend bused for Unit Risk Estimate
Dose Levels (uq/kq/week)
Low 0.01
Medium 0.05
12/49 (24%) 9/49 (18%)
13/49 (26%) 8/49 (16%)
High 0.5
27/50 (54%) (p=l.31xl0-4)
17/50 (34%) (p=0.002)
TABLE B-5
NCI 2,3,7,8-TCDD (Gavage) Bioassay (No. 80-1765) B6C3F1 Female Mice (2 years)3
B-10
Tissue and Diagnosis
1. Subcutaneous tissue Fibrosarcoma
2. Hematopoietic system Lymphoma or leukemia
3. Liver Hepatocellular adenoma or carcinoma
Hepatocellular carcinoma
4. Thyroid Follicular cell adenoma
Vehicle Control 0
1/74 (IX)
18/74 (24%)
3/73 (4%) (p=0.0050) 1/73 (1%) (p=0.008)6
0/69
Total (1, 2, 3 or 4 above) (each mouse had at least one tumor above)
22/74 (30%)
aAverage body weight of female mouse = 40 g bCochran-Armltage test for trend
Dose Levels (uQ/kq/week)
Low 0.04
Hedlum 0.2
1/50 (2%)
1/48 (2%)
12/50 (24%)
13/48 (27%)
6/50 (12%) 2/50 (4%)
6/48 (12%) 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=l.84xl0~3 )
6/47 (13%) (p=0.014)
5/46 (11%) (p=8.93xl0~3 )
31/47 (66%) (p=8.94xl0_s)
TABLE B-6
Curve Fit of the Multistage Model Parameters to Experimental Data by Study and Pathologist Linear Parameter q-|, Maximized to Give Upper 95% Limit q-|*
Compound................... 2,3,7,8-TCDD Study...................... Koclba - Dow Sex-species................ Male rat Height (wa )..... ........... 600 g Tumor sites (one or more)---Tongue - squamous cell carcinomas
Nasal turb1nates/hard palate - stratified squamous cell carcinoma (ref. Table B-l)
Pathologist - Koclba
B-l 1
Exposure level (mg/kg/day)
0
1 x 10 6
l x 10~5
1 x 10"*
+r/n
0/76
2/49
1/49
7/42
+r = number of animals with one or more of the tumors n = total number of animals examined
Estimated multistage parameters
qg
q-| q2 <13
a^l*
Goodness of fit X2
When all dose groups are used
1.40 x 10-2 1.10 x 103 0
5.86 x 1010 3.01 x 103 3.34 (d.f. = 2)
When the highest dose group Is not used
Above fit Is satisfactory
adl* =
the maximum linear component from the model with adequate goodness of fit (p>0.01) = 3.01xT03 (mg/kg/day)-1
qi* = aqi* (70/wa )1/3 = 1.47xl04 (mg/kg/day)-1, the upper 95% limit slope factor associated with human dose response.
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-|*
B--12
Compound................... 2,3,7,8-TCDD Study...................... Dow Sex-spec1es........;....... Hale rat Height (wa )................ 600 g T u m o r sites (one or more)....Nasal turblnates/hard palate - squamous cell carcinoma
Tongue - squamous cell carcinoma (ref. Table B-l)
Pathologist - Squire
Exposure level (mg/kg/day)
0
1 x 10-*
1 x 10-5
1 x 10"*
fr/n
0/77
2/44
1/49
9/44
+r = number of animals with <one or more of the tumors n = total number of animals examined
Estimated multistage parameters
When all dose groups are used
qg
<ll q2
<13
afll*
Goodness of fit X2
0.015
1.05 x 103
0
109.40 x 10
3.53 x 103
3.90 (d.f. = 1)
When the highest dose group 1s not used
Above fit 1s satisfactory
aqi* =
the maximum linear component from the model with adequate goodness of fit (p>0.01) = 3.53x103 (mg/kg/day)-1
fll* = afll* (^O/Wa)1^3 = 1-73x10* (mg/kg/day)-1, the upper 95% limit slope factor associated with human dose response.
