xvEPA
United States
Environmental Protection
Agency
Office of Water
Regulations and Standards
Washington, DC 20460
EPA 440/5-84-007
February 1984
Water
440584007
Ambient
Water Quality
Criteria for
2, 3, 7, 8 - Tetrachloro-
dibenzo - p - dioxin
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AMBIENT WATER QUALITY CRITERIA FOR
2,3,7,8-TETRACHLORODIBENZO-P-DIOXIN
Prepared By
U.S. ENVIRONMENTAL PROTECTION AGENCY
Office of Water Regulations and Standards
Criteria and Standards Division
Washington, D.C.
Office of Research and Development
Environmental Criteria and Assessment Office
Cincinnati, Ohio
Carcinogen Assessment Group
Washington, D.C.
Reproductive Effects Assessment Group
Washington, D.C.
Environmental Research Laboratories
Corvallis, Oregon
Duluth, Minnesota
Gulf Breeze, Florida
Narragansett, Rhode Island
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DISCLAIMER
This report has been reviewed by the Environmental Criteria
and Assessment Office, U.S. Environmental Protection Agency, and
approved for publication. Mention of trade names or commercial
products does not consititute endorsement or recommendation for
use.
11
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FOREWORD
Section 304 (a)(l) of the Clean Water Act of 1977 (P.L. 95-217),
requires the Administrator of the Environmental Protection Agency to publish
criteria for water quality accurately reflecting the latest scientific
knowledge on the kind and extent of all Identifiable effects on health and
welfare which may be expected from the presence of pollutants 1n any body of
water. Including groundwater. Proposed water quality criteria for the 65
toxic pollutants listed under section 307 (a)(l) of the Clean Water Act were
developed and a notice of their availability was published for public
comment on March 15, 1979 (44 FR 15926), July 25, 1979 (44 FR 43660), and
October 1, 1979 (44 FR 56628). This document 1s a revision of those
proposed criteria based upon a consideration of comments received from other
Federal Agencies, State agencies, special Interest groups, and Individual
scientists. The criteria contained 1n this document replace any previously
published EPA criteria for the 65 pollutants. This criterion document 1s
also published 1n satisfaction of paragraph 11 of the Settlement Agreement
In Natural Resources Defense Council, et al. vs. train. 8 ERC 2120 (D.D.C.
1976), modified, 12 ERC 1833 (D.D.C. 1979).
The term "water quality criteria" Is used In two sections of the Clean
Water Act, section 304 (a)(l) and section 303 (c)(2). The term has a
different program Impact 1n each section. In section 304, the term repre-
sents a non-regulatory, scientific assessment of ecological effects. The
criteria presented 1n this publication are such scientific assessments.
Such water quality criteria associated with specific stream uses when adopt-
ed as State water quality standards under section 303 become enforceable
maximum acceptable levels of a pollutant in ambient waters. The water
quality criteria adopted 1n the State water quality standards could have the
same numerical limits as the criteria developed under section 304. However,
in many situations States may want to adjust water quality criteria
developed under section 304 to reflect local environmental conditions and
human exposure patterns before incorporation into water quality standards.
It is not until their adoption as part of the State water quality standards
that the criteria become regulatory.
Guidelines to assist the States 1n the modification of criteria present-
ed In this document, 1n the development of water quality standards, and in
other water-related programs of this Agency, have been developed by EPA.
STEVEN SCHATZOW
Director
Office of Water Regulations and Standards
111
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ACKNOWLEDGEMENTS
Aquatic Toxicology:
Charles E. Stephan (author)
Environmental Research Laboratory,
Duluth
U.S. Environmental Protection Agency
Gary A. Chapman
Environmental Research Laboratory,
CorvalUs
U.S. Environmental Protection Agency
David J. Hansen (reviewer)
Environmental Research Laboratory,
Narragansett
U.S. Environmental Protection Agency
Mammalian Toxlclty and Human Health Effects*:
Debdas Mukerjee (document manager)
Environmental Criteria and Assessment
Office, Cincinnati
U.S. Environmental Protection Agency
Roy Albert
Institute of Environmental Medicine
New York University Medical Center
Donald G. Barnes
Office of Pesticides and Toxic
Substances
U.S. Environmental Protection Agency
Steven P. Bayard
Carcinogen Assessment Group
U.S. Environmental Protection Agency
David L. Bayliss
Carcinogen Assessment Group
U.S. Environmental Protection Agency
Dipak K. Basu
Syracuse Research Corporation
Randall J.F. Bruins
Environmental Criteria and Assessment
Office, Cincinnati
U.S. Environmental Protection Agency
K. Diane Courtney
Health and Effects Research
Laboratory, Research Triangle Park
U.S. Environmental Protection Agency
Frederick Coulston
Coulston International Corporation
Michael L. Dourson
Environmental Criteria and Assessment
Office, Cincinnati
U.S. Environmental Protection Agency
David Firestone
Food and Drug Administration
S. Garattlni
Institute d1 Recerche
Farmacologic "Mario Negrl"
Milan, Italy
Richard Grelssmer
Oak Ridge National Laboratory
Bernard H. Haberman
Carcinogen Assessment Group
U.S. Environmental Protection Agency
Lennart Hardell
University Hospital
Umea, Sweden
*An additional 60 participants from EPA's headquarters, Research Triangle
Park, Cincinnati, and regional offices and 135 observers from industries,
academia, environmental groups and news media also attended the meeting at
which this chapter was reviewed.
1v
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Robert Harless
Environmental Monitoring Systems
Laboratory
U.S. Environmental Protection Agency
Rolf Hartung
University of Michigan
AUstalr W.M. Hay
University of Leeds, U.K.
Charallnggayya Hlremath
Carcinogen Assessment Group
U.S. Environmental Protection Agency
Otto Hutzinger
University of Amsterdam
The Netherlands
R.D. Klmbrough
Centers for Disease Control
Richard 3. Kociba
Dow Chemical Company
Marvin Legator
University of Texas Medical Branch
Ruth L1l1s
Mt. Sinai School of Medicine
Prab D. LotHkar
Temple University School of Medicine
Fumio Matsumura
Michigan State University
E. McConnell
National Institute of Environmental
Health Sciences
W.P. McNulty
Oregon Regional Primate Research
Center
Robert Miller
National Cancer Institute
Ralph Nash
U.S. Department of Agriculture
Charles H. Nauman
Exposure Assessment Group
U.S. Environmental Protection Agency
Michael W. Neal
Syracuse Research Corporation
James Olsen
School of Medicine
State University of New York
F. Pocchiarl
Institute Superlore d1 Sanlta
V1ale Regina, Rome, Italy
Shane Que Hee
University of Cincinnati Medical
Center
C. Rappe
University of Umea, Sweden
Sheila L. Rosenthal
Reproductive Effects Assessment Group
U.S. Environmental Protection Agency
Steven H. Safe
Texas A&M University
Marvin Schneiderman
Environmental Law Institute
Ellen Silbergeld
Environmental Defense Fund
David Stalling
Columbia National Fisheries Research
Laboratory
Jerry F. Stara
Environmental Criteria and Assessment
Office, Cincinnati
U.S. Environmental Protection Agency
Lewis Thibodeaux
University of Arkansas
Thomas Tiernan
Wright State University
Technical Support Services Staff: J.A.
Mann, E. Durden, C.A. Cooper
Olsen, B.L. Zwayer, P.A. Daunt, K.S.
Clerical Staff: N.C. Bauer, S.J. Faehr, T. Highland, L.A. Schwaegerle
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TABLE OF CONTENTS
Page
INTRODUCTION .....
..................... A- 1
Physical Properties ........... . ,
Cocontamlnants of 2,3,7,8-TCDO In Chlorinated Products ...... A ?
Synthesis ........ ...... V"^
Chemistry ........... [ [ [ .............. ?~~
Analytical Methods for TCDD ... ................ J "f
Summary of Health Effects ..... .............. 2 R
References ............... ."!.'.'.'.*!!.'.''' A-9
AQUATIC TOXICOLOGY ...... B ,
................... B- 1
Introduction ............... R ,
Acute Toxicity to Aquatic Animals ....... ......... B~i
Chronic Toxicity to Aquatic Animals ..... . . [ ....... B"2
Toxicity to Aquatic Plants ........... ...... . " ' ' B"3
Bioaccumulation ...... ............. R~
Other Data ............ '.'.'.'.'.'.'. ......... B 6
Unused Data ........... .".....'.'. ........ n 6
Summary .............. ............... R"
National Criteria ............ '.'.'.'.'.'.'.'.'''' B-10
References ....... ........... D~n
................... b- 1 I
MAMMALIAN TOXICOLOGY AND HUMAN HEALTH EFFECTS ............. C-l
EXPOSURE
Water and Soil Related. ................ c_-,
Ingestion from Food ........... .'.'.* ......... r~6
'"" ............ '
Inhalation
Dermal
(._1
-,
6
........... C-l 7
PHARMACOKINETICS
c_18
Absorption .......... r 1R
Distribution ........ " ........... r",5
Metabolism .............. .'.'.*.'.'."!!! ..... C-28
Excretion ................ ....... ..... r-31
EFFECTS .......... r „,
...................... L-JO
Acute, Subacute and Chronic Toxicity ............... C_35
Synerglsm and/or Antagonism ........... ....... C-69
Teratogenicity ................. . . . . . C-72
Mutagen1c1ty ............... ......... C-102
Carcinogenlcity ................ .'!!!.'.' C-115
vi
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TABLE OF CONTENTS
CRITERION FORMULATION ......................... c-176
Existing Guidelines and Standards ................ C-176
Current Levels of Exposure ....................
Special Groups at Risk ......................
Basis and Derivation of Criterion ................
Estimates by Others of Carcinogenic Potency and Criteria .....
REFERENCES .............................. C"185
APPENDIX
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LIST OF TABLES
No. Title paqe
1. Predicted B1oconcentrat1on Factors for 2,3,7,8-TCDD
Based on Estimated and Measured Values of the Octanol-
Water Partition Coefficient B-4
2. Other Data on Effects of 2,3,7,8-TCDD on Aquatic Organisms. . B-7
1. Levels of 2,3,7,8-TCDD 1n F1sh and Shellfish C-10
2. Percentage of 2,3,7,8-TCDD 1n the Liver 24 Hours After
Oral Administration of 0.5 ma of Various Formulations
Containing TCDD C-20
3. Tissue Distribution of 2,3,7,8-TCDD C-23
4. Elimination of 2,3,7,8-TCDD C-33
5. Lethality of 2,3,7,8-TCDD Following Acute Exposure C-37
6. Toxic Responses Following Exposure to 2,3,7,8-TCDD:
Species Differences C-42
7. Hepatocellular Fatty Change Observed In Rats Following
Subchronlc Exposure to 2,3,7,8-TCDD C-56
8. Effects of Chronic Exposure to 2,3,7,8-TCDD In
Laboratory Rodents C-59
9. Studies on the Potential Teratogenlc Effects of
2,3,7,8 TCDD-Contamlnated 2,4,5-T C-74
10. Studies on the Potential Teratogenlc Effect of
2,3,7,8-TCDD C-79
11. The Results of Mutagenlclty Assays for 2,3,7,8-TCDD 1n
Salmonella typhlmurlum C-103
12. Distribution of Tumor Types In Two Case-Control Studies
of Soft-Tissue Sarcoma C-122
13. Exposure Frequencies 1n Two Case-Control Studies of
Soft-Tissue Sarcoma C-123
14. Relative Risks of Soft-Tissue Sarcoma 1n Relation to
Exposure to Phenoxyacetlc Adds and Chlorophenols In Two
Case-Control Studies C-125
15. Distribution of Hlstologlcal Types of Soft-Tissue
Sarcomas C-130
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Therefore, the levels that may result In an Increase of cancer risk over the
lifetime are estimated at 10-, 1(T« and 10-. The corresponding
recommended criteria are 1.3x10-, 1.3x10- and 1.3x10- yg/l.
respectively. If the above estimates are made for consumption of aquatic
organisms only, excluding consumption of water, the levels are 1.4x10-.
1.4x10- and 1.4x10- vg/l, respectively. If these estimates are made
for consumption of water only, the levels are 2.2x10-, 2.2x10- and
2.2x10— pg/l, respectively.
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CRITERIA DOCUMENT
2,3,7,8-TETRACHLORODIBENZO-P-DIOXIN
CRITERIA
Aquatic Life
Not enough data are available concerning the effects of 2,3,7,8-TCDD on
aquatic life and Its uses to allow derivation of national criteria. The
available Information Indicates that acute values for some freshwater animal
species are >1.0 pg/&; some chronic values are <0.01 pg/n, and the
chronic value for rainbow trout Is <0.001 yg/i. Because exposures of
some species of fishes to 0.01 pg/s. for <6 days resulted 1n substantial
mortality several weeks later, derivation of aquatic life criteria for
2,3,7,8-TCDD may require special consideration. Predicted bloconcentratlon
factors (BCFs) for 2,3,7,8-TCDD range from 3000-900,000, but the available
measured BCFs range from 390-13,000. If the BCF Is 5000, concentrations
>0.00001 yg/j. should result In concentrations in edible freshwater and
saltwater fish and shellfish that exceed levels Identified in a U.S. FDA
health advisory. If the BCF is >5000 or 1f uptake 1n a field situation is
greater than that in laboratory tests, the value of 0.00001 yg/a. will be
too high.
Human Health
For the maximum protection of human health from the potential carcino-
genic effects due to 2,3,7,8-TCDD exposure through Ingestlon of contaminated
water and contaminated aquatic organisms, the ambient water concentration
should be zero. This criterion is based on the non-threshold assumption for
2,3,7,8-TCOD. However, zero may not be an attainable level at this time.
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LIST OF TABLES
No. litle
16 Midland County Soft and Connective Tissue Cancer
Deaths 1960-1981
17. Other Occupations (Minus Forestry/Agriculture) ........ C-145
18. Other Occupations (Minus Forestry/Agriculture/
Woodworkers) ......................... c"14b
19 Analysis of Stomach Cancer Mortality In a Group of
West German Factory Workers Exposed to 2,3,7,8-TCDD ..... C-lbU
20 Reanalysls of Stomach Cancer Mortality in a Group of
West German Factory Workers Exposed to 2,3,7,8-TCOD ..... C-153
21. Stomach Cancer Mortality In Three Studies of Workers
Exposed to Phenoxyacetlc Acid Herbicides and/or
2,3,7,8-TCDD ......................... c-'bt)
22 Incidence of Primary Tumors In Female Swiss-Webster
Mice by Dermal Application of 2,3,7,8-TCDD or
2,3,7,8-TCDD Following DMBA ................. c-'60
23 Incidence of Primary Tumors in Male Swiss-Webster
Mice by Dermal Application of 2,3,7,8-TCDD or
2,3,7,8-TCDD following DMBA ................. c-'61
24 Summary of Neoplastic Changes After 2,3,7,8-TCDD
in Rats ........................... C-163
25. Summary of Neoplastic Lesions Produced by 2,3,7,8-TCDD
in Sprague-Dawley Rats, Spartan Substrain, that are
Statistically Significant in at Least One Sex ........ C-166
26 2,3,7,8-TCDD Oral Rat Study by Dr. Kodba, with Dr.
Squire's Review (8/15/80) Female Sprague-Dawley Rats -
Spartan Substrain (2 years) ................. c-'67
27. 2,3,7,8-TCDD Oral Rat Study by Dr. Kociba, with Dr.
Squire's review (8/15/80) Male Sprague-Dawley Rats -
Spartan Substrain (2 years) ................. c-'68
28. Incidence of Primary Tumors in Male Osborne-Mendel Rats . . . C-170
29. Incidence of Primary Tumors 1n Female Osborne-Mendel Rats . . C-172
30. Incidence of Primary Tumors in Female B6CF1 Mice ....... C-173
31. Incidence of Primary Tumors in Male B6CF1 Mice ........ C-174
32. Summary of Human Potency Estimates for 2,3,7,8-TCDD ..... C-242
1x
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INTRODUCTION
The major source of 2,3,7,8-tetrachlorod1benzo-p_-d1ox1n (2,3,7,8-TCDD)
(CAS Number 1746-01-6) appears to be as a contaminant formed during the pro-
duction of 2,4,5-tMchlorophenol (2,4.5-TCP) from 1,2,4,5-tetrachlorobenzene
(Mllnes, 1971; Klmmlg and Schulz, 1957; Firestone et al., 1972). 2,4.5-TCP
1s the major chemical feedstock 1n the production of several herbicides
including 2,4,5-trlchlorophenoxyacetlc add (2,4,5-T). 2,4,5-T esters and
SHvex. Each of these chemicals may contain 2,3,7,8-TCDD as a contaminant
(Buser and Bosshardt, 1974; Courtney et al., 1970; Edmunds et al., 1973;
ZUko and Choi, 1971). It has also been reported that 2,3,7,8-TCDD may be
formed during the pyrolysls of chlorinated phenols (Buu-Ho1 et al.,
1971a,b), chlorinated benzenes (Buser, 1979) and polychloMnated dlphenyl
ethers (Llndahl et al., 1980), and thus can also be emitted by municipal
Incinerators (Rappe et al., 1983a; Lustenhouwer et al., 1980; 011 e et al.,
1982, 1983). There Is no clear evidence that 2,3,7,8-TCDD 1s a typical con-
taminant In the herbicide 2,4-d1chlorophenoxyacet1c add (2,4-D) (Woolson et
al., 1972; Henshaw et al., 1975; Cochrane et al., 1981).
Physical Properties
2,3,7,8-TCDD 1s a symmetrical, nearly planar molecule with the empirical
formula C H Cl^. The four chlorine atoms are Indistinguishable
from one another (Poland and Glover, 1973). 2,3,7,8-TCDD 1s a white crys-
talline solid with a melting point range of 302-305°C (Sparschu et al.,
1971; Elvldge, 1971) and has a molecular weight of 321.9. The vapor pres-
sure of this compound Is estimated to be 10~6 mm of Hg (0.1 mPa) at 1
atmosphere and 25°C (Mabey et al., 1981). The Henry's constant has been
estimated to be 2.1xlO"3 atmosphere m3 mol"1 (Mabey et al., 1981).
2,3,7,8-TCDD 1s I1poph1l1c, exhibiting some solubility 1n fats, oils and
A-l
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other relatively nonpolar solvents, and Is only slightly soluble in water
(0.2 pg/i) (Grummet and Stehl. 1973; Norrls, 1981). The solubility of
2,3,7,8-TCDD In various organic solvents 1s given below (Grummet and Stehl.
1973):
So1vent Solubility (ppm)
lard oil 44
benzene 57Q
o-d1chlorobenzene 1400
chloroform 37Q
acetone 110
n-octanol 5Q
methanol IQ
The partition coefficient of 2,3,7,8-TCDD in a water:hexane system has
been reported to be 1000 (Matsumura and Benezet, 1973). The octanol/water
partition coefficient (K^) has been calculated by the methods of Hansch
and Leo (1979) and has been experimentally measured. Calculated values for
log KOW range from 6.84-7.28, and a measured value of 6.15 has been
reported (see Section B, B1oaccumulat1on).
Cocontamlnants of 2.3.7.8-TCDD In Chlorinated Products
2,3,7,8-TCDD Is only one of many trace contaminants found In some
chlorinated Industrial products Including a few chlorinated phenols, a few
chlorinated phenoxy acids (especially the herbicides 2,4,5-T and Sllvex) and
hexachlorophene. Among the other trace contaminants found In these products
are members of the polychlorlnated d1benzo-p_-d1ox1ns (PCDDs), polychlorl-
nated dlbenzofurans (PCDFs), polychlorlnated dlphenylethers (PCDPEs), poly-
chlorlnated phenoxyphenols (PCPPs), polychlorlnated blphenyls (PCBs) and
polychlorlnated benzenes (PCBz). Of these, some possess properties that
make them difficult to separate analytically from the 2,3,7,8-TCDD Isomer
(Klmbrough, 1974; U.S. EPA, 1980; Bumb et a!., 1980; Rappe et al., 1983b).
A-2
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Synthesis
2,3,7,8-TCDD has been synthesized by several methods In moderate yield
(e.g., reaction of dlchlorocatechol salts with o-chlorobenzene by refluxlng
In alkaline dlmethylsulfoxlde; chlorlnation of dibenzo-p_-d1oxin 1n the pres-
ence of ferric chloride and Iodine; UV irradiation of PCOOs of high chlorine
content; Ullman reaction of chlorinated phenolates at 180-400°C; pyrolysis
of chlorinated phenolates and chlorinated phenols; heating 1,2,4-trichloro-
5-n1trobenzene and 4,5-d1chlorocatechol in the presence of base). These
processes have been reviewed in U.S. EPA (1980).
Chemistry
2,3,7,8-TCDD is considered to be relatively stable toward heat, acids
and alkalies. It begins to decdmpose at 500°C, and at a temperature of
800°C, virtually complete degradation occurs within 21 seconds (Stehl et
a!., 1973). From a theoretical equation for thermal dissociation constant
K = 1015'5 exp(-80,000/RT) sec"1
K = dissociation constant, R = universal gas constant, T = temperature
for tetrachlorod1benzo-j)-d1ox1ns formulated by Staub and Tsang (1983), the
99.99% gas phase dioxln dissociation at 727°C will require about 15.4
minutes. The same equation predicts a 99.99% decomposition of tetrachloro-
*
dibenzo-p_-dioxins in 0.3 seconds at 977°C. Gamma radiation degrades the
molecule (Fanelll et al., 1978). 2,3,7,8-TCDD can be perchlorinated
(Hutzinger et al., 1972).
2,3,7,8-TCDD 1s transformed very slowly in aquatic systems. Of the four
transformation processes (photoreactlon, biotransformation, hydrolysis and
radical oxidation) that control the fate of a chemical in aquatic media only
the first two processes are throught to effect the transformation of
2,3,7,8-TCDD (Matsumura et al., 1983). In organic solvents, 2,3,7,8-TCDD
undergoes reductive photodechlorinatlon at wavelengths <320 nm (Crosby et
A-3
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al., 1971; L1bert1 et al., 1978). In aqueous solution hydroxylatlve dechlo-
Mnatlon probably occurs, although this has not been seen. L1bert1 et al.
(1978) showed that 2,3,7,8-TCDD spread over silica gel, aluminum, glass,
ceramic tile and marble 1n the absence of an organic solvent showed various
decomposition rates on UV Irradiation. Little decomposition occurred on
glass or marble, but substantial degradation occurred on silica gel and
aluminum. Also, 1:1 ethyl oleate/xylene was found to be a satisfactory H
donor. pilmmer et al. (1973) reported that a 2,3,7,8-TCDD suspension 1n
distilled water remained unchanged when Irradiated with a sunlamp. Simi-
larly, 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 photodegradation after Irradiation with
sunlamps (Crosby et al., 1971). In contrast, 2,3,7,8-TCDD 1n methanol solu-
tion, or a benzene solution of 2,3,7,8TCDD 1n water 1n the presence of a
surfactant underwent substantial photodegradation under sunlamp or sunlight
Irradiation (Pllmmer et al., 1973; Crosby et al., 1971). NestMck et al.
(1980) experimentally determined the photolytlc half-life of 2,3,7,8-TCDD 1n
n-hexa-dccane under sunlamp Irradiation to be -57 minutes. The surfactant,
l-hexyldecylpyr1o1n1um chloride, sensitized the photodecompos1t1on 1n aque-
ous solution (Botre et al., 1978). The evolution of 2,3,7,8-TCDD from more
highly chlorinated PCDDs on UV Irradiation from sunlight Is unlikely since
dechlorlnatlon 1n organic solvents and 1n the presence of artificial UV
sources occurs preferentially at the 2,3,7,8-pos1t1ons (Buser and Rappe,
1978; NestMck et al., 1980).
Analytical Methods for TCDD
Most of the current analytical methods used for the Identification and
quantHatlon of 2,3,7,8-TCDD are based upon gas chromatography/mass spectro-
metry (GC/MS). This method provides both high sensitivity (detection at
A-4
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low-ppt levels) and required selectivity (Crummett and Stehl, 1973; Tlernan
et al.. 1975; Taylor et al.. 1975; Buser and Bosshardt, 1976; Buser, 1977;
U.S. EPA, 1980; Tlernan, 1983). Unfortunately, the GC/MS method Is expen-
sive, time consuming and difficult. Elaborate quality control and quality
assurance of analytical methods are necessary. Radlolmmunoassay and elec-
tron capture-GC have also been developed; both were essentially screening
techniques (Karasek and Onuska, 1982).
Sampling Method — Two types of sampling methods can be used for col-
lecting aqueous samples for TCOD. In the first method, no preconcentratlon
of the samples during collection 1s made. Grab samples are collected 1n
clean amber glass bottles of 1 I or 1 quart capacity fitted with screw
caps lined with clean Teflon or aluminum foil (U.S. EPA, 1982). The sample
containers must be kept refrigerated at 4°C and protected from light during
collection and shipment of grab and composite samples. All samples must be
extracted within 7 days and completely analyzed within 40 days of extraction
(U.S. EPA, 1982).
The second method 1s the preconcentratlve method of sample collection
(D1Domen1co et al., 1980). In tals method, 2-20 ft of water are allowed to
pass through a 12 cm long x 1.5 cm Internal diameter XAD-2 column. The
XAD-2 columns containing the polychlorlnated dloxlns should be protected
from light and kept at 4°C during transportation and storage.
Analysis — An appropriate volume of water (depending on the desired
13
detection limit) with added Internal standard of either C-,2 or
37C14 2,3,7,8-TCOO In the amount of 2.5-25 ng (Harless et al., 1980;
U.S. EPA, 1982) can be extracted with hexane (D1Domen1co et al., 1980),
dlchloromethane (U.S. EPA, 1982; Harless et al., 1980) or petroleum ether
A-5
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(Van Ness et al., 1980). Judging from the recovery data (U.S. EPA, 1982;
DIDomenico et al., 1980; Harless et al., 1980). dlchloromethane appears to
be a better solvent.
The extract containing TCODs can be cleaned up by add and base wash
(Harless et al., 1980; U.S. EPA, 1982; Van Ness et al., 1980) and by subse-
quent liquid chromatography with an alumina column (Harless et al., 1980;
Van Ness et al., 1980); however, U.S. EPA (1982) recommends another 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 is performed by low resolution
GC-HRMS (Van Ness et al., 1980; Harless et al., 1980) or high resolution
GC-HRMS or LRMS (U.S. EPA, 1982). The U.S. EPA (1982) method derived from
the method of Buser and Rappe (1980) seems to be an appropriate one because
it recommends using a 50 m Silar IOC capillary column that resolves 2,3,7,8-
TC110 from Us other Isomers. This same column can resolve 1,2,3,7,8-penta-
COO from other penta-CDDs, and 1,2,3,6,7,8-, 1,2,3,7,8,9- and 1,2,3,4,7,8-
hexa-CDDs from other hexa-CODs (Rappe et al., 1983a). Other suitable col-
umns include SP-2330, SP-2340 and DB-5 (Tinman, 1983). Harless et al.
(1980) reported that TCOD In water can be accurately determined down to a
concentration of 0.03 ppt. However, for determination of <1 ppt, rigorous
measures must be taken to avoid the possibility of sample contamination dur-
ing collection, storage, transportation or analysis.
Gas Chromatography/Mass Spectrometry (GC/MS): The mass spectrometral
pattern of 2,3,7,8-TCDO Is very similar to the spectra of other tetrachloro-
dibenzo-£-d1ox1ns. Since other compounds (e.g., certain polychlorinated
blphenyls) present In the sample extract can also give rise to mass spectral
A-6
-------
Ions at the same nominal masses as TCDDs (m/e 320 and m/e 322), two
approaches are being used to Increase specificity (U.S. EPA, 1980).
The first approach of applying high resolution mass spectrometry (M/AM
>9000) to Increase the selectivity makes use of the small difference In the
"exact" masses of TCDOs (C12H4C14°2 nav1ng an "exact" mass of
321.8936) compared with compounds of similar molecular weight. Application
of the optimum chromatographlc conditions and columns to maximize the reso-
lution of compounds 1s necessary before the MS step.
The second approach to avoid the problem of Interferences from closely
related compounds Is to make use of low-resolution mass spectrometry Incor-
porated with a more selective separation step such as capillary column GC
(Buser and Rappe, 1980; Rappe et a!., 1983b) or high performance liquid
chromatography followed by GC (Nestrick et al., 1979). The former method
can be used for all PCCOs and PCDFs; the latter method Is selective In char-
acterizing only the TCDDs (Bumb et al., 1980).
The following criteria have been outlined by Harless et al. (1980) for
confirmation of 2,3,7,8-TCDD residues:
1. Correct GC/MS 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 jnolecular Ions 320, 322
and 328.
4. Correct responses for the co-Injection of sample fortified with
37C1-TCDD and 2,3,7,8-TCDD standard.
5. Response of molecular Ions 320 and 322 must be >2.5 times the
noise level.
Supplemental criteria that Harless et al. (1980) suggested for highly
contaminated extracts are:
1. COC1 loss Indicative of TCDD structure
2. GC/MS peak-matching analysis of molecular Ions 320 and 322 1n
real time to confirm the 2,3,7,8-TCDD elemental composition.
A-7
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Summary of Health Effects
2,3,7,8-TCDD 1s one of the most toxic substances known. It exhibits a
delayed biological response 1n many species and 1s highly lethal at low
doses to aquatic organisms, birds and mammals. It has been shown to be
acnegenlc, fetotoxlc, teratogenlc, mutagenlc (1n a limited number of muta-
genldty tests) and carcinogenic, and affects the Immune responses 1n
mammals.
These "findings, In conjunction with the wide distribution of contami-
nated products and Us extreme stability 1n the environment, lead to the
conclusion that 2,3,7,8-TCDD represents a potential hazard to both aquatic
and terrestrial life, and makes 2,3,7,8-TCDD one of the major concerns for
public health.
A-8
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Llndahl, R.. C. Rappe and H.R. Buser. 1980. Formation of polychlorlnated
d1benzo-E-d1ox1ns (PCDOs) from the pyrolysls of polychlorlnated dlphenyl
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Lustenhouwer, 3.W.A., K. Olle and 0. Hutzlnger. 1980. Chlorinated dlbenzo-
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Hatsumura, F. and H.3. Benezet. 1973. Studies on the bloaccumulatlon and
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Health Perspect. 5: 253-258.
Matsumura, F., 0. Quensen and G. Tsushlmoto. 1983. Mlcroblal degradation
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mines. M.H. 1971. Formation of 2,3,7,8-tetrachlorod1benzo-P_-d1ox1n by
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NestMck, T., et al. 1979. Synthesis and Identification of the 22 tetra-
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gas chromatography. Anal. Chem. 51: 2273.
A-13
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NestMck, T.J., L.L. Lamparsk! and D.I. Townsend. 1980. Identification of
tetrachlorod1benzo-p_-d1ox1n Isomers at the 1 ng level by photolytlc degrada-
tion and pattern recognition techniques. Anal. Chem. 52: 1865-1875.
Norrls, L.A. 1981. The movement, persistence and fate of the phenoxy
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Ol1e, K., M.V.O. Berg and 0. Hutzlnger. 1983. Formation and fate of PCOO
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Poland, A. and E. Glover. 1973. Chlorinated d1benzo-p_-d1ox1ns: Potent
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A-14
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Rappe, C., S. Marklund, P.-A. Bergqvlst and M. Hansson. 1983a. Polychlori-
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Taylor, M.L., et al. 1975. Determination of tetrachlorodibenzop-dioxlns in
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Tlernan, T.O., et al. 1975. Measurement of tetrachlorod1benzo-p,-d1ox1ns 1n
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Tlernan, 1.0. I9B3. AnalyMc«il chemistry of polychlorlnated d1benzo-p-
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A-16
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Aquatic Toxicology*
Introduction
Most of the available data related to effects of 2,3,7,8-tetrachlorodl-
benzo-p-dloxln (2,3,7,8-TCOD) on aquatic life have been generated by Morris
and co-workers, Isensee and co-workers, Matsumura and co-workers, and
Helder. Much of the available Information Is from studies Involving fresh-
water microcosms. Although such studies are Intended to provide Information
on fate of a test material, some data concerning effects on aquatic life are
also obtained. No tests have been conducted using saltwater organisms.
The last literature search for Information that could be used In this
chapter was conducted 1n November, 1983.
Acute Toxldty to Aquatic Animals
Although the data available concerning 2,3,7,8-TCDD do not allow calcu-
lation of an acute value for any species, some useful Information does
exist. Data published by Miller et al. (1973) and Norrls and Miller (1974)
Indicate that the 96-hour LC s for a worm, Paranals sp., a snail, Physa
sp., and larvae of the mosquito, Aedes aegyptl. would be >0.2 vg/8.,
whereas those for the coho salmon, Oncorhynchus klsutch, and the guppy,
Poedlla retlculata. would be >1 and >10 yg/8., respectively. Based on
microcosm studies 1n which concentrations In water were measured at 2-day
Intervals, the 96-hour LC for flngerUng channel catfish, Ictalurus
punctatus. would be >0.24 yg/a, whereas those for Daphnla maqna and a
*An understanding of the Guidelines for Deriving Numerical National Water
Quality Criteria for the Protection of Aquatic Life and Its Uses (Stephan
et al., 1983) Is necessary In order to understand the following text and
tables.
B-l
-------
snail, Phjfsa sp., would be >1.3 yg/a (Isensee and Jones, 1975; Isensee,
1978). Yocklm et al. (1978) did not observe acute toxldty to D. magna. a
snail, Helosoma sp., or the mosqultofIsh, Gambusla afflnls. exposed for over
96 hours to a measured concentration of 0.0024-0.0042 vg/i. Helder
(1980, 1981, 1982a) found that the 96-hour LC5()s for embryos of northern
pike, Esox luclus, and embryos and yolk-sac fry of rainbow trout, Sal mo
galrdnerl. Wou1d be >0.01 pg/i; the 96-hour LC50 for juvenile rainbow
trout would be >0.1 vg/a. Although no 48- or 96-hour LC s or EC s
can be calculated, the available data Indicate that those for the coho sal-
mon, guppy, 0. maqna. and a snail, Physa sp., are >1.0 yg/si.
Chronic Toxlcity to Aquatic Animals
No standard chronic toxldty tests have been conducted on 2,3,7,8-TCDD
with aquatic animals, but several exposures that have been conducted for
other purposes do provide some Information concerning chronic toxldty.
Because Miller et al. (1973) used static long-term exposures, no conclusions
can be drawn concerning chronic toxldty from their exposures of A. aeqyptl
or a snail, Physa sp., but 1t can be concluded that 0.2 vg/s. would cause
chronic toxldty to a worm, Paranals sp. A 96-hour exposure to an Initial
concentration of 0.0056 yg/a. resulted In 55% mortality among coho salmon
within 60 days (MUler et al., 1973, 1979); thus 0.0056 vgA would cause
chronic toxldty to this species. S1m1lar1ly, 0.1 vg/a. would cause
chronic toxldty to the guppy, "because exposure to 0.1 vgA for 5 days
killed all Individuals within 40 days (Norrls and Miller, 1974). In micro-
cosms In which the concentrations of 2,3,7,8-TCDD were measured at 2-day
Intervals, both D. maqna and a snail, Physa sp., reproduced at 1.3 vq/i
(Isensee and Jones, 1975; Isensee, 1978). Exposure to a measured concentra-
tion of 0.0024-0.0042 yg/a killed all exposed mosqultoflsh and channel
B-2
-------
catfish within 20 days (Yocklm et al.. 1978). Based on effects caused by
96-hour exposures, 0.001 vg/l would cause chronic toxlclty to rainbow
trout and 0.01 vg/l would chronically affect northern pike {Helder,
1980, 1981, 1982a). Branson et al. (1983) reported that a 6-hour exposure
to 0.1 yg/8. adversely affected rainbow trout after 64-139 days.
Apparently 0.001 vg of 2,3,7,8-TCDD/l would cause unacceptable chronic
toxlclty to rainbow trout and 0.01 vg/8. would be chronically toxic to
coho salmon, mosqultof1sh, channel catfish and northern pike; 1.3 vg/i
may not be chronically toxic to D. magna or a snail, Phisa sp.
Toxlclty to Aquatic Plants
The few data available on the toxlclty of 2,3,7,8-TCDD to aquatic plants
are also from microcosm studies. The alga, Oedogonlum cardlacum, and the
duckweed, Lemna minor, were not affected by 30-day exposures to 1.3 vg/a
and 0.71 yg/l, respectively (Isensee and Jones, 1975; Isensee, 1978).
Yocklm et al. (1978) did not observe any adverse effects on 0. cardlacum
exposed to a measured concentration of 0.0024-0.0042 vg/«. for 32 days.
Bloaccumulatlon
Several equations have been developed for predicting the steady-state
bloconcentratlon factor (BCF) for an organic compound from Us octanol-water
partition coefficient (Kenaga and Goring, 1980; velth et al., 1980; VeHh
and Koslan, 1983). Several estimated values (Leo, 1979; Mabey et al., 1982;
Neely, 1983) and one measured value (Neely, 1979, 1983; Kenaga, 1980; Bran-
son, 1983) have been reported for the octanol-water partition coefficient
for 2,3,7,8-TCDD. Use of various equations with four available values for
the partition coefficient, KOW, results 1n the predicted BCFs shown 1n
Table 1. The predicted BCFs range from 3000-68,000 using the measured value
of the partition coefficient and from 7000-900,000 using the calculated
values.
B-3
-------
TABLE 1
Predicted Bloconcentratlon Factors for 2,3,7,8-TCDD Based on
Estimated and Measured Values of the Octanol-Water Partition Coefficient (Kow)
log BCF
log BCF
CD
1
* log BCF
log BCF
log BCF
BCF = 0.
Equation
= 0.542 log Kow + 0.124
= 0.76 log Kow - 0.23
= 0.79 log Kow - 0.40
= 0.635 log Kow + 0.7285
= 0.85 log Kow - 0.70
048 Kow
Reference
e,f
f
g
e
f.g
g
Measured
6.15a
2,870
27,800
28,700
43,000
33,700
67,800
log
6.84b
6,780
93,000
101,000
118,000
130,000
332,000
KOW
Estimated
7.14C
9,860
157,000
174,000
183,000
234,000
663,000
7.28d
11,700
201,000
224,000
225,000
308,000
915,000
Branson, 1983
bMabey et al., 1982
CNeely, 1983
dLeo, 1979
eKenaga and Goring, 1980
fVe1th et al., 1980
9Ve1th and Koslan, 1983
-------
Several measured BCFs have been reported for 2,3,7,8-TCDD. Using micro-
cosm studies In which the concentrations In water were measured at 2-day
Intervals for 30-33 days, Isensee and Jones (1975) and Isensee (1978) ob-
tained BCFs of 390-13,000 for the alga, 0. cardlacum. a snail, Physa sp.,
and D. maqna. In a separate 32-day microcosm study 1n which the measured
concentrations of 2,3,7,8-TCDD ranged from 0.0024-0.0042 V9/l. BCFs for
0. cardlacum. Physa sp., and D. maqna ranged from 660-7070 from the seventh
day to the end of the test.
In a different kind of test channel catfish were held 1n a cage In a
discharge plume 1n a river for 28 days. Four 24-hour composite water sam-
ples were analyzed for 2,3,7,8-TCDD. A whole-body BCF of 2,000 was obtained
(U.S. EPA, 1983; Thomas, 1983). In a laboratory bloconcentratlon test rain-
bow trout were exposed to 0.107 yg/8, for 6 hours and followed through a
139-day depuration period. The resulting projected steady-state BCF was
5450 If growth dilution was not taken Into account, and 9270 1f growth
dilution was taken Into account. These values are for the whole body; the
concentration of 2,3,7,8-TCDD In muscle was about one-half that 1n the whole
body (Branson et al., 1983).
Corbet et al. (1983) conducted bloconcentratlon tests on 1,3,6,8-TCDD
with the fathead minnow, Plmephales promelas. and rainbow trout, using a
4-day uptake phase and 48-day depuration phase. All results were based on
radioactivity measurements, because no confirmatory analyses were performed.
The projected steady-state BCFs were 1061 with the fathead minnow and 469
with the rainbow trout. The authors concluded the environmental behavior of
1,3,6,8-TCDD Is quite different from that of 2,3,7,8-TCDD, based on a 10- to
15-fold difference 1n measured clearance rate constants and the fact that
the projected BCF for 1,3,6,8-TCDD was much less than a predicted BCF for
2,3,7,8-TCDD.
B-5
-------
Information on maximum permissible tissue concentrations 1s available
from two sources. Hawkes and Norris (1977) found that feeding activity and
growth decreased and fin erosion and liver pathology Increased when a
portion of the diet fed to young rainbow trout for 105 days contained 2.3 mg
of 2,3,7,8-TCDD/kg of food. These effects were not observed when a portion
of the diet contained 0.0023 mg/kg. A diet containing 2.3 mg of 2,3.7.8-
TCDD/kg 1s obviously unacceptable for rainbow trout, but It Is not known how
low the average concentration In the diet would have to be to prevent
unacceptable effects on survival, growth and reproduction.
The U.S. FDA Issued a human health advisory on fish containing 0.000050
mg of 2,3,7,8-TCDD/kg; the FDA believed there was little cause for concern
1f the average concentration In fish was <0.000025 mg/kg (Hayes, 1981).
Other Data
Because delayed effects had been observed In tests on 2,3,7,8-TCDD with
mammals, several studies were conducted to determine whether delayed effects
also occurred with fishes. In tests with a number of species, exposures
that lasted <6 days caused substantial mortality several weeks later
(Table 2).
The bullfrog was less sensitive to Injected 2,3,7,8-TCDD than many
mammalian species (Beatty et al. 1976).
Unused Data
Publications, such as those by Lamparskl et al. (1979), Nlemann et al.
(1983) and Ryan et al. (1983), that only dealt with analytical methodology
for measuring 2,3,7,8-TCDD in aquatic life were not used. Publications by
Baughman and Meselson (1973), Young et al. (1975), Harrison et al. (1979),
Harless and Lewis (1982), O'Keefe et al. (1983), Harrison and Crews (1983),
B-6
-------
TABLE 2
Other Data on Effects of 2,3,7,8-TCDD on Aquatic Organisms
00
I
—J
Species
Coho salmon,
Oncorhynchus klsutch
Rainbow trout (embryo),
Salmo qalrdnerl
Rainbow trout (yolk-sac fry),
Salmo qalrdnerl
Rainbow trout (yolk-sac fry),
Salmo qalrdnerl
Rainbow trout (Juvenile),
Salmo qalrdnerl
Rainbow trout (juvenile),
Salmo qalrdnerl
Rainbow trout,
Salmo qalrdnerl
Northern pike (embryo),
Esox luclus
Northern pike (embryo),
Duration
96 hours
96 hours
96 hours
96 hours
16 hours/day
for e4 days
16 hours/day
for 4 days
6 hours
96 hours
96 hours
Effect
50% dead 1n 56 more days
Some mortality In 24 weeks
All dead 1n 24 weeks
Growth retarded for 23 weeks
All dead 1n 27 days
Growth reduced for 10 weeks
F1n rot, hemorrhaglng and
death after 64 days
Nearly all dead 1n 23 days
Slight reduction 1n growth
Result
(yg/i)
0.0056
0.01
0.01
0.001
0.1
0.01
0.1
0.01
0.0001
Reference
Miller et al..
1973, 1S~"
Helder,
Helder,
•I f\ Q f\ _
198
-------
TABI E 2 (cent.)
I
CD
Spedes
Guppy,
Poedlla retlculata
Guppy,
Poedlla retlculata
Guppy,
Poedlla retlculata
Bullfrog (tadpole),
Rana catesbelana
Bullfrog (adult),
Rana catesbelana
Duration Effect
120 hours Killed 18%
120 hours All dead 1n 17 more days
24 hours 10% dead 1n 41 more days
50 days No deaths
35 days No deaths
t
Result
10
0.1
0.01
1000 pg/kg
d.p.)
500 yg/kg
d.p.)
Reference
Norrls and
Miller, 1974
Norrls and
Miller, 1974
Miller et al.,
1979
Beatty et al..
1976
Beatty et al..
1976
l.p. = 1ntraper1toneal
-------
Harless et al. (1983) and Stalling et al. (1983) reported concentrations 1n
aquatic organisms but did not report enough data on the concentrations In
water to allow calculation of a BCF. Botre et al. (1978) and Ward and
Matsumura (1978) only dealt with fate of 2,3,7,8-TCDO and presented no data
on effects on aquatic life.
The bloconcentratlon tests of Matsumura and Benezet (1973) and Matsumura
(1977) were static and usually lasted for only a few days. The concentra-
tion of 2,3,7,8-TCOD 1n water was not measured adequately by Tsushlmoto et
al. (1982). Isensee and Jones (1975) reported BCFs based on dry weight;
fortunately Isensee (1978) reported results of the same tests based on wet
weight. Helder et al. (1982) exposed rainbow trout to fly ash and an
extract of fly ash and no conclusions can be drawn concerning effects of
2,3,7,8-TCDD on aquatic life. The data of Zullel and Benecke (1978) were
not used because the test species was not Identified well enough to allow a
determination of whether It Is resident In North America. Young et al.
(1976, 1978), Esposlto et al. (1980), Helder (1982b), Kenaga and NorMs
(1983) and a report of the National Research Council of Canada (1981) only
contained data published elsewhere.
Summary
The data that are available concerning the effects of 2,3,7,8-TCDD on
aquatic organisms and their uses do not allow the calculation of an acute or
chronic toxldty value for any freshwater animal species. Data available
from various studies do Indicate, however, that the acute values for several
freshwater species are >1.0 vg/i. Similar data Indicate that the
chronic value for rainbow trout 1s <0.001 vg/«. and that chronic values
for several other species are <0.01 vg/a. Effects were not observed on
the two plant species exposed to 1.3
B-9
-------
Estimates of the bloconcentratlon factor for 2,3,7,8-TCDD range from
3000-900,000. Measured BCFs have been reported for a variety of species and
range from 390-13,000. The U.S. FDA Issued a health advisory for fish con-
taining more than 0.000050 mg of 2,3,7,8-TCDO/kg. A concentration of 2.3 mg
of 2,3,7,8-TCDD/kg 1n a portion of the diet affected rainbow trout.
Exposures of <6 days resulted In deaths among four species of fishes several
weeks later.
No tests have been conducted on 2,3,7,8-TCDD with saltwater species.
National Criteria
Not enough data are available concerning the effects of 2,3,7,8-TCDD on
aquatic life and Its uses to allow derivation of national criteria. The
available Information indicates that acute values for some freshwater animal
species are >1.0 pg/i; some chronic values are <0.01 pg/i, and the
chronic value for rainbow trout is <0.001 vq/i. Because exposures of
some species of fishes to 0.01 vg/i for <6 days resulted In substantial
mortality several weeks later, derivation of aquatic life criteria for
2,3,7,8-TCDD may require special consideration. Predicted bioconcentratlon
factors (BCFs) for 2,3,7,8-TCDD range from 3000-900,000, but the available
measured BCFs range from 390-13,000. If the BCF is 5000, concentrations
>0.00001 yg/a. should result in concentrations in edible freshwater and
saltwater fish and shellfish that exceed levels identified in a U.S. FDA
health advisory. If the BCF is >5000 or if uptake 1n a field situation is
greater than that in laboratory tests, the value of 0.00001 yg/j. will be
too high.
B-10
-------
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4
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B-11
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Harless, R.L. and R.G. Lewis. 1982. Quantitative determination of 2,3,7.8-
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Compounds, R.E. Tucker, A.L. Young and A.P. Gray, Ed. Plenum Press, NY.
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Hawkes, C.L. and L.A. Norrls. 1977. Chronic oral toxldty of 2,3,7,8-
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106: 641-645.
Hayes, A.H., Or. 1981. Letter to W.G. Hllllken. U.S. FDA, Rockville, MD.
August 26.
B-12
-------
Helder, T. 1980. Effects of 2,3,7,8-tetrachlorod1benzo-p-d1ox1n (TCOO) on
early life stages of the pike (Esox ludus L.J. Scl. Total Environ. 14:
255-264.
Helder, T. 1981. Effects of 2,3,7,8-tetrachlorod1benzo-p-d1ox1n (TCDD) on
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Helder, T. 1982a. Effects of 2,3,7,8-tetrachlorod1benzo-p-d1ox1n (TCDD) on
early life stages of two fresh-water fish species. In: Chlorinated Dloxlns
and Related Compounds, 0. Hutzlnger, R.W. Frel, E. Merlan and F. Pocchlarl.
Ed. Pergamon Press, NY. p. 455-462.
Helder, T. 1982b. Effects of TCDD on early life stages of fresh water
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Procedures 1n Ecotoxlcology. EUR 7549. Commission of the European Communi-
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Helder, T., E. Stutterheim and K. Olie. 1982. The toxlclty and toxic
potential of fly ash from municipal Incinerators assessed by means of a fish
early life stage test. Chemosphere. 11: 965-972.
Isensee, A.R. 1978. Bloaccumulatlon of 2,3,7,8-tetrachlorod1benzo-para-
dloxln. Ecol. Bull. (Stockholm). 27: 255-262.
Isensee, A.R. and G.E. Jones. 1975. Distribution of 2,3,7,8-tetrachloro-
d1benzo-p-d1ox1n (TCDD) 1n aquatic model ecosystem. Environ. Scl. Techno!.
9: 668-672.
B-13
-------
Kenaga, E,E, 1980. Correlation of bloconcentratlon factors of chemicals In
aquatic and terrestrial organisms with their physical and chemical proper-
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Kenaga, E.E. and C.A.I. Goring. 1980. Relationship between water solubil-
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Hendricks, Ed. ASTH STP 707. Am. Soc. Test. Materials, Philadelphia, PA.
p. 78-115.
Kenaga, E.E. and L.A. Norris. 1983. Environmental toxlclty of 1COD. In.:
Human and Environmental Risks of Chlorinated Dloxins and Related Compounds,
R.E. Tucker, A.L. Young and A.P. Gray, Ed. Plenum Press, NY. p. 277-299.
Lamparski, L.L., T.J. Nestrlck and R.H. Stehl. 1979. Determination of
part-per-tMlllon concentrations of 2,3,7,8-tetrachlorod1benzo-p-d1oxin 1n
fish. Anal. Chetn. 51: 1453-145C.
Leo, A.J. 1979. Letter to C. Stephen. Pomona College, Claremont, CA,
May 11.
Mabey, W.R., J.H, Smith, R.T. Podoll, et al. 1982. Aquatic fate processes
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Matsumura, F. 1977. Absorption, accumulation, and elimination of pesti-
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B-14
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Matsumura, F. and H.J. Benezet. 1973. Studies on the bloaccumulatlon and
microbial degradation of 2,3,7,8-tetrachlorodibenzo-p-diox1n. Environ.
Health Perspect. 5: 253-258.
MUler, R.A., L.A. Morris and C.L. Hawkes. 1973. Toxlclty of 2,3,7,8-
tetrachlorod1benzo-p-d1oxin (TCDD) In aquatic organisms. Environ. Health
Perspect. 5: 177-186.
Miller, R.A., L.A. Norris and B.R. Loper. 1979. The response of coho
salmon and guppies to 2,3,7,8-tetrachlorodlbenzo-p-dloxin (TCOD) In water.
Trans. Am. Fish. Soc. 108: 401-407.
National Research Council of Canada. 1981. Polychlorlnated dibenzo-p-
dioxins: Criteria for their effects on man and his environment. NRCC No.
18574. Ottawa, Canada.
Neely, W.B. 1979. Estimating rate constants for the uptake and clearance
of chemicals by fish. Environ. Sci. Technol. 13: 1506-1510.
Neely, W.B. 1983. Letter to C.E. Stephan. Dow Chemical U.S.A., Midland,
HI, June 10.
Niemann, R.A., W.C. Brumley, D. Firestone and J.A. Sphon. 1983. Analysis
of fish for 2,3,7,8-tetrachlorod1benzo-p-dioxin by electron capture capil-
lary gas chromatography. Anal. Chem. 55: 1497-1504.
B-15
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Norrls, L.A. and R.A. Miller. 1974. The toxlclty of 2,3,7,8-tetrachloro-
dlbenzo-p-dloxln (TCDO) In gupples (PoeclHa retlculatus Peters). Bull.
Environ. Contam. Toxlcol. 12: 76-80.
O'Keefe, P., C. Meyer, D. Milker, et al. 1983. Analysis of 2,3,7,8-tetra-
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^
laboratory study on determination of 2,3,7,8-tetrachlorod1benzo-p-d1oxin 1n
fish. J. Assoc. Off. Anal. Chem. 66: 700-707.
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chlorinated d1benzo-p-d1ox1ns and dlbenzofurans 1n Laurentlan Great Lakes
fish. In: Human and Environmental Risks of Chlorinated Oloxlns and Related
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Stephan, C.E., D.I. Mount, D.J. Hansen, J.H. Gentile, G.A. Chapman and W.A.
Brungs. 1983. Guidelines for deriving numerical national water quality
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July 22.
Tsushlmoto, G., F. Matsumura and R. Sago. 1982. Fate of 2,3,7,8-tetra-
chlorodlbenzo-p-dloxln (TCDD) 1n an outdoor pond and 1n model aquatic eco-
systems. Environ. Toxlcol. Chem. 1: 61-68.
B-16
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U.S. EPA. 1983. Dow Chemical Company - Midland Plant Wastewater Character-
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Services D1v., Eastern District Office. March 28.
Velth, G.D. and P. Koslan. 1983. Estimating bloconcentratlon potential
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Ed. Ann Arbor Science, Ann Arbor, MI. p. 269-282.
Velth, G.D., K.3. Macek, S.R. Petrocelll and J. Carroll. 1980. An evalua-
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Toxlcol. 7: 349-357.
Yocklm, R.S., A.R. Isensee and G.E. Jones. 1978. Distribution and toxlclty
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B-17
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Young, A.L., C.E. Thalken, E.L. Arnold, J.M. Cupello and L.G. Cockerham.
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Environ. Contam. Toxlcol. 20: 786-792.
B-18
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Mammalian Toxicology and Human Health Effects
EXPOSURE
Water and Soil Related
The amount of human exposure of 2,3.7,8-TCDD attributable to drinking
water alone cannot be readily determined. A National Academy of Sciences
(NAS) document states that 2,3,7,8-TCDD has never been detected In drinking
water using methods with limits of detection In the parts per trillion (ppt)
range (NAS, 1977). The two most likely sources of 2,3,7,8-TCDD contamina-
tion are discharge of contaminated Industrial effluents, and washouts from
contaminated disposal sites. However, even after contamination 2,3,7,8-TCDD
should remain strongly sorbed to sediments and biota (Isensee and Jones,
1975). In one study, >90% of 2,3,7,8-TCDD In the aquatic media was present
In the sorbed state (Ward and Matsumura, 1978). The possibility of 2,3,7,8-
TCDD leaching Into the groundwater appears remote. Helling (1971), Kearney
et al. (1972) and Helling et al. (1973) found that 2,3,7,8-TCDD tended to
remain on or near the surface of the soil. The mobility of 2,3,7,8-TCDD in
five different soil types was examined by Kearney et al. (1973). They found
that decreasing mobility of 2,3.7,8-TCDD was associated with Increasing
organic content of the soil. Based on this observation, and the fact that
dioxlns were relatively Immobile in all soils tested, they concluded that
underground water supplies probably would not be contaminated with 2,3,7,8-
TCDD. Similar conclusions were made by Matsumura and Benezet (1973) who
hypothesized that any movement in the soil environment would most likely
occur via horizontal transfer of soil and dust particles.
Nash and Beall (1980) conducted studies on the fate of 2,3,7,8-TCDD In a
mlcroagroecosystem and found that 80% of the applied 2,3,7,8-TCDD remained
C-l
-------
in the upper 2 cm of soil. Trace amounts of 2,3,7,8-TCOD detected at depths
of 8-15 cm suggested that some movement of dloxln Into the soil had
occurred. Analysis of water leachate samples showed no detectable
2,3,7,8-TCDO following two applications (days 0 and 35) of Sllvex containing
44 ppb 2,3,7,8-TCDD. However, similar analyses of Teachable samples taken
42 days after a third application of SHvex containing 7500 ppb 2,3,7,8-TCDD
Indicated a maximum concentration of 0.05-0.06 ppt of 2,3,7,8-TCOD.
The downward vertical migration of 2,3,7,8-TCDD Into the first 1.5 cm of
soil was reported around Seveso, Italy (DIDomenlco et al., 1980a,b). 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 migration could be saturation of sorptlon sites 1n soil, solvatlon
of 2,3,7,8-TCDD by organic solvents (NRCC, 1981), or blotlc mixing by earth-
worms or other soil Invertebrates. Nevertheless, both studies support the
view that 2,3,7,8-TCDD does not migrate readily 1n soils.
The photodecomposltlon of 2,3,7,8-TCDD on wet or dry soil under artifi-
cial and natural sunlight was studied by Crosby et al. (1971). The photo-
decomposition was found to be negligible 1n soils. Similarly, P11 inner et
al. (1973a,b) determined that photodecomposltlon of 2,3,7,8-TCDD on soils
was too slow to be detected. In a later experiment, Pllmmer (1978) found
that although 2,3,7,8-TCDD decomposed significantly on a precoated silica
plate (-22%) 1n 8 hours of sunlight Irradiation, practically no decomposi-
tion of 2,3,7,8-TCDD was observed from 2,3,7,8-TCDD sorbed on soil under
similar conditions.
C-2
-------
The photodegradatlon of 2,3,7,8-TCDD In combination with other pesticide
mixtures was studied by Crosby and Wong (1977). When Agent Orange contain-
ing 15 ppm of 2,3,7,8-TCDD was applied on the surface of glass plates (5
mg/cm2), rubber plant (Hevea brasHlensIs) (6.7 mg/cm2), and on the sur-
face of sieved Sacramento loam soil (10 mg/cm2) and exposed to sunlight,
2,3,7,8-TCDD was found to photodecompose. The loss of 2,3,7,8-TCDD In 6
hours was >50% from the glass plates, -100% from the surface of leaves and
-10% from the surface of soil. The rapid photolysis of 2,3,7,8-TCDD from
these surfaces Indicates that the herbicide formulation provided a hydrogen
donor which probably allowed the photolysis to occur. The authors attribut-
ed the slower photolysis of 2,3,7,8-TCDD 1n soil to a shading effect by the
soil particles.
The overall half-life of 2,3,7,8-TCDD In soil was first reported to be
1-3 years (Kearney et al., 1972). Studies performed by the U.S. A1r Force
suggested that the half-life of this chemical In soils under relatively dry
conditions (Utah test area) was -330 days. In more moist soils and under
warm conditions (Florida test area), the half-life was -190 days. This 1s
consistent with the blodegradatlon half-life of -0.5 year for 2,3,7,8-TCDD
determined from the soil 1n rural Missouri after the accidental spraying of
TCDD-contamlnated oil (IARC, 1977). However, physical removal may be an
Important factor also. More recent data (Young, 1983; Wlpf and Schmld,
1983) Indicate that the half-life Is closer to 10 years.
The half-life of 2,3,7,8-TCDD following an accidental 2,3,7,8-TCDD
release from a trichlorophenol manufacturing plant at Seveso, Italy, was
studied by DIDomenlco et al. (1980a). The disappearance of 2,3,7,8-TCDD
from the topmost soil layer after 1 year was speculated to be due to photo-
degradation, volatilization, or vertical movement through the soil. These
C-3
-------
Investigators estimated the first half-life of 2,3,7,8-TCDD 1n soil at the
time of Us release to be 5 months. One month after release, the next
2,3,7.8-TCDD half-life was estimated to be 1 year, whereas 17 months later
It was estimated to be >10 years. It has recently been shown that radio-
labeled TCDD adsorbed to soil becomes progressively more resistant to
extraction (Ph1l1pp1 et al., 1981; Huetter and Ph1l1pp1, 1982) and, there-
fore, the persistence of 2,3,7,8-TCDD residues In aged soil 1s probably
greater as well.
2,3,7,8-TCDD exhibits relatively strong resistance to mlcroblal bio-
degradation. Only 5 of -100 mlcroblal strains that have the ability to
degrade persistent pesticides show slight ability to degrade 2,3.7,8-TCDD
(U.S. EPA, 1980d).- Ward and Matsumura (1977) reported that the half-life of
2,3,7,8-TCDD in sediment-containing Wisconsin lake waters was 550-590 days.
In lake water alone, -70% of the 2,3,7,8-TCDD remained after 589 days.
Using an outdoor pond as a model aquatic ecosystem, Tsushimoto et al. (1982)
and Matsumura et al. (1983) estimated the apparent half-life of 2,3,7,8-TCDD
to be -1 year. Although blodegradatlon may have been responsible for part
of the degradation, other Investigators (Huetter and PhUippi, 1982) have
reported the virtually complete lack of blodegradabUHy of 2,3,7,8-TCDD.
The blodegradatlon half-life of 2,3,7,8-TCDD can be estimated from the
theoretical rate constant values based on relative rates of transformation
reported in the literature or on structure-activity analogy values given by
Mabey et al. (1981). Assuming the blotransformatlon rate constant of
IxlO^o ms. cell"1 hr'1 (Mabey et al., 1981) and the concentration of
microorganisms capable of degrading TCDD as 5xl05 cell ml"1 (Burns et
al., 1981), the half-life of blodegradatlon is estimated to be >1 year.
C-4
-------
2,3,7,8-TCDD on dry and wet soils showed negligible photodegradatlon
after irradiation with sunlamps (Crosby et a!., 1971). In order to explain
the longer half-life of 2,3,7,8-TCDD in a model laboratory ecosystem than In
an outdoor pond, Matsumura et al. (1983) and Tsushimoto et al. (1982) specu-
lated photolysis as the most likely cause. In the outdoor environment where
the intensity of sunlight was higher compared to the laboratory experiments,
algae-mediated photosensitization of 2,3,7,8-TCDD may cause some photodecom-
position of this compound. From the available information, it is difficult
to predict the fate of 2,3,7,8-TCDD in aquatic media under environmental
photolytlc conditions. In the presence of hydrogen atom donating sub-
strate(s) in surface waters, photolysis may be a significant fate process.
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, little quantitative information regarding
the volatilization of 2,3,7,8-TCDD from aquatic media is available.
2,3,7,8-TCDD may undergo some water-mediated evaporation in aquatic media
(Matsumura et al., 1983). A transport model to estimate TCDD volatilization
from a cooling pond on an industrial site on the basis of measured concen-
trations In the pond bottom sediment and pond surface area led to an esti-
mated rate of 15-16 mg/year (Thlbodeaux, 1983). Using the formulas of Liss
and Slater (1974), a vapor pressure value of *~10~6 torr (0.1 m Pa) and a
solubility value of 6.2xlO~10 mole/l, NRCC (1981) calculated the vola-
tilization half-life for 2,3.7,8-TCDD to be 6 minutes from water of 1 cm
depth and 10 hours from water of 1 m depth. Evaporation half-life is
directly proportional to water depth and inversely proportional to mass
transfer coefficient (Thlbodeaux, 1979). The limitations of the Liss-Slater
theory to predict the rate of volatilization have been discussed in the NRCC
C-5
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(1981) document. The L1ss-Slater model does not consider terrestrial
matrices (suspended solids, sediments, biota, etc.) normally encountered In
natural surface water. A computerized EXAMS model, considering sorptlon of
TCOO on the suspended and bottom sediments and otherwise employing the
L1ss-Slater model, gave the result that may account for the 100% of the
Fraction lost due to volatilization under the most favorable conditions
(NRCC, 1981). The volatilization half-life for 2,3,7,8-TCOO has been
estimated to be 5.5 and 12 years from pond and lake water, respectively.
However, It should be remembered that these are estimated values and no
experimental confirmation of these values 1s yet available.
Ingestion from Food
The occurrence of 2,3,7,8-TCDO 1n food could result from (1) contamina-
tion of plant crops with 2,3,7,8-TCOO as a result of using herbicides such
as SHvex and 2,4,5-T (for weed control); (2) consumption by livestock of
2,3,7,8-TCDD-contam1nated forage; or (3) magnification of residues through
the food chain. Conceivably, 2,3,7,8-TCDD could also be deposited on food
crops after being formed during certain combustion processes (NRCC, 1981).
Galston (1979) has speculated that under certain conditions 2,3,7,8-TCDD
might enter the human body from a 2,4,5-T-treated food chain and might
accumulate in the fat and be secreted 1n the milk. 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 spraying nor taken up from the soil into
the mature plants (Isensee and Jones, 1971; Matsumura and Benezet, 1973).
However, young plants accumulated up to 40 ppb of 2,3,7,8-TCDD (Isensee and
Jones, 1971). From the analysis of several parts of fruit trees and
kitchen-garden plants such as carrots, onions, potatoes and narcissuses
collected from the contaminated (400-1000 vg/m2 of 2,3,7,8-TCDD in soil)
C-6
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Seveso area In 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 Investigations of W1pf et al. (1982) 1n which vegeta-
tion samples analyzed from the Seveso area from 1976 through 1979 strongly
suggested that the contamination In vegetation was from local dust and not
from plant uptake.
Unlike the Seveso Incident where release of 2,3,7,8-TCDD Into the
environment took place, normal use of herbicides containing 2,3,7,8-TCDD
Impurity may not cause detectable 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 2,3,7,8-TCDD) at
a maximum rate of 2.25 Ibs/acre. No 2,3,7,8-TCDD residues (detection limit
2-10 ppt) were found In these rice grains nor were any found In 30 samples
of rice purchased In retail stores throughout the United States. Contamina-
tion of fruits, vegetables or grains In the United States with 2,3,7,8-TCDD
has never been Investigated.
The presence of polychlorlnated dloxlns In the fat of cattle that had
grazed on pasture treated with 2,4,5-T has been reported (U.S. EPA, 1980d).
The levels of 2,3,7,8-TCDD ranged from 4-70 ppt. Other Investigators have
failed to detect 2,3,7,8-TCDD (detection limit 1 ppt) In fats of cattle
grazing on pasture or rangeland treated with normal applications of 2,4,5-T
(Kocher et al., 1978).
C-7
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Bovine milk collected after the accident In the Seveso area was analyzed
by FanelH et al. (1980). The concentration of 2,3,7,8-TCDD was found to
vary from none detected (detection limit <40 opt) to as high as 7.9 ppb.
Other Investigators have failed to detect 2,3.7,8-TCDD (detection limit
1 ppt) In surveillance samples of milk (after normal application of 2,4,5-T
on pasture) from the states of Oklahoma, Arkansas and Missouri, or quaran-
tined milk In the state of Michigan (Lamparskl et al., 1978; Mahle et al.,
1977). Firestone et al. (1979) fed pentachlorophenol containing several
dloxlns (not 2,3,7,8-TCDD) to lactatlng cows for 70 days. The concentration
factor for 1,2,3,6,7,8-hexachlorod1benzo-p_-d1ox1n, the dloxln of highest
concentration, in milk fat was -2.4 times relative to Us concentration In
the diet.
The analysis of human milk and urine for 2,3,7,8-TCDD has been reported.
A study of 103 samples of breast milk from mothers living 1n sprayed areas
in the United States revealed no 2,3,7,8-TCDD at a detection limit of 1-4
ppt (U.S. EPA, 1980d). About 6 of the 9 human milk samples analyzed by
Langhorst and Shadoff (1980) may have contained 2,3,7,8-TCDD at levels
slightly higher than the detection limits (0.2-0.7 ppt). Because of the
lack of validation of the precision and accuracy of data, however, It was
concluded that 2,3,7,8-TCDD was not present.
Bumb et al. (1980) analyzed charcoal-broiled steak in order to detect
any 2,3,7,8-TCDD formed as a result of the broiling process. No 2,3,7,8-
TCDD was detected (detection limit 1-10 ppt).
C-8
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2,3,7,8-TCDO has been reported 1n several species of commercial and non-
commercial fish 1n several rivers and lakes In the United States and Canada.
The levels of 2,3,7,8-TCDO In fish and shellfish as determined by various
authors are given 1n Table 1. In some cases the values listed are means or
composites of more than one organism, species or location. Values for
Individual analyses as high as 695 ppt in fish tissue have been reported
(Harless and Lewis, 1982). The efficiency of various extraction and cleanup
procedures for 2,3,7,8-TCDD analysis in fish has been discussed by Brumley
et al. (1981).
Results of analyses shown in Table 1 indicate that the 2,3,7,8-TCDO
levels in fish and shellfish depend not only on the sites from which they
are collected, but also on the type of species collected. For example, fish
and shellfish collected from Bayon Meto/Arkansas River, Tittabawassee/Sag1-
naw River, Saglnaw Bay, Lake Ontario, Lake Huron, and Cayuga Creek showed
higher levels of 2,3,7,8-TCDD than those collected from Lake Erie, Lake
Michigan, Lake Superior and the Atlantic Ocean. In addition, certain types
of aquatic species that are bottom feeders, have high fat content or are
carnivorous, such as catfish, carp, trout and salmon, showed higher levels
of 2,3,7,8-TCDD than bass, bullhead or suckers. The influence of 2,3,7,8-
TCDD levels in surrounding water on the bloconcentratlon of fish species Is
not known.
Cordle (1983) estimated the potential maximum human daily intake of
2,3,7,8-TCDD for residents of the Great Lakes region who regularly consume
Great Lakes fish. Within the population subset consuming these species (-17
million Individuals), daily consumption of fish tissue was 15.7 g at the
90th percentile, and 36.8 g at the 99th percentile. Within the smaller sub-
set consuming pike (number of individuals not specified), daily consumption
at the 99th percentile was 83.95 g. For hypothetical mean 2,3,7,8-TCDD
C-9
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TABLE 1
Levels of 2,3,7,8-TCDD 1n F1sh and Shellfish
Type/Section
of F1sh
Sampling Site
Concentration3
(ppt)
Reference
Edible fleshb
Catfish
Buffalo
Predatorb
Bottom feederb
Rock bass/muscle
Eel, Smelt and Catfish/muscle
Crayfish
Catfish, Bass and Walleyed pike
Lake trout/whole body
Chinook salmon/skinless fillet
Coho salmon/skinless fillet
Rainbow trout/skinless fillet
Bayou Meto/Arkansas River 480
Bayou Meto/Arkansas River ND (7)-50
Bayou Meto/Arkansas River ND (7-13)
Bayou Meto/Arkansas River 15-230
Bayou Meto/Arkansas River 77
Lake Ontario/Lake Erie/ ND (<2)
Wei land Canal
Lake Ontario/Lake Erie/ 2-39
Wei land Canal
Bergholtz Creek, Love Canal 3.7
2,4,5-T contaminated water- ND (5-10)
shed 1n Arkansas and Texas;
TUtabawassee and Saglnaw
Rivers
Lake Ontario 51-107
Lake Ontario 26-39
Lake Ontario 20-26
Lake Ontario 17-32
MHchum et al., 1980
MHchum et al., 1980
MUchum et al., 1980
MHchum et al., 1980
MHchum et al., 1980
Josephson, 1983
Josephson, 1983
Smith et al., 1983b
Shadoff et al., 1977;
U.S. EPA, 1980d;
Buser and Rappe, 1980
O'Keefe et al., 1983
O'Keefe et al., 1983
O'Keefe et al., 1983
O'Keefe et al., 1983
-------
TABLE 1 (cont.)
o
i
Type/Section
of F1sh
Brown trout/skinless fillet
White perch/skinless fillet
White sucker/skinless fillet
Smallmouth bass/skinless fillet
Brown bullhead/skinless fillet
Carp, Goldfish/skinless fillet
Northern pike/skinless fillet
Pumpkin seed/skinless fillet
Rock bass/skinless fillet
Coho salmon/skinless fillet
Walleye p1ke/sk1nless fillet
Smallmouth bass/skinless fillet
Carp, Goldfish/skinless fillet
Lake trout/whole body
Carp/skinless fillet
Channel Catfish/skinless fillet
Sampling Site
Lake Ontario
Lake Ontario
Lake Ontario
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
Concentration3
(ppt)
8-162
17-26
NO (3.2J-10
5.9
3.6
87
32
31
12
1.4-<3.5
2.6
1.6-<2.4
NO (2.6)
21
26
20
Reference
O'Keefe et al..
O'Keefe et al.,
O'Keefe et al.,
O'Keefe et al.,
O'Keefe et al.,
O'Keefe et al.,
O'Keefe et al.,
O'Keefe et al.,
O'Keefe et al.,
O'Keefe et al.,
O'Keefe et al.,
O'Keefe et al..
O'Keefe et al.,
O'Keefe et al.,
O'Keefe et al.,
O'Keefe et al.,
1983
1983
1983
1983
1983
1983
1983
1983
1983
1983
1983
1983
1983
1983
1983
1983
-------
TABLE 1 (cent.)
Type/Section
of F1sh
Sampling Site
Concentration3
(ppt)
Reference
Sucker/skinless fillet
Yellow Perch/skinless fillet
Coho salmon/skinless fillet
Rainbow trout/skinless fillet
Perch, Sucker
Catfish
Carp
Catfish
Bottom feeders**
Lake trout
Rainbow trout
Ocean haddock
Carp
Channel catfish
Carp
Lake Huron
Lake Huron
Lake Michigan
Lake Superior
Saglnaw Bay
Saglnaw Bay
Saglnaw Bay
Bayou Meto/Arkansas River
Bayou Meto/Arkansas River
Lake Ontario
Lake Ontario
Atlantic Ocean
Lake Huron
THtabawassee, Saglnaw and
Grand Rivers
THtabawassee, Saglnaw and
Grand Rivers
25
ND (8.7)
NO (3.8)
1.0
ND (3.8)-25
14-37
23-47
ND (3.8)
ND (6.7)-12
34-54
43
ND (4.6)
3-28
28-695
ND (7J-153
O'Keefe et al., 1983
O'Keefe et al., 1983
O'Keefe et al., 1983
O'Keefe et al., 1983
Nlemann et al., 1983C
Nlemann et al., 1983C
Nlemann et al., 1983C
Nlemann et al., 1983C
Nlemann et al., 1983C
Nlemann et al., 1983C
Nlemann et al., 1983C
Nlemann et al., 1983C
Stalling et al., 1983
Harless and Lewis,
1982
Harless and Lewis,
1982
-------
TABLE 1 (cent.)
o
i
Type/Section
of F1sh
Yellow perch
Smallmouth bass
Sucker
Lake trout
I -* L* +L 4-**.-vti4- /i.iKrtlia KnHw
Sampling Site
THtabawassee and Saglnaw
Rivers
Grand River
THtabawassee River
and Saglnaw Bay
Lake Michigan
1 ake Ontario at
Concentration3
(ppt)
NO (5)-20
7-8
NO (4)-21
ND (5)
61.2
Reference
Harless and Lewis,
1982
Harless and Lewis,
1982
Harless and Lewis,
1982
Harless and Lewis,
1982
Ryan et al., 1983
Rainbow trout/whole body
Lake trout/whole body
Ocean haddock/fillet
Burlington, Canada
Lake Ontario at Toronto
Harbor, Canada
Lake Huron at
Burnt Island, Canada
East Coast, Canada
32.3
30.4
ND (1-10)
Ryan et al., 1983
Ryan et al., 1983
Ryan et al., 1983
aWhen not detected, the detection limit is Indicated within the parentheses.
bOrganisms not further identified in the report.