TABLE B-8
Curve Fit of the Multistage Model Parameters to Experimental Data by Study and Pathologist Linear Parameter q-|, Maximized to Give Upper 95% Limit q^*
Compound................... 2,3,7,8-TCDD Study...................... Dow Sex-species................ Female rat /Weight (wa )................ 450 g Tumor sites (one or more)___Liver, lung, hard palate, or nasal turbinates (ref. Table B-2)
Pathologist - Koclba
B-13
Exposure level (mg/kg/day)
0
1 x 10-6
1 x 10-5
1 x 10-4
+r/n
9/86
3/50
18/50
34/49
+r = number of animals with one or more of the tumors n = total number of animals examined
i Estimated
multistage parameters
qg 91
92
When all dose groups are used
0.12
1.23 x 104
0
93 a9l* 0 1.67 x 104
Goodness of fit X2
6.67 (d.f. = 2) 0.025 < p<0.05
When the highest dose group Is not used
0.09
0
Above fit Is satisfactory
3.5 x 109
0
4.69 x 104
0.92 (d.f. = 1) p>0.25
When the two highest dose groups are not used
Above fit Is satisfactory
aqi* =
the maximum linear component from the model with adequate goodness of fit (p>0.01) = 1.67xl04 - 4.69xl04 (mg/kg/day)-1
qi* =
afll* (70/wa
= 8.98xl04 - 2.52xl05 (mg/kg/day)-1, the upper 95% limit slope
factor associated with human dose response depending on Inclusion or exclusion of the highest dose
data.
TABLE B-8A
Curve 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-spec1es................. Female rat Weight (wa )................. 450 g Tumor sites (one or more)___ Liver, lung, hard palate, or nasal turbinates
(ref. Table B-2)
Pathologist - Koclba (Eliminating first year's data to adjust for high early mortality 1n the high-dose group.)
Exposure level (mg/kg/day)
0
1 x 10"
1 x 10"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 multistage parameters qg
<n 32 <13 a<Il*
Goodness of fit X2
When all dose groups 0.11 2.08 x 104 0 are used
0 2.82 x 104 3.38 (d.f. = 2) 0.25 < p < 0.10
When the highest dose group 1s not used
Above fit Is satisfactory
p > 0.25
aq-|* =
the maximum linear component from the model with adequate good ness of fit (p>0.01) = 2.82xl04 (mg/kg/day) 1
qi* = aq-|* (70/wa )1/3 = 1.51x10s (mg/kg/day)"1, the upper 95% limit slope factor associated with human dose response.
B-14
Curve Fit of the Multistage Model Parameters to Experimental Data by Study and Pathologist Linear Parameter q-|, Maximized to Give Upper 9554 Limit q-|*
Compound................... 2,3,7,8-TCDD Study...................... Koclba - Dow Sex-species................ Female rat Weight (wa )................ 450 g Tumor sites (one or more)___ Liver, lung, hard palate, or nasal turbinates (ref. Table B-2)
Pathologist - Squire
Exposure level (mg/kg/day)
0
1 x 10-6
1 x 10-s
1 x 10"*
+r/n
16/86
8/50
27/50
34/47
+r = number of animals with ione or more of the tumors n = total number of animals examined
B-15
Estimated multistage parameters
qg
<11
Goodness of fit
<12 <13 a<ll*
X2
When all dose groups are used
0.26
1.25 x 10
0
0
9.8 (d.f. = 2) p<0.01
When the highest dose group Is not used
0.19
0
5.83 x 109
7.90 x 10
0.209 (d.f. = 1)
When the two highest dose groups are not used
Above fit Is satisfactory
a<11* =
the maximum linear component from the model with adequate goodness of fit (p>0.01) = 7.90x10* (mg/kg/day)-1
qi* = aq-|* (70/wa )1/3 = 4.25x10s (mg/kg/day)-1, the upper 9554 limit slope factor associated with human dose response.
i
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....................... Koclba - Dow Sex-spec1es................. Female rat Weight (wa ).................450 g Tumor sites (one or more)....Liver, lung, hard palate, or nasal turbinates
(ref. Table B-2)
Pathologist - Squire (Eliminating first year's data to adjust for high early mortality 1n the high-dose group.)
Exposure level (mg/kg/day)
0
1 x 10"*
1 x 10-5
1 x 10"4
+r/n
16/85
8/48
27/48
34/40
+r = number of animals with one or more of the tumors n = total number of animals examined
Estimated multistage parameters qo
q-| q2 q3 aq-|*
Goodness of fit X2
When all dose groups
are used
0.24 2.12 x 10 0 0 3.00 x 10 6.41 (d.f.= 2)
0.025 < p < 0.05
When the highest dose group 1s not used
Above fit 1s satisfactory
aq-|* =
the maximum linear component from the model with adequate good ness of fit (p>0.01) = 3.00xl04 (mg/kg/day)_1
qi* = aq-|* (70/wa )1/3 = 1.61x10s (mg/kg/day)-1, the upper 95% limit slope factor associated with human dose response.