C0nly the GC/MS results of these authors are Included in tabulation
dThese are the mean concentrations in samples showing detectable levels of 2,3,7,8-TCDD.
ND = Not detected
-------
residue levels of 25-100 ppt, estates of dally Intake would thus range
from 0.39-8.4 ng 2,3,7,8-TCDD/day. As shown 1n Table 1, tissue residues for
species 1n certain areas do fall within this range.
In order to derive an ambient water quality criterion for the protection
of human health fom the harmful effects of 2,3,7,8-TCDO, H 1s necessary to
estimate the average level of exposure of the U.S. population which would
result from a particular concentration of 2,3,7,8-TCDD 1n ambient fresh or
estuarlne waters (45 FR 79348). Data from a recent survey on fish and
shellfish consumption 1n the United States were analyzed by SRI Internation-
al (U.S. EPA, 1980a). The results were used to estimate that the per capita
consumption of freshwater and estuarlne fish and shellfish in the United
States Is 6.5 g/day (Stephan, 1980).
A bloconcentratlon factor (BCF) relates the concentration of a chemical
in aquatic species to the concentration 1n water. Several regression
equations can be used to estimate a BCF value for 2,3,7,8-TCDD from Us
octanol-water partition coefficient (K^). Using three calculated values
of Kow, the regression equations estimate the BCF 1n the range of
7000-900,000. Using the only measured KQW value, the regression equations
predict a BCF for 2,3,7,8-TCDD in the range of 3000-68,000. The available
measured BCFs, however, range from 390-13,000. The sources of the theo-
retical and experimental BCF values cited here can be found 1n detail 1n
Section B. Until further Information 1s available, the U.S. EPA's best
current estimate for the BCF of 2,3,7,8-TCDD in aquatic organisms is 5000.
Thus a BCF of 5000 will be used in the "Criterion Formulation" section to
estimate the human exposure to 2,3,7,8-TCDD which would result from consump-
tion of aquatic organisms taken from 2,3,7,8-TCDD-contam1nated waters. If
the BCF 1s actually >5000 or if uptake 1n a field situation 1s greater than
in laboratory tests, human exposure will be underestimated.
C-14
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Inhalation
No data pertaining to the Inhalation exposure of 2.3,7,8-TCDD were
found. However, the spraying of older formulations of 2,4,5-T containing
2,3,7,8-TCOO Impurity may lead to a concomitant exposure to 2,3,7,8-TCDD.
Exposure could be through spray drift and through the vapor phase. From
mlcroagroecosystem chamber and field studies, Nash and Beall (1980) deter-
mined 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 SHvex)
containing 44 ppb and 7.5 ppm 2,3,7,8-TCDD Impurity, respectively. Using
trltlated 2,3,7,8-TCDD, these authors found that atmospheric concentrations
of 2,3,7,8-TCDD vary not only with the number of days elapsed after applica-
tion (lower concentration at longer time period), but also with formulation
(granular form gave lower concentration than emulslflable concentrate), and
the 2,3,7,8-TCDD Impurity present 1n SHvex (higher Impurity levels produced
higher atmospheric concentrations). Depending on these variables, the atmo-
spheric concentration In mlcroagroecosystem chambers, expressed 1n fg/m3
(10~15 g/m3), was found to vary from 0.09 fg/m3 (granular Sllvex
applied at 1.3 kg/ha, concentration measured 35 days after application) to
79,800 fg/m3 (emulsified Sllvex applied at 2.0 kg/ha, concentration
measured during application).
A1r filter samples collected from Elizabeth, NO, after an Industrial
fire on April 22, 1980, were analyzed for 2,3,7,8-TCDD by Harvan et al.
(1981). Of the nine samples analyzed by coll1s1on-1nduced-d1ssodat1on
mass-analyzed 1on kinetic energy spectrometry by these authors, one con-
tained 20 pg of 2,3,7,8-TCDD, four contained <9 pg, and. four others probably
contained 5-12 pg of unspecified TCDD Isomer which was not the 2,3,7,8-TCDD
Isomer.
C-15
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The atmospheric concentrations of 2.3.7,8-TCDD near two hazardous waste
sites have been monitored. In one study, U.S. EPA (1982) failed to detect
any 2,3,7,8-TCDD 1n the atmosphere (detection limit 1-20 ppt) at the Love
Canal, NY, area. In another study of a waste disposal site near Jackson-
ville, AR, Thlbodeaux (1983) reported an average concentration of 1100 ppt
of 2,3,7,8-TCDD 1n two air partlculate samples collected near the disposal
site.
The levels of 2,3,7,8-TCDD in atmospheric dust was monitored in the
Seveso, Italy, area between 1977 and 1979. The concentrations of 2,3,7,8-
TCDD were found to be 1n the range of 0.06-2.1 ppb of dust using dustfall
jars, and 0.17-0.50 ppb of dust by high volume sampling (DIDomenlco et al..
1980c).
Another source of atmospheric emission of polychlorlnated dloxlns 1s
Incineration. The concentrations of TCDDs In fly ash from municipal Incin-
erators have been studied by several authors (Elceman et al., 1979, 1980;
NestMck et al., 1982; Karasek et al., 1982; Bumb et al., 1980; Buser and
Bosshardt, 1978; Tlernan et al., 1982; Taylor et al., 1983). The TCDD
Isomer known to be the most toxic (e.g., 2,3,7,8-TCDD) was either not
detected or detected at a low level. The quantities emitted In 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 stm Inadequate. Whereas Bumb et
al. (1980) and Buser and Rappe (1980) detected 0.4 ng/g of 2,3,7,8-TCDD 1n
the fly ash from a United States municipal Incinerator, the U.S. EPA con-
cluded that emissions from five municipal waste combustors did not present a
public health hazard for residents living 1n the Immediate vicinity (CEQ,
1981). 2,3,7,8-TCDD has been detected In the emissions of some municipal
C-16
-------
waste Incinerators \n Europe (Gizzl et al., 1982; Benfenatl et al., 1983;
Taylor et al., 1983; Ol1e et al., 1982, 1983; Lustenhouwer et al., 1980;
Barnes, 1983). For an Industrial boiler in the United States where penta-
chlorophenol (PCP) was known to have been burned, Rappe et al. (1983)
reported ~5 ppm PCDDs in the baghouse and bottom ash. However, >90% of the
PCDOs were lower chlorinated congeners than octa-CDD, the expected dlmeriza-
tion product of PCP. Among the large number of isomers found, only a small
amount of 2,3,7,8-TCDO could be quantified.
Analyses of soot samples from a transformer fire in Binghamton, NY, in
February 1981, revealed that 2,3,7,8-TCDO (0.6 ppm) and 1,2,3,7,8-penta-COD
were the dominating isomers of the PCDDs formed (Buser and Rappe, 1983;
Rappe et al., 1983). The origin of the polychlorinated dioxins was probably
the chlorobenzenes in the transformer oil (Buser, 1979). Analyses of wipe
tests from a garage adjacent to this accident site did reveal the presence
of polychlorinated d1benzo-p_-d1ox1ns prior to the cleaning of the garage.
Following the clean-up, no contamination was found (Tlernan et al., 1982;
Tiernan, 1983).
Dermal
Dermal exposure to 2,3,7,8-TCDD is likely to be most significant during
the spraying of 2,4,5-T. Lavy et al. (1980) determined the exposure levels
of applicators spraying 2,4,5-T (ESTERON 245) during typical applications in
a forest. The average dermal exposure to 2,4,5-T was estimated to be 0.6
mg/kg bw. If the 2,3,7,8-TCDD content in 2,4,5-T is assumed to be <0.1 ppm
and the absorption rate is assumed to be the same, an exposure of 0.6 mg/kg
of 2,4,5-T will correspond to <60 pg/kg bw of 2,3,7,8-TCDD for dermal
exposure. Lavy et al. (1980) found a slightly lower level of 2,3,7,8-TCDD
concentration (-12.5% lower) than the predicted value. No 2,3,7,8-TCDD was
detected 1n any of the urine samples (detection limit 1.7 ng/a).
C-17
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PHARHACOKINETTrs
The pharmacoklnetlcs of 2,3,7.8-TCDD has been Investigated In a number
of laboratory anl^s, and there are several recent reviews on this subject
(Neal et al., 1982; Gas1ew1cz et al., 1983a; Olson et al.. 1983,. This
section will examine our current understanding of the absorption, distribu-
tion, metabolism and excretion of 2,3.7.8-TCDD 1n various mammalian species.
Absorption
The Dermal and gastrointestinal absorption of 2,3,7,8-TCOD have been
investigated in several species. No studies are available on the pharmaco-
klnetlcs of 2,3,7,8-TCDD through the Inhalation route of exposure.
Absorption From the Gastrointestinal Tract
Experimentally, 2,3,7,8-TCDD 1s generally administered In the diet or by
gavage 1n an oil vehicle. In Sprague-Dawley rats given a single oral dose
of 1-0 Pg ["C]2,3,7,8-TCDD/kg of bw, absorption from the Intestinal
tract was estimated at -83% (Rose et al., 1976}. WHh repeated oral dosing
at 1.0 vg/kg/day (5 days/week x 7 weeks), absorption was observed to be
approximately that observed for the single oral dose. WHh a much larger
single oral dose, 50 ,g/kg bw, -70% of the dose was absorbed by Sprague-
Dawley rats (Piper et al., 1973). In these studies, the chemical was admin-
istered by gavage 1n acetone:corn oil (1:25 or 1:9). One study In the
guinea pig reported that -50% of a single oral dose (quantity not mentioned,
of 2,3,7,8-TCDD In acetone:corn oil was absorbed (Nolan et al., 1979). The
gastrointestinal absorption of 2,3,7,8-TCDD was also examined 1n the
hamster, the species most resistant to the acute toxldty of this toxin
(Olson et al., 1980a). Olson et al. (1980b, administered hamsters a single,
sublethal, oral dose of [1,6-3H]-2,3,7,8-TCDO In olive oil (650 vg/kg,
and reported that 74% of the dose was absorbed. When 2,3,7,8-TCDD was
C-18
-------
administered to rats 1n the diet at 7 or 20 ppb (0.5 or 1.4 vg/kg/day) for
42 days, 50-60% of the consumed dose was absorbed (Fries and Marrow, 1975).
These findings Indicate that over a wide range of doses and under these
experimental conditions, 2,3,7,8-TCOO Is generally well absorbed from the
gastrointestinal tract of the three species that have been examined.
Contact with 2,3,7,8-TCDO In the environment would most often Involve
exposure to a complex mixture containing the toxin, as opposed to the above
experimental situation, where 2,3,7,8-TCDD was administered 1n the diet or
through an oil vehicle.
The Influence of dose and vehicle or adsorbent on gastrointestinal
absorption has been Investigated 1n rats by Polger and Schlatter (1980),
using hepatic concentrations 24 hours after dosing as an Indicator of the
amount absorbed. They found a linear relationship between ng 2,3,7,8-TCDD
administered 1n 50% ethanol (for doses of 12-280 ng, equivalent to 0.06-1.4
vg/kg) and the percentage of the dose 1n hepatic tissues (36.7-51.5%). At
the next higher dose of 1070 ng, however, the percentage fell off to about
42%. Their results regarding the Influence of vehicle or adsorbent on
gastrointestinal absorption have been summarized 1n Table 2. Administration
of 2,3,7,8-TCDD 1n an aqueous suspension of soil resulted In a decrease In
the hepatic levels of 2,3,7,8-TCDD as compared with hepatic levels resulting
from administration of 2,3,7,8-TCDD 1n 50% ethanol. The extent of the
decrease was directly proportional to the length of time the 2,3,7,8-TCDD
had been In contact with the soil. When 2,3,7,8-TCDD was mixed In an aque-
ous suspension of activated carbon, absorption was almost totally eliminated
(<0.07% of the dose 1n hepatic tissues).
C-19
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TABLE 2
Percentage of 2,3,7,8-TCDD In the Liver of Rats 24 Hours After Oral
Administration of 0.5 ml of Various Formulations Containing TCDD*
Formulation TCDD Dose No. of Percentage of Dose
(ng) Animals in the Liver
50% Ethanol 14.7 7 3& ? + 1 2
Aqueous suspension of soil
(37%, w/w) that had been
In contact with TCDD for:
10-15 hours 12.7, 22.9 17 24.1 * 4.8
8 days 21.2, 22.7 10 16.0 I 2.2
Aqueous suspension of
activated carbon
(25%, w/w) 14.7 6 <0.07
*Source: Poiger and Schlatter, 1980
C-20
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Ph1l1pp1 et al. (1981) and Mutter and Ph1l1pp1 (1982) have shown that
radlolabeled 2,3,7,8-TCDO becomes progressively more resistant with time to
extraction from soil. Similarly, the feeding of fly ash, which contains
PCDOs, to rats 1n the diet for 19 days resulted In considerably lower
hepatic levels of PCDDs than did the feeding of an extract of the fly ash at
comparable dietary concentrations of PCDDs (van den Berg et al., 1983). The
PCDDs were tentatively Identified as 2,3,7,8-TCDD, 1,2,3,7,8-PeCDD,
1,2,3,6,7,8-HxCDD and 1,2,3,7,8,9-HxCDD and the difference 1n hepatic levels
noted between fly ash-treated and extract-treated rats was greater for the
more highly chlorinated Isomers than H was for 2,3,7,8-TCDD. These results
Indicate the Importance of the formulation or vehicle containing the
tox1n(s) on the relative bloavaHablHty of 2,3,7,8-TCDD, PeCDD and HxCDDs
following oral exposure.
Information on the absorption of 2,3,7,8-TCDD through the skin Is found
only In a study by Poiger and Schlatter (1980). The authors administered 26
ng 2,3,7,8-TCDD In 50 ys. methanol to the skin of six rats. After 24
hours, the liver contained 14.8^2.6% of the dose. By comparing to the
hepatic levels obtained after oral administration In 50% ethanol (1n the
same study), the amount absorbed from a dermal application can be estimated
at -4054 of the amount absorbed from an equivalent oral dose. This compari-
son assumes that hepatic levels are valid estimates of the amount absorbed
from both oral and dermal routes and that absorption from methanol 1s equiv-
alent to absorption from 50% ethanol. As compared with dermal application
1n methanol, dermal application of 2,3,7,8-TCDD to rats In vaseline or poly-
ethylene glycol reduced the percentage of the dose In hepatic tissue to 1.4
and 9.3%, respectively, but had no observable effect on the dose of 2,3,7,8-
TCDD required to Induce skin lesions (~1 yg/ear) 1n the rabbit ear assay.
C-21
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Application of 2,3.7,8-TCOD 1n a soil/water paste decreased hepatic 2,3.7,8-
TCOO to -2% of the administered dose and Increased the amount required to
produce skin lesions to 2-3 yg 1n rats and rabbits, respectively.
Application In an activated carbon/water paste essentially eliminated
absorption, as measured by percent of dose 1n the liver, and Increased the
amount of 2,3,7.8-TCDD required to produce skin lesions to -160 yg. These
results suggest that the dermal absorption and acnegenlc potency of 2,3,7,8-
TCDD are dependent on the formulation (vehicle or adsorbent) containing the
toxin.
Distribution
The tissue distribution of 2,3,7,8-TCDD In a number of species 1s summa-
rized In Table 3. From these data H 1s apparent that 2,3,7,8-TCDD dis-
tributes preferentially to the liver and adipose tissue of most species that
have been examined. Piper et al. (1973) used a single oral dose of
[14C]2,3,7.8-TCDD to study distribution and excretion In male Sprague-
Dawley rats. Most of the radioactivity (53.2%) was excreted via the feces,
but the urine and expired air accounted for 13.2 and 3.2%, respectively.
Analysis of the tissues after 3 days showed liver and adipose tissue to
contain the highest percent of the dose per gram of tissue, with 3.18 and
2.60%, respectively.
Rose et al. (1976) also examined the distribution of [14C]2,3,7,8-TCDD
In the rat. Twenty-two days after a single oral dose of 1.0 vg/kg, liver
and adipose tissue had retained most of the 14C activity, with 1.26 and
1.25% of the label retained per gram of tissue, respectively. With repeated
oral doses, the activity was again localized mainly in the liver and adipose
tissue, but the liver had five times as much radioactivity as did the fat.
C-22
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TABLE 3
Tissue Distribution of 2,3,7,8-TCOD
Species
Route of
Administration
Tissues with the Highest Concentration
of 2,3,7,8-TCOD
References
Rat
Rat
Rat
Rat
Rat
Rat
Mouse
Mouse
Rhesus monkey
Golden Syrian
hamster
Guinea pig
Guinea pig
oral
oral
oral
oral
oral
1.p.
oral
1.p.
l.p.
1.p. or oral
oral
Hver
liver > fat
liver > fat
liver > fat
liver > fat
liver > fat
liver > fat > kidney > lung
liver > fat > kidney > lung > spleen
fat > skin > liver > adrenal = thymus
liver > fat
fat > liver > adrenals > thymus > skin
fat > liver > skin > adrenals
Fries and Marrow, 1975
Rose et al., 1976
Piper et al., 1973
Kodba et al., 1978
Allen et al., 1975
Van Miller et al., 1976
Manara et al., 1982
Manara et al., 1982
Van Miller et al., 1976
Olson et al., 1980a
Nolan et al., 1979
Gas1ew1cz and Neal, 1979
NA = Not applicable
-------
With the single oral dose, no radioactivity was detected 1n either the urine
or expired air. Indicating that most If not all of the elimination of
2,3,7,8-TCOO and/or Us metabolites was through the feces. With repeated
oral doses, the J*C activity was also excreted primarily through the
feces, but significant amounts were found 1n the urine, especially of the
female rats. Male rats given 1.0 yg/kg/day of 2,3,7,8-TCDD for 7 weeks
excreted an average of 3.1% of the cumulative dose In the urine while the
female rats excreted an average of 12.554 1n the urine (Rose et a!., 1976).
Fries and Marrow (1975) have also reported evidence of sex differences 1n
tissue distribution 1n rats. During 42 days of administration of 2,3,7,8-
TCDD, -85% of the total body residue of male rats was located 1n the liver,
while 70% of the total body residue of female rats was located 1n this organ.
Studies performed by Van Miller et al. (1976) on rhesus monkeys and rats
using single l.p. doses of Initiated 2,3,7,8-TCDD (400 vg/kg bw) showed
that while rats had over 40% of the 2,3,7,8-TCDD in the liver 7 days after
dosing, the monkeys had only about 10% in the same organ at that time. In
two strains of mice, the liver contained -35% of an administered dose of
2,3,7,8-TCDD 1 day after oral or l.p. administration (Manara et al., 1982).
The liver was also found to be the major site of accumulation of 2,3,7,8-
TCDD In the hamster, with 20% of the dose localized in the liver (5.3% of
dose/g liver) at 3 days following a sublethal dose of 650 ^g 3H-2,3,7,8-
TCDD/kg (Olson et al., 1980a). In all three species, 1-22 days after
single-dose oral or i.p. administration, levels of 2,3,7,8-TCDD in adipose
tissue were generally slightly lower than levels in the liver, and were con-
siderably higher than concentrations 1n other tissues (Piper et al., 1973;
Rose et al., 1976; Van Miller et al., 1976; Olson et al., 1980a; Manara et
al., 1982), Including the thymus (Rose et al., 1976; Van Miller et al.,
1976; Olson et al., 1980a).
C-24
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Koclba et al. (1978) found that female rats maintained on a dally diet-
ary 2,3,7,8-TCDD intake of 0.1 yg/kg/day for 2 years had an average
2,3,7,8-TCOD content of 8100 ppt in fat and 24,000 ppt in the liver. Rats
given 0.01 yg/kg/day had an average of 1700 ppt of 2,3,7,8-TCDD In the fat
and 5100 ppt in the liver. For both of these daily dosages the I1ver:body
fat ratio of 2,3,7,8-TCDD was 3:1. At the lowest dose level of 0.001
yg/kg/day, both fat and liver contained an average of 540 ppt 2,3,7,8-
TCDD. Kociba et al. (1976) presented evidence that steady-state had been
reached after <13 weeks of feeding of 2,3,7,8-TCDD.
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 100 ppt in adipose tissue and 15
ppt In liver. These results indicate that prolonged retention of 2,3,7,8-
TCDD may occur in this species. The tissue distribution of 2,3,7,8-TCDD 1n
the guinea pig appears to be similar to the monkey, with the highest concen-
tration of the toxin being found in adipose tissue (Gaslewicz and Neal,
1979; Nolan et al., 1979). The Interspecies difference in the tissue dis-
tribution of 2,3,7,8-TCDD may be related to the relative adipose tissue
content of a given species and/or the affinity of 2,3,7,8-TCDO for the
hepatic microsomal fraction; however, the significance of these differences
remains in doubt. For example, the hepatotoxicity of 2,3,7,8-TCDD in a
given species does not appear to be related to the hepatic concentration of
the toxin (Neal et al., 1982).
2,3,7,8-TCDD has been demonstrated to be teratogenic and fetotoxic in
the rat (see Teratogenlcity section); 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 [1*C]2,3,7,8-TCDD was Investigated by Moore et al. (1976). They
C-25
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found low concentrations of 2,3,7,8-TCDD 1n the fetus at gestation days 14.
18 and 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 liver on day 21. Nau and Bass (1981, (more recently
reported by Nau et al.. 1982) Investigated the fetal uptake of 2,3,7,8-TCDD
In NMRI mice following oral. l.p. or s.c. administration of the compound at
dose levels of 5. 12.5 or 25 vg/kg 1n dlmethylsulfoxlde (DMSO):corn oil or
acetone:corn oil. The chemical was usually administered as a single dose 2
days prior to sacrifice. All three modes of administration produced similar
maternal and embryonic or fetal levels of 2,3,7,8-TCDD at 5 and 12.5
vg/kg. At 25 yg/kg, higher maternal and fetal tissue levels were
obtained with s.c. administration, and much higher levels were obtained with
1.p. administration, than were obtained with oral administration. Embryonic
2,3,7,8-TCDD concentrations were maximal on gestatlonal days 9 and 10;
however, low levels were found 1n the embryo and fetus between gestatlonal
days 11 and 18. This sharp decrease 1n 2,3,7,8-TCDD concentration coincides
with placentatlon. 2,3,7,8-TCDD concentrations 1n the placenta were an
order of magnitude greater than 1n the fetus Itself. The affinity of fetal
liver for 2,3.7,8-TCDD was relatively low, as compared to maternal liver;
however, 2,3,7,8-TCDD levels 1n fetal livers were 2-4 times higher than the
levels In other fetal organs. An attempt was made to correlate 2,3,7,8-TCDD
levels In the fetuses with the observed Incidence of cleft palate, but no
clear relationship was observed.
Autorad1ograph1c studies of tissue localization following 1.v. adminis-
tration of p«C]2,3,7,8-TCDD 1n DMSO to three strains of mice Indicated
that the liver had the highest concentration and longest retention of radio-
activity In the body, followed by the nasal mucosa (Appelgren et al., 1983).
C-26
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In pregnant mice, the concentration of radioactivity In the fetuses was
lower than In the dams, but a similar, selective labelling of the liver 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 liver at 1 hour after dosing, but thereafter was much lower than In the
liver. Labelling of the thymus, lymph nodes, bone marrow and prostate were
low at all observation times (I.e.. 5 minutes to 61 days after Injection).
Very few data are available on the tissue distribution of 2,3,7,8-TCDO
in humans. Facchettl et al. (1980) reported tissue concentration of
2,3,7,8-TCDO at levels of 1-2 ng/g In liver and <0.1 ng/g In thyroid, brain,
lung, kidney and blood In 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 adenocardnoma (which was not considered related
to the accident) Involving the pancreas, liver and lung.
In addition Young et al. (1983) reported preliminary results of the
analysis 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 321.8936 and 319.8965. Three groups were
studied consisting of 20 veterans claiming health problems related to Agent
Orange exposure, 3 Air Force officers with known heavy exposure to Agent
Orange during disposal operations, and 10 control veterans with no known
herbicide exposure. In the first group, 10 of the 20 had measurable levels
of 2.3,7,8-TCDD (5 with 5-7 ppt, 3 with 9-13 ppt and 1 with 23 and 35 ppt
and another with 63 and 99 ppt). In the second group only two officers had
measurable 2,3,7,8-TCDO levels and these did not exceed 3 ppt. In the 10
control veterans, 4 had 2,3,7,8-TCDD levels between 7 and 14 ppt. Levels of
C-27
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2,3,7,8-TCDD In 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-TCOD 1n adipose tissue was too
limited to draw any firm conclusions.
Metabolism
Vlnopal and Caslda (1973, found no evidence of water soluble metabolites
of 2,3,7,8-TCOD following Incubation with mammalian liver mlcrosomes or 1.P.
Injection Into mice. In the same experiment, only unmetabollzed 2,3,7,8-
TCDD was extractable from mouse liver 11-20 days after treatment. Van
Miller et al. (1976) claimed that the slow elimination of 2,3,7,8-TCDD they
observed In both rats and monkeys after l.p. Injections suggested that
2,3,7,8-TCDD was not readily metabolized. Metabolites of 2,3,7,8-TCDD have
been detected 1n the bile and urine of Syrian Golden hamsters after single
oral or l.p. doses (Olson et al., 1980a) and in the bile of dogs following
repeated direct Introduction of the chemical Into the duodenal lumen (Polger
et al., 1982a).
Polger and Schlatter (1979), Ramsey et al. (1979) and Ramsey et al.
(1982) demonstrated biliary excretion of several metabolites of
["C]2.3.7.8-TCDD by rats after repeated oral dosing. The metabolites
were tentatively Identified as glucuronldes of hydroxylated 2,3,7,8-TCDD.
The amounts of metabolites found were small. Indicating that 2,3,7.8-TCDD 1s
only slowly metabolized in the liver. Previous work by Piper et al. (1973)
using single oral doses of 2,3,7,8-TCDO concluded that, since small amounts
of radioactivity were found in the urine and expired air of male rats during
the first 10 days, metabolic alteration or breakdown must occur. The study
by Rose et al. (1976) using oral doses stated that while the «c activity
In the rat livers appeared to be present as unchanged 2,3,7,8-TCDD, a
C-28
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significant amount of radioactivity found In the feces appeared to come from
substances other than 2,3,7,8-TCDD; the excretion of 14C 1n the urine also
Indicated that metabolism had occurred.
Polger et al. (1982b) Investigated the toxlclty of 2,3,7,8-TCDD metabo-
lites by administering extracts of bile from 2,3,7,8-TCDD-treated dogs to
male guinea pigs 1n single oral doses equivalent to 0.6, 6.0 and 60 yg/kg
of parent compound. Other groups of guinea pigs received bile extract from
untreated dogs or 2,3,7,8-TCDD Itself. A comparison of the mortality data
at 5 weeks after dosing Indicated that the acute toxldty of 2,3,7,8-TCDD to
guinea pigs was at least 100 times higher than was the acute toxldty of Its
metabolites.
More recently, Olson et al. (1983) reported that all of the radioactiv-
ity 1n urine and bile from 14C-2,3,7,8-TCDD-treated rats, hamsters and
guinea pigs corresponded to metabolites of 2,3,7,8-TCDD. The enzymatic
hydrolysis of the 2,3,7,8-TCDD metabolites from the rat and hamster altered
the chromatographlc profile of the metabolites, Indicating the presence of
glucuronlde conjugates In bile and sulfate conjugates 1n urine (Olson and
Blttner, 1983). The apparent absence of these metabolites In extracts of
hamster and rat liver suggest that once formed, the metabolites of 2,3,7,8-
TCDD are readily excreted (Olson et al., 1980a; Rose et al., 1976). These
results also Indicate that urinary and biliary elimination of 2,3,7,8-TCDD
1s dependent upon metabolism of the toxin. Although urine and bile appear
to be free of unmetabollzed TCDD, data from the hamster and rat Indicate
that from 10 to 40% of the 2,3,7,8-TCDD-deMved radioactivity 1n feces
represents unchanged 2,3,7,8-TCDD (Olson et al., 1983; Olson and BHtner,
1983). The dally presence of unchanged 2,3,7,8-TCDD 1n feces and Us
absence In bile suggests that direct Intestinal elimination may be the
C-29
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source for the Fecal excretion of 2,3,7.8-TCDD. This finding demonstrates
that the half-life for elimination of 2,3,7,8-TCDD may not directly reflect
the in vivo rate of 2,3,7,8-TCDD metabolism in a given animal. Neverthe-
less, the metabolism of 2,3,7,8-TCDD does in part regulate Us elimination
or relative persistence 1n a given animal.
Several metabolites of 2,3,7,8-TCDD have recently been Identified.
Sawahata et al. (1982) Investigated the jn vitro metabolism of 2,3,7,8-TCDD
In Isolated rat hepatocytes. The major product was deconjugated with
B-glucur«n1dase, derlvatlzed with dlazomethane, and separated Into two com-
pounds by high performance liquid chromatography (HPLC). These metabolites
were subsequently Identified as l-hydroxy-2,3,7,8-TCDD and 8-hydroxy-2,3,7-
tr1chlorod1benzo-p_-d1ox1n. Polger et al. (1982b) Identified six metabolites
In the bile of dogs that were given a lethal dose of [3H]2,3,7,8-TCDD. The
major metabolite was 1,3,7,8-tetrachloro-2-hydroxyd1benzo-p_-d1oxin; 2,3,7,8-
tr1chlor-3-hydroxyd1benzo-p_-d1ox1n and 1,2-d1chloro-4,5-hydroxybenzene were
also Identified as minor metabolites. The structures of the three remaining
metabolites were not determined; however, two appeared to be trlchloro-
hydroxyd1benzo-p_-d1ox1ns and the third was apparently a chlorinated
2-hydroxyd1phenyl ether.
Data on the metabolism of 2,3,7,8-TCDD suggests that reactive epoxlde
Intermediates may be formed. Poland and Glover (1979) have Investigated the
In vivo binding of [1,6-3H]-2,3,7,8-TCDD derived radioactivity to rat
hepatic macromolecules. They found maximum levels equivalent to 60 pmol
2,3,7,8-TCDD/mole of amlno acids 1n protein, 12 pmol 2,3,7,8-TCDD/mole of
nucleotlde In rRNA, and 6 pmol of 2.3,7,8-TCDD/mole of nucleotlde 1n DNA.
This corresponds to one 2,3,7,8-TCDD-DNA adduct/35 cells. Poland and Glover
(1979) suggest that 1t 1s unlikely that 2,3,7,8-TCDD-lnduced oncogenesls 1s
through a mechanism of covalent binding to DNA and somatic mutation.
C-30
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Further studies In other species, possibly with [14C]-2,3,7,8-TCDD, are
needed to confirm these results and assess the relationship between covalent
binding and the short and long-term toxlclty of 2,3,7,8-TCOD.
Isolated rat hepatocytes 1n suspension have been used as an in vitro
system for assessing 2,3,7,8-TCDD metabolism under various conditions (Olson
et al., 1981). Data Indicate that the rate of 2,3,7,8-TCOD 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 (Neal et al., 1982). Pretreatment of rats with
2,3,7,8-TCDD has been shown to enhance the rate of 2,3,7,8-TCDD metabolism
In Isolated hepatocytes, demonstrating that 2,3,7,8-TCDD can Induce Us own
rate of metabolism. Beatty et al (1978) also found a correlation between
hepatic mixed-function oxldase (MFO) activity and the toxlclty of 2,3,7,8-
TCDD 1n rats. In both naturally occurring age and sex-related differences
In MFO activity, and following administration of Inducers and Inhibitors of
MFO enzyme systems, hepatic MFO activity was directly correlated with the
20-day LD5Q.
Olson and Blttner (1983) reported that the rate of 2,3,7,8-TCDD metabo-
lite formation in vitro was higher 1n hepatocytes from the hamster than 1n
hepatocytes from the rat. Qualitative evaluation of j_n vivo and in vitro
metabolites by HPLC also suggested significant Interspedes variability.
The authors suggested that such differences 1n metabolism may partially
explain the differences 1n toxlclty among species.
Excretion
The following discussion assumes that elimination Is a first order
process. With the exception of the guinea pig, which may follow zero order
kinetics (Gaslewlcz and Neal, 1979), elimination data yield a straight line
C-31
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on a semnogarlthmlc plot, Indicating that elimination Is a single, first
order process. Miles and Bruce (1976) have pointed out that the studies of
Allen et al. (1975) and Piper et al. (1973) can be Interpreted equally well
by either zero or first order kinetics. The majority of the data, however,
seem to support the assumption of a first order elimination process.
The excretion of 2,3.7,8-TCDD and Us metabolites has been Investigated
in a number of species. Table 4 summarizes results on the elimination of
2,3,7,8-TCOD-der1ved radioactivity, following a single exposure to >H- or
[*«C]-2.3,7.8-TCDD. These studies show that 2,3,7,8-TCDD was slowly
excreted from the bodies of all species tested, with a half-life in the body
of 10-43 days. In the Syrian Golden hamster, the least sensitive mammalian
species to the acute toxldty of 2,3,7,8-TCDD, excretion occurred readily
through both the urine (35% of administered dose, 41% of total excreted
radioactivity) and feces (50% of the administered dose, 59% of total excret-
ed radioactivity) (Olson et al., 1980b; Gas1ew1cz et al., 1983a). The high
levels found 1n the urine of Infant monkeys were probably due to the Incom-
plete separation of urine and feces (Van Miller et al., 1976). In all the
other species tested so far, excretion occurred mainly through the feces
(80-100% of total urinary and fecal radioactivity) with only minor amounts
of 2,3,7,8-TCDD metabolites found in the urine (Piper et al., 1973; Allen et
al., 1975; Rose et al., 1976; Gasiewicz and Neal, 1979). Only Piper et al.
(1973) reported the excretion of metabolites in the expired air. During 21
days following administration of a single oral dose of [14C]2,3,7,8-TCDD
to rats, 3.2% of the administered radioactivity (4.6% of the excreted radio-
activity) was recovered 1n the expired air.
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£-)
I
CO
CO
TABLE 4
Elimination of 2,3,7,8-TCDD
Species
Guinea pig
Guinea pig
Rat
Rat
Rat
Rat
Monkey
(adult)
Monkey
(Infant)
Mouse
C57BL/65
OBA/2J
B6D2FT/J*
Hamster
Hamster
Single Treatment
vg/kg (route)
2 d.p.)
1.45 (oral)
1.0 (oral)
50 (oral)
50 (oral)
400 (1.p.)
400 (1.p.)
400 (1.p.)
10 (1.p.)
10 (1.p.)
10 (1.p.)
650 (1.p.)
650 (oral)
Relative % of TCDD-Der1ved
Half-Life for Radioactivity
Elimination
(days)
30.2 i 5.8
22 - 43
31+6
17.4 + 5.6
21.3 + 2.9
NT
NT
NT
11.0 + 1.2
24.4 + 1.0
12.6 + 0.8
10.8 ± 2.4
15.0 i 2.5
Feces
94.0
NT
>99
80.0
95.5
91.0
78.0
39.0
72.0
54.0
72.0
59.0
NT
Urine
6.0
NT
<1
20.0
4.5
9.0
22.0
61.0
28.0
46.0
28.0
41.0
NT
Reference
Gas1ew1cz 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 MUler et al., 1976
Van Miller et al., 1976
Gas1ew1cz et al., 1983a,b
Gas1ew1cz et al., 1983a,b
Gas1ew1cz et al., 1983a,b
Olson et al., 1980a
Olson et al., 1980a
Offspring of C57BL/6J and DBA/2J which are heterozygous at the Ah locus
NT = Not tested
-------
Rose et al. (1976) Investigated the elimination of [14C]2,3,7,8-TCDD
In rats given repeated oral doses of 0.01, 0.1 or 1.0 vg/kg/day Monday
through Friday for 7 weeks, or a single dose of 1.0 vg/kg. In the single-
dose study, no *«C was excreted 1n the urine or expired air; In the
repeated-dose study, however, 3-18% of the cumulative dose was excreted In
the urine by 7 weeks. This study Indicated that steady-state concentrations
will be reached 1n the bodies of rats In -13 weeks. The rate constant
defining the approach to steady-state concentrations was Independent of the
dosage of 2,3,7,8-TCOO over the range studied. This 1s consistent with the
observations of Fries and Marrow (1975) who found that the total retention
In the bodies of rats was proportional to total Intake. When rats were
maintained on a diet containing either 7 or 20 ppb 2,3,7,8-TCDO, the amount
of 2,3,7,8-TCOD retained 1n the body was 5.5 times the dally Intake of
2,3,7,8-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 In Table 4 suggest some Interspedes differences 1n the half-
life for elimination (t 1/2) of 2,3,7,8-TCOO. In the hamster, the least
sensitive species to the acute toxldty of 2,3,7,8-TCDD, a mean t 1/2 of
10.8 days was observed (Olson et al., 1980a,b), and 1n the guinea pig, the
most sensitive species to the acute toxldty of 2,3,7,8-TCDD, the mean t 1/2
was 30.2 days (Gas1ew1cz and Neal, 1979). The observed Interspedes differ-
ences 1n the t 1/2 of 2,3,7,8-TCDD may 1n part be related to the relative
sensitivity of a given species to the acute toxldty of 2,3,7,8-TCDD.
The Intraspedes differences In the t 1/2 of 2,3,7,8-TCDD 1n three mouse
strains may be due to the finding that the DBA/23 strain possesses ~2-fo1d
greater adipose tissue stores than the C57BL/6J and B6D2F,/J strains
(Gaslewlcz et al., 1983b). The sequestering of the Upophlllc toxin In
C-34
-------
adipose tissue stores of the DBA/2J mouse may contribute to the greater
persistence of 2,3,7,8-TCDO 1n this strain.
In all of the rat studies shown In Table 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 Blttner (1983) examined the elimination
of 2,3,7,8-TCOD-derlved radioactivity 1n rats over a 35-day period following
a single 1.p. exposure at 1 vg 3H-2,3,7,8-TCDD/kg. They observed first
order kinetics for elimination, with a fast component having a t 1/2 of 7
days (representing 13% of total elimination) and a slow component having a
t 1/2 of 75 days (87% of total). The second, slow component for elimination
was evident only when urinary and fecal elimination were monitored for >30
days. This study suggests that 2,3,7,8-TCDD may be more persistent than
earlier studies suggested. A preliminary study 1n the rhesus monkey Indi-
cates 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-TCOD In
the fat of a monkey to be ~1 year.
Studies In the rat, guinea pig, hamster and mouse have found that all of
the 2,3,7,8-TCDD derived radioactivity excreted 1n the urine and bile corre-
sponds to metabolites of 2,3,7,8-TCDD (Olson et al., 1983). The apparent
absence of 2,3,7,8-TCDD metabolites In liver and fat suggests that, 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 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 (Olson et
al., 1983). Unmetabollzed 2,3,7,8-TCDD thus appears to enter the Intestinal
C-35
-------
lumen by some route other than bile (direct Intestinal elimination) for a
number of days following treatment. Studies In lactatlng rats have also
found that unchanged 2,3,7,8-TCDO may be excreted 1n the milk of lactatlng
animals {Moore et al., 1976; Luder et al., 1975). Lactation, direct Intes-
tinal elimination, and perhaps sebum may serve as routes for excretion of
2,3,7,8-TCDO, which are not dependent upon metabolism of the toxin. These
data suggest that the \n 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).
EFFECTS
Acute. Subacute and Chronic Toxlcity
The acute LD5Q for 2,3,7,8-TCDD 1n several species 1s shown 1n
Table 5. The oral LD values range from 0.6 yg/kg bw for guinea pigs
to 5051 yg/kg bw for hamsters (Schwetz et al., 1973; Vos et al., 1974;
McConnell et al., 1978a,b; Henck et al., 1981; Olson et al., 1980b). The
dermal LD for rabbits was 275 yg/kg of body weight (Schwetz et al.,
1973); death was sometimes delayed as long as 40 days following acute
exposure. Of the laboratory animals studied, the guinea pig was the most
susceptible to the toxic effects of 2,3,/,8-TCDD (Schwetz et al., 1973;
Gupta et al., 1973; Greig et al., 1973).
The acute toxiclty has also been found to vary with the sex, age and
strain of the test animal. Schwetz et al. (1973) found male Sherman rats
more sensitive to 2,3,7,8-TCDD than females, while Beatty et al. (1978)
found female Sprague-Dawley rats more sensitive than adult male rats. Thus,
no general sex difference 1s apparent in the rat, perhaps due to strain dif-
ferences in sensitivity to 2,3,7,8-TCDD. A significant sex difference was
observed In the C57BL/10 mouse, with the oral L05_ In females being 3-fold
C-36
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TABLE 5
Lethality of 2,3,7,8-TCDD Following Acute Exposure
o
i
Species/Strain
Guinea pigs/
Hartley
Guinea pigs/
Hartley
Guinea pigs/
Hartley
Rats/Sherman
Rats/Sherman
Rats/Sprague-
Oawley
Rats/Sprague-
Dawley
Monkey/rhesus
H1ce/C57Bl
Sex/No./
Group
M/NR
H/NR
H/9
M/5-10
F/NR
M/6
F/6
F/3
H/14
Route/Vehicle
gavage/corn oil-
acetone (9:1)
gavage/corn oil-
acetone (9:1)
gavage/corn oil
gavage/corn oil-
acetone (9:1)
gavage/corn o1l-
acetone (9:1)
1 .p. /olive oil
1 .p. /olive oil
gavage/corn oil
gavage/corn oil-
acetone (9:1)
Dose
Tested
NR
NR
NR
8
16
32
63
NR
NR
NR
0
70
350
0
100
150
200
Duration of
Observation
2-8 weeks
2-8 weeks
30 days
2-8 weeks
2-8 weeks
20 days
20 days
>35 days
60 days
(vg/kg)
0.6
(0.4-0.9)*
2.1
(1.5-3)*
2
22
45
(30-66)*
60
25
<70
114
Comments
time to death was 5-34 days, the
2,3,7,8-TCDD was 91X pure
time to death was 9-42 days, the
2,3,7,8-TCDO was 99X pure
median time to death was 17-20 days,
marked weight loss, thymus atrophy,
Intestinal hemorrhage, no porphyrla
and only mild liver Injury
time to death was 9-27 days, the
2,3,7,8-TCDD was 91X pure
time to death was 13-43 days, the
2,3,7,8-TCDO was 91X pure
LOso (»ig/kg, mean + SE) adult male,
60.2 i 7.8; weanling male, 25.2 + 1.4
LDso (pg/kg, mean + SE) adult
female, 24.6 ± 2.0
weight loss, edema, severe thymus
atrophy, loss of hair, mild liver
damage
time to death 1n the high dose group
was 15-20 days, body weight loss,
edema 1n 25X of treated animals,
severe thymlc and spleen atrophy.
Reference
Schwetz et al. ,
Schwetz et al. ,
HcConnell et al
1978a
Schwetz et al..
Schwetz et al..
Beatty et al. ,
Beatty et al.,
McConnell et al
1978b
1973
1973
1973
1973
1978
1978
• t
Vos et al.. 1974
and small Intestine, liver necrosis
1n the centrllobular region
-------
TABLE 5 (cont.)
Sex/No./
Species/Strain Group
M1ce/C57Bl M/9
HUe/C57BL/10 M/5
H1ce/C57BL/10 F/5
o
i
co
oo
H1ce/C57BL/6J M/NR
M1ce/OBA/2J M/NR
M1ce/B602F!/J M/NR
Rabbits/ H&F/NR
New Zealand
Rabbits/ M&F/5
New Zealand
Route/Vehicle
gavage/corn oil
gavage/arachls oil
gavage/aracMs oil
1. p. /olive oil
1 .p. /olive oil
1 .p. /olive oil
gavage/corn o1l-
acetone (9:1)
1. p. /corn oil
Dose Duration of 1050
Tested Observation {wg/kg)
(tig/kg)
NR 30 days 283.7
85 45 days 146
107
135
170
213
85 45 days >450
107
135
170
213
269
338
426
536
NR 30 days 132
NR 30 days 620
NR 30 days 300
NR 2-8 weeks 115
(38-345)*
32 4 weeks NR
63
126
252
500
Comments
median time to death was 22-25 days,
dose-related body weight loss, thyralc
atrophy, Increased liver weight and
porphyMa, gross and historic liver
alterations, subcutaneous edema.
Intestinal hemorrhage
95X confidence limits of 111-211
yg/kg. Host deaths occurred from
22-26 days after dosing. Signs of
porphyHa, edema, hemorrhage.
1 of 4 animals died at dose of
426 ng/kg
BGD2F]/J mice are the offspring of
C57BL/6J and DBA/2J and are hetero-
zygous at the Ah locus.
time to death was 6-39 days, the
2,3,7,8-TCDO was 91X pure
time to death was 6-23 days,
2-3 animals/group died 1n all but
the low exposure group
Reference
McConnell et al.,
1978a
Smith et al., 1981
Smith et al., 1981
Gaslewlcz et al.,
1983a.b
Gaslewlcz et al. ,
1983a,b
Gaslewlcz et al.,
1983a,b
Schwetz et al., 1973
Schwetz et al., 1973
-------
TABLE 5 (cont.)
Sex/No./
Species/Strain Group
Rabbits/ M&F/NR
New Zealand
Hamster/ H/6
golden Syrian
Hamster/ M&F/5-6
golden Syrian
^
i
CO
10
Hamster/ N/5
golden Syrian
Dogs/beagle M/2
Dogs/beagle F/2
Route/Vehicle
dermal/acetone
gavage/corn oil-
acetone (9:1)
1. p. /olive oil
gavage/ollve oil
gavage/corn oil-
acetone (9:1)
gavage/corn oil-
acetone (9:1)
Dose
Tested
31.6
63
126
252
500
0
300
600
1000
3000
6000
0
500
1000
2000
3000
500
1000
2000
3000
3000
30
100
Duration of 1050
Observation (yg/kg) Comments
3 weeks 275 time to death was 12-22 days
(142-531)
55 days 5051 (3876- time to death was 26-43 days, the
18,487; 95X liver and thymus appeared to be the
confidence) primary target organs, only 1 death
occurred 1n the 300 and 3000 yg/kg
group
50 days >3000 significant dose-related decrease 1n
thymus weight starting at 500 yg/kg,
only 2 deaths occurred out of 11
hamsters In the 3000 yg/kg group.
50 days 1157 death generally occurred between 24
and 45 days, decrease In body weight
above 2000 yg/kg, prollferatlve
1le1t1s with mild to severe Inflam-
mation
2-8 weeks NA all animals died
2-8 weeks NA all animals survived
Reference
Schwetz et al., 1973
Henck et al., 1981
Olson et al.. 1980b
Olson et al., 1980b
Schwetz et al., 1973
Schwetz et al., 1973
•The number In parentheses appears to Indicate the range of lethal doses; however, the studies did not specify what these numbers represented.
NA = Not applicable; NR = not reported
-------
greater than that In males (Smith et al.. 1981). In a study of age-related
differences, Beatty et al. (1978) reported weanling male rats to have an
acute LD5Q of 25 yg/kg 1n contrast to the value of 60 yg/kg 1n adult
males. Vos et al. (1974) found 0, 17 and 44% mortality in mice of 4, 2 and
1 months of age, respectively, following 4 weekly doses of 25 yg 2,3,7,8-
TCDO/kg. These limited studies suggest that young animals may be more
susceptible to the acute toxlclty of 2,3,7,8-TCDD. Various strains of mice
have been used to study the mechanism of action of 2,3,7,8-TCDD, based on
the ability of the toxin to Induce enzymes that have been shown to segregate
with a single genetic locus, the Ah locus (Poland et al., 1974, 1976a,b).
The "non-responsive" strains (e.g., DBA/2J) appear to be less responsive to
enzyme 1nduct1en due to an altered receptor with lower affinity for
2,3,7,8-TCDD, In comparison to the "responsive" strains (e.g., C57BL/6J).
Gaslewlcz et al. (1983b) reported that the "responsive" C57BL/6J mice have
an acute LD5Q for 2,3,7,8-TCDD of 132 yg/kg, compared with an LD of
620 yg/kg In the "non-responsive" DBA/2J mice. An Intermediate LD. of
300 yg/kg was also reported for B6D2F1/J mice, which are offspring of
C57BL/6J and DBA/2J (B6D2F-./J mice are heterozygous at the Ah locus).
These results suggest that the acute LDcn for 2,3,7,8-TCDD varies with the
DU
strain of mouse and the relative activity "responsiveness" at the Ah locus.
The hepatotoxldty of 2,3,7,8-TCDD 1s well established, especially 1n
rats, mice and rabbits where the hepatic lesions are particularly severe
(Mllnes, 1971). Sublethal doses of 2,3,7,8-TCDD 1n rats produced signifi-
cant liver damage, characterized by fatty changes, centrllobular necrosis
(Cunningham and Williams, 1972), megalocytosls, and unusual numbers of
multlnucleated giant hepatocytes (Gupta et al., 1973). A single dose of 0.1
yg/kg In rats produced Increased liver weights (Harris et al., 1973).
C-40
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In rats given single doses of 5 and 25 vg/kg 2,3,7,8-TCDO, Fowler et al.
(1973) reported extensive proliferation of the smooth and rough endoplasmlc
retlculum, especially near the bile ducts. Twenty-eight days after dosing,
the electron micrographs of the livers were Indistinguishable from controls.
Similar results were observed by Jones and Butler (1974) and Jones and Grelg
(1975). In mice, exposure to 1-10 vg/kg 2,3,7,8-TCDD/day produced liver
damage as Indicated by elevated SCOT, SGPT, serum LOH, alkaline phosphatase
and bH1rub1n levels (Z1nkl et al., 1973).
A number of toxic responses have been observed following exposure to
2,3,7,8-TCDO and these have been summarized for a number of species In
Table 6. 2,3,7,8-TCDO toxldty exhibits marked Interspecles variability,
with some responses being highly species specific and confined to one or a
few species. Loss of body weight or reduced weight gain and thymlc atrophy
are the most consistent toxic responses of 2,3,7,8-TCDD exposure In various
species, with the latter being one of the most sensitive Indicators of
toxldty. In general, the toxlcologic pattern observed with 2,3,7,8-TCDO 1s
not unique; 1t also occurs with certain halogenated dlbenzofurans, chlori-
nated biphenyls, naphthalenes, and bromlnated dloxins (McConnell, 1980).
An extended period was observed between treatment and death. During
this period the animals had poor weight gain or loss of weight and appeared
to be "wasting away". At death, loss 1n body weight was reported to be as
great as 50% for some species (McConnell, 1980). In female Wlstar rats
Intubated with 2,3,7,8-TCDD at a dose of 100 vg/kg, the weight loss was
blphaslc (Courtney et al., 1978). The Initial weight loss occurred rapidly
during the first 7-10 days after treatment and was associated with decreased
food and water consumption. This Initial phase of weight loss was reversed
with the resumption of normal food Intake for 4 or 5 days, only to be
C-41
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TABLE 6
Toxic Responses Following Exposure to 2,3,7,8-TCDO: Species Differences3
Monkey
Guinea
Pig
Cowb
Rat
Mouse Rabb4tb Ch1ckenb Hamster
o
I
Hyperplasla and/or metaplasia
Gastric mucus
Intestinal mucosa
Urinary tract
B1le duct and/or
gall bladder
Lung: focal alveolar
Skin
Hypoplasla, Atrophy, or Necrosis
0
0
0
0
0
0
*d
0
0
Thymus
Bone marrow
lestlcle
Other
Liver lesions
PorphyMa
Edema
+ 4. 4.
+ 4.
4. +
+ +•
0 0
+ 0
+ + +
+ 4.
•*•+ * <••«• + 4-
+ n- +0
• w
0 + +.+ +
aReferences: Monkey (McConnell et al., 1978a; Norback and Allen, 1973; Allen et al.. 1977); Guinea p1q
(McConnell et al., 1978a; McConnell, 1980; Moore et al., 1979; Turner and Collins, 1983); Cow (McConnell
1980); Rat (McConnell, 1980; Kodba et al., 1978, 1979); Mouse (Schwetz et al., 1973; McConnell et al '
1978a; Vos et al., 1973); Rabbit (K1mm1g and Schultz, 1957; Schwetz et al., 1973; Vos and Beems 1971)'
Chicken (Schwetz et al., 1973; Norback and Allen, 1973; Allen and Lallch, 1962; Vos and Koeman 1970)'
Hamster (Olson et al., 1980b; Henck et al., 1981) '
Responses followed exposure to 2,3,7,8-TCDD or structurally related chlorinated aromatic hydrocarbons.
cSymbols: 0, lesion not observed; +, lesion observed (number of "+" denote severity); +, lesion observed
to a very limited extent; blank, no evidence reported 1n literature.
Skin lesions 1n cattle are observed, but they differ from the skin lesions observed 1n other species.
Source: Adapted from Poland and Knutson, 1982
-------
followed by a second, more gradual, decline 1n food and water Intake and
weight until death. Providing animals with an adequately nutritious liquid
diet by Intubation did not appreciably alter the pattern of weight loss nor
affect survival. In contrast, 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. Also, severe thymlc atrophy has been
universally observed 1n all species given lethal doses of 2,3,7,8-TCDO, and
since weight loss and thymlc atrophy are both associated with malnutrition,
van Logten et al. (1981) Investigated the effects of dietary protein on the
toxldty of 2,3,7,8-TCDD. Groups of female Fischer 344 rats administered
2,3,7,8-TCDD (20 yg/kg) and maintained on low (3.5%), normal (26%) or high
(55%) protein diets maintained approximately the same body weight (gains
were -0.2^3, 7^6 and 7*-3 g for each dietary group, respectively) during the
subsequent 10-day period. The weight gain In treated animals was 10-18 g
less than that 1n the respective control rats. Dietary protein also had no
effect on preventing or enhancing the 2,3,7,8-TCDD Induced thymlc atrophy.
In yet another study, Seefeld and Peterson (1983) suggest that a reduc-
tion 1n food Intake caused by 2,3,7,8-TCDD 1s primarily responsible for the
loss of body weight or depressed growth rate of rats. Pair-fed control rats
lost weight at the same rate and to the same extent as their weight matched
2,3,7,8-TCDD-treated partners (25 or 50 yg/kg) until day 10 after treat-
ment. 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. They pro-
pose a hypothesis that 2,3,7,8-TCDD lowers a regulated level of "set-point"
for body weight control 1n the rat. The ensuing change 1n food Intake 1s
C-43
-------
thought to occur secondarily to the change 1n "set-point". Thus, the pre-
cise mechanism for the 2,3,7,8-TCOD Induced weight loss remains uncertain;
however, It Is evident that weight loss 1s a contributing factor to
2,3,7,8-TCDD Induced mortality and morbidity.
Feeding a diet containing 7 ppb of 2,3,7,8-TCDD to rats caused an
Increase of liver weight while unexpectedly, 20 ppb caused less of a liver
weight gain. After the feeding of 2,3,7,8-TCDD was discontinued, recovery
was greater 1n the 7 ppb groups (Fries and Marow, 1975). The hepatotoxldty
of 2,3,7,8-TCDD was most severe In rats, mice and rabbits (Vos and Beems,
1971; Gupta et al., 1973; Schwetz et al., 1973; Vos et al., 1974). 2,3,7,8-
TCDD-1nduced liver alterations 1n the guinea pig and hamster were generally
limited to the responses accompanying liver hypertrophy (Turner and Collins,
1983; Olson et al., 1980b). Limited steatosls, focal necrosis, and cyto-
plasmic hyalln-like bodies were also observed In the guinea pig (Turner and
Collins, 1983). Comparative studies Indicate that the guinea pig and
hamster were the least sensitive to 2,3,7,8-TCDD-induced hepatotoxldty,
which 1s In contrast to the 5000-fold difference in the acute LD for
2,3,7,8-TCDD in these species.
2,3,7,8-TCDD affects porphyMn metabolism and causes significantly
elevated excretion of porphyrins and S-am1nolevul1nic acid (see Metabolism
section). Goldstein et al. (1978) showed that a-am1nolevul1nic acid
synthetase, a rate-limiting enzyme in porphyrin synthesis, was slightly
Increased (2-fold) in male C57B1 mice given 4 weekly doses of 2,3,7,8-TCDD
at 25 yg/kg. This dose of 2,3,7,8-TCDD Increased liver prophyrln levels
2000-fold. Catabolism of porphyrin by uroporphyrlnogen decarboxylase (UD)
also appeared to be decreased in 2,3,7,8-TCDD-treated mice. Smith et al.
(1981) reported a decrease in UD activity from -25 to 7 nmoles/hr/g liver In
C-44
-------
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 In DBA/2 mice, which were Insensitive to the Induction of prophy-
rla. A time course of changes In DO 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% In C58B1 mice and only 4% In DBA/2 mice. Other
factors besides the Increase In a-amlnolevullnlc acid synthetase and the
decrease In UD activity may also participate In the dramatic Increase 1n
liver porphyrln In mice, associated with exposure to near lethal doses of
2,3,7,8-TCDD.
A number of biochemical studies have resulted from the observation that
2,3,7,8-TCDD produces fatty livers and a resulting Increase 1n total hepatic
llpld content In several species. A sublethal dose of 2,3,7,8-TCDD In the
rat produced an Increase 1n trlglycerldes and free fatty adds and a
decrease In sterol esters, while a lethal dose Increased cholesterol esters
and free fatty adds (Albro et al., 1978). Poll et al. (1980) treated rats
with a single 1.p. Injection of 2,3,7,8-TCDD at doses of 2.5, 5, 10 and 20
vg/kg. At day 21 after treatment there was a dose-related Increase 1n
total plasma cholesterol and high density Upoproteln cholesterol, while no
change was observed In trlglycerldes or very low and low density Upo-
protelns (VLDL and LDL, respectively). At a dose of 20 vg/kg the maximum
Increase In HDL cholesterol and total cholesterol occurred 30 days after
treatment, and a significant elevation was still present at 60 days after
treatment when the study was terminated. Slight changes 1n the apoprotelns
C-45
-------
of HDL from 2,3,7,8-TCDD rats and control rats were Indicative of new apo-
proteln synthesis. Although the Increase 1n HDL cholesterol may be 1n
response to eliminating excess I1p1ds, the exact function has not been
clearly shown.
In contrast to rats, male Hartley strain guinea pigs given a single 1.p.
Injection of 2,3,7,8-TCDD at a dose of 2 yg/kg had Increased hyperllpldema
characterized by Increases In VLDL and LDL (Swift et a!., 1981). In animals
sacrificed 7 days after exposure to 2,3,7,8-TCDD, there was an Increase 1n
total serum I1p1d, cholesterol esters, trlglycerldes and phosphollplds when
comparison was made to pair-fed, weight-paired or ad_ libitum fed control
groups. Serum-free fatty adds were not changed quantitatively; however,
some qualitative changes occurred, reflecting an Increase 1n the types of
fatty adds which were abundant 1n the adipose tissue of guinea pigs.
Analysis of Upoprotelns revealed a 19-fold Increase In VLDL and a 4-fold
Increase 1n LDL, with no change observed 1n the levels of HDL. The VLDL was
also qualitatively different 1n the 2,3,7,8-TCDD treated animals, containing
less cholesterol ester and an altered C apoproteln. The Importance of these
qualitative changes 1s unclear. The hyper!1p1dem1a may result from the
2,3,7,8-TCDD Induced mobilization of free fatty acids, which are then used
In the synthesis of VLDL and are subsequently formed Into LDL. The rela-
tionship of the changes In serum I1p1d levels to the mechanism of 2,3,7,8-
TCDD toxldty needs further study.
Gupta et al. (1973) reported slight to moderate thymlc atrophy In guinea
pigs after 8 weekly oral doses of 0.2 v9/kg. The thymlc atrophy was
characterized by a decrease 1n the number of cortical thymocytes, reduction
In size of the thymlc lobules, and the absence of a demarcation between
cortex and medulla. There was a relative depletion of lymphold cells 1n the
C-46
-------
spleen and the lymph nodes. In addition, moderate thymlc atrophy was
observed In rats after 31 dally oral doses of 1 yg/kg 2,3,7.8-TCDD.
Thymlc atrophy has also been noted 1n monkeys (Norback and Allen, 1973). In
later studies 2,3,7,8-TCDD was found to suppress cell-mediated Immune func-
tion 1n young rats without affecting humoral Immune function. Suppression
of T-cell function was selective In that "helper" cell function was not
suppressed (Faith and Moore, 1977). Recently, the effects of 2,3,7,8-TCDD
on thymus Involution In rats were found not to Involve the adrenal or pitui-
tary glands and were not prevented by treatment with growth hormone (van
Logten et al., 1980).
Increased susceptibility to Salmonella Infection was found 1n mice
treated 1ntragastr1cally with 2,3,7,8-TCDD at doses between 1 and 20 yg/kg
bw once weekly for 4 weeks. Such Increased susceptibility after 2,3,7,8-
TCDD administration was not seen with Herpes virus Infection (Thlgpen et
al., 1975). Thymus atrophy with consequent suppression In cell-mediated
Immunity as measured by several parameters was found by Vos et al. (1978) In
mice after various doses of 2,3,7,8-TCDD up to 50 yg/kg bw. The effects
were dose related. Juvenile and adult mice treated with 2,3,7,8-TCDD 1n
their feed at 10 and 100 ppm displayed several dose related changes, Includ-
ing depression 1n total serum protein, gamma globulin and albumin. Primary
and secondary antibody responses to both tetanus toxold and sheep erythro-
cytes were also reduced, as well as resistance to challenge with either
Salmonella typhlmurlum or Llsterla monocytoqenes (Hlnsdlll et al., 1980).
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). In the bone marrow, hypocellularlty
C-47
-------
and depressed macrophages-granulocyte progenitor cells and p^UMpotent stem
cells were associated with 2,3,7,8-TCOD exposure at the 5.0 and 15.0 yg/kg
dose levels. Host susceptibility to L. monocytogenes and PYB6-tumor cells
was tested In the 2,3,7,8-TCDO-exposed neonates. Death occurred 1n 73% and
40% of the L_. monocytoqenes Inoculated (1.2xl06 viable organisms) mice 1n
the 5.0 and 1.0 vg/kg dose groups, respectively, compared to 28% of
controls. Tumor development occurred 1n 44, 60 and 22% of the neonates
Inoculated with 5xl04 tumor cells from the 5.0 wg 2,3,7,8-TCDD/kg, 1.0
yg 2,3,7,8-TCDD/kg and control groups, respectively. While thymlc atrophy
may be one of the most sensitive Indicators of experimental exposure to
2,3,7,8-TCDD, animals given a lethal dose of 2,3,7,8-TCDD do not appear to
die from Infections, nor does a germ-free environment protect them from
death (Grelg et al., 1973).
In the Gupta et al. (1973) study, rats also showed degenerative changes
1n the renal collecting tubules, degenerative changes of the thyroid folli-
cles, necrosis and ulceratlon of the glandular stomach and hemorrhage Into
the adrenals. This latter set of findings generally occurred at higher dose
levels than the minimum dose needed to provide thymlc atrophy or liver
enlargement. More recent studies on the effects of 2,3,7,8-TCDD on renal
functions have been carried out. Analzl and Cohen (1978) reported an In-
crease In the renal secretion of phenolsulfonphthaleln (PCP) and a signifi-
cant decrease 1n glomerular filtration rate (GFR) compared with controls 1n
rats treated with 10 vg/kg (1.p.) of 2,3,7,8-TCDD. These authors attrib-
uted these effects to the toxlcity of 2,3,7,8-TCDD on glomerular structures.
However, other reports concluded that 2,3,7,8-TCDD causes no specific func-
tional lesions in the kidney, rather that the effects on renal functions
reflect a general toxicosis (Pegg et al., 1976).
C-48
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Pronounced dermal effects with 2,3,7,8-TCDD treatment have been reported
for rabbits by Schwetz et al. (1973). Milnes (1971) observed chloracne in
rabbits after a single oral dose of 1 yg/kg. Dogs, though apparently less
sensitive to 2,3,7.8-TCDD than rabbits (lethal effects) following oral
administration, have exhibited hair loss (Schwetz et al., 1973). McConnell
et al. (1978b) found facial alopecia with acne-like eruptions, blepharitis,
weight loss and anemia in rhesus monkeys after single oral doses of 70 or
350 vg/kg 2,3,7,8-TCDD. Allen et al. (1977) observed loss of facial hair
and eyelashes, accentuated hair follicles, dry scaly skin and gastric
mucosal dysplasla in eight female rhesus monkeys fed a diet containing 500
ppt 2,3,7,8-TCDD for up to 9 months. Eventually 5 of the 8 monkeys died
from severe pancytopenia. In humans, the most characteristic and frequently
observed lesion produced by 2,3,7,8-TCDD and other chlorinated aromatic
hydrocarbons is chloracne (Crow, 1981; Taylor, 1979). This lesion consists
of hyperplasia and hyperkeratosis of the interfolllcular epidermis, hyper-
keratosis of the hair follicle, especially at the infundlbulum, and squamous
metaplasia of the sebaceous glands which form keratlnaceous comedones and
cysts (Kimbrough, 1974).
A number of studies have been directed toward evaluating the mecha-
nism(s) for the toxlcity of 2,3,7,8-TCDD. Such studies will ultimately
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 marked Interspecies differences in
relative sensitivity to 2,3,7,8-TCDD, and thus help establish man's relative
sensitivity. These studies may also some day provide for the better treat-
ment of human exposure to these toxins.
C-49
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Pharmacogenetlc studies have played an Important role In understanding
the biologic and toxic effects of drugs and xenoblotlcs. Nebert and co-
workers have shown that carcinogenic polycycllc aromatic hydrocarbons (PAHs)
Induce the cytochrome P-450-dependent monooxygenase, aryl hydrocarbon
hydroxylase (AHH), 1n certain responsive strains of mice (e.g., C57BL/6J,
BALBc, C3HF/He), whereas this PAH Induction activity Is minimal or non-
existent In non-responsive strains (DBA/2J) (Nebert, 1979, 1982; Nebert and
Jensen, 1979; Nebert et al., 1981, 1983). The gene complex responsible for
the Induction of AHH and several other enzymes has been designated the Ah
locus, which comprises regulatory, structural and possible temporal genes.
Extensive studies on genetically Inbred responsive and non-responsive mice
(and their backcrosses) Indicate that these differences a-re related to the
Ah regulatory gene and Us gene product, the Ah cytosollc receptor protein.
This receptor protein Interacts with PAH llgands and the resultant PAH:Ah
receptor complex translocates Into the nucleus and presumably Initiates the
Induction of AHH via a process comparable to that proposed for the steroid
hormones.
Since the carcinogenic and toxic effects of PAHs are dependent on their
oxldatlve metabolism to reactive electrophlUc forms, 1t Is not surprising
that the Ah receptor plays an Important role 1n mediating their tox1c1ty and
carc1nogen1dty (Kourl, 1976; Kourl et al., 1974; Benedict et al., 1973;
Shum et al., 1979; Thomas et al., 1973; Legraverend et al., 1980; Robinson
et al., 1975; Mattlson and Thorgelrsson, 1979). Responsive mice are more
susceptible to the toxic (Inflammation, fetotoxldty, primordial oocyte
depletion) and carcinogenic effects of PAH at organs/tissues 1n direct
contact with the applied chemical; In contrast, non-responsive mice are more
susceptible to the tumorlgenlc effects of PAHs at tissue/organ sites remote
C-50
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from the Initial site of exposure to the PAHs. These differences In suscep-
tibility are due to several factors Including AHH-med1ated toxlcatlon and
detoxlcatlon.
Genetic studies also support the role of the Ah_ receptor In 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; Poland and Knutson, 1982) demonstrated that the mlcrosomal AHH-
Induclng activity of 2,3,7,8-TCDD and 3-methylcholanthrene (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 (e.g.,
C57BL/6J); In contrast to MC, 2,3,7,8-TCDD Induced mlcrosomal AHH 1n the
DBA/2J non-responsive mice; however, the ED5 for this biologic response
was significantly higher than values reported for the responsive mice. In
genetic crosses between responsive C57BL/6 and non-responsive DBA/2 mice 1t
was also shown for both 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 MC
and 2,3,7,8-TCDD are related to their relative Ah receptor affinities
(Poland and Knutson, 1982) and pharamcoklnetlc and metabolic factors, which
would more rapidly diminish the "available" concentrations of MC due to
metabolism and excretion.
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, a-am1nolevu!1n1c add, glutath1one-S-transferase B,
T-aldehyde dehydrogenase and chollne klnase by 2,3,7,8-TCDD or MC 1n
genetically Inbred mice has also been shown to segregate with the Ah locus
(Beatty and Neal, 1976a; Owens, 1977; Klrsch et al., 1975; Dietrich et al.,
C-51
-------
1978; Ishldate et al., 1980; Poland and Glover. 1973). Toxicology studies
with genetically Inbred mice confirm the role of the Ah locus In mediating
several toxic effects Including porphyrla, 1mmunotox1c1ty, a wasting syn-
drome, thymlc atrophy and cleft palate formation (Jones and Sweeney, 1980;
Poland and Glover, 1980; Courtney and Moore, 1971; Vecchl et al., 1983).
Poland et al. (1982) have also linked the tumor-promoting activity of
2,3,7,8-TCOO In hairless mice to the cytosollc receptor, J^n 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 which resembles some of the
characteristics of chloracne (Knutson and Poland, 1980). This cell line 1s
also responsive to AHH Induction and contains a cytosollc receptor binding
protein.
Although the murlne Ah receptor has not been characterized, several
studies confirm that a protein with high affinity for MC and 2,3,7,8-TCDD 1s
present 1n low concentrations 1n the hepatic (-30-50 fmolar) and extra-
hepatic tissues of responsive C57BL/6J mice (Greenlee and Poland, 1979; Okey
et al., 1979, 1980; Poland et al., 1976b; Mason and Okey, 1982; Gas1ew1cz
and Neal, 1982; Okey and Vella, 1982; Okey, 1983; Nebert et al., 1983).
Although the Ah receptor has not been detected In the cytosol of DBA/2J
mice, after the administration of radlolabeled 2,3,7,8-TCDD to these mice,
some of the radiolabel is detected in the nuclei of the non-responsive mice.
Moreover, the sedimentation characteristics of the [3H]-2,3,7,8-TCDD:
nuclear protein complex in DBA/20 mice are similar to those observed with
the bound Aji cytosollc receptor protein in C57BL/6J mice using a sucrose
density gradient centrlfugation separation technique (Okey, 1983). Several
reports have also demonstrated that the cytosollc Ah_ receptor protein
migrates into the nucleus of the cell only after binding with 2,3,7,8-TCDD
C-52
-------
(Greenlee and Poland, 1979; Okey et al., 1979, 1980) and this parallels the
observations noted for the Interactions between steroids and their receptor
proteins.
It should be noted, however, that the binding affinity and concentration
of the cytosol receptor for 2,3,7,8-TCOD In liver from guinea pig, rat,
C57BL/6 mouse, rabbit and hamster are very similar despite a 5000-fold
difference In LD for 2,3,7,8-TCOD between the guinea pig and hamster
(Poland and Knutson, 1982; Gaslewlcz et al., 1983a). Thus the affinity and
concentration of hepatic cytosol receptors does not alone explain the
profound Interspedes variability 1n sensitivity to TCDD.
In a subchronlc study, Koclba et al. (1976) fed rats 0, 0.001, 0.01, 0.1
or 1.0 yg 2,3,7,8-TCDD/kg of body weight by gavage for 5 days/week for 13
weeks. The dosing at 1.0 vg/kg/day caused some mortality, lethargy,
decreased body weights, liver pathology, biochemical evidence of liver
damage, thymic atrophy, decreased lymphatic tissues, disturbances of
porphyrin metabolism and slight alterations 1n the hematopoietic system.
There was also evidence of mild adverse effects on the male and female
reproductive systems. The effects on the reproductive system Included
decreased size of the testis and secondary sex organs In 2 of 5 males and
uteri in 4 of 5 females. The 0.01 pg/kg/day level was considered by the
authors to be the no-observed-adverse-effect level (NOAEL) and the 0.001
vg/kg/day level was the no-observed-effect level (NOEL) for this treatment
regimen.
The dietary administration of 2,3,7,8-TCDO to rats at dose levels equiv-
alent to 0, 0.001, 0.01 or 0.1 wg/kg/day for three generations (Murray et
al., 1979) produced effects on liver, thymus and reproduction (discussed In
C-53
-------
the TeratogenlcHy section) at 0.01 and 0.1 yg/kg/day. According to the
authors, the 0.001 wg/kg/day exposure was a NOAEL (however, equivocal
effects were noted In some generations at this dose).
Liver toxldty was the only effect of treatment observed by hlstologlc
examination of Osborne-Mendel rats and B6C3F mice administered 2,3,7,8-
TCDO for 13 weeks 1n a preliminary subchronlc toxldty study designed to
define an acceptable dose for a chronic toxldty study (U.S. DHHS, 1980b).
The anliffals In groups of 10 males and 10 females were administered the com-
pound 1n corn o1l:acetone (9:1) twice a week at doses for rats of 0.0, 0.5,
1, 2, 4 and 8 vg/kg/week, and for mice at doses of 0.0, 1, 2, 5, 10 and 20
vg/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 vg/kg/week group died. Deaths were accompanied
by severe toxic hepatitis. Hepatic lesions were observed 1n all other rats
examined In groups administered 1-8 yg/kg/week; however, not all animals
1n each group were submitted to necropsy. Normal liver histology was
observed 1n the two male rats examined from the low dose groups and only
threshold toxic effects occurred 1n the low dose female rats.
Similar effects of treatment were observed In mice, with a single death
occurring 1n each sex at the high exposure level along with reports of
hepatic lesions on hlstologlc examination. In contrast to rats, female mice
were less sensitive to the hepatotoxlc effect of 2,3,7,8-TCDD than were the
male mice. Hepatic lesions were observed 1n all dose groups of male mice,
while the 1 and 2 yg/kg/week dose groups of female mice had normal livers.
Although the group sizes were small, making conclusions tenuous, 1t appeared
that sex differences In the sensitivity to the toxic effects of 2,3,7,8-TCDD
occurred, and that the more sensitive sex may vary with species tested.