B--16
TABLE B-10
Curve Fit of the Multistage Model Parameters to Experimental Data by Study and Pathologist Linear Parameter q-j, Maximized to Give Upper 95% Limit qi*
Compound................... 2,3,7,8-TCDD Study...................... NCI Sex-species................ Female rat Weight (wa )..... ........... 450 g } Tumor sites (one or more)---Liver neoplastic nodules or hepatocellular carcinoma (ref. Table B-3)
Pathologist - NCI Reviewed
Exposure level (mg/kg/day)
0
1.43 x 10"*
7.14 x 10"*
7.14 x 10"5
r/n
5/75
1/49
3/50
14/49
8-- 17
+r = number of animals with one or more of the tumors n = total number of animals examined
Estimated multistage parameters
When all dose groups are used
10 11
0.05
0
<12 5.65 x 107
13 air 0 6.09 x 103
Goodness of fit X2
1.44 (d.f. = 2)
When the highest dose group 1s not used
Above fit Is satisfactory
aq-|* =
the maximum linear component from the model with adequate goodness of fit (p>0.01) = 6.09xl03 (mg/kg/day)"1
qi* = aqi* (70/wa )l/3 = 3.28xl04 (mg/kg/day)"1, the upper 95% limit slope factor associated with human dose response.
TABLE B--11
Curve Fit of the Multistage Model Parameters to Experimental Data by Study and Pathologist Linear Parameter q-|, Maximized to Give Upper 9554 Limit q-|*
Compound................... 2,3,7,8-TCDD Study...................... NCI Sex-species................ 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"*
7.14 x 10"*
7.14 x 10-5
*r/n
8/73
9/49
8/49
17/50
B-18
+r = number of animals with one or more of the tumors n = total number of animals examined
Estimated multistage parameters
When all dose groups are used
qg 0.15
11 3.80 x 10;
92 93
a9l*
0 0 6.63 x 103
Goodness of fit X2
2.43 (d.f. = 2)
When the highest dose group Is not used
Above fit Is satisfactory
a9l* =
the maximum linear component from the model with adequate goodness of fit (p>0.01) = 6.63x103 (mg/kg/day)-1
91* = aq-|* (70/wa )1/3 = 7.52xl04 (mg/kg/day)-1, the upper 95% limit slope factor associated with human dose response.
TABLE B-12
Curve Fit of the Multistage Model Parameters to Experimental Data by Study and Pathologist Linear Parameter q-|, Maximized to Give Upper 95% Limit q-|*
Compound................... 2,3,7,8-TCDD Study...................... NCI Sex-spec1es................ Female mice Weight (wa )................ 40 g Tumor sites (one or more)---Subcutaneous tissue - Fibrosarcoma, hematopoietic system lymphoma, or leukemia
Liver - hepatocellular adenoma or carcinoma (ref. Table B-5)
Pathologist - NCI Reviewed
B--19
Exposure level (mg/kg/day)
0
5.71 x 10"
2.86 x 10"5
2.86 x 10"
+r/n
22/74
20/50
19/48
31/47
+r = number of animals with one or more of the tumors n = total number of animals examined
Estimated multistage parameters
When all dose groups are used
qg 0.41
<11 2.38 x 103
fl2 <13
a<Il*
0 0 3.78 x 10
Goodness of fit Xa
1.20 (d.f. = 2)
When the highest dose group Is not used
Above fit Is satisfactory
aq-|* =
the maximum linear component from the model with adequate goodness of fit (p>0.01) = 3.78xl03 (mg/kg/day)-1
Ql* = a<ll* (70/Wa)1/3 = 4.56x10 (mg/kg/day)-1, the upper 95% limit slope factor associated with human dose response.
TABLE B-13 Summary of Human Slope Estimates for 2,3,7,8-TCDD
B-20
Species
Study
Sex
Pathologist
Human Slope Estimate q-|* In (mg/kg/day)-1
Rat Rat Rat
Rat
Rat Mice Mice
Dow Male
Koclba
1.47 x 10*
Squire
1.73 x 10*
Female
Koclba - unadjusted
8.98 x 10* - 2.52 x 10s
- adjusted for early deaths
1.51 x 105+
Female
Squire - unadjusted
4.25 x 105
- adjusted for early deaths
1.61 x lost
NCI
Female
NCI - Reviewed
3.28 x 10*
NCI Male
NCI - Reviewed
7.52 x 10
Female
NCI - Reviewed
4.56 x 10
+Values used to determine geometric mean of 1.56 x 10s (mg/kg/day) 1
Ref. Table No.
B6 B7 B8 B8A
B9 B9A
BIO Bll B12
APPENDIX C COMPARISON OF RESULTS BY VARIOUS EXTRAPOLATION MODELS
The estimate of unit risk from animals presented 1n the body of this document 1s calculated by the use of the linearized multistage model, for the reasons given herein. The use of this nonthreshold model 1s part of a methodology that estimates a conservative linear slope at low extrapolation doses that 1s 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 doseresponse relationship.