C-54
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In a more extensive subchronlc study 1n rats, King and Roesler (1974)
followed the development of toxldty by a series of Interim sacrifices
during 28 weeks of exposure to 2,3,7,8-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 1n corn o1l:acetone (9:1) at doses
of 0.0, 0.1 and 1.0 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 treat-
ment. In addition, 3 rats of each sex were killed 4 and 12 weeks after
termination of exposure. Animals were monitored for gross changes during
the study and were examined for gross and hlstologlc changes at necropsy.
Besides a dose-related decrease In body weight gain 1n male rats and a
decrease In body weight gain 1n the high dose female rats, the only effect
of exposure to 2,3,7,8-TCDO was hlstologlc changes 1n the liver. Liver
pathology was normal 1n all treated groups up through the Interim kill at 10
weeks. Fatty changes 1n the liver were considered the most Important obser-
vation and the data Is summarized 1n Table 7. The fatty changes ranged from
single large I1p1d droplets In a few centrHobular heptocytes to I1p1d drop-
lets 1n all centrllobular hepatocytes with extension Into the mldzonal
hepatocytes. No clear dose-response pattern was observed 1n this study;
however, 1t did appear that the severity of fatty changes was greater 1n
female rats. During the recovery period fatty changes progressively de-
creased In severity, but were still present 1n some treated animals 12 weeks
after cessation of exposure. Other hlstologlc changes observed 1n the liver
of a small number of animals, predominantly In the animals killed at 28
weeks, Included single cell or very small areas of necrosis, Increased
nuclear size, subtle distortion of liver architecture, and hyperchromatlc
C-55
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TABLE 7
Hepatocellular Fatty Change Observed 1n Rats Following Subchronlc
Exposure to 2,3,7,8-TCDDa
Treatment Groupb
28 Weeks
N
S
M1
Mo
N
4-Weeks Recovery
S M1 Mo Ma
12-Weeks Recovery
N
M1 Mo Ma
o
I
en
Male Control
Males at 0.1 vg/kg
Males at 1.0 yg/kg
Female Control
Females at 0.1 vg/kg
Females at 1.0 yg/kg
7/10 3/10 NS MS NS
2/10 1/10 1/10 5/10 1/10
0/10 1/10 2/10 5/10 2/10
10/10 NS NS NS NS
4/9 4/9 1/9 NS NS
1/10 4/10 4/10 1/10 NS
2/3 1/3 NS NS NS
2/3 1/3 NS NS NS
NS 1/3 1/3 1/3 NS
3/3 NS NS NS NS
3/3 NS NS NS NS
1/3 1/3 NS 1/3 NS
3/3 NS NS NS NS
2/3 NS NS 1/3 NS
2/3 NS NS 1/3 NS
3/3 NS NS NS NS
3/3 NS NS NS NS
1/4 3/4 NS NS NS
aSource: King, and Roesler, 1974
bAn1mals were treated twice weekly by gavage with 2,3,7,8-TCOD dissolved 1n corn ollracetone (9:1)
N = None
S = Slight: random hepatocyte containing a solitary, large I1p1d droplet-equivocal
M1 = M1ld: several centrllobular hepatocytes contain I1p1d
Mo = Moderate: most centrllobular hepatocytes contain I1p1d
Ma = Marked: all centrllobular and some mldzonal hepatocytes contain I1p1d
NS = Not specified
-------
nuclei. All of these lesions were considered to be slight or mild, and less
toxicologlcally relevant than the fatty changes. The data on hepatic
steatosls Indicated that the liver was a sensitive organ to the toxic effect
of 2,3,7,8-TCDD, and although some recovery occurred after termination of
treatment, the recovery process was slow and not complete by the time the
study was terminated.
The recovery time was also demonstrated to be long In a subchronlc study
by Goldstein et al. (1982) of 2,3,7,8-TCOD-1nduced porphyMa. Groups of
eight female Sprague-Dawley rats were given 2,3,7,8-TCDD In corn o1l:acetone
(7:1) weekly by gavage for 16 weeks at doses of 0.0, 0.01, 0.1 or 10.0
vg/kg/week and killed 1 week after the last treatment. Additional groups
of rats received doses of 0.0 or 1.0 vg/kg/week for 16 weeks and were
allowed to recover for 6 months. The high dose level was lethal to all
animals within 12 weeks, while the only other gross sign of toxldty was a
decrease in body weight gain In the group receiving 1.0 yg/kg/week. After
16 weeks of exposure to 2,3,7,8-TCDD, liver porphyMns were elevated
-1000-fold In 7 of 8 animals receiving 1.0 yg/kg/week, but only 1 of 8
animals In the 0.1 vg/kg/week group had elevatod porphyrln levels. No
effect was observed 1n the low dose animals. After a 6-month recovery
period the porphyrln level 1n animals exposed to 1 vg/kg/week was still
100-fold higher than values 1n the control group. A similar pattern was
observed for urinary excretion of uroporphyrln. The rate limiting enzyme 1n
heme synthesis, 5-amlnolevullnlc 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 treat-
ment, cytochrome P-450, aryl hydrocarbon hydroxylase, and glucuronyl trans-
ferase, returned to near normal levels by 6 months. It was clear that a
C-57
-------
6-month recovery period from subchronlc exposure to 2,3,7,8-TCDO at a dose
of 1.0 yg/kg/week was not sufficient for complete reversal of 2,3,7,8-
TCDD-1nduced porphyMa.
In rats, Increased urinary porphyMn was also observed after subchronlc
exposure to 2,3,7,8-TCDO (Cantonl et al., 1981). Female CD rats were orally
administered weekly doses of 2,3,7,8-TCDD at levels of 0.01, 0.1 and 1.0
pg/kg/for 45 weeks. The Initial Increase was observed In the high dose
group at 3 months, and in the other two groups at 4 months, after the start
of exposure. Not only did the absolute amount of porphyrln Increase, but
the relative distribution also changed to compounds containing more carboxyl
groups. Only 1n the high dose group did the livers, at the terminal
necropsy, show signs of excess prophyrln under examination by ultraviolet
light.
The toxic effects, other than neoplasla, of long-term exposure to
2,3,7,8-TCDD have been studied 1n rats, mice and monkeys. The primary
purpose of many of the studies 1n rodents was to assess the cardnogenlclty
of 2,3,7,8-TCDD. (These effects are discussed 1n detail 1n the Carclnogene-
sls section.) The observation of non-neoplast1c systemic toxic effects 1n
these studies was often limited, and observations were made near the end of
the natural Hfespan when conditions associated with aging may have obscured
some effects produced by 2,3,7,8-TCDD. Table 8 summarizes the toxic effects
of chronic exposure to 2,3,7,8-TCDD and provides Information on the exposure
levels which result 1n the observed effects.
Human health effects related to excessive exposures to 2,3,7,8-TCDD have
been noted 1n several Instances. However, 1n many of these cases H 1s
difficult to quantify the exposure to 2,3,7,8-TCDD leading to the observed
symptoms. Most of the exposures occurred In relation to the manufacture of
C-58
-------
TABLE 8
Effects of Chronic Exposure to 2.3.7.8-TCOD In Laboratory Rodents
I
en
Species/ Sex/No. Dose
Strain '
Rat/Sprague- H/10 0.0 ppt
Oawley
H/10 1 ppt
H/10 5 ppt
H/10 50 ppt
H/10 500 ppt
H/10 1000 and 5000 ppt
H/10 50,000, 500,000 and
Treatment Schedule
NA
continuous 1n diet
for 78 weeks
continuous In diet
for 78 weeks
continuous 1n diet
for 78 weeks
continuous 1n diet
for 78 weeks
continuous 1n diet
for 78 weeks
continuous 1n diet
Duration of
Study
95 weeks
95 weeks
95 weeks
95 weeks
95 weeks
95 weeks
95 weeks
Parameters
Honltored
survival
survival
survival
survival
survival
survival
survival
Effects of Treatment
40X survived until 95 weeks, the
first death occurred at week 68
BOX survived until 95 weeks, the
first death occurred at week 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
Reference
Van Hlller
et al.. 1977a
Same
Same
Same
Same
Same
Same
as above
as above
as above
as above
as above
as above
1.000.000 ppt for 78 weeks
Rat/Sprague- H&F/508.50 -2193 ppt
Oawley (0-1 wg/kg/day)
continuous 1n diet 2 years
for 2 years
extensive hlsto-
pathology, hema-
tology, urine
analyses, and
clinical chemistry
Cumulative mortality Increased (F);
Body weight gain decreased (H,F);
Red blood cell count decreased
(H.F); Packed cell volume decreased
(M,F); Hemoglobin decreased (H,F);
RetUulocytes Increased (H,F);
White blood cell count decreased (F);
SGPT Increased (F); G-Glutamyl trans-
ferase Increased (F); Alkaline phos-
phatase Increased (F); Urinary copro-
porphyrln Increased (F); Urinary uro-
porphyrln Increased (F); Urinary *-
am1nolevul1n1c acid Increased hepatic
degeneration Increased (H.F)
Kodba et al..
1978, 1979
-------
TABLE 8 (cont.)
Species/ Sex/No.
Strain
Dose Treatment Schedule Duration of
Study
Parameters
Monitored
Effects of Treatment Reference
o
I
o>
o
Rat/Sprague- M&F/50&50 -208 ppt
Dawley (0.01 pg/kg/day)
Rat/Sprague- MiF/508,50 -22 ppt
(0.001 pg/kg/day)
Rat/Osborne- M&F/75&75 0.0 ng/kg/week
Mendel
Rat/Osborne- M&F/50&50 0.5 pg/kg/week
Mendel
Rat/Osborne- M&F/50&50 0.05 pg/kg/week
Mendel
Rat/Osborne- M&F/50&50 0.01 pg/kg/week
Mendel
M1ce/B6C3Fl M&F/75J.75 0.0 pg/kg/week
M1ce/B6C3Fl M&F/50&50 0.5 pg/kg/week (M)
2.0 pg/kg/week (F)
M1ce/B6C3Fl M&F/50&50 0.05 pg/kg/week (M)
0.2 pg/kg/week (F)
M1ce/B6C3Fl M&F/50&50 0.01 pg/kg/week (M)
0.04 pg/kg/week (F)
continuous 1n diet
for 2 years
continuous 1n 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
administered by
gavage biweekly
for 104 weeks
administered by
gavage biweekly
for 104 weeks
2 years
2 years
106 weeks
107 weeks
107 weeks
extensive hlsto-
pathology. hema-
tology, urine
analyses and
clinical chemistry
extensive hlsto-
pathology, urine
analyses and
clinical chemistry
extensive hlsto-
pathology
extensive hlsto-
pathology
extensive hlsto-
pathology
107 weeks extensive hlsto-
pathology
105-106 weeks extensive hlsto-
pathology
107 weeks extensive hlsto-
pathology
107 weeks extensive hlsto-
pathology
107 weeks
extensive hlsto-
pathology
Urinary coproporphyMn Increased (F); Kodba et al.
Urinary uroprophyMn Increased (F); 1978, 1979
Hepatic degeneration Increased (H.F)
No differences from values obtained Same as above
from control animals
Toxic hepatitis; 0/74 (M), 0/75 (F) U.S. OHHS.
1980b
Toxic hepatitis; 14/50 (M), 32/50 Same as above
The Incidence of toxic hepatitis Same as above
was not elevated 0/50 (M), 1/50 (F)
The Incidence of toxic hepatitis Same as above
was not elevated 1/50 (M), 0/50 (F)
Toxic hepatitis; 1/73 (M), 0/73 (F) Same as above
Toxic hepatitis; 44/50 (M), 34/47 Same as above
The Incidence of toxic hepatitis Same as above
was not elevated 3/49 (M), 2/48 (F)
The Incidence of toxic hepatitis Same as above
was not elevated 5/44 (M), 1/50 (F)
-------
TABLE 8 (cont.)
Species/ Sex/No.
Strain
Mice/Swiss H/38
Nice/Swiss M/44
Mice/Swiss M/44
Mice/Swiss M/43
Dose Treatment Schedule Duration of Parameters
Study Monitored
0.0 pg/kg/week NA 588 days
0.007 pg/kg/week administered by 649 days
gavage weekly for
1 year
0.7 pg/kg/week administered by 633 days
gavage weekly for
1 year
7.0 pg/kg/week administered by 424 days
gavage weekly for
1 year
histology on all
organs
histology on all
organs
histology on all
organs
histology on all
organs
Effects of Treatment
Dermatitis and amyloldosls; 0/38
Dermatitis and amyloldosls; 5/44
Dermat1t1s-and amyloldosls; 10/44
Early mortality, dermatitis and
amyloldosls; 17/43
Reference
Toth et al.,
1978. 1979
Same as above
Same as above
Same as above
NA = Not applicable
o
I
-------
2,4,5-tMchlorophenol or 2,4,5-tr1chlorophenoxyacet1c add. The best known
among these may be the release of 2,3,7,8-TCDD due to the 1976 explosion of
a malfunctioning 2,4,5-tMchlorophenol reactor 1n Seveso, Italy (Carrerl,
1978). The area closest to the plant received exposures of up to 5000
vg/m2 of soil; more remote areas received 1-75 vg/m2, and within
these areas 12 cases of chloracne developed (Regglanl, 1980). Other symp-
toms Included acute dermatitis. All but the most severe cases of chloracne
recovered within 26 months. No neurological, visceral or Immune effects
were noted 1n these reports.
Pocch1ar1 et al. (1979) reported a more extensive study of the Seveso
Incident. These authors showed 75 cases of chloracne due to 2,3,7,8-TCDD
exposure; 15 of those cases were severe or very severe. After 18-24 months,
19 cases had fully recovered while 1 case was still listed as severe. A
subsequent survey 1n children by these authors uncovered an additional 137
cases of mild to serious chloracne. As Indicated 1n the Pocch1ar1 et al.
(1979) report, signs of liver damage were also found after the Seveso Inci-
dent 1n Italy. Raised serum transamlnase and y-glutamyl transferase
levels were found in -20% of the people living in or near the area of great-
est deposition of 2,3,7,8-TCDD (Bert et al., 1976; Hay, 1976). In addition,
some neurological effects were also noted among the exposed people. Immuno-
logical and cytogenetlc Investigations yielded normal results.
The most commonly reported symptom related to 2,3,7,8-TCDD exposure 1n
man has been chloracne (Bauer et al., 1961; K1mm1g and Schulz, 1957; Schulz,
1957; Firestone, 1977; Dugols and Colomb, 1956, 1957; Dugois et al., 1958;
Blelberg et al., 1964; Oliver, 1975; Poland et al., 1971; Klmbrough, 1980).
The acneform lesions of the skin may develop a few weeks after the exposure
and may persist for over 1 year following the cessation of exposure. Other
C-62
-------
skin problems which have been reported Include hyperp1gmentat1on, hlrsutlsm,
Increased skin fragility and veslcobullar eruptions on exposed areas of the
skin.
Most cases of chloracne have been reported for Industrial workers.
Bauer et al. (1961) reported that 31 cases of chloracne occurred within a
few months after a factory manufacturing 2,4,5-T near Hamburg, Germany,
Instituted a change 1n Us Industrial process. On this occasion, the Inves-
tigators were able to show that the chloracne was not caused by purified
2,4,5-T, but that H was attributable to 2,3,7,8-TCOO which was a contami-
nant In the technical grade 2,4,5-T. A similar outbreak of chloracne
affected 60 workers at a 2,4,5-T plant In Michigan In 1964 (Firestone, 1977).
In addition to chloracne, some- occupational exposures to 2,3,7,8-TCOD
have provided evidence for liver damage and for neurological effects.
Hyperl1pem1a and hypercholesterolemla were reported (1n addition to chlor-
acne) for 17 workers at a plant producing trlchlorophenol In France (Dugols
and Colomb, 1956, 1957; Dugols et al., 1958). Among 29 subjects who exhib-
ited chloracne after working 1n a plant which produced 2,4-D and 2,4,5-T 1n
Newark, New Jersey, 11 had Increased excretion of uroporphyrlns. Three of
these were diagnosed as porphyrla cutanea tarda, and two of these also
exhibited elevated serum glutamlc-oxalacetlc transamlnase levels (Blelberg
et al., 1964; Firestone, 1977).
Pazderova-Vejlupkova et al. (1981) reported that 80 workers developed
chloracne, nausea, fatigue and weakness In the lower extremities while
engaged 1n the production of 2,4,5-sodlum trlchlorophenoxyacetate and
trlchlorophenoxyacetate butylester 1n a plant located In Prague, Czechoslo-
vakia. Prominent clinical symptoms among 55 of the 80 workers Included
hypercholesterolemla, hyperllpemla and hyperphosphol1pem1a, Increased plasma
C-63
-------
alpha and gamma globulins, and decreased plasma albumin. PorphyMa cutanea
tarda was observed 1n 11 of the 55 workers tested. In some cases, Illness
subsided while other cases became more severe during a 3 to 4-year follow-up
period. Long-term pathological symptoms (remaining evident 5 years after
exposure) Include deviations 1n I1p1d metabolism, abnormal glucose toler-
ance, and high urinary excretion of uroporphyrlns. Polyneuropathy, usually
of the lower extremities, occurred during the period of Illness and the
symptoms remained after 4 years. Singer et al. (1982) also Indicated a
decrease In nerve conduction velocities of sural nerves 1n workers exposed
to phenoxy add herbicides (average exposure, 7 years) when compared to a
similar group of non-exposed workers (40.3 m/sec 1n exposed vs. 42.8 m/sec
1n non-exposed, p=0.02). Although the causative agent was not known, dloxln
contaminants are suggested.
Poland et al. (1971) re-examined all of the employees of the Newark
factory In 1969, after the level of 2,3,7,8-TCDD 1n the tMchlorophenol had
been reduced from 10-25 mg/kg to <1 mg/kg. Chloracne was still found 1n 13
of 73 workers. A number of employees exhibited hyperplgmentatlon or facial
hypertrlchosls. No definite diagnoses of porphyMa were made, and only one
worker had a mild case of uroporphyMnurla. The authors suggested that
chloracne and porphyMa cutanea tarda are essentially Independent syndromes.
In the Newark workers, Poland et al. (1971) noted that serum cholesterol
was elevated 1n 10% of the cases and serum lactic dehydrogenase was elevated
In 29% of the cases. Seven persons (10% of the workers) had a white blood
cell count of <5000/mma. In addition, -30% of the workers reported suf-
fering from gastrointestinal symptoms (nausea, vomiting, diarrhea, abdominal
pains, blood 1n the stools); -10% of the workers had other symptoms, such as
weakness of the lower extremities, headaches and decreased auditory acuity.
C-64
-------
Some hypomanla was observed, with the degree of hypomanla (as measured on
the Minnesota Multlphaslc Personality Inventory Hypomanlc Scale) showing an
association with the severity of chloracne.
Among the workers exposed In Hamburg, many also showed clinical signs of
systemic toxldty, mainly muscular weakness, loss of appetite and weight,
sleep disturbances, orthostatlc hypotension, abdominal pain and liver
Impairment. Most of the workers presented psychopathologlcal changes that
were Interpreted to be a specific syndrome (Bauer et a!., 1961; Klmmlg and
Schulz, 1957; Schulz, 1957).
Teleglna and Blkbulatova (1970) reported that the production of 2,4,5-T
In the USSR began In 1964. At a specific site, 128 workers showed skin
lesions, and among 83 examined, 69 had chloracne. Many, especially those
with severe skin lesions, also presented evidence of liver Impairment. In
addition, 18 workers had a neurasthenic syndrome.
Similar findings were described by Jirasek et al. (1973, 1974), who
reported 76 cases of chloracne following exposure to 2,3,7,8-TCOD between
1965 and 1968 In a plant 1n Czechoslovakia which produced 2,4,5-T and penta-
chlorophenol. Fifty-five of these cases were followed medically for over 5
years; some had symptoms of porphyrla cutanea tarda, uroporphyrlnurla,
abnormal liver tests (elevated blllrubln levels, Increased SGOT or SGPT
activities, and elevated bromsulphthaleln clearance times) and liver
enlargement. The majority of the patients also suffered from neurasthenia
and a depressive syndrome. In 17 persons, signs of a peripheral neuropathy,
especially 1n the lower extremities, were confirmed by electromyographlc
examinations. More than half of the patients showed raised blood levels of
cholesterol and total llplds.
C-65
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Three scientists were poisoned In the course of an experimental prepara-
tion of 2,3,7,8-TCDD made by heating potassium trlchlorophenate (Oliver,
1975). Two scientists developed typical chloracne 6 and 8 weeks after the
exposure. Delayed symptoms, possibly due to 2,3,7,8-TCDD, developed ~2
years after the Initial exposure 1n two of the scientists. These symptoms
were personality changes, Including loss of energy and drive, Impairment of
vision, taste and muscular coordination, disturbance of sleep, gastrointes-
tinal symptoms and hlrsutlsm. Hypercholesterolemla (1n excess of 300 mg/100
ml) occurred 1n all three patients.
In November 1953, an accident occurred at a factory 1n Ludwlgshafen,
Federal Republic of Germany, during the manufacture of trlchlorophenol
(Goldman, 1972; Hofmann, 1957). As a consequence, 53 workers were affected
by chloracne, 42 In a severe form. The son of one of the workers developed
chloracne following contact with his father's clothes. Twenty-one of the 42
workers with severe chloracne showed signs of damage to Internal organs or
disturbances of the nervous system. The most relevant features were poly-
neuritis, sensory Impairment and liver damage.
In a follow-up study of these workers, Thelss and Goldmann (1977) re-
ported that of the 53 workers exposed to 2,3,7,8-TCDD In the 1953 accident,
22 were still working at the factory, 16 had retired and 15 had died (6
while still employed at the factory and 9 while 1n retirement). Of the 22
workers still employed, 2 still had acne of the face and scrotum, 1 had
paralysis of the left leg and 1 had permanent loss of hearing. The remain-
Ing workers were well, except for scars left by the chloracne. Of the 15
deaths, 7 were from cardiovascular disease, 2 of which were myocardlal
Infarcts, 1 due to mitral stenosis, 4 from cancer, 2 from suicide, 1 from
necrotlc pancreatitis and 1 from esophageal hemorrhage. The 16 men who had
C-66
-------
retired and were still alive were well. No abortions or miscarriages were
reported 1n the wives of exposed workers still employed at the factory.
2,3,7,8-TCDD has also been Identified as the cause of an outbreak of
poisoning In humans, horses and other animals In 1971 (Carter et al., 1975;
Klmbrough et al., 1977). Exposure was related to the spraying of waste oil
contaminated with 2,3,7,8-TCDD on riding arenas for dust control. The most
severe effects occurred 1n a 6-year-old girl who played 1n the arena soil.
Her symptoms Included headache, nosebleedlng, diarrhea, lethargy, hemorrhag-
1c cystitis and focal pyelonephritis. Three other children and one adult
who were frequently In the arena complained of skin lesions. In two of the
children, the described lesions were similar to chloracne.
Exposure to 2,3,7,8-TCDD and other dloxlns occurring as contaminants In
Agent Orange have been associated with many health effects reported 1n
veterans and residents of Vietnam. Symptoms Include numbness of extremi-
ties, skin rashes and Irritation, liver dysfunction, weakness, loss of sex
drive, and psychological changes (Holden, 1979).
The toxic effects attributed to 2,3,7,8-TCDD exposure were studied over
a 10-month period In a group of 78 Vietnam veterans who claimed to have been
exposed to Agent Orange (Bogen, 1979). Symptoms reported by the veterans
Included gastrointestinal complaints (anorexia, nausea, diarrhea, constipa-
tion, abdominal pain), joint pain and stiffness, and neurological complaints
(numbness, dizziness, headaches, depression and bouts of violent rage). It
Is mentioned that these patients had previously been chronically 111 and had
frequent Infections and allergies. This study was apparently based on
personal 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. Most of these complaints were non-specific, judgmental and occur
commonly 1n the general public.
C-67
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In an effort to evaluate the toxic effects attributed to 2,3,7,8-TCDD as
a contaminant of Agent Orange, Stevens (1981) established 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 stud-
ies 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-
chlorodibenzo-p_-furan (TCDF), the cumulative minimum toxic dose of 2,3,7,8-
TCDD 1n man was calculated to be 0.1 vg/kg. 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 TCDD on sprayed surfaces of Vietnam
was ~8 yg/m2. Based on a comparison of the development of toxic systems
1n humans exposed to 2,3,7,8-TCDD 1n the Seveso accident and a child exposed
In an Eastern Missouri horse arena, the measured environmental levels of
2,3,7,8-TCDD, and estimates of the absorbed dose necessary to produce these
symptoms, the author calculated an average intake transfer factor (ratio of
absorbed compound to environmentally available compound) of 1:2050 for
2,3,7,8-TCDD. Assuming this absorption-to-exposure ratio and even assuming
that a soldier was directly sprayed (exposed to 8 vg/m2) for each day of
his 1 year service 1n Vietnam, his cumulative Intake would be only 1.4 ^g
or 0.02 yg/kg of 2,3,7,8-TCDD. Based on these calculations, Stevens
(1981) reports 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 feels that claims of
Illness caused by 2,3,7,8-TCDD 1n Agent Orange are without merit. Exception
Is made, however, for certain workers (forest Industries) who could be
exposed to 2,4,5-T and 2,3,7,8-TCDD for many years.
C-68
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Synerglsm and/or Antagonism
2,3,7,8-TCDD elicits diverse toxic and biologic effects and therefore
can be expected to alter the activities of other chemicals. For example,
many compounds, Including 2,3,7,8-TCDD, which Induce drug-metabolizing
enzymes or act as cancer promoters, can greatly Influence the activity of
other carcinogens and toxins. This type of Interaction can result In non-
additive effects which could be called synerglsm or antagonism. However,
when the mechanism of action of the Interacting chemical 1s different (such
as Initiators and promoters of carclnogenesls), the Interaction effects
should be called modulation. Thus a cancer promoter modulates the effects
of a carcinogen. An example of a synerglstic or antagonistic effect would
occur when two chemicals that elicit the same toxic effect are coadmlnls-
tered and the resultant magnitude of the toxic response Is non-additive. It
1s clear that 2,3,7,8-TCDD Interacts with chemicals via modulation and
synerglsm/ antagonism as Indicated below.
Results from several cocardnogenldty studies appear to give only
limited support to the modulating effect of 2,3,7,8-TCDD. The DBA/2N mouse
strain, which responds only weakly to the sarcomatogenlc action of MC,
becomes susceptible after treatment with 2,3,7,8-TCDD (Kourl, 1976; Kourl
and Nebert, 1977). As an extension of this%study, Kourl et al. (1978)
demonstrated In two Inbred strains of mice, C57BL/6Cum and DBA/2Cum, that
administration of 2,3,7,8-TCDD simultaneously with MC appears to enhance the
carcinogenic response. The authors concluded that their results suggest
that 2,3,7,8-TCDD acts as a cocarclnogen, possibly as an inducer of AHH at
the site of inoculation. In contrast, when mice were painted with 1 vg
2,3,7,8-TCDD prior to 7,l2-d1methylbenz(a)anthracene (DMBA) Initiation and
12-0-tetradecanoylphorbol-13-acetate (TPA) promotion, tumor formation was
C-69
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inhibited (Berry et al.. 1979). The greatest degree of Inhibition (94%) was
seen when the 2,3,7,8-TCDD was applied 5 days prior to Initiation by DMBA;
when pretreatment was at 3 days and 1 day, the Inhibition was 86 and 3%,
respectively.
2,3,7,8-TCDD did not promote the carclnogenlcHy of DMBA 1n a two-stage
skin tumor 1 genes 1s assay with CD1 female mice (Berry et al., 1979).
2,3,7,8-TCDD was applied twice weekly for 30 weeks at 0.1 vg 1n acetone to
both DMBA treated and untreated control rats. No papHlomas were observed
In either group.
The recent results from the NCI cardnogenesls testing program (U.S.
DHHS, 1980a) Indicate that 1n female Swiss-Webster mice the Incidence of
Mbrosarcoma 1n the Integumentary system Is higher when 2,3,7,8-TCDD 1s
applied alone or following DMBA application than 1n the control. The Inci-
dence between the two experimental groups Is comparable. This report has
been discussed 1n detail 1n the carclnogenlcHy section of this document.
In order to test the potential of 2,3,7,8-TCDD as a promoter of hepato-
cardnogenesls, rats which had received a single 10 mg/kg dose of dlethyl-
nHrosamine (DEN) following partial hepatectomy were given 2,3,7,8-TCDD
(0.14 and 1.4 vg/kg s.c. once every 2 weeks) for 7 months. Animals which
received (a) only a single Initiating dose of DEN after partial hepatectomy
and no further treatment, or (b) 2,3,7,8-TCDD alone with no Initiating dose
of DEN exhibited relatively few enzyme-altered fod and no hepatocellular
carcinomas. However, animals Initiated with DEN and then given 2,3,7,8-TCDD
had a marked Increase In enzyme-altered fod. At the higher dose of
2,3,7,8-TCDD, hepatocellular carcinomas were present In 5 of 7 rats. By
means of three different enzyme markers used to evaluate the phenotypes of
the enzyme-altered fod, a distinct phenotype heterogeneity of the fod was
C-70
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noted with a shift towards phenotypes exhibiting a greater deviation from
normal liver when 2,3,7,8-TCDD was given following DEN-part1al hepatectomy.
Quantltatlon of the numbers of enzyme-altered fod was performed by relating
measurements made from two-dimensional tissue sections to the number of foci
per unit volume of liver using relationships established 1n the field of
stereology. The total volume of the liver occupied by the enzyme-altered
foci, but not their number, Increased with the dose of 2,3,7,8-TCDD adminis-
tered following DEN-partlal hepatectomy. These studies demonstrate that
2,3,7,8-TCDD Is a potent promoting agent for hepatocardnogenesls (PHot et
al., 1980).
D1G1ovann1 et al. (1979) noted an Inhibitory effect of 2,3,7,8-TCDD on
mice Initiated with benzo(a)pyrene [B(a)P] and promoted with TPA. Again,
the greatest Inhibition of skin tumor formation (65%) was seen when 2,3,7,8-
TCDD was applied 5 days prior to B(a)P Initiation. Inhibition was 57 and
13% at 3 and 1 days pretreatment, respectively. D1G1ovann1 et al. (1979)
and Berry et al. (1979) suggested that this antlcardnogenlc effect was
related to the ability of 2,3,7,8-TCDD to Induce monooxygenase systems of
the skin.
2,3,7,8-TCDD pretreatment has been observed to modify the effects of
barbltuates and other xenoblotlcs (Grelg, 1972). Adult male Porten rats
were given a single oral dose of 200 jig 2,3,7,8-TCDD/kg bw 1-3 days
preceding treatment with 100 mg/kg zoxazolamlne hydrochlorlde or 150 mg/kg
hexabarbltone sodium. 2,3,7,8-TCDD pretreatment resulted In a 54% decrease
1n the duration of the paralysis Induced by zoxazolamlne and a 2-fold
Increase 1n the sleeping time produced by hexabarbltone.
C-71
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The synergUtlc or antagonistic effects of chemical combinations have
not been well documented. A recent report compares the ImmunotoxIcHy of
2,3,7,8-TCDD, 2.3,7,8-TCDF, and 2,3,7,8-TCDF plus 2,3,7,8-TCDD (coadmlnls-
tered) (Rlzzardlnl et al., 1983). Seven days after administration of 1.2
vg/kg of 2,3,7,8-TCDD to C57BL/6J mice, sheep red blood cells were Inject-
ed by l.p. administration and plaque-forming cells (RFC) 1n the spleen were
counted 5 days later. 2,3,7,8-TCDD Inhibited antibody production by 80%.
In a parallel study, a dose of 2,3,7,8-TCDF was administered (10 yg/kg)
and no significant 1mmunotox1c effects were observed. Coadmlnlstratlon of
2,3,7,8-TCDD (1.2 yg/kg) plus 2,3,7,8-TCDF (10 yg/kg) resulted In 50%
reduction 1n antibody production and demonstrates a significant antagonistic
effect by 2,3,7,8-TCDF. Coadmlnlstratlon of these two Isostereomers result-
ed 1n antagonistic effects with respect to the Induction of hepatic mlcro-
somal cytochrome P-450 and 7-ethoxycoumar1n 0-deethylase. Sweeney et al.
(1979) found that Iron deficiency protected mice against the development of
hepatocellular damage (Including porphyrla) normally caused by 2,3,7,8-TCDD
exposure. In contrast, the teratogenlc and fetoxlc data reported by Neubert
and Dlllmann (1972) and Courtney and coworkers (see Teratogen'dty section)
suggests that Coadmlnlstratlon of phenoxy herbicides and 2,3,7,8-TCDD may
also result 1n synerglstlc effects.
TeratoqenlcUy
Courtney et al. (1970a,b) were the first to report that 2,4,5-T was
capable of causing teratogenlc effects 1n rats and mice. In these studies,
rats and two strains of mice were exposed s.c. or orally to 2,4,5-T contain-
ing 30 ppm 2,3,7,8-TCDD. The mixture was teratogenlc and fetotoxlc to mice
at >46.4 mg/kg. Rats were more sensitive, exhibiting fetotoxlc responses at
10 mg/kg for this 2,4,5-T/2,3,7,8-TCDD mixture. Since this Initial report,
C-72
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research has focused on determining the role of 2,3,7,8-TCDO contamination
In eliciting the teratogenlc response. These studies are summarized 1n
Table 9.
Neubert and Dlllmann (1972) conducted a detailed study to determine the
significance of 2,3,7,8-TCDD contamination. These Investigators assayed
three 2,4,5-T samples: a highly purified sample containing <0.02 ppm
2,3,7,8-TCDD (referred to as Sample A), a purified sample Identical to that
used by Roll (1971) that contained 0.05+0.02 ppm 2,3,7,8-TCDD (Sample B),
and a commercial sample containing an undetermined quantity of 2,3,7,8-TCDD
(Sample C). All three samples Induced cleft palates In NMRI mice at suffi-
ciently high doses. 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
Dillmann (1972) also assayed a sample of 2,3,7,8-TCDD alone and 1n various
combinations with the highly purified sample of 2,4,5-T. This approach
allows at least partial quantification of the significance of 2,3,7,8-TCDD
contamination in 2,4,5-T-1nduced cleft palates. When the litter is used as
the basic experimental unit, the incidences of cleft palate (number of
litters with cleft palate/total numbers of Utters) versus the dose can be
plotted on log dose/probit response paper, correcting for central response
using Abbott's equation. According to this method, the ED5Q (by eye-fit)
for cleft palate Induction are:
2,3,7,8-TCDD: 4.6 yg/kg bw
2,4,5-T (Sample A): 115 mg/kg bw
2,4,5-T (Sample B): 46 mg/kg bw
C-73
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TABLE 9
Studies on the Potential Teratogenlc Effects of 2,3,7,8-TCDD-Contam1nated 2.4.5-T
Species/Strain Vehicle
Form of
2,4,5-T TCDO Level
Treat- Obser-
Oally Dose ment vatlon Maternal Response
Days Day
Fetal Response
Reference
MIce/NMRl Rape-seed oil
o
i
-j
HIce/NMRI
HIce/NMRI
M1ce/NNRI
HIce/NMRI
Mlce/CO-1
acid <0.02 ppm
(Sample A)
Rape-seed oil
Rape-seed oil
Rape-seed oil
add
NR
Corn o1l:acetone
(9:1)
8, 15, 30, 45. 6-15
60, 90
and 120 mg/kg
acid 0.05+0.02 ppm 30. 60 and
90 mg/kg
(Sample B)
NR
(Sample C)
butyl NR
ester
90 mg/kg
12 and 17
mg/kg
add 0.05^0.02 ppm 20, 35, 60.
90 and 130
mg/kg
add <0.05 ppm
115 mg/kg
6-15
6-15
6-15
6-15
10-15
18 No toxic effects;
decreased maternal
weight at doses of
90 mg/kg and
greater
18 No toxic effects;
decreased maternal
weight at 90 mg/kg
18 No toxic effects
but decreased
maternal weight
18 No toxic effects
NR Toxic effects
observed at 90
and 130 mg/kg
18 No significant
effect on weight
gain or I1ver-to-
body weight ratios
Significant Increases 1n the Neubert and
Incidence of cleft palates Olllmann, 1972
at doses above 30 mg/kg
(see text for additional
details). Significantly
decreased (p<0.005) fetal
weight at all dose levels.
Increases In the Incidence Same as above
of cleft palate at 60 and 90
mg/kg; significantly decreased
(p<0.005) fetal weight at all
dose levels
Increase 1n the Incidence Same as above
of cleft palate; significant
(p<0.005) decrease In fetal
weight
Significant decrease In
fetal weight but no effect
on mortality; Increase 1n
the frequency of cleft
palate similar to that seen
with add (see text)
Increases In the percentage
of resorptlons and/or dead
fetuses at 90 and 130 mg/kg;
Increases 1n the Incidence
of cleft palate and retarda-
tion of skeletal development
at 35 mg/kg and above
No effect on fetal mortality
or fetal weight but an In-
crease In the Incidence of
cleft palate
Same as above
Roll, 1971
Courtney, 1977
-------
TABLE 9 (cont.)
Species/Strain
Vehicle
Form of
2.4,5-T TCOD Level
Dally Dose
Treat-
ment
Days
Obser-
vation
Day
Haternal Response
Fetal Response
Reference
M1ce/C57BL/6 Honeyrwater
(1:1)
add 30 ppm
M1ce/AKR
Honeyrwater
(1:1)
Rat/Sprague- Gavage/hydroxy-
Dawley (groups propyl-methyl-
of 25 rats) cellulose
add 30 ppm
acid 0.5 ppm
o
i
Rat/W1star Gavage/aqueous acid <0.5 mg/kg
gelatin or corn
oil
Rat/W1star Gavage/aqueous butyl <0.5 mg/kg
gelatin or corn ester
oil
46.4 and 113
mg/kg
6-14
18
113 mg/kg
1, 3. 6. 12
or 24 mg/kg/
day
6-15
6-15
19
20
25, 50, 100
or 150 mg/kg/
day
50 or 150
mg/kg/day
6-15
22
6-15
22
NR
Increase In Uver-
to-body weight
ratio
No effect on body
weight and no
observable signs
of toxldty
Some maternal mor-
tality and de-
creased body weight
gain at 150 mg/kg;
no signs of toxU-
1ty at 100 mg/kg
or below
NR
Significant (p<0.01) In-
creases In the Incidence of
cleft palate 1n the high dose
group and cystic kidney In
both dose groups; Increased
fetal mortality also observed
1n the high dose group
Significant (p<0.05) In-
creases In the Incidence of
cleft palate and fetal
mortality
A slight but statistically
significant (p<0.05)
decrease 1n Implantations
and Utter size 1n lowest
dose group only; no frank
teratogenlc effects based
on a detailed examination
of the control and 24 mg/kg
dose group; the only effect
noted was an Increase 1n
the Incidence of 5th par-
tially ossified sternebrae
At 100 or 150 mg/kg de-
creased fetal weight. In-
creased fetal mortality and
an Increase 1n the Incidence
of skeletal anomalies; no
significant effect at the
two lower dose levels
No significant effect on
fetal mortality, fetal
weight, or the Incidence
of anomalies
Courtney
et al., 1970a,b
Same as above
Emerson et al.,
1970, 1971
This appears to
be a full pub-
lication of the
abstract
summary by
Thompson et
al., 1971
Khera and
HcKlnley, 1972;
Khera et al.,
1971
Khera and
HcKlnley, 1971;
Khera et al..
1971
-------
TABLE 9 (cont.)
Species/Strain Vehicle
Form of
2.4,5-T TCOO Level
Treat-
Dally Dose merit
Days
Obser-
vation Maternal Response
Day
Fetal Response
Rat/Holtzman Gavage/l:l solu- add 30 ppm
tlon of honey and
water
Rat/CD
Gavage/15X add 0.5 ppm
sucrose solution
Rat/Strain Gavage/methocel add 0.5 ppm
not specified
Rat/Strain Gavage/methocel add 0.5 ppm
not specified
Syrian hamster/ Gavage/acetone. add
Hesocrlcetus corn oil and car-
euratus boxymethyl cellu-
lose 1n ratio of
1:5.8:10
<0.1-4.5 ppm
4.6, 10.0 and
46.4 mg/kg/day
10-15
20
10.0. 21.5.
46.4 and 80.0
mg/kg/day
6-15
20
50 mg/kg
6-15
NS
100 mg/kg 6-10
20. 40, 80 6-10
and 100 mg/kg
NS
14
NR
Reduced maternal
weight gain at the
two higher dose
levels (p<0.05)
and Increased
llver-to-body
weight ratio at
the highest dose
level (p<0.05)
No effect on mor-
tality or body
weight gain
Increased mor-
tality and
decreased body
weight gain
NS
Significant (p<0.01) In-
creases In fetal mortality
at the two higher dose
levels; dose related In-
creases 1n the percent of
abnormal fetuses per litter;
a high Incidence of cystic
kidneys 1n treated groups
Increase 1n the Incidence of
ktdney anomalies, but no
Increase 1n cleft palate
Reference
Courtney
et al., WOa.b
Courtney and
Moore, 1971
No significant effect on
fetal mortality or fetal
weight; a significant
(p<0.05) Increase 1n the
Incidence of delayed
ossification
Increase 1n the Incidence
of delayed ossification and
poorly ossified or mal-
allgned sternabrae (p<0.05)
Dose-related Increases 1n
fetal mortality, gastro-
intestinal hemorrhages and
fetal abnormalities; see
text for discussion of effect
of TCDO level on development
Sparschu
et al.. 1971a
Same as above
Collins et al.,
1971
NS = Not specified; NR = not reported
-------
If the assumption were made that all teratogenlc activity 1n the 2,4,5-T
samples were attributable to 2,3,7,8-TCDD contamination, the expected E05Q
for Samples A and B would be 230,000 mg/kg (0.0046 mg/kg x 0.02 ppnf1) and
92,000 mg/kg (0.0046 mg/kg x 0.05 ppnf1), respectively. Since the
observed ED was lower by a factor of over 1000, 2,3,7,8-TCDD appears not
to be the sole factor 1n 2,4,5-T-1nduced cleft palate.
The nature of possible Interaction between 2,4,5-T and 2,3,7,8-TCDD 1s
more difficult to define. Based on assays of five mixtures of 2,3,7,8-TCDD
and the highly purified 2,4,5-T, Neubert and Dlllmann (1972) noted a greater
than additive effect on the Induction of cleft palates. A similar conclu-
sion can be reached 1f one assumes that Sample A was a "totally pure" sample
of 2,4,5-T. According to the assumption simple similar action (Finney,
1971) and by treating Sample B as a mixture of 2,3,7,8-TCDD and 2,4,5-T, the
expected EDr,. for Sample B would be 119.8 mg/kg. The observed value of 46
50
mg/kg again suggests a greater than additive effect. A more detailed
statistical analysis of these data, however, would be required to support
the assumptions of simple similar action or Independent joint action that
are implicit 1n these analyses. Furthermore, the inability to define
precisely the levels of 2,3,7,8-TCDD in the 2,4,5-T samples and the possible
«
significance of other contaminants would preclude an unequivocal Interpreta-
tion of the results of the analysis.
Nevertheless, three of the studies summarized in Table 6 (Neubert and
Dlllmann, 1972; Roll, 1971; Courtney, 1977) have demonstrated the induction
of cleft palate in 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 un-
doubtedly a factor in the teratogenlc activity of 2,3,7,8-TCDD contaminated
2,4,5-T, the above analysis suggests that 2,3,7,8-TCDD contamination is not
C-77
-------
the sole factor, and that some teratogenlc activity must be attributed to
2,4,5-T Itself or other contaminants 1n 2,4,5-T.
Effects on reproductive success and fertility have also been studied 1n
four groups of C57BL/6 male mice (25 animals/group) following oral Ingestlon
of mixtures of 2,4-D, 2,4.5-T and 2,3,7.8-TCDD. Dally doses of -40 mg/kg
2,4-D, 40 mg/kg 2,4,5-T and 2.4 yg/kg 2,3,7,8-TCDD 1n Group II; 20 mg/kg
2,4-D, 20 mg/kg 2,4,5-T and 1.2 vg/kg 2,3,7,8-TCDD 1n Group III; and 40
mg/kg 2,4-D, 40 mg/kg 2,4,5-T and 0.16 vg/kg 2,3,7,8-TCDD 1n Group IV
animals were given. Vehicle control animals 1n Group I had corn oil added
to the feed. At the conclusion of an 8-week dosing period, treated animals
were mated to untreated virgin females. No significant decrease 1n fertil-
ity, reproduction and germ cell toxldty were noted. Survival of offspring
and the development of the newborns apparently were not affected (Lamb et
al., 1980).
Courtney and Moore (1971) tested a purified sample of 2,3,7,8-TCDD for
teratogenlc potential. A summary of this study and others assessing the
teratogenlc potential of purified 2,3,7,8-TCDD are presented 1n Table 10.
CD-I, DBA/2J and C57B1/6J mice were given s.c. 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 toxldty,
although an Increase 1n the maternal I1ver/bw ratio was observed In DBA/2J
and C57B1/6J mice. 2,3,7,8-TCDD had no measurable effect on fetal mortal-
ity; however, anatomical abnormalities were observed 1n all strains and at
all dose levels, with C57B1/6J being the most sensitive strain. The abnor-
malities observed were cleft palate and unspecified kidney anomalies.
C-78
-------
TABLE 10
Studies on the Potential Teratogenlc Effect of 2.3.7.8-TCDO
Species/Strain
Mouse/C57BL/6
Mouse/AKR
Mouse/CD-I
Mouse/DBA/2J
Mouse/C57Bl/6J
Mouse/C57Bl/6
Mouse/CD-I
o
to Mouse/CF-1
House/NMRI
Rat/CD
Rat/Sprague-
Dawley
Rat/Wlstar
Vehicle
DMSO or
honey:water
(1:1)
DMSO
Acetone:
corn oil
(1:9)
DMSO or
corn oil
corn oil:
acetone
(98:2)
rape-seed
oil
OMSO
corn oil/
acetone
corn oil/
anlsole
Dally Dose Treatment Days
21.5. 46.4. 6-14 or 9-17
113.0 mg/kg
0.5, 1. 3 vg/kg 6-15
1, 3 vg/kg 10-13 or 10
25. 50, 100, 7-16
200, 400 vg/kg
0.001, 0.01, 6-15
0.1. 1.0,
3.0 tig/kg
0.3, 3.0, 4.5, 6-15
9.0 vg/kg
0, 0.5, 6-15, 9 and 10,
2.0 vg/kg or 13 and 14
0, 0.03, 6-15
0.125, 0.5, 2.0
and 8.0 pg/kg
0.0, 0.125, 6-15
0.25, 1, 2, 4.
8, 16 vg/kg
Observation
Day
19a
17a or 18
18a
18b
18a
18
20a
20a
22
Maternal Response
Increased liver/body
weight ratio
Increased liver/body
weight ratio
none reported
Increased liver/
body weight ratio
none reported
no effect observed
none reported
vaginal hemorrhage at
2.0 and 8.0 yg/kg
maternal toxlclty ob-
served at or above
1 vg/kg
Fetal Response
fetoddal. cleft
palate, cystic kidney
cleft palate, kidney
anomalies
cleft palate, kidney
anomalies
cleft palate, hydro-
nephrotlc kidneys,
hydrocephalus, open
eyes, edema, petechlae
cleft palate, dilated
renal pelvis
fetoddal at the high
dose, cleft palate at
doses at or above
5 vg/kg
kidney malformations
at both dose levels
Intestinal hemorrhage
at 0.125 and 0.5 vg/kg,
fetal death at higher
doses, subcutaneous
edema
Increased fetal death
observed at or above
1 vg/kg, subcutaneous
oHonu anrf hemnrrhaaes
Reference
Courtney et al. ,
1970b
Courtney and Moore,
1971
Moore et al., 1973
Courtney, 1976
Smith et al., 1976
Neubert and Dlllmann,
1972
Courtney and Moore,
1971
Sparschu et al. ,
1971b
Khera and Ruddlck.
1973
1n the 0.25-2 Mg/kg
groups
-------
TABLE 10 (cont.)
o
i
oo
o
Species/Strain
Rat/Sprague-
Dawley
Rat/Sprague-
Dawley
Rabbit/
New Zealand
Vehicle
corn oil/
acetone
(9:1)
diet
corn oil/
acetone
/ rt 1 »
(9:1 )
Dally Dose Treatment Days
0.1. 0.5. 1-3
2.0 pg/kg
0.001, 0.01 throughout
and 0.1 pg/kgc gestation
0.0. 0.1, 0.25. 6-15
0.5 and 1 pg/kg
Observation
Day
21
post-
parturition
28
Maternal Response
decrease In body
weight gain In the
high dose group
low fertility at 0.01
and 0.1 pg/kg decreased
body weight at 0.01
and 0.1 pg/kg dilated
renal pelvis
maternal toxldty at
doses of 0.25 pg/kg
and above
Fetal Response
decreased fetal weight
1n the 0.5 and 2 pg/kg
group
low survival at 0.01
and 0.1 pg/kg, de-
creased body weight at
0.01, slight dilated
renal pelvis at 0.001
pg/kg 1n the F] but not
succeeding generations
Increases 1n extra
Mbs and total soft
tissue anomalies
Reference
G1av1n1 et al., 1982a
Murray et al., 1979
61av1n1 et al., 1982b
aF1rst day of gestation designated day zero
bF1rst day of gestation designated day one
cThe high dose level (0.1 yg/kg/day) was discontinued due to very low fertility In adults
-------
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 (hydronephrosls) were observed on
an average of 95.1% of the fetuses/Utter, 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 In 36.3% of the fetuses/
Utter. A single dose of 1 yg/kg on day 10 of gestation produced kidney
anomalies In 34.3% of the fetuses; however, no cleft palates were observed.
When C57B1/6 mice were treated with 1 vg/kg on day 10 of gestation and
were then allowed to Utter, the detection of kidney lesions on postnatal
day 14 was found to depend largely on whether the pups nursed on a
2,3,7,8-TCDD-treated mother. When pups from a 2,3,7,8-TCOD-treated mother
nursed on control mice, kidney anomalies were found in only 1/14 Utters.
In contrast, when pups from control mothers nursed on 2,3,7,8-TCOD-treated
mice, kidney anomalies were observed In 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 Utters. Kidney anomalies observed following
in utero exposure or exposure through the milk were similar, and these
kidney anomalies may not be considered a purely teratogenlc response.
Neubert et al. (1973) reviewed what was known of the embryotoxlc effects
of 2,3,7,8-TCDD In 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
C-81
-------
anomalies were reported. Neubert and Dlllmann (1972) administered 2,3,7.8-
TCDD by gavage to 20 female mice on days 6 through 15 of gestation at doies
of 0.3, 3.0, 4.5 and 9.0 yg/kg. At day 18 of gestation, extensive
reabsorptlon was observed In the high dose group with 6/9 Utters totally
resorbed. In the few surviving fetuses, there was an 81% Incidence of cleft
palate. At lower doses, there were 9 and 3% Incidences at doses of 4.5 and
3.0 vg/kg, respectively, and no cleft palates were observed In 138 fetuses
examined In the 0.3 wg/kg group. Fetal mortality was Increased at the 9.0
yg/kg dose if animals were treated only on days 9 through 13; however, the
Incidence of cleft palate remained high at a frequency of 60%. In a series
of experiments to determine the time of gestation at which 2,3,7.8-TCDD was
effective 1n Inducing cleft palate, mice were treated for a single day
between days 7 and 13 of gestation with 2,3,7,8-TCDD at a dose of 45
yg/kg. A maximum number of Inducted cleft palates occurred when animals
were treated on either day 8 or 11 of gestation, while exposure to 2,3,7.8-
TCOD after day 13 of gestation produced no cleft palates in the fetuses.
Courtney (1976) compared the teratogenlc potential of 2,3,7,8-TCDD
administered orally with 2,3,7,8-TCDD administered s.c. 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 s.c. 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 hydro-
nephrotlc kidneys being most common. Other abnormalities observed Included
hydrocephalus, open eye, edema and petechlae. Subcutaneous administration
of 2,3,7,8-TCDD produced a greater teratogenlc response at a lower dose than
C-82
-------
oral administration, with abnormalities observed 1n 87% of the fetuses
following s.c. 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) In CF-1 mice. The mice were given
0.001-3.0 vg 2,3,7,8-TCOD/kg/day by gavage from day 6 through 15 of gesta-
tion. The Incidence of cleft palate was found to be significantly Increased
In 1.0 and 3.0 vg/kg/day dose groups, and the Incidence of kidney
anomalies was significantly Increased at 3.0 wg/kg/day. There were no
observable teratogenlc effects In the study at 0.1 yg/kg/day; however,
some were noted at lower dose levels, although not statistically signifi-
cantly elevated.
Poland and Glover (1980) compared cleft palate formation by 2,3,7,8-TCDD
In the responsive C57BL/6J, the non-responsive DBA/2J and the hybrid
B6D2F /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 s.c. dose of 3.0, 10.0 or 30.0 wg/kg of 2,3,7,8-TCDD dissolved 1n
p-dloxane 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 Pg/kg of 2,3,7,8-TCDD, cleft palates
(3?4 Incidence among live fetuses) were only observed 1n the C57BL/6J mice at
the higher dose level. At a dose of 30 vg/kg, the Incidence of cleft
palates among live fetuses for the C57BL/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 non-responsive mice. At a dose level of 30 yg/kg of
C-83
-------
2,3,7,8-TCDD, the Incidence of cleft palates among live fetuses for the
responsive C57BL/6J, A/J, BALB/cByJ and SEC/1REJ mice was 54, 73, 65 and
95%, respectively. The only responsive mouse (CBA/J) strain that was
resistant to 2,3,7,8-TCDD-med1ated cleft palate was also resistant to the
teratogenlc effects of cortisone. In contrast, the Incidence of cleft
palates In the non-responsive DBA/2J, RF/J, AKR/J, SWR/J and 129/J mice was
between 0-3% at the 30 vg/kg dose level. Thus the responsive mice.
containing high levels of the Ah receptor, are highly susceptible to the
effects of 2,3,7.8-TCDO in producing cleft palate, whereas the non-
responsive mice, which contain low (or 0) levels of the Ah receptor protein,
are resistant to this teratogenlc effect of 2,3,7,8-TCDD. These data and
other results (Hassoun and Dencker, 1982) suggest that cleft palate forma-
tion elicited by 2,3,7,8-TCDD segregates with the Ah locus.
In an early study, Courtney and Moore (1971) tested the teratogenlc
potential of 2,3,7,8-TCDD 1n pregnant rats (CD) Injected s.c. on a dally
basis with 2,3,7,8-TCDD (0.5 or 2 yg/kg) in dimethyl sulfoxlde on days 6
through 15, days 9 and 10, or days 13 and 14 of gestation. The only remark-
able anomaly was kidney malformations 1n fetuses exposed to 2,3,7,8-TCDD.
In the group exposed transplacentally at a dose of 0.5 yg/kg, 4/6 Utters
had fetuses with kidney malformations (average number of kidney defects/
Utter was 1.8). An 11 and 34% Incidence of kidney anomalies occurred In
groups exposed to 2,3,7,8-TCDD on days 9 and 10, and 13 and 14, respective-
ly. In addition, six hemorrhaglc gastrointestinal 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.
C-84
-------
2,3,7,8-TCDO was administered by gavage to groups (10-14 animals/group)
of pregnant Sprague-Dawley rats at dose levels of 0, 0.03, 0.125, 0.5, 2.0
and 8.0 yg/kg/day on days 6 through 15 of gestation (Sparschu et al.,
1971b). No adverse teratogenlc effects were reported In fetuses exposed
transplacentally at the 0.03 vg/kg level. At the 0.125 vg/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,
resorptlons were Increased, 6 dead fetuses were reported, and 36 of 99
fetuses suffered an Intestinal hemorrhage. In the 2.0 yg/kg group, only 7
live fetuses were reported (occurring In only 4/11 Utters), 4 having
Intestinal hemorrhage. Early and late resor-ptions were prevalent. No live
fetuses, but many early resorptlons, were reported in the group exposed to
8.0 vg 2,3,7,8-TCDD/kg/day. Subcutaneous edema appeared dose-related,
occurring In a considerable number of fetuses from the higher dose groups.
Male fetuses appeared to be more susceptible to 2,3,7,8-TCDD exposure; how-
ever, there was no significant difference In the sex ratio of live fetuses.
Khera and Ruddick (1973) tested a wide range of doses of 2,3,7,8-TCDD
for teratogenlc and fetotoxic 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 and 16
*
vg/kg on days 6 through 15 of gestation. At day 22 of gestation, there
were no live fetuses in groups exposed to >4 wg/kg, and reduced Utter
size was observed in the 1 and 2 yg/kg group. Unspecified maternal toxic-
1ty was reported In all groups where there was fetal mortality. In groups
exposed to 0.25-2 vg/kg, there were fetal anomalies observed as either
gross or microscopic lesions consisting of subcutaneous edema of the head
and neck, and hemorrhages in the intestine, brain and subcutaneous tissue.
C-85
-------
The Incidences of grossly observed lesions were 0/18, 2/11, 7/12 and 11/14
In 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). WHh 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 vg/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 vg/kg on days 6 through 15 of gestation and allowed
the dams to Utter and wean the pups. In this experiment, maternal toxUHy
was reported 1n the 0.5 and 1 yg/kg group. At birth, there were fewer
viable pups, and the pups had lower body weights in all but the 0.125
wg/kg 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 from
delayed toxldty from jm utero exposure to 2,3,7,8-TCDD.
G1av1n1 et al. (1982a) assessed the effect of small doses of 2,3,7,8-
TCDD administered during the prelmplantatlon period 1n Sprague-Dawley rats.
The animals, In groups of 20, were treated by gavage with 2,3,7,8-TCDD at
doses of 0.0, 0.1, 0.5 and 2 yg/kg on days 1-3 of gestation. (The legends
to the tables In this paper Indicated that the low dose was 0.125 yg/kg.)
C-86
-------
At day 21 of gestation, no toxic effects were observed In the dams except
for a decrease from 19.3-12.9 g In average maternal weight gain In the high
dose animals as compared to controls. In the fetuses, weight was signifi-
cantly reduced (p<0.05) In the 0.5 and 2 vg/kg groups. Malformed Utters
and malformation/fetuses examined were 2, 5, 5 and 6, and 2/270, 8/260,
5/255 and 8/253, respectively, 1n the control 0, 0.1, 0.5 and 2 vg/kg
groups; however, these Increases In the treated animals were not statistic-
ally significant. The anomalies observed were restricted to cystic kidney.
This exposure to 2,3,7,8-TCDD early In pregnancy did not affect Implantation
frequency, and the decrease In fetal weight was considered a result of
2,3,7,8-TCDD delayed Implantation.
In a second study, G1av1n1 et al. (1983) administered the same doses of
2,3,7,8-TCDD (0.0, 0.125, 0.5 and 2 yg/kg) dally to 15 female CRCD rats
per group by gavage 1n corn olliacetone (9:1) for 2 consecutive weeks prior
to mating. Females that did not become pregnant during three estrous cycles
were necropsied to determine signs of toxlclty, 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 toxlclty 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 toxlclty was llstlessness during the
treatment period 1n the animals of the high-dose group. The only signifi-
cant (p<0.01) fetal effect observed 1n the 0.5 vQ/kg group was an Increase
In Post1mplantat1on losses from 2.9% In the control group to 10.2%. In the
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high-dose group, there were decreases In corpora lutea and Implantations
(averages of 17.6% 1n control and 14.9% 1n treated animals, and 15.5% In
control and 12.0% In treated animals, respectively), and Increases In both
pre- and postlmplantatlon losses of 11.7% for controls and 19.5% (p<0.05) 1n
treated animals, and 2.9% in control and 30.3% (p<0.001) 1n treated animals,
respectively. In addition to these signs of fetal toxldty, 9 of 10 Utters
In the high-dose group contained at least one malformed fetus as compared
with 1/13, 2/13 and 2/13 In the control, 0.125 and 0.5 vg/kg groups. The
predominant fetal malformations were cystic kidney and dilated renal pelvis,
which have been observed In other studies In which 2,3,7,8-TCDD was adminis-
tered during gestation.
The reproductive effects of 2,3,7,8-TCDD were also examined In a 3-gen-
eratlon study using Sprague-Dawley rats (Murray et a!., 1979). Throughout
the study, animals were continuously maintained on diets providing doses of
0, 0.001, 0.01 or 0.1 vg 2,3,7,8-TCDD/kg/day. The parental group (f )
was maintained for 90 days on the test diets prior to mating. The f rats
were mated twice, producing the filial generations (f and f1D).
IA ID
Selected f1B and f^ rats were mated at -130 days of age to produce the
f and f 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 produced 1n this group. At the Intermediate dose (0.01 vg/kg/day),
2,3,7,8-TCDD caused lower body weights In exposed rats of both sexes (f
and f»). At the low dose, no toxic effects were discerned.
Fertility was greatly reduced 1n the f_ generation exposed to 0.1 vg
2,3,7,8-TCDD/kg/day. At 0.01 vg 2,3,7,8-TCDD/kg/day, fertility was
significantly (p<0.05) reduced 1n the f and f rats. Fertility In rats
(of any generation) exposed to 0.001 yg 2,3,7,8-TCDD/kg/day was not
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different from that of control rats. Decreases In Utter size were noted 1n
the f group exposed to 0.1 yg/kg/day and the f? and f3 Utters
I A
exposed at 0.01 yg/kg/day. Statistically significant decreases 1n fetal
survival throughout gestation were noted In f^ and f3 Utters of the
0.01 yg 2,3,7,8-TCOO/kg/day exposed dams. At 0.001 yg 2,3,7,8-TCDO/kg/
day, a decreased gestatlonal survival was reported for the f^ Utters, but
not for other generations. Decreased neonatal survival was noted among
f and f pups exposed to 0.01 yg 2,3,7,8-TCDD/kg/day, but not among
1A 2
fn_ or f~ pups. Postnatal body weights of the ?2 and f3 Utters at
ID o
0.01 yg 2,3,7,8-TCDD/kg/day were significantly depressed. At the low dose
(0.001 vg 2,3,7,8-TCDD/kg/day) necropsy of 21-day-old pups revealed a
statistically significant (p<0.05) Increase In dilated renal pelvis In the
f 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
Increased liver weight were reported In the fg generation, but not In the
f generation (f- generation data not obtained) of the Intermediate dose
group. Murray et al. (1979) concluded that 2,3,7,8-TCDD Ingested at 0.01 or
0.1 yg/kg/day Impaired reproduction among rats, and NOAELs were associated
with 0.001 vg 2,3,7,8-TCDD/kg/day. Nlsbet and Paxton (1982) Devaluated
the primary data of Murray et al. (1979) using different statistical
methods. From this revaluation It was concluded that 2,3,7,8-TCDD signifi-
cantly reduced the gestatlonal Index, decreased fetal weight, and Increased
llver-to-body weight ratios and the Incidence of dilated renal pelvis In
both lower dose groups. Nlsbet and Paxton (1982) concluded that the dose of
0.001 yg/kg/day was not a NOAEL In this study.
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A single report by Glavlnl 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 administered 2,3,7,8-TCDD by gavage at doses of
0.0, 0.1. 0.25, 0.5 and 1 yg/kg on days 6 through 15 of gestation. The
dams were examined for Implantation sites, resorptlons, and live fetuses,
and the fetuses were examined for malformations on day 28 of gestation.
Decreased maternal weight gain and unspecified signs of maternal toxlclty
occurred. In dams exposed to 2,3,7,8-TCDD at doses of >0.25 yg/kg. At
doses of 0.5 and 1 yg/kg, there were 2 and 4 deaths, respectively, among
the dams. There were Increases In abortions and resorptlons at a dose of
>0.25 yg/kg with no live fetuses detected In the high dose group. In the
fetuses, the most common observation 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
ug/kg dose groups. Although there was no significant Increase In specific
soft tissue anomalies, there was an Increase from 0/87 to 3/78, 2/33
(p<0.05) and 2/28 (p<0.05) in total soft tissue anomalies In the control,
0.1, 0.25 and 0.5 yg/kg groups. The most prevalent soft tissue anomaly
was hydronephrosls, which the authors point out was a common finding In rat
fetuses exposed to 2,3,7,8-TCDD \n_ utero. These effects were considered to
be signs of embryotoxldty rather than a teratogenlc effect.
In addition to the fetotoxlc effects of prenatal exposure to 2,3,7,8-
TCDD, Norman et al. (1978) demonstrated that 2,3.7,8-TCDD could Induce Hver
mlcrosomal enzymes following ±n utero exposure. Pregnant New Zealand
rabbits were given s.c. 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 exam-
ined for enzyme activity within 12 hours after birth. While this treatment
Increased the liver cytochrome P-450 levels In the adults ~2-fold, from
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1.8-3.7 nmol/mg protein, the Increase \n the newborns was ~5-fold, from
0.3-1.6 nmol/mg protein. SDS-polyacrylam1de gel electrophoresls revealed
that 2,3,7,8-TCDD Induced a single form (form 6) of cytochrome P-450, and
that this form was one of the two that were also Induced by 2,3,7,8-TCDD In
the adult liver. The Identity of form 6 was confirmed by 1mmunolog1c reac-
tion and Its peptlde fingerprint. It was shown that Induction of cytochrome
P-450 In newborns resulted In levels of benzo(a)pyrene hydroxylase and
7-ethoxyresorufln-O-deethylase activity similar to adult levels. The conse-
quence to the newborn of these changes In the development of liver mlcrosom-
al enzymes has not been established.
Dougherty et al. (1975) failed to find evidence for teratogenldty or
embryotoxlclty 1n rhesus monkeys which were given on days 22-38 of gestation
dally oral doses (1n gelatin capsules) of up to 10 mg/kg/day of 2,4,5-T
containing 0.05 ppm 2,3,7,8-TCDD. The 2,3.7,8-TCDD dose at the highest dose
level of 2,4,5-T administered (10 mg/kg/day) would correspond to 0.5 vg
2,3,7,8-TCDD/kg/day. However, 1t should be noted that palate closure In the
monkey occurs on gestatlonal days 42-44 and the kidney Is also a late
developing organ.
Adverse effects of exposure to 2,3,7,8-TCDD on reproductive success In
monkeys have also been described. Schantz et al. (1979) fed a diet contain-
ing 50 ppt 2,3,7,8-TCDD to rhesus monkeys for 20 months. Seven months Into
the study the female monkeys were bred to control males. There were four
abortions and one stillbirth, two monkeys did not conceive even though they
were mated repeatedly, and two monkeys carried their young to term. The
total 2,3,7,8-TCDD Intake over the seven months was estimated by the authors
to be 0.35 yg/kg, corresponding to a calculated dally dose of 0.0015 yg
2,3,7,8-TCDD/kg/day.
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Allen et al. (1979) fed adult female rhesus monkeys on diets containing
50 or 500 ppt of 2,3,7,8-TCDD for 7 months. These exposure levels corre-
spond to total doses per animal at the end of 7 months of 1.8 and 11.7 vg
2,3,7,8-TCDD. respectively. Although menstrual cycles were not affected 1n
either treatment group, 5/8 animals 1n the high dose group had decreased
serum estradiol and progesterone levels. Hormone levels were normal 1n the
low dose animals. At 7 months, the females were bred to non-exposed males,
and 6/8 and 3/8 females 1n the low and high dose groups, respectively, were
Impregnated. Of the Impregnated animals, 4/6 and 2/3 had spontaneous
abortions, while the remaining Impregnated animals had normal births.
McNulty (U.S. EPA, 1980b) 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 a total dosage of 5 vg/kg bw (two animals), 1
vg/kg bw (four animals) and 0.2 yg/kg bw (four animals) 1n nine divided
doses, 3 times/week during weeks 4, 5 and 6 (days 20 through 40) after
conception. Group II, consisting of 12 animals, received single doses of 1
vg/kg bw of 2,3,7,8-TCDD on days 25, 39, 35 and 40 after conception.
Three animals were exposed 1n each of these 4 days. The vehicle control
group, consisting of 11 animals, was treated with corn olltacetone only, on
the same schedule as Group I animals. Both the females, who received the
highest dose (5 yg/kg), had fetal losses. In the next lower dosed animals
(1 vg/kg In both groups) 12 of 16 females had fetal losses; and In the
lowest dosed animals (0.2 vg/kg 1n Group I) one abortion occurred 1n four
pregnancies. Maternal toxldty was observed 1n many of these treated
females. The difference In 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
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short exposure to 1 jig/kg bw of 2,3,7,8-TCDD during early pregnancy
results In fetal loss 1n rhesus monkeys and the results appear to be related
to the adverse effects of 2,3,7,8-TCDD on the fetus (U.S. EPA, 1980b).
A positive association between 2,4,5-T exposures and Increases In birth
defects or abortions has been reported 1n human populations In Oregon (U.S.
EPA, 1979), New Zealand (Hanlfy et a!., 1981) and Australia (Field and Kerr,
1979). A lack of any such association has been reported 1n human popula-
tions 1n Arkansas (Nelson et al., 1979), Hungary (Thomas, 1980), New Zealand
(Dept. of Health, New Zealand, 1980; McQueen et al., 1977) and Australia
(Aldred, 1978). Almost all of the reports are geographic correlation stud-
ies, and because of the uncertainties Inherent 1n this type of epldemlologlc
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 abortlfa-
dents In humans.
Based on a report of a high Incidence of abortions In a small group of
women living around Alsea, Oregon, who may have been exposed to the herbi-
cide 2,4,5-T from aerial spraying (Smith, 1979), the U.S. EPA (1979) Initi-
ated a study, often referred to as the "Alsea II study", to determine 1f
spontaneous abortion rates differed between the exposed and unexposed popu-
lation, If spontaneous abortion rates evidenced seasonal variation In these
two groups, and 1f such seasonal variations were associated with 2,4,5-T
spray application.
Spontaneous Abortion Rate Index, as defined by the U.S. EPA, 1s
"basically the ratio of the number of hospitalized spontaneous abortions to
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the number of births corresponding to the spontaneous abortions, based on
the residence zip code of the women contributing to each event." Upon com-
pletion of the study, the EPA concluded that (1, the 1972-1977 Spontaneous
Abortion Rate Index for the study area was significantly higher than In the
Rural Control Area or the Urban area; (2) there was a statistically signifi-
cant seasonal cycle In the abortion Index In each of the areas with a period
of about 4 months. In particular there was an outstanding peak 1n the study
area 1n June; and (3) there was a statistically significant correlation
between-the Spontaneous Abortion Rate Index and spray patterns in the study
area when a lag-time of 2 or 3 months was Included. The EPA concluded
however, "This analysis 1s a correlational analysis, and correlation does
not necessarily mean causation."
miby et al. (1980), citing three critiques of the Alsea II study that
were 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 in human risk assessment. The Alsea II study has also been
reviewed by a panel of epidemiologists who, in 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 biased and would likely result in the
underestimation of abortions, particularly in the urban area {the Incidence
of abortions 1n all three groups was within the expected background rate of
8-15%); only a small portion of the area from which the exposed subjects
were selected was actually sprayed with 2,4,5-T; and the study was not con-
trolled for other factors such as age, smoking habits and alcohol consump-
tion, which may affect the spontaneous abortion rate. Based on a new report
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by Smith (1979), the U.S. EPA 1s attempting or has attempted to correlate
2,3,7,8-TCDD levels In the affected areas with the observed rate of abor-
tion. No published reports have been encountered on the outcome of this
effort.
In the only other report encountered on a population 1n the United
States, Nelson et al. (1979) noted a general Increase In the reported
Incidence of facial cleft In both high and low exposure groups In Arkansas
from 1948 to 1974. In this study, exposure estimates were based on average
rice production In different areas of Arkansas, and the Incidence of cleft
palate was determined by screening birth certificates and checking records
of the Crippled Children's Services. No consistent exposure/effect correla-
tions were noted, and the general Increase with time 1n the Incidence of
facial clefts was attributed to better reporting procedures; however, there
does not have to be a direct correspondence of malformations In 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 Dept. of Health Is essentially anecdotal, Involv-
ing two women who gave birth to malformed children (one with an atrlal
septal defect and a malformation of the trlcuspld valve of the heart and the
other with biliary atresla). In both cases, exposure to 2,4,5-T could not
be ruled out. Based on an analysis of spraying records, the time course of
the pregnancies and plant damage near the women's homes, however, the
Department of Health, New Zealand (1980) concluded that there was Insuffi-
cient 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 In 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.
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The study by McQueen et al. (1977) Is not published 1n the open litera-
ture but Is summarized by M1lby et al. (1980). According to the summary,
McQueen et al. (1977) "...examined the epidemiology of neural-tube defects
In three areas In New Zealand and concluded 'there 1s no evidence to Impli-
cate 2,4,5-T as a causal factor 1n human birth defects."' No additional
details are provided.
Hanlfy et al. (1981) performed an epldemlologlc study in Northland, New
Zealand, In areas where spraying of 2,4.5-T was carried out by various
companies for a number of years. The rate of birth defects was obtained
from an examination of hospital records In seven mutually exclusive 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 non-exposed 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) In comparisons between the
exposed (1972-1976) and control (1959-1965) populations. Exposure estimates
were made for the seven areas and for different years using company records
of aerial spraying and a model that factored In 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 statis-
tically significant association between the occurrence of talipes and expo-
sure when the fractional removal rate was assumed to be 0.25. There was,
however, no statistically significant association where 1.0 was used as the
fractional removal rate.
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Smith et al. (1982a) Investigated the outcome of pregnancy In families
of professional 2,4,5-T applicators and agricultural contractors 1n New
Zealand. Agricultural contractors were chosen as the control population
since both sprayers and contractors were of the same economic group with
similar outdoor occupations. The survey was conducted by mall with 89% of
the chemical applicators responding and 83% of the agricultural contractors
responding to questions asking whether they used 2,4,5-T and its temporal
relationship to reproductive histories regarding birth, miscarriages, still-
births and congenital defects. The relative risks of congenital defects and
miscarriages were 1.19 (0.58-2.45% confidence limits) and 0.89 {0.61-1.30%
confidence limits) for the wives of chemical sprayers as compared to the
wives of agricultural contractors. These data Indicate that exposure of
fathers and mothers (e.g., while cleaning clothes) had no effect on the out-
come of pregnancy. Biases that may have affected the results, such as the
age of the mother at childbirth, smoking habits and birth to Maori parents
were investigated and eliminated as possible confounders.