Other nonthreshold models that have been used for risk extrapolation are the one-h1t, the log-Prob1t, and the Welbull models. The one-h1t model 1s characterized by a continuous downward curvature, but Is linear at low doses. Because of Its functional form, the one-h1t model can be considered the linear form or first stage of the multistage model. This fact, together with the downward curvature of the one-h1t model, means that 1t will always yield low-level risk estimates which are at least as large as those of the multistage model. In addition, whenever the data can be fitted adequately by the one-h1t model, estimates based on the one-h1t model and the multi stage model will be comparable.
The log-Problt and the Welbull models, because of their general "Sn curvature, are often used for the Interpretation of toxicological data 1n the observable range. The low-dose upward curvatures of these two models usually yield lower low-dose risk estimates than those of the one-h1t or multistage models. The log-Prob1t model was originally used 1n biological assay problems such as potency assessments of toxicants and drugs, and 1s
C-l
generally used to estimate such values as percentile lethal dose or percen tile effective dose. The development of the model occurred along strictly empirical lines, l.e., 1t was observed 1n these studies that several log dose-response relationships followed the cumulative normal probability distribution function, $. In fitting the cancer bioassay data, assuming an Independent background, this becomes
P(D;a,b,c) = c + (1-c) i> (a+blog1Q 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-Prob1t response slope.
The one-h1t 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-(a+bd) a,b > 0 where a and b are the parameter estimates. The estimate a represents the background or zero dose rate, and the parameter estimated by b represents the linear component or slope of the dose-response model. In discussing the added risk over background, Incorporation of Abbott's correction leads to
P(D;b) = l-exp-(bd) b > 0 Finally, a model from the theory of carcinogenesis arises from the multihit model applied to multiple target cells. This model has been termed here the Welbull model. It 1s of the form
P(D;b,k) = l-exp-(bdk) b,k > 0 For the power of dose only, the restriction k > 0 has been placed on this model. When k > 1, this model yields low-dose estimates of risks usually significantly lower than either the multistage or one-h1t models, which are
C-2
linear at low doses. When 0 < k < 1, the model yields low-dose estimates of risk that are greater than the one-hit and multistage models; this Is generally regarded as biologically Implausible. All three of these models usually project risk estimates that are significantly higher at low exposure levels than those projected by the log-Problt model.
The Dow Chemical Company data for female Sprague-Dawley rats were fitted to the above models, after adjusting for early mortality by eliminating all animals dying before 1 year. The results are Identical for the multistage and one-h1t models, as shown In Tables C-l and C-2. The log-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-h1t or the multistage model, since k, determined by best fit to the data, 1s <1. As discussed 1n the text and shown 1n Tables B-8 and B-9* dropping the highest dose resulted in a larger upper-Umlt slope estimate for the multistage model. However, without the highest dose points, neither the log-Prob1t nor the Welbull models could be fitted to the data, for the reason that the control group response was higher than that of the lowest dose group.
C-3
A toxicity-based criterion has bben caiculated Tbr comparisort with the cancer-based criterion 1n accordance, with public comments. 1>1nce the data
from the limited study by Schantz jet a l . (1979) are supportive of the find
ings by Hurray et al. (1979), it seems reasonable to determine an ADI based on the LOAEL. if one selects an uncertainty factor of 100 based on the Existence of. lifetime animal studies a/td knowledge of effects 1n mar\ as per U.S. EPA methodologies (Federal Register, 1980b), and then an additional 10 because a LOAEL 1s used as the basis of this calculation,* then the ADI for a 70 kg man would be:
-10' pg/kg/day (LOAEL)
ADI =
= 7 . 0 x 10~5 pg/kg/day.
100 x 10
However, th1$ concentration may not be sufficiently protective of human health since 1t does not take Into account the demonstrated carcinogenic effects of 2,3,7,8-TCDD 1n anlm&ls and the probability that 2,3,7,8-TCDD 1s a human carcinogen as discussed 1n Section 11.6.1.
According to the methods published by U.S. EPA (Federal Register, 1980b), an additional uncertainty factor between 1 and 10 must be used because the calculation ,1s, .based on a LOAEL. / An uncertainty factor of 10 was chosen because of the adverse effects seen 1n rhesus monkeys at 0.0015 pg/kg/day, despite the equivocal nature of the effects 1n rats seen at "the 0.001 pg/kg/day dose level.
6 U.S. GOVERNMENT PRINTING OFFICE:! 9 8 7 - 7 4 8 - 1 2 1 / 6 7 0 2 4
D-2