The two reports from Australia (Aldred, 1978; Field and Kerr, 1979) also
present apparently conflicting results. The report by Aldred (1978) is not
published in the open literature, but the following summary Is taken from
Milby et al. (1980): "The report concluded that birth defects 1n a group of
babies born in the [Yarram] district in 1974 and 1976 could not be attribut-
ed to exposure to 2,4,5-T or 2,4-D." Additional details that might be
useful in assessing the rationale for this statement are not provided in the
summary. The report by Field and Kerr (1979) plotted the incidence of
neural-tube defects (anencephaly and meningomyelocele). 1n New South Wales,
Australia, over the years 1965-1975, and the previous years usage of 2,4,5-T
In all of Australia. The authors noted a decrease In the Incidence of
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neural-tube defects expected on the basis of the plotted line In 1975 and
1976, when Australia Instituted monitoring of 2,4,5-T to ensure a 2,3,7,8-
TCDO level <0.1 ppm. The data were not tested for significance, although
Field and Kerr (1979) Indicate that they consider the epidemlologlcal data
on neural-tube defects to be "relatively complete," they do not comment on
the Increasing Incidence of neural-tube defects with time and whether or not
an Increase In the thoroughness of reporting neural-tube defects could have
contributed to the apparent correlation of 2,4,5-T exposure with these
defects. A visual replotting of the data suggests that the Incidence of
cleft palate correlates better with 2,4,5-T usage than with time. Nonethe-
less, the appropriateness of correlating 2,4,5-T usage In all of Australia
with the Incidence of -defects In one area of Australia Is questionable.
Thomas (1980) used an approach similar to that of Field and Kerr (1979)
on data from Hungary. One major difference, however, is that Thomas (1980)
compared the incidence of stillbirths, cleft lip, cleft palate, splna
biflda, anencephalus and cystic kidney disease in all of Hungary between
1976 and 1980 with 2,4,5-T use in 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 in rural areas where 2,4,5-T exposure may be expected to be
greatest, and because annual use of 2,4,5-T 1n Hungary had risen from 46,000
kg in 1969 to 1,2000,000 kg 1n 1975, Thomas (1980) considered Hungary to be
"...probably the best country 1n which to examine possible health effects of
this herbicide." In any event, all Indices of birth defect rates decreased
or remained stable over the period of study.
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 is also an inadvertant contaminant of
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2,4,5-trlchlorophenol (TCP). Chronic exposure to 2,3,7,8-TCOD may occur
during the manufacture of TCP and high level acute exposure to 2,3,7,8-TCDD
has occurred after an accident In July, 1976 at the ICHESA TCP chemical
factory In Seveso, Italy (Bonaccorsl et al., 1978). In this accident, the
reaction used to produce TCP became uncontrolled, producing conditions
favorable for 2,3,7,8-TCDD formation prior to venting the contents of the
chemical reactor Into the atmosphere. The resulting cloud of chemicals
settled over a heavily populated area. Although the amount of 2,3,7,8-TCDD
released was not known, the reported cases of chloracne, a symptom of acute
exposure to 2,3,7,8-TCDD, Indicated that exposure to 2,3,7,8-TCDD had
occurred. Some preliminary results are available from ep1dem1olog1c studies
of reproductive events 1n the Inhabitants of Seveso, and recently a study
has become available on the reproductive history of men employed 1n the
chemical manufacturing Industry with possible chronic exposure to
2,3,7,8-TCDD (Townsend et al., 1982).
Ep1dem1olog1c studies to determine the reproductive effects In Individ-
uals exposed to 2,3,7,8-TCDD and TCP following the accidental contamination
of a populated area around Seveso, Italy, are not completed. The Incidence
of spontaneous abortions occurring between March 1976 and January 1978 have
been reported for Inhabitants 1n the area around Seveso by Bonaccorsl et al.
(1978), Reggianl (1980) and B1sant1 et al. (1980). The spontaneous abortion
rate In the contaminated area for the three trimesters following the acci-
dent was 13.1, 11.0 and 13.05%, which was similar to the worldwide 15-20%
frequency of spontaneous abortion. Subdividing the contaminated area Into
highly, moderately, and least contaminated, and examining the rates for each
area Individually, also failed to demonstrate any change 1n the spontaneous
abortion rate. The Incidence rates of malformations also were examined;
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however, the numbers were too small for meaningful assessment. There are
several reasons why these studies would not Indicate that the effect of
2,3,7,8-TCDD exposure 1n this accident had no effect on human reproduction.
The authors note that there are many difficulties in Interpreting these
data. The Incidence rates of spontaneous abortions and birth defects were
not adequately available for the region prior to the accident as a result of
suspected under-reporting. There was Inadequate reporting even after the
accident due to 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 Induced birth defects. In a recent review of the
progress of epidemlologic investigations of the Seveso accident, Tognonl and
Bonaccorsi (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 produc-
tion. A total of 930 potentially exposed males were identified who had
worked for >1 month between January 1939 and December 1975 in a job with
potential 2,3,7,8-TCDD exposure. Exposure estimates of low, moderate and
high were made by an industrial hyglenist primarily from job description and
surface contamination data; however, the high potential exposure group was
reserved for process workers during 1963-1964 when changes in operations
resulted in a number of cases of chloracne. The control population was an
equal number of male employees not involved in any process that might cause
exposure to 2,3,7,8-TCDD and matched for date of hire. In these groups, 586
wives were identified and 370 agreed to participate as the exposed group,
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while 345 wives In the control group agreed to participate. After Identifi-
cation 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 (concep-
tion which occurred 1n the exposed group prior to work records Indicating
potential exposure to 2,3,7,8-TCDD were placed 1n the control group), there
was no statistically significant Increase 1n spontaneous abortions, still-
births, Infant deaths or selected congenital malformations. Sample sizes
were too small to provide meaningful data 1f the populations were subdivided
by extent of exposure. It was suggested that many confounding factors could
account for these negative results, such as the Inappropriate selection of
the populations, unidentified covarlables and insufficient power; however,
It was maintained 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), In testimony before the House Committee on Science and
Technology, described a re-analysis of the primary data used by Towsend et
al. (1982). In this re-analysis, the rate of cleft palate and cleft lip
were reported to be elevated by 1.9 (90% confidence Intervals of 1.0-3.6) in
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 Center
for Disease Control on the yearly rate of cleft palate alone or cleft lip
with or without cleft palate for births in Midland County, Michigan (the
site of a chlorophenol production facility) during the years 1970-1981. The
data indicated an Increased rate for these defects of between 50 and 10054 in
the years 1971-1975, with the rate returning to expected from 1976-1981.
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The observed Increase was only statistically significant 1f the rates for
cleft palate alone and cleft Up with or without cleft palate were combined;
however, 1t was the opinion of Houk (1983) that these defects should not be
combined since the causal mechanism may be different. The Michigan Depart-
ment of Public Health (1983a) also reported these results and, 1n addition,
demonstrated that the same results occurred If the comparison was made with
other counties 1n Michigan as well as with the general population of the
United States. It was noted 1n this report that "runs" of Increases 1n oral
cleft for successive years have occurred In six other counties with no
obvious potential for chemical exposure described. The Michigan Department
of Public Health (1983a) Interpreted the data to Indicate that a more
detailed case control study was necessary to determine 1f any common factors
may exist, such as exposure to chemicals contaminated with 2,3,7,8-TCDD.
Mutaqenldty
Short-term Yn vitro test systems have been developed to assess the bio-
logic, toxic and genotoxlc effects of chemicals. These assays have proven
to be useful Indicators of potential activity of diverse Industrial chemi-
cals, a broad range of drugs and xenoblotlcs, carcinogens and crude environ-
mental extracts. The most widely used short-term test system, the Ames test
for bacterial mutagenesls, employs several strains of Salmonella typh1mur1um
which are highly susceptible to the effects of mutagenlc chemicals. Despite
the obvious utility of the Ames test and related short-term assays, their
predictive capabilities (I.e., the correlation between bacterial mutagenlc-
1ty and cardnogenlclty) have not been fully assessed (Bartsch et al., 1982).
Mutagen1c1ty assays 1n microorganisms have been used to assess the geno-
toxlc effects of 2,3,7,8-TCDD; however, the results of most of these assays
have Indicated little potential for mutagenlc effects (Table 11).
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TABLE 11
o
1
__J
o
to
Type of Assay
Spot test
Plate
Incorporation
Plate
Incorporation*
Fluctuation
test
Spot test
Plate
Incorporation
Plate
Incorporation
Suspension
assay
Suspension
assay
S-9 TA98
+/- NT
+/- NT
+/- 0
+/- 0
NT
+ 0
NT
NT
+/- 0
TA1530
NT
NT
0
0
0
NT
NT
0
NT
TA1535
0
0
0
0
NT
0
NT
NT
0
TA1537
0
0
0
0
NT
0
0
NT
0
Strains
TA1538
0
0
0
0
NT
0
NT
NT
NT
of Salmonella tvpMmurlum
TA1532 TA1950
0 NT
0 NT
0 0
0 0
+ NT
NT NT
NT NT
* NT
NT NT
TA1975
NT
NT
0
0
NT
NT
NT
NT
NT
TA1978
NT
NT
0
0
NT
NT
NT
NT
NT
G46
NT
NT
0
0
0
NT
NT
NT
NT
TA100
NT
NT
0
0
NT
0
NT
NT
0
TA1531
NT
NT
NT
NT
QR
NT
NT
NT
NT
TA1534
NT
NT
NT
NT
QR
NT
NT
NT
NT
Reference
McCann, 1978
McCann. 1978
Gilbert et al.,
Gilbert et al..
Seller, 1973
Gelger and Neal
Gelger and Neal
Hussaln et al. ,
Zelger, 1983
1980
1980
, 1981
, 1981
1972
*The assay was performed under both aerobic and anaerobic conditions.
NT = Not tested; QR * Questionable response
-------
Hussaln et al. (1972) exposed Salmonella typhlmurlum hlstldene-dependent
strains TA1530 and TA1532 In liquid suspension to 2,3,7,8-TCDD followed by
plating Into selective medium to observe reversion to prototypes. No
Increase In the reversion rate was observed with strain TA1530 at exposure
levels of 1 and 10 wg/m!i. These exposures resulted In cell survivals of
90 and <1%, respectively. In strain TA1532 Increased reversion frequency
was not observed at 2,3,7,8-TCDD concentrations of 2-3 vg/rai, which
resulted 1n a 0-50% decrease 1n survival; however, at 2,3,7,8-TCDD levels
which resulted in a 99% decrease 1n survival, there was an Increased number
of revertant colonies/surviving cells. The dose levels were not specified.
The source of the 2,3,7,8-TCDD sample studied in this paper was the Food and
Drug Administration, and Its reported purity was 99%. Also, Seller (1973)
observed a positive mutagenlc response 1n a spot test of 2,3,7,8-TCDD per-
formed 1n the absence of a metabolic activation system. However, the purity
of the sample studied was not provided. In tester strains G46 and TA1530,
the ratio of revertants/108 cells In the treated plates divided by spon-
taneous revertants/108 cells was <1. In strains TA1531 and TA1534, the
ratio was between 1 and 2, which was considered a "doubtful" mutagenlc
response, while 1n strain TA1532, the ratio was >10. There was no mention
of the 2,3,7,8-TCDD levels tested 1n this assay. The positive controls,
dlethylsulfate, 2-am1nopur1ne and 2-am1nofluorene, produced ratios of 2 to
5, <1 and 5 to 10, respectively, 1n strain TA1532. In both the study by
Hussaln et al. (1972) and the study by Seller (1973), 2,3,7,8-TCDD produced
a positive mutagenlc response only 1n the j>. typhlmurlum strain TA1532,
which 1s sensitive to frameshlft mutagens.
Hussaln et al. (1972) also performed a mutagenldty test of 2,3,7,8-TCDD
In two other mlcroblal test systems. A positive response was observed 1n
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Escherichia coll Sd-4 as Indicated by a reversion to streptomycin Indepen-
dence. In this assay, cells were treated In suspension for 1 hour with
2.3,7,8-TCDD at 0.5-4 vg/ml. The greatest mutation frequency (256
mutants x 10~8, as compared to the control frequency of 2.2 mutants x
1(TB) occurred at a dose level of 2 Pg/ml. The absolute number of
colonies/plate was 7 for the control and 46 for the treated plate. The dose
of 2 vg/ml caused an 89% decrease In cell survival. In the second test
system, the ability of 2,3,7,8-TCDD to Increase prophage Induction in E_.
col_L K-39 cells was examined. The vehicle control, DMSO, Inhibited prophage
Induction as compared to the untreated controls, while the most effective
dose level of 2,3,7,8-TCDD (0.5 vg/mfc) resulted in an increased prophage
Induction as compared to vehicle control but not as compared to the untreat-
ed controls. Hussaln et al. (1972) concluded that 2,3,7,8-TCDD was capable
of causing Increases In the reverse mutation rate 1n E_. coll Sd-4 and that
2,3,7,8-TCDD had a weak ability to induce prophage 1n E.. coll K-39 cells.
The studies which followed these two early reports of Hussain et al.
(1972) and Seller (1973) failed to detect mutagenic activity of 2,3,7,8-TCDD
In S. tvphimurium. Wasson et al. (1978) reported on a personal communica-
tion from McCann (1978) that 2,3,7,8-TCDD was Inactive in both the spot test
and plate Incorporation assay with S. tvphimurium strains TA1532, TA1535,
TA1537 and TA1538. Doses and other experimental protocols were not men-
tioned except that the tests were performed both with and without metabolic
activation. Gilbert et al. (1980) reported that 2,3,7,8-TCDD gave "substan-
tially negative results" with S. tvphimurium 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
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of Aroclor 1254 pretreated rats. In the plate Incorporation assay the test
compound was tested at 1-2000 yg/plate under both aerobic and anaerobic
conditions. Details were not provided for the fluctuation assay. It 1s
difficult to assess possible reasons for the conflicting results between the
earlier studies and these later mutagenlcHy assays, since Information on
experimental conditions was limited In the negative studies.
In an attempt to resolve the conflicting results and observe a mutagenlc
response, Gelger and Neal (1981) tested 2,3,7,8-TCDD 1n the standard plate
Incorporation assay using 8-9 prepared from different sources. In order to
maximize the amount of compound tested, dloxane, a better solvent for
2,3,7,8-TCDD than the commonly employed DMSO, was used. Even with the use
of dloxane, the limited solubility of 2,3,7,8-TCDD allowed only 20
vg/plate to be tested, a dose which was shown to be non-toxic 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 vg/plate, and regardless of the source
of the S-9, there was no observed mutagenlc response. In further attempts
to duplicate the previous positive results, Gelger and Neal (1981) tested
the same concentrations of 2,3,7,8-TCDD 1n strain TA1537, a more sensitive
direct descendent of strain TA1532, for mutagenlc activity In the absence of
S-9. Again, no Increase 1n the number of revertants was observed. In
assays either with or without S-9, positive controls had predictable
Increases 1n the number of revertant colonies. The authors concluded that
2,3,7,8-TCDD was not active under the conditions of this assay; however,
testing at higher concentrations may elicit a positive response. It was
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also noted that many other polychlorlnated aromatic compounds are not muta-
genic in the Ames test, even though there 1s positive evidence of cardno-
genldty.
Mutagenlc effects of 2,3,7,8-TCDO 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 llv
revertants. An S10 metabolic activation system was required. Exposure of
the yeast to 2,3,7,8-TCDO at the highest level tested (10 pg/ml) result-
ed in 16% survival and yielded 4-fold Increases in reversion and gene
conversion.
In the host-mediated assay, male mice were exposed to 25 yg of
2,3,7,8-TCDO/kg (Bronzettl et al., 1983). After 5, 10, 20 or 30 days, 0.2
ma, of a yeast culture (4xl08 cells) was instilled retroorbitally. Four
hours later, the liver and kidneys were removed and the yeast cells in these
organs were assayed for mutagenic responses. Increases (4- to 6-fold) in
reversion and gene conversion were observed 1n yeast cells obtained from the
livers and kidneys. The toxic response of the animals to an exposure of 25
yg/kg was not described in this report. ToxicUy should be expected at
this high dose. The positive results described 1n this paper may suggest
that 2,3,7,8-TCDD 1s mutagenic 1n yeast, but more definitive studies are
needed before a firm conclusion can be drawn.
Hay (1982) has found that 2,3,7,8-TCDO dissolved in DMSO transformed
baby hamster kidney cells (BHK) in vitro. The dioxln Isomers 2,8-d1chloro-
and !,3,7-tr1chlorod1benzo-p_-d1ox1n also transformed BHK cells but the
response was weak. The unchlorlnated d1benzo-£-d1ox1n and the fully chlori-
nated octachlorod1benzo-p_-d1oxin were both negative in the BHK assay (I.e.,
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there was no cell transformation). More recently, Rogers et al. (1982)
reported that 2,3,7,8-TCOD Induced mutations In the excess thymldlne, thio-
guanlne and methotrexate selective systems In L5178Y mouse lymphoma cells 1n
culture.
The National Toxicology Program (Zleger, 1983) provided data on TCDO
from four assay systems: the S. typhlmuMum (strain TA98, TA100, TA1535 and
TA1537) h1st1d1ne reversion assay, the sex-linked recessive lethal test 1n
Drosophlla, and cytogenetlc studies (sister chromatld exchange and chromo-
some aberrations) 1n Chinese hamster ovary cells. Negative results were
obtained in all these assays. However, these studies cannot be evaluated
because the procedures used to obtain the data were not described.
The solubility of 2,3,7,8-TCDD in water is only 0.2 Pg/s. '(Grummett
and Stehl, 1973). Therefore, negative in vitro results must be viewed with
caution unless precise descriptions of the preparation of each test sample
are supplied (e.g., were the samples predissolved and, if so, In what
solvent).
In vitro reactions of 2,3,7,8-TCDD with bacteriophage QB RNA were evalu-
ated by Kondorosi et al. (1973). Active RNA was purified from QB phage fol-
lowed by incubation for 1 hour at 37°C with 0.0, 0.2, 2.0 or 4.0 vg/ms,
of 2,3,7,8-TCDD. At all concentrations tested, 2,3,7,8-TCDD had no effect
on the transfectivity of QB RNA. Other compounds tested Included the alkyl-
atlng agents methyl, ethyl and isopropyl methanesulfonate, and diethyl pyro-
carbonate, all of which inactivated QB RNA under the same experimental con-
ditions. The authors suggested that 2,3,7,8-TCDD inactivity in this assay
indicated that 2,3,7,8-TCDD was an intercalating agent, and hence would
require double stranded DNA in order to interact. The data presented in
this study, however, were insufficient to support this conjecture.
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In vivo binding of radlolabeled 2,3,7,8-TCDD to liver macromolecules was
studied In Sprague-Dawley rats by Poland and Glover (1979). Both male and
female animals were administered [1,6->H]2,3,7,8-TCDD by l.p. Injection at
a dose of 7.5 vg/kg. This dose corresponded to a tritium level of 0.87
mCl/kg. The animals were killed either 12, 48 and 168 hours after treat-
ment, or 24 hours after treatment when the animals were pretreated with the
enzyme Inducers phenobarbHal or unlabeled 2,3,7,8-TCDD. Following sacri-
fice, Isolation of macromolecules, and removal of free labeled 2,3,7,8-TCDD,
the amount of label bound to protein, RNA and DNA was determined. The
greatest non-extractable binding of labeled 2,3,7,8-TCDD occurred to
protein; however, the amount of label bound was small and only amounted to
0.03-0.1% of the total radioactivity administered. The total amount of
label associated with RNA and DNA was, respectively, only 50 and 4 cpm above
background. Time after exposure, sex, or prior enzyme Induction had no
significant effect on 2,3,7,8-TCDD binding. As a result of the extremely
low levels of radioactivity associated with RNA and DNA, it is uncertain
whether 2,3,7,8-TCDD truly binds covalently to these macromolecules and if
so, whether there is any biological significance to this low level of
apparent binding.
The effects of 2,3,7,8-TCDD exposure on the extent of chromosomal
aberrations In the bone marrow of male rats were reported in an abstract by
Green and Moreland (1975). In the Initial experiment, no Increase In
chromosomal aberration was observed after 5 daily gavage treatments at a
2,3,7,8-TCDD dose of 10 vg/kg. In the second portion of this study, rats
were exposed by a single l.p. Injection of 2,3,7,8-TCDD at 5, 10 or 15
vg/kg or a single gavage treatment at 20 vg/kg. The animals at the two
highest exposure levels were killed 24 hours post-treatment, while the
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remaining animals were killed 29 days post-treatment. Again, no Increase in
chromosomal aberrations was observed, except In the positive control group
exposed to tr1ethylenemelam1ne.
In a later report, a small but significant Increase 1n chromosomal aber-
rations was observed in the bone marrow cells of male and female Osborne-
Mendel rats (Green et al., 1977). Bone marrow cells for cytogenetlc analy-
sis were obtained from Osborne-Mendel rats used 1n a range-finding study
preliminary to a chronic bloassay (Green et al., 1977). The animals in
groups of 8 males and 8 females received twice weekly Intubations of
2,3,7,8-TCDD at respective doses of 0.25, 1.0, 2.0 and 4.0, or 0.25, 0.5,
2.0 and 4.0 yg/kg for 13 weeks. Because it was not required for the
range-finding study, a control group was not Included. Bone marrow cells
were analysed for abnormalities and cells 1n mitosis In the animals which
survived to the end of the study (4-8 animals/group). The only significant
Increases In chromosomal aberrations In comparison to the low dose group
were in males at 2 and 4 yg/kg and females at 4 pg/kg. The greatest
Incidence observed was 4.65% of the cells with chromosomal breaks in the
high-dose males, and this was considered only weakly positive. The weak
response, as well as the lack of data from control animals and the reported
difficulty of obtaining cells from the high-dose animals as a result of
2,3,7,8-TCDD tox1c1ty, makes the conclusion from this study that 2,3,7,8-
TCDD produced chromosomal breaks tenuous.
Czelzel and Klraly (1976) reported an Increased Incidence (p<0.001) of
chromatld-type and unstable chromosome aberrations 1n the peripheral lympho-
cytes of workers exposed to the herbicides 2,4,5-tMchlorophenoxyethanol
(2,4,5-TCPE) and Bumlnol. The 2,3,7,8-TCDD levels In the final product were
<0.1 mg/kg; however, the exposure levels for Individual workers were not
available.
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Hulcahy (1980) reported In a letter no Increased Incidences of chromo-
somal aberrations 1n lymphocytes of 15 soldiers exposed to "Agent Orange".
The exposure was for 6-15 months and all subjects complained of symptoms,
Including skin eruptions, which they associated with "Agent Orange". The
analyses were performed with lymphocytes obtained -10 years after the last
exposure, and comparisons were made with eight subjects who had no history
of exposure to 2,3,7,8-TCDD. Neither sister chromatld exchange nor struc-
tural aberrations Including both gaps and breaks were Increased. The
authors note that the long time between exposure and analysis may have
accounted for the negative results.
Also, both Regg1an1 (1980) and Mottura et al. (1981) have studied
Inhabitants 1n Seveso, Italy, exposed to 2,3,7,8-TCDD from an accident 1n a
trlchlorophenol manufacturing plant. Regg1an1 (1980) examined 4 adults and
13 children (3-13 years) for chromosomal aberrations within 2 weeks of the
accident. These 17 Individuals were examined to support claims of, and
determine extent of, Injury before an Inquest judge. Although burnllke skin
lesions In these 17 Individuals Indicated chemical exposure, no Increase 1n
chromosomal aberrations 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, subjects were chosen from the area of heavy contami-
nation following the accident (acute high level exposure), from the working
population of the plant (chronic low level exposure) and a non-exposed con-
trol population. The number of subjects 1n each group was not enumerated.
The specimens were examined by three Independent laboratories and no labora-
tory reported an Increase 1n chromosomal aberrations, although there was a
significant difference 1n the reported scores between laboratories. There
C-lll
-------
was no Information In this abstract on the extent of Individual exposure or
the length of time that elapsed between the accident and obtaining samples
for analyses of chromosomal aberrations.
DUernla et al. (1982) conducted additional studies on lymphocytes
prepared 1n 1976 and 1979 from 8 persons considered acutely exposed to
2,3,7,8-TCDD 1n the Seveso accident, 8 ICMESA factory workers (considered
chronically exposed), and 14 control subjects (8 had chromosomes prepared 1n
1976 and 6 In 1979). Cells were examined for average number of satellite
associations (SAs) (evidence for functional rlbosomal genes), both on a cell
basis and for the large acrocentMc chromosomes (D group chromosomes).
There was no change In the frequency of SAs on a per cell basis In any of
the groups as compared to control values, nor 1n D group chromosomes from
acutely exposed subjects examined Immediately after the accident. There
was, however, a decrease 1n the average frequency of SAs 1n group D chromo-
somes of acutely exposed subjects examined in 1977 and in ICMESA workers at
both the 1976 and 1979 examinations. Although the biologic relevance of
these observations has not yet been confirmed, OiLernia et al. (1982) ob-
served a similar decrease 1n SAs after exposure of lymphocytes to x-1rrad1a-
tion. It was concluded that the decrease 1n SAs may have resulted from
mutagenic damage to functional nucleolar organizing regions.
The potential of exposure to 2,3,7,8-TCDD to result in chromosomal
damage has been studied in experimental animals and humans. Host of the
studies in experimental animals gave no evidence that 2,3,7,8-TCDD may
result in chromosomal aberrations; however, there is a report of a single
positive response which was weak and little detail was provided in the
report to assess the quality of the results. In the studies of humans,
exposure occurred to chemicals which contain 2,3,7,8-TCDD as a contaminant.
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In two of these studies Involving Individuals exposed 1n the Seveso acci-
dent, there was no observed Increase In the Incidence of chromosomal aberra-
tions. In a third report of Individuals exposed at Seveso, there were
changes observed 1n lymphocyte chromosomes from exposed workers which were
suggested to have risen from mutation 1n functional nucleolar organizing
regions; however, this bioassay has yet to be validated. In the only posi-
tive study, workers in a chemical plant were exposed to the herbicides,
2,4,5-TCPE and Buminol, as well as 2,3,7,8-TCOO. The participation of the
herbicides in the resulting Increase in the workers of chromosomal aberra-
tions cannot be excluded. At present, the data from experimental animals
and humans are too limited to designate 2,3,7,8-TCDD as a clastogenic agent.
In summary, a limited number of initial studies of the mutagenicity of
2,3,7,8-TCOO in bacteria reported positive results in S. tvphimurium strain
TA1532 In the absence of a mammalian metabolic activation system (Hussain et
a!., 1972; Seller, 1973). More recent attempts to repeat these results with
strain TA1532 or related strains have failed (Geiger and Neal, 1981; Gilbert
et al., 1980; McCann, 1978). These authors have also reported no Increase
In mutation rate when 2,3,7,8-TCDD was tested in the presence of a mammalian
metabolic activation system. In other In vitro assays, 2,3,7,8-TCDD has
produced a positive response In reversion to streptomycin independence in £.
sd-4 cells and questionable positive response with prophage induction
in £. coll K-39 cells (Hussain et al., 1972). Also, 2,3,7,8-TCDD has been
reported to be mutagenic in the yeast S. cerevisiae in both the in vUro
assay with S-10 and the host-mediated assay (Bronzetti et al., 1983).
Rogers et al. (1982) have also reported positive mutagenicity results in the
mouse lymphoma assay. In the E. coli studies, the poor survival of the
cells or the Interference of the vehicle solvent, DMSO, with the assay makes
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the evaluation of the studies difficult. WHh the data available. H Is not
possible to resolve the conflicting reports on the mutagenlc potential of
2,3,7,8-TCDD.
Overall, the data Indicate little potential for the Interaction of
2,3.7,8-TCDD with nucleic adds or the ability of 2,3,7,8-TCDD to produce
chromosomal aberrations. Kondorosl et al. (1973) demonstrated that 2,3,7,8-
TCDD did not react with RNA in vitro in the absence of a metabolic activa-
tion system, in vivo studies using radlolabeled 2,3,7,8-TCDD Indicated some
association of non-extractable label with RNA and DNA (Poland and Glover,
1979); however, the very low level of bound label observed suggest that the
"binding" may have been merely an artifact. Similar marginal data were
available on the clastogenlc effect of 2,3,7,8-TCDD. Although one In vivo
study 1n rats (Green and Moreland, 1975) failed to demonstrate any
treatment-related chromosomal aberration, a second study by the same authors
(Green et al., 1977) using a longer exposure period reported a small
Increase 1n the number of aberrations. In humans exposed to 2,3,7,8-TCDD
during the manufacture of 2,4,5-TCPE and Bumlnol, Czelzel and Klraly (1976)
reported an Increase 1n the number of chromosomal aberrations, while no
Increase was detected In Individuals exposed to 2,3,7,8-TCDD following an
Industrial accident 1n Seveso, Italy (Regglani, 1980; Mottura et al.,
1981). The studies of the clastogenlc effect of 2,3,7,8-TCDD were presented
with little or no experimental detail to assist 1n evaluating the merits of
the reports. The data available are too limited to Indicate whether
2,3,7,8-TCDD can Interact with nucleic adds or produce chromosomal
aberrations.
The differences among the results described above could be due to sev-
eral factors, such as treatment protocols, solubility problems, purity of
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the samples tested and the high toxlclty of 2,3,7,8-TCOD. This chemical may
be a weak mutagen, but because H 1s very toxic, the dose range for detect-
ing a positive genetic effect may be very narrow. Therefore, additional
experimentation Is necessary before any conclusive determination can be
made. Suggested further testing Includes the ability of 2,3,7,8-TCDD to
Induce forward mutations In mammalian cells In culture, additional yeast and
bacterial studies and the sex-linked recessive lethal test 1n DrosophHa.
Cardnogenlclty
Ep1dem1olog1ca1 Studies
Case Reports. Observations of an unusual occurrence of relatively rare
soft-tissue sarcomas were first made by Harden (1977). Of some 87 patients
seen from 1970-1976 at the Department of Oncology, University Hospital,
Umea, Sweden, seven Individuals with soft-tissue sarcomas were Identified.
All seven had had occupational exposure to phenoxy adds 10-20 years
earlier. The tumors were 2 lelomyosarcomas, 1 llposarcoma, 1 rhabdomyosar-
coma, 1 myxofIbrosarcoma and 2 additional sarcomas of which the hlstopathol-
ogy was uncertain but was probably a neurofIbrosarcoma 1n one and a rhabdo-
myosarcoma In the other. The clustering of this rare tumor type among these
patients prompted the author to suggest that ep1dem1olog1cal studies be done
to determine If exposure to phenoxy adds and the Impurities they contain
are related to the occurrence of soft-tissue sarcomas.
Zack and Susklnd (1980) reported the finding of a soft-tissue sarcoma
death 1n a cohort study of workers exposed to 2,3,7,8-TCDD 1n a trlchloro-
phenol process accident In NHro, West Virginia. This tumor, a fibrous
hlstlocytoma, was noted by the author as a rare event. . This study, referred
to as the NHro study, 1s discussed later.
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Cook et al. (1980) 1n a cohort mortality study of 61 male employees of a
trlchlorophenol manufacturing area, who acquired chloracne following a 1964
Incident, noted four deaths by the end of his study period, but one of the
four was 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, revealed no soft-tissue
sarcomas among 11 deaths that had occurred by 1976. But only 1 of these 11
was a malignant neoplasm.
In a discussion of the cohort studies of Zack and Susklnd, Cook, a third
unpublished study by Zack (1n which a llposarcoma was found), and a fourth
study by Ott et al. (1980), Honchar and Halperin (1981) noted 3 (2,9%)
soft-tissue sarcomas In a total of 105 deaths, compared roughly to 0.0754
deaths 1n U.S. males aged 20-84 years (ICD 171, 8th Revision, 1975)* Indi-
cating an unusual excess of such tumors. This may be somewhat of an under-
estimate due to the possiblHy that some soft-tissue sarcomas may be coded
to categories other than ICO 171. Separately none of the reported case
studies reported a significant excess of soft-tissue sarcomas. The number
of soft-tissue sarcomas noted by Honchar and Halperin was Increased by a
fourth when Cook (1981a) found a malignant fibrous hlstlocytoma after a
later review of the medical records from his earlier cohort study. Cook,
who was familiar with the earlier three cases, went on to say that frank
chloracne occurred previously 1n two cases of the 4 having a diagnosis of
*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.
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malignant fibrous histlocytoma. A third case diagnosed as a flbrosarcoma
worked In a trlchlorophenol (TCP) process area contaminated with 2,3,7,8-
TCOO. This Individual exhibited facial dermatitis, but no diagnosis of
chloracne was made. The last case was diagnosed as a Uposarcoma, and the
Individual had been employed earlier 1n a plant producing 2,4,5-T. Cook
noted that although chloracne was not reported, 1t could not be discounted.
He also noted that all four were 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.
Hardell and Eriksson (1981) discounted this hypothesis by citing that
only one of Hardell's seven cases exhibited chloracne prior to the appear-
ance of the soft-tissue saromcas, and that In his later case control study,
he found no difference in smoking habits between his cases and controls.
Moses and Selikoff (1981) reported discovering a fifth soft-tissue sar-
coma In a worker employed at the Monsanto Chemical Company at a time when
trlchlorophenol and 2,4,5-T were being produced. He died of a retroperi-
toneal neurogenic sarcoma (malignant schwanoma) in 1980 at the age of 58.
The employee, prior to his death, in a detailed occupational history said
that he was potentially exposed to these chemicals while he was a truck
driver, hauler and maintenance worker, but that he did not work In the pro-
duction of either chemical. He was a non-smoker and did not have a history
of chloracne.
Johnson et al. (1981) treated a father and son with soft-tissue sarcomas
(the 33-year-old son was diagnosed as having a 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-dlchlorophenol
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can be a precursor of 2,4-D and 2,4,5-T. The father had had prolonged expo-
sure prior to his disease. The son supposedly had a shorter latency,
according to the author. In neither case Is the follow-up time given.
Sarma and Jacops (1981) reported three cases of thoracic soft-tissue
sarcoma 1n Individuals who were exposed to Agent Orange while serving 1n
Vietnam. The diagnoses were fibrous hlstlocytoma, medlastlnal flbrosarcoma,
and a pleural/dlaphragmatic lelomyosarcoma. All three served 1n areas where
defoliants were used at the time. One was drenched with the material 1n one
spraying.
Bishop and Jones (1981) found two cases of non-Hodgk1n's lymphomas of
the scalp 1n a related clinical study of 158 employees of a pentachloro-
phenol manufacturing plant In Wales. Homologues of 2,3,7,8-TCDD occurred as
contaminants at up to 300 ppm at Intermediate manufacturing stages and 5 ppm
In 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 (1982) noted that of 123 male patients seen at his
clinic 1n Sweden with a recent diagnosis of non-Hodgk1n's lymphoma (NHL), 5
had cutaneous lesions as the only clinically detectable manifestation of
NHL. Four of the five had repeatedly sprayed large areas with phenoxy add
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 PO.001. Olsson and Brandt suggested that a relationship
C-118
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exists between cutaneous presentation of NHL and occupational exposure to
phenoxy acids, and believed their observations were similar to those of
Bishop and Jones.
Adding these case studies together, the total number of workers exposed
to phenoxy acids and/or chlorophenols Is small, but considering the rarity
of this cancer, It Is unusual that so many cases of soft-tissue sarcomas
have occurred. A Lancet editorial (Anonymous, 1982) calls this phenomenon
"disturbing." It Is suggestive of an association of cancer with exposure to
phenoxy adds and/or chlorophenols, and consequently with the dloxln Impuri-
ties found In these herbicides.
Soft-Tissue Sarcomas. Soft-tissue sarcomas {STS) constitute a collec-
tion of heterologous lesions that Include both malignant and non-malignant
tumors. Not all of them have their origin In primordial mesenchymal cells.
Some exceptions are tumors of peripheral nerves, and neuroectodermal tumors
which are classified as STS, but are derived from non-mesenchymal cells.
Classification, grading and staging of STSs Is difficult because of the
capacity of such cells to differentiate Into many different tissues. Fairly
precise histogenetlc classification of such tumors is 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 fol-
lowing six well-defined tissue complexes: fibrous tissue, tendosynovlal
tissue, adipose tissue, muscle, vessels and bone. STSs can be Induced in
any of these tissue types (Hajdu, 1983). The classification of STSs for
cause of death coding in 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
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and other soft-tissue" (ICD 171). Lymphosarcomas, retroperltoneal sarcomas
and extra skeletal STSs of the bone are coded elsewhere. In some Instances.
1f site Is mentioned, H 1s coded to the site, e.g., lelomyosarcoma of the
stomach (ICD 151.9), neuroflbroma of the chest wall (215.4).
Questions have been raised concerning the appropriateness of lumping
together malignant tumors of different sites and tumor types 1n order to
derive risk estimates. It may not be scientifically appropriate to do so
because an elevated risk cannot readily be ascribed to a particular site or
type as 1s usual with most carcinogenic chemicals and substances. Unfortu-
nately, with respect to STSs, tallies of deaths due to STSs of particular
sites and types are not maintained separately by the vital statistics
offices because of their rarity, and therefore, H 1s Impossible to derive
risk estimates for particular types at given sites. Altogether, -2000
deaths/year can be attributed to STSs 1n the United States, most of which
are coded to ICD category 171 for purposes of developing Incidence and mor-
tality rates for this composite cause. Within ICD 171, Individual types
that may be correlated with exposure cannot be Identified.
A separate problem that potentially could arise from assigning STSs to
multiple ICD codes 1s that Incidence and death rates due to STSs may be
underestimated. Furthermore, risk estimates derived from dividing observed
cases (or deaths) by expected cases (or deaths) could be biased upward.
This could happen when observed STSs classified to ICD codes other than ICD
171 are lumped together while expected STSs are based upon ICD 171 only.
Thus, action of this sort, especially with respect to cohort studies of
Individuals exposed to dloxln-contalnlng herbicides and/or chlorophenols,
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could lead to risk estimates that may be biased upward by the Inclusion of
STSs in the observed category for risk estimation that should be coded to
categories other than 171.
Prompted by clinical observations over a 7-year period of malignant sar-
comas In seven men with previous occupational exposure to phenoxyacetic acid
herbicides (Hardell, 1977), researchers at the Department of Oncology, Uni-
versity Hospital, Umea, Sweden, initiated epidem1olog1c studies to test the
hypothesis of an etiologic association (Hardell and Sandstrom, 1979). The
investigators elected to conduct case-control studies, a type of epidemic-
logic research particularly well suited for rare diseases with long periods
of induction (Cole, 1979). Cases were defined as male patients with sar-
comas of soft connective tissue, such as smooth muscle (leiomyosarcoma) and
fat (liposarcoma). The distribution of tumor types in the two studies 1s
shown in Table 12. Sarcomas of harder connective tissues, such as bone and
cartilage, were excluded. According to the authors, these tumors may have a
different etiology and there occurred a different age-distribution in pat-
ients with these tumors as compared to that of STS (Hardell, 1983).
Two case-control studies were conducted, the first in northern Sweden
(referred to below as Study A), and the second in the southern part of the
country (Study B). The frequencies of exposure to the substances of primary
Interest are shown in Table 13. In the north, occupational exposure to
phenoxyacetic acids took place in both forestry and agricultural work. In
the south, these exposures were predominantly agricultural. The phenoxy-
acetic acids to which exposure occurred consisted predominantly of 2,4,5-T
and 2,4-D in both studies. Exposure to 2,4,5-T in the absence of 2,4-D was
rarely reported in either study. Exposure to chlorophenols, which contain
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TABLE 12
Distribution of Tumor Types In Two Case-Controls Studies
of Soft-Tissue Sarcoma
Diagnosis
Tissue of Origin
Percent of Cases
Study Aa Study Bb
(n=52) (n=110)
Lelomyosarcoma
Fibrous hlstlocytoma
Llposarcoma
Neurogenlc sarcoma
Anglosarcoma
Hyxosarcoma
Flbrosarcoma
Other sarcomas
Total
Smooth muscle
Subcutaneous connective
tissue
Fat tissue
Nerve tissue
Blood vessels
Primitive connective
tissue
Fibrous tissue
30
17
14
10
8
6
4
11
100
23
25
6
4
2
8
8
24
100
aUnpub!1shed Information supplied by Hardell to EPA (Harden and Sand
strom, 1979)
bEr1ksson et al., 1979, 1981
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TABLE 13
Exposure Frequencies In Two Case-Control Studies of Soft-Tissue Sarcoma
Substance(s)
Phenoxyacetlc adds only
Chlorophenols only
Both
Total
Study
Cases
(n=52)
23.1
11.5
1.9
36.5
Percent
A
Controls
(n=206)
6.3
2.4
0.5
9.2
Exposed
Study
Cases
(n=110)
12.7
10.0
0
22.7
B
Controls
(n=219)
2.3
3.6
0
5.9
'Sources: Study A, Hardell and Sandstrom, 1979; Study B, Eriksson et al.,
1979, 1981
C-123
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chlorinated dlbenzodloxln 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 adds, only 2,4,5-T 1s known to be contaminated
with 2,3,7,8-TCDD. In Study B, a relative risk of 4.9 (90% confidence
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 14.* Studies A and B Indicate a risk of developing STSs
among workers exposed to phenoxyacetlc adds only, chlorophenols only, or
phenoxyacetlc adds and/or chlorophenols several times higher than among
persons not exposed to these chemicals. In each comparison, the point esti-
mate of relative risk 1s high and unlikely to have resulted by chance alone.
Since IHtle 1s known of the etiology of STSs, the consideration of con-
founding 1n these studies was largely a hypothetical matter. The authors
prevented the effects of age, sex, and place of residence as possible con-
founding factors in the selection of controls.t Because of the high cor-
relation between exposure to the substances of Interest and employment 1n
agriculture and forestry, a reasonable hypothesis could be developed that
some other unknown factor present In these occupations was responsible for
the elevated relative risks.
*In the analyses considering phenoxyacetlc adds only and chlorophenols
only, persons exposed to the other categories of substances were excluded.
In Study A, the three persons exposed to both chlorophenols and phenoxy-
acetlc adds were Included 1n all comparisons.
tControls were matched Individually to cases on the basis of these factors.
Unmatched analyses are presented In Table 24 for the sake of simplicity.
The matched-method relative risks for exposure to phenoxyacetlc acids and/or
chlorphenols were 6.2 (P<0.001) In Study A and 5.1 (P<0.001) in Study B.
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TABLE 14
o
I
cv>
Ul
Relative Risks of Soft-Tissue Sarcoma 1n Relation to Exposure to
Phenoxyacetlc Adds and Chlorophenols 1n Two Case-Control Studies
Relative r1skb
90% Confidence Interval0
Significance leveld
Phenoxyacetlc Adds
Only
Study A Study B
5.3 6.8
2.7-10.2 3.1-14.9
<0.001 <0.001
Chlorophenols
Only
Study A Study B
6.6 3.3
2.8-15.6 1.6-7.0
<0.001 <0.005
Phenoxyacetlc Adds
and/or
Chlorophenols
Study A Study B
5.7 4.7
3.2-10.2 2.7-8.3
<0.001 <0.001
Source: Study A, Hardell and Sandstrom, 1979; Study B, Eriksson et al., 1979, 1981
bUnmatched odds ratio
cTest-based method of M1ett1nen, 1976
square statistic, no continuity correction, one-tailed test
-------
To test this hypothesis, 1t 1s possible to calculate the relative risk
1n relation to the phenoxyacetlc add exposure In Study B, restricting the
analysis to workers within agriculture and forestry. The result Is a rela-
tive risk of 6.1 (90X confidence Interval 2.4-15.4). This finding strongly
suggests that some confounding risk factor for STS distributed throughout
agriculture and forestry work was not responsible for the overall Increase
In risk found 1n relation to phenoxyacetlc add exposure.
Because exposure histories were obtained by means of questionnaires and
Interviews, the major potential source of bias 1n these studies stems from
the need to rely upon the personal recollection of cases and controls for
exposure histories. The published papers Indicate that the researchers paid
a great deal of attention to this potential problem and state that they took
all reasonable precautions to avoid H 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
chlorophenols and comparing them to unexposed persons 1n other occupations
was 0.9 (90% confidence Interval 0.3-2.4) 1n Study B. This suggests that a
great deal of recall bias was not present (Axelson, 1980).
In an update of his earlier study, Eriksson et al. (1981) obtained
Information on the effects of phenoxy acids In the absence of the Impuri-
ties—polychlorlnated d1benzod1ox1ns and dlbenzofurans. The risk ratio
given exposure to phenoxy adds free of polychlorlnated dlbenzodloxlns and
dlbenzofurans equaled 4.2 based upon 7 out of 14 respondents who Indicated
exposure to phenoxy add herbicides. When consideration was given to only
phenoxy acids that contain such Impurities, the risk was 17.0. A descrip-
tion of the basis for the determination of exposure or non-exposure to
dloxlns Is not well presented 1n this study.
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The author concluded that exposure to phenoxy acids and chlorophenols
"might oori'J'Mute a ri-A factor in Uic tlovr>"Uy ,-nt uT .nft-Mssue sarcomas."
Ihis risk relates nol. only l.o ?,4,!>-tr1chlorophenoxy adds contalniny -1'oxln
Impurities, but to other phenoxy acids as well. Some doubt was raised con-
cerning the possible mlsclasslf1cat1ons of Individuals who were exposed to
phenoxy acids free of polychlorInated dlbenzodloxlns (I.e., 1n particular,
"dlchoroprop" in the Eriksson study). In a recent communication from
Hardell (1983), Eriksson recalculated his risk estimates after reclasslfylng
his dlchoroprop-exposed cases and controls Into the category of probable
exposure to phenoxy acids contaminated with polychlorlnated d1benzod1ox1ns
and removing them from the non-exposed category. His new estimates were 4.0
based upon 5 out of 8 respondents who were exposed to phenoxy adds free of
contamination and 10.9 for those exposed to contaminat<•(.! jihonoxy a<'^ TVI:'
first estimate was of only borderline significance utilizing the Mletlnen
test based statistic, thus, weakening any finding that the risk of STS
extends to phenoxy acids free of dloxln.
In a cohort mortality study (Cook et a!., 1980a) of 61 males Involved 1n
a 1964 chloracne Incident, employees 1n a trlchlorophenol manufacturing area
were found to have chloracne due to skin absorption of 2,3,7,8-TCOO. The
skin lesions characterizing chloracne ranged from a few comedones on thr-
back of one employee (predating his entry Into the process area where expo-
sure 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 measure-
ments of dloxln 1n the air were provided by the author. On the other hand,
the author did subjectively divide the cohort of 61 males Into potentially
"high" vs. "low" exposure by place of work based upon dermal exposure,
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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 vs. 1.6 expected. The
remaining death was hypersensitive heart disease vs. 3.8 expected. The
hlstopathologic causes of death of the three cancer victims were 1) fibro-
sarcoma, 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."
Cancer mortality is slightly elevated in this cohort despite its rela-
tive low sensitivity, the lack of a sufficient latent period, and the pres-
ence of the healthy worker effect. This increased mortality was not attrib-
utable to any particular cause. 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 chlor-
acne." The authors furthermore concluded that the latency period was suffi-
cient to "allow the identification of a potent human carcinogen," since it
"exceeded 14 years." Orris (1981) criticized this conclusion with a refer-
ence to the Hardell and Sandstrom (1979) study in which the authors noted
that the latent period for soft-tissue tumors may be as long as 27 years and
for many, over 14 years. Cook (1981b) countered that the Hardell and
Sandstrom (1979) conclusions were based upon questionable data in that the
self-administered questionnaires used in that study provided neither valid
quantitative nor qualitative estimates of exposure. Therefore, it could not
be used to determine latent periods. In any case, Hueper and Conway (1964)
noted that the latent period for the chemical induction of solid malignant
tumors in man exceeds 15 years and Is probably <30 years.
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Although the Hardell and Sandstrom (1979) study has some deficiencies,
the Cook et al. (1980a) study provides little evidence to support the
premise advanced by the authors that dloxln "cannot be considered to be a
potent human carcinogen with organ or tissue specificity." There 1s a dis-
tinct likelihood that the latent period for the development of STSs and
related tumors due to exposure to dloxln may not have been achieved within
the 14-year follow-up period specified In the study. Furthermore, a much
larger cohort may be needed 1n order to detect a significantly Increased
cancer risk.
Smith et al. (1982b) conducted a case-control study of 102 males Identi-
fied from the New Zealand Cancer Registry as having STSs (ICO 171) between
1976 and 1980." For each case, three controls each with another form of can-
cer were matched by age and year of registration. The selection of cancer
controls from the same registry was done to eliminate recall bias and/or
Interviewer bias. The distribution of hlstological types In the cases 1s
given In Table 15. The Interview to elicit occupational history Information
was accomplished via the telephone either with the next of kin to the pat-
ient or the patient himself if he was well enough. Anxiety was alleviated
by the mailing of a letter prior to the Interview, the purpose of which was
to Inform the person of the Intention of the interviewer to ask some ques-
tions about his occupational history.
Apparently, the questions asked were not specific enough to Identify
definite exposure to phenoxy herbicides and/or chlorophenols. The authors
asked only about current occupation or last occupation if retired. Compari-
sons 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
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TABLE 15
Distribution of Hlstologlcal Types of Soft-Tissue Sarcomas
Cell Type
Fibrosarcoma
Llposarcoma
Rhabdomyosarcoma
Lelomyosarcoma
Malignant H1st1ocytoma
Other
Unspecified
Total
Number of Cases
25
20
9
7
6
22
13
102
Percent
24
20
9
7
6
21
13
100
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phenoxy herbicides and chlorophenols. Expected cases for each major occupa-
tional classification were derived based upon the occupational distribution
of the controls. The authors found no unusual excess of cases of STS In any
major occupational category. In agriculture, forestry and fishing, 14 cases
were observed vs. 14.0 expected. In laborers, production and transport
workers, 35 cases were observed vs. 37.0 expected. A further breakdown of
these two broad categories Into finer subcategorles within the major occupa-
tional categories revealed no significant excesses. The study, however, 1s
not useful In assessing the risk of STS from exposure to phenoxy adds
and/or chlorophenols for several reasons. First, as was pointed out by the
authors, but subsequently dismissed by them as having not much of an Influ-
ence, 1s the possibility that switching from one major occupational category
to another over the time period Involved for latent conditions to manifest
themselves could Introduction a negative bias Into any estimates of relative
risks. The latency for STS Is felt 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 In the "first 20 Interviews" In which a change could be noted
Is not necessarily Indicative of fidelity to the same job over long periods
1n all 408 cases and controls. Information Identifying a switch may be
lacking In those cases and controls 1n which a switch did occur only because
the switch resulted 1n separation of the earlier work history from the lat-
ter. Besides the "first 20 Interviews" where a change could be noted 1s not
representative of the entire cohort 1n any case.
Furthermore, the authors do not know absolutely that any of their cases
and controls were exposed to phenoxy adds and/or chlorophenols since
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apparently no effort was made to confirm "potential" exposures. Only dif-
ferences In occupational classification were noted where "potentially" cases
or controls could have had exposure to the dloxln-contalnlng herbicides. It
was pointed out that the risk estimates noted do not "preclude" the possi-
bility that an association may be found 1n this study when the cases and
controls (or surviving kin) are Interviewed for chemical spraying at a later
time. The authors themselves conclude that the preliminary study results
"should not be taken as substantial evidence against the hypothesis that
phenoxy herbicides and chlorophenols may cause human cancer."
It should be noted that the distribution of tumor types differed con-
siderably from the Hardell and Eriksson study to the Smith study. Lelomyo-
sarcomas, malignant hlstocytomas, neurogenlc sarcomas and myxosarcoma seem
to predominate In the Harden and Eriksson study, whereas fIbrosarcomas and
Uposarcomas appear prominently 1n the Smith study. More attention should
be devoted to the study of the dlsbrlbutlons of STS types 1n registry data
everywhere in order to determine if such variations 1n the reporting of STS
types are random occurrences. It is possible that the cancer effect of
exposure to phenoxy herbicides may be narrowed to just certain types of
STSs, the predominant ones in 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-kln or patient if
alive. Telephone interviews were conducted by only one Interviewer who had
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no knowledge of the patients cancer history and were completed on 80 cases
and 92 controls. Because some 32 potential cases (14 Ineligible) and 20
controls were excluded or lost from the study for various reasons, 1t raises
a question whether control of confounding by age and year of registration
was maintained In the final group of 172 cases and control Included In the
analysis. Presumably the corresponding "matched" case or control to each of
the 52 lost members of the total study group were not excluded.
Patients were classified as having had potential exposure to phenoxy-
acetlc acids 1f they had definite, probable or possible exposure to phenoxy-
acetlc add through spraying or hand contact. The actual chemical was
Identified only In some Instances. The authors concluded In all remaining
situations that 1f the member sprayed "gorse" and/or "blackberries" this was
tantamant to potential exposure to phenoxyacetlc add. Smith calculated
elevated but non-significant relative risks of exposure to phenoxyacetlc
acid ranging from 1.3 In those Individuals who were "probably exposed" for a
minimum of 5 days not In the previous 10 years prior to cancer registration
to 1.6 In Individuals "probably exposed" for a minimum of 1 day not In the
previous 5 years prior to cancer registration. When risk ratios were calcu-
lated after stratifying by year of birth and whether or not the patient or a
relative was Interviewed, the rates Increased to 1.7 (from 1.6) 1n the
latter and 1.4 (from 1.3) 1n the former calculation, although still nonsig-
nificant. It would be of Interest to repeat the above calculations exclud-
ing only those with potential exposure occurring only within the 15-year
period just prior to cancer registration. Furthermore, the categories of
exposure "probably or definitely" exposed for >1 day or. even 5 days raises a
question whether any of the cases or controls could really be said to have
ever come In contact with enough phenoxyacetlc add to justify such a
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designation. It could be that, In fact, potentially exposed Individuals 1n
New Zealand have had little or no contact with the herbicide.
The authors did conclude that the finding of a relative risk of 1.7 1n
Individuals with >1 day exposure not 1n the last 5 years cannot be entirely
discounted. But then the authors state that 1f exposures of >5 days prior
to 10 years before cancer registration are not Included they would expect an
Increase, and since they do not see an Increase, there Is no evidence of a
"real causal link." One might question whether this 1s a suitable criterion
for providing evidence of a causal association. Perhaps a more valid group
for study would be one where the potential exposure was considerably longer
than "5 days" and >15 years prior to Initial cancer registration. As kind
of a subtle justification for the finding of no significant risk 1n workers
exposed In phenoxy adds, the author alludes to the fact that there are
currently 500 full-time workers registered In New Zealand who do full time
ground spraying and altogether some 2000 workers who were at some time
professionally Involved In phenoxyacetlc add herbicide spraying from the
air or ground with exposure "very much greater" than that of patients In
this study. This kind of argument has appeal 1f these workers could be
shown to have had their exposure sufficiently far 1n the past that latency
considerations could be adequately addressed. However, the real question
again remains how much real exposure did those patients 1n the study really
have 10-15 years earlier, and 1n what numbers. The author remarks that 1t
is surprising that he found no STS victims who had ever worked full-time 1n
phenoxyacetlc acid herbicide spraying. Perhaps they have not yet been 'ob-
served for a long enough period. However, as was pointed out by the author,
the findings do not support the hypothesis that exposure to phenoxyacetlc
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acid herbicides causes STS. But neither do they support a negative finding
without better documentation regarding actual exposure and time of actual
exposure.
Pazderova-Vejlupkova et al. (1981) studied 80 workers Involved In the
production of 2,4,5-sodlum trlchlorophenoxyacetate and butylester of tr1-
chlorophenoxyacetlc add who subsequently became 111 from exposure to
2,3,7,8-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 porphyrla cutanea tarda. Chief chemical signs were
metabolic disturbances, pathologically elevated liplds with abnormalities 1n
the Upoproteln spectrum, and "pathological" changes In glucose tolerance.
Other symptoms noted were biochemical deviations consistent with "a mild
liver lesion," light steatosls, perlportal Hbrosls or activation of Kupffer
cells, or nervous system focal damage (peripheral neuron lesion In lower
extremetles). Altogether six patients were reported to be deceased during
this 10-year period, 2 from bronchogenlc carcinoma, 1 from cirrhosis, 1
atherosclerosis preclpue cerebl and 2 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 six deaths that occurred during the
10-year observation period 1n the 55 cannot be construed to be associated
with exposure to the 2,4,5-T. Because of the small number of cases and the
short follow-up period, nothing can be said concerning the association of
exposure with cancer, especially specific types of cancer such as STS or
non-Hodgk1n's lymphoma.
C-135
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Rllhlmakl et al. (1982, 1983) studied a cohort 1926 herbicide appli-
cators formed In 1972 from personnel records of four Finnish employers
(I.e., the Forestry Authority, Highway Authority, State Railways and a
state-owned electric power company). Chlorinated phenoxyadds had been used
since the 1950's In Finland for spraying. They constituted 2:1 mixtures of
emulsified esters of 2,4-D and 2,4,5-T dissolved 1n water. Analyses from
old herbicide formulations dating back to the 1960's revealed that these
mixtures contained 0.1-0.9 mg/kg of 2,3,7,8-TCDO).
This cohort of male workers was exposed a minimum of 2 weeks during at
least one growing season from 1955-1971. Follow-up continued 9 years
through 1980 for mortality but only until 1978 for morbidity. Fifteen
Individuals could not be traced by 1980. Expected deaths were generated
based upon cause- and age-specific national Finnish death rates for 1975.
Expected cases were similarly calculated based upon national Incidence rates
of 1975.
By 1980, 144 deaths had occurred vs. 184.0 expected, a deficit of 22% 1n
observed mortality. Only 26 cancer deaths had occurred vs. 36.5 expected, a
29% deficit. The authors separated out "natural" deaths from the total.
The observed residual deaths equaled 39 while the expected deaths equaled
28.7. This excess was of borderline significance. The authors also con-
sidered 10-year and 15-year latent periods. Even after 15 years, the defi-
cit of deaths continued to manifest itself both 1n categories of all causes
and total cancers; 35 observed vs. 53.6 expected and 5 observed vs. 11.3
expected, respectively. Similarly, the 7-year follow-up of cancer morbidity
revealed 26 cases of cancer vs. 37.2 expected. After.10 years latency, 16
cancer cases were observed vs. 20.1 expected. None of the 26 cancer deaths
C-136
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or 26 cancer cases were of the STS or lymphoma type. (However, only 0.1 STS
and 0.5 lymphomas were expected.) In no Instance was cancer of any site
significantly elevated.
The authors note that this unusual deficit of mortality and morbidity of
between 70-82% (even after 15 years from Initial exposure) Is probably a
consequence of the "healthy worker effect" In that only able-bodied and
healthy Individuals were selected Into the Industry. The fact that the
cohort was assembled In 1972 from records of persons who were exposed as
early as 1955 (17 years prior) raises the likelihood that In 1972 a "survi-
vor" population remained (45 deaths prior to 1972 were eliminated from the
cohort) that was relatively healthy. Furthermore, the unusually large num-
ber 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 note 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 Is needed in 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 is too small." It was suggested that more
valid conclusions could be made only with the passage of time.
Recently, the Michigan Department of Public Health (1983b), produced an
ecological study of soft and connective tissue cancer mortality rates In
Midland and other selected Michigan counties. They found that mortality
C-137
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rates for this cause were 3.8-4.0 times the national average for the periods
1960-1969 and 1970-1978, respectively, for white females In Midland. These
estimates are based upon 5 deaths and 7 deaths, respectively, and are listed
In Table 16. 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. It Is unlikely to be a chance happen-
ing. At the same time the age-adjusted male and female cancer mortality
rates for Midland were below that of the State of Michigan in the period
1970-1979. Midland County 1s the home of a major chemical company that pro-
duced phenoxyacetlc add herbicides until recently. The authors state that
a detailed review of death certificates, hospital records, residency and
occupational histories of the 20 male and female cases revealed no "common-
alities" suggesting a "single causative agent" although a majority or their
spouses had worked at this chemical facility. They recommend that a case-
control study should be Instituted to evaluate possible Influences, such as
lifestyle, occupation or location of residence on the risk of STS.
In a separate review of the ep1dem1olog1cal evidence for STS from expo-
sure to 2,4,5-T-conta1n1ng herbicides, the United Kingdom Ministry of Agri-
culture, Fisheries and Food (1983) concluded that there was no evidence ,o
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 too readily discounts the
positive studies of Hardell and Eriksson as being biased, and 1t makes no
reference to the later validity study by Hardell (1981) of his own work
utilizing colon cancer controls (see Section on Malignant Lymphoma). In
this report Hardell effectively answered these early criticisms that were
reiterated by the British 1n their report. At the same time, the British
C-138
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TABLE 16
Midland County Soft and Connective Tissue Cancer Deaths 1960-1981*
o
I
OJ
Identlf Oration
Year of
Death
1961
1963
1964
1968
1969
1970
1970
1974
1976
Sex
F
F
F
F
1-
F
F
F
F
Age
24
75
51
37
45
59
56
1
77
Type
Hemanglosarcoma
Llposarcoma
Leiomyosarcoma
Llposarcoma
Flbrosarcoma
Leiomyosarcoma
Kaposl sarcoma
Flbrosarcoma
Leiomyosarcoma
Rhabdomyosarcoma
Llposarcoma
Type of
Primary Site
Face
Right gluteal
Uterus
Spine
Right thigh
Uterus
Right leg
Right thigh
Abdominal wall
Inguinal area
Right thigh
Mallqnancy
Metastases Month and Year
Diagnosed
Skull and upper lobe
of lung
Unknown
Widespread
Lungs, pelvis
Lung, liver
Adrenal gland and skin
Lymph nodes
Spine
Lung
Unknown
Buttock, lung, rib,
5-58
Unknown
11-63
1-66
10-68
8-68
1960
1967
8-73
12-74
1970
64
Leiomyosarcoma
Left knee
Liver, lymph nodes,
lung, bone
7-70
-------
TABLE 16 (cent.)
Ident
Year of
Death
1978
1978
1979
r> 1962
i
1967
1967
1969
1971
1972
1976
1f1cat1on
Sex
F
F
F
M
M
M
M
M
M
M
Age
26
88
27
63
77
20
32
76
89
53
Type
Rhabdomyosarcoma
Flbrosarcoma
Lelomyosarcoma
Rhabdomyosarcoma
Mesothelloma
Rhabdomyosarcoma
Llposarcoma
Lelomyosarcoma
Lelomyosarcoma
Flbrosarcoma
Type of
Primary Site
Rectum
Right cheek
Left thigh
Left lower leg
Lung
Pharynx
Left arm
Small
Intestine
Retro-
perl tonal
region
Per1t1oneum
Malignancy
Metastases
Lung, neck, Inguinal
region
Facial area
Lung
Lung and right outer
chesfwall
Lung, peritoneum and
diaphragm
Per1orb1tal area and
1 1 ver
Perineum and buttock
Liver
Hepatic system
Lung, 11 ver
Month and Year
Diagnosed
6-76
6-78
3-78
8-61
6-67
1-67
6-64
10-69
7-72
3-75
*Source: Michigan Department of Public Health, 1983b
-------
report appears to put undue emphasis on non-positive studies that do not
demonstrate a risk, although most of them have methodological limitations
(I.e., low power, Insufficient latency and Inappropriate study method). In
short, the British review appears to be overly optimistic about the safety
of 2,4,5-T herbicides.
In summary, the associations reported in the two Swedish soft-tissue
sarcoma studies are great enough to make it unlikely that they have resulted
entirely from random variation bias or confounding, even though the possi-
bility cannot be dismissed that bias or confounding was present. Therefore,
the studies provide a strong suggestion that phenoxyacetic acid herbicides,
chlorophenols or their impurities are carcinogenic in humans.
Malignant Lvmphoma. A separate series of clinical observations at the
Department of Oncology in Umea, Sweden (Harden. 1979), led the researchers
to conduct a case-control study of malignant lymphoma In relation to phen-
oxyacetic acid, chlorophenols, and other organic compounds (Hardell et al.,
1980, 1981). Approximately 33% of the cases in this study were patients
with Hodgkin's disease; the remainder of the lymphomas were non-Hodgkin's
forms.
This study employed essentially the same methods and produced results
closely comparable to these from the STS studies: statistically significant
5-fold to 6-fold relative risks in relation to phenoxyacetic adds and
chlorophenols. In addition, an elevated relative risk was found in connec-
tion with exposure to organic solvents, such as benzene, trichloroethylene,
and styrene. In the published report, the methods and results were Incom-
pletely documented, especially the possibility of confounding by exposure to
the organic solvents.
C-141
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In the update of the earlier 1980 study. Harden et al. (1981), utiliz-
ing the same basic data source, found that 36.1% of the cases had been
exposed to phenoxy herbicides or chlorophenols, while only 9.6% of their
controls were so exposed. The estimated relative risk was 6.0 when matching
was considered and 5.3 when matching was eliminated. When cases and con-
trols who were exposed to chlorophenols only were excluded, the relative
risk of lymphoma from phenoxy adds alone was 4.8 (95% C.I. 2.9-8.1). On
the other hand, if phenoxy adds are excluded and consideration Is given to
just chlorophenols (which Includes combined exposure to phenoxy adds and
chlorophenols), then the relative risk equaled 4.3 (95% C.I. 2.7-6.9). The
author further subdivided this group Into "low-grade" vs. "high-grade" expo-
sures to chlorophenols. A continuous exposure of not more than 1 week or
repeated Intermittent exposures totaling not more than 1 month was classi-
fied as low-grade. The relative risk for high-grade exposure was 8.4 (95%
C.I. 4.2-16.9), while that for low-grade exposure equaled 9.2 (95% C.I.
1.6-5.2). If exposure to organic solvents 1s examined, given that cases and
controls exposed to only phenoxy adds and/or chlorophenols were excluded
except for combined exposure to organic solvents, H 1s found that high-
grade and low-grade relative risks were 2.8 (95% C.I. 1.6-4.8) and 1.2 (95%
C.I. 0.5-2.6), respectively. However, the author notes that exposure to
phenoxy acids and high-grade organic solvents (exposure to chlorophenols
excluded) produced a relative risk of 11.2 (95% C.I. 3.2-39.7) based upon a
few cases and controls with exposure to both. The authors concluded that
"exposure to organic solvents, chlorophenols and/or phenoxy adds consti-
tutes a risk factor for malignant lymphoma."
C-142
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This latter study Is still subject to the same methodological criticisms
to which the earlier study was subjected. Chief among those 1s the possi-
bility of observational and/or recall bias creeping Into the responses that
are elicited from self-administered questionnaires on kind and length of
exposure. Secondly, confounding by exposure to potentially carcinogenic
organic solvents and other agents could have had an effect, although the
author assures the reader that they did not.
Other research has tentatively suggested that lumberjacks may be at
Increased risk of lymphoma (EdUng and Granstam, 1979). The NHro study
found three deaths from cancers of the lymphatic and hematopoletlc system,
against only 0.88 expected (P = 0.06, one-tailed Polsson test).
The lymphoma case-control study (Hardell et a!., 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 methodologlc flaw 1n
all these studies.
The two Swedish case control studies on STSs and a later case control
study of malignant lymphoma {Hardell et al., 1981) were subjected to a
validity analysis with respect to the assessment of exposure by Hardell and
Eriksson (1981). To answer the question raised regarding the recall of
occupation 1n a forestry/agriculture job, secondary to the recall of expo-
sure to phenoxy adds and/or chlorophenols, the cases and controls were
divided Into three groups: those who worked their entire time since 1950 In
an agriculture/forestry job, those who worked some time In an agriculture/
forestry job but not exclusively, and the remainder who never worked 1n a
forestry/agriculture job. The study found that the risk ratio was still 8.2
for STS 1n exclusively agriculture/forestry workers who were exposed to
phenoxy adds compared to workers found In other occupations having no
apparent exposure to phenoxy acids or chlorophenols. Even when comparing
C-H3
-------
phenoxy acid and/or chlorophenol exposed agricultural/forestry workers
exclusively with non-exposed agricultural/forestry workers, the risk ratio
was still 7.1. This argument seems to answer effectively questions regard-
Ing recall of occupation secondary to exposure.
On the other hand, the relative risk remains 5.4 when comparing phenoxy
acid and/or chlorophenol exposed workers exclusively In occupations other
than agriculture/forestry with non-exposed workers in those same occupa-
tions, thus, suggesting the presence of either recall bias or still another
occupation with potential exposure to phenoxy adds and/or chlorophenols
(Table 17).
When woodworkers are separated out (possible exposure to chlorophenols
1n treatment of wood) the risk ratio becomes 9.7 (Table 18). These data
suggest the presence of some recall bias.
Another focus of this study was to determine If observational bias on
the part of the investigators could explain the significantly high risk
estimates. To answer the question, the study compared the exposure data
derived from the Interviewee's returned questionnaires only with the com-
bined Information from both the phone interviews and questionnaires. The
study found no substantial differences 1n the frequency of reporting
exposure.
Still a third consideration of possible bias Involves recall of exposure
to phenoxy acids and/or chlorophenols because of subject knowledge of having
cancer 1n the cases versus no knowledge of cancer in the referent popula-
tion. The study chose as a referent group for the 52 STS cases (Harden and
Sandstrom, 1979) and the 169 malignant lymphomas (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.
C-144
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TABLE 17
Other Occupations (Minus Forestry/Agriculture)*
Group Phenoxy Acids/Chlorophenols Non-exposed
Cases n 68
Referents 5
RR = 5.4 X' = 11.01 (P<0.01)
*Source: Hardell and Erlkkson, 1981
C-145
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TABLE 18
Other Occupations (Minus Forestry/Agriculture/Woodworkers)*
GPOUP Phenoxy Aclds/Chlorophenols' Non-exposed
Cases h
4 66
Referents -.
1 160
RR = 9-7 X* = 5.98 (P<0.05)
*Source: Hardell and Erlkkson, 1981
C-146
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Utilizing a Mantel-Haenszel rate ratio, the study found the risk of
exposure to phenoxy adds remaining significantly high at 5.5 and to chloro-
phenols 5.4 In the STS cases compared to the colon cancer controls. Simi-
larly, with the malignant lymphomas, the Identically derived risk ratios
remain significantly high at 4.5 with respect to phenoxy adds and/or
chlorophenol exposure 1n the cases, hence, the study concludes, no "sub-
stantial observational bias" exists. If the study 1s assuming that recall
bias was and Is the same as observational bias, then such a conclusion 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 possibility that
recall bias can still be present In their data for other reasons. Hardell
refers to an Intense "debate about phenoxy adds and their presumptive risk"
1n Sweden at the time the colon cancer study was conducted. But, there Is
no reason to think that colon cancer victims would assume their disease was
brought about from exposure to dloxln containing chemicals 1f no connection
was suggested.
It seems plausible that STS and/or non-Hodgkln's lymphoma patients would
either learn at the time of their diagnosis that exposure to dloxln contain-
ing chemicals was the likely cause of this rare type of tumor or quickly
learn from other sources, such as the news media, that exposure to herbi-
cides containing dloxln could cause their rare form of cancer. Whereas,
colon cancer victims (a rather common form of cancer) would not necessarily
be led to believe that exposure to the same dloxln containing chemicals
caused their disease. Hence, It 1s not difficult to Imagine that such
unusual victims of cancer could better "remember" exposure to such chemicals
than could colon cancer patients.
C-147
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Therefore, although this study may explain any biases Introduced from
secondary recall of occupation, observational bias Introduced from the tele-
phone Interviewer and recall bias due to subject knowledge of cancer, H
does not adequately answer questions of recall bias Introduced through the
acquired awareness on the part of the victim of STS or non-Hodgkln's lym-
phoma that his condition may have been caused by exposure to dloxln contain-
ing herbicides.
Stomach Cancer. Studies of two of the oldest cohorts of workers known
to have been exposed to phenoxyacetlc add herbicides and/or 2,3,7,8-TCOO
report stomach cancer mortality rates significantly higher than expected,
but the results 1n each study were based on small numbers of deaths. In one
study (Axelson et al., 1980), 348 Swedish railroad workers with at least 46
days of herbicide exposure between 1955 and 1972 were followed through
October 1978. The workers were grouped on the basis of their primary herbi-
cide exposures: those primarily exposed to phenoxyacetlc acids (2,4-D and
2,4,5-T) only, to amitrole (aminotriazole) only, and to both types of herbi-
cides. After a 10-year latency was achieved, 3 stomach cancer deaths were
observed vs. 0.71 expected (P<0.05). None were attributable to amitrol
alone, but two were assigned to phenoxy acids alone while the remaining
stomach cancer death occurred 1n a worker exposed to both amitrol and
phenoxy acids in combination. The excess was more pronounced (3 observed
vs. 0.57 expected, PO.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).
C-148
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Axelson et al. (1980) also notes an excess 1n total "tumors" after 10
years latency as well (15 observed vs. 6.87 expected, PO.005). This 1s
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" 1n Sweden are
analogous to malignant neoplasms 1n the United States. The author states
that no specific type of tumor predominates and no breakdown by tumor type
Is provided.
The other study showing Increased stomach cancer mortality 1s the
follow-up of 75 workers exposed to 2,3,7,8-TCDD during and after a 1953 run-
away reaction at a trlchlorophenol manufacturing facility In Ludwlgshafen,
Federal Republic of Germany (Thelss and Frentzel-Beyme, 1977). Two sources
were used to calculate expected deaths: national mortality rates for the
period 1971-1974, and 1972-1975 rates for Rhlnehessen-Palatlnate, the region
1n which Ludwlgshafen Is located.*
The results, shown in Table 19, 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 should be noted that could have
Influenced these results. First, the available report does not Include an
analysis allowing for a minimum period of cancer Induction. It 1s known
that all three stomach cancer deaths in the Ludwlgshafen cohort occurred
more than 10 years after initial exposure. Employing a 10-year restriction
to follow-up (as in the Swedish cohort study) would result In a higher rela-
tive risk estimate by reducing the number of expected deaths.
*The report originally included expected deaths using rates for the dty of
Ludwlgshafen, which were later shown to be Inaccurate.
C-149
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TABLE 19
Analysis of Stomach Cancer Mortality 1n a Group of
West German Factory Workers Exposed to 2,3,7,8-TCDD*
*™:*5 Selths Stomach Cancer Deaths Relative Significance
Risk Level
Observed Expected
Federal Republic
of Germany
1971-1974 3 0.559 5.4 0.02
Rhlnehessen-
Palatlnate
1972-1975 3 0.495 6.1 o.Ol
*Source: Thelss and Frentzel-Beyme, 1977
C-150
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Secondly, national and regional mortality rates from the 1970's were
used to generate expected deaths to compare with observed mortality over a
much longer period (1953-1977). Strong temporal trends 1n stomach cancer
mortality In West Germany during the late 1950's and 1960's would make these
expected figures Inaccurate. Without knowledge of such trends, the direc-
tion and magnitude (If any) of this possible source of bias cannot be
estimated.
The researchers also used an Internal control group which does not raise
the second concern discussed above. This group consisted of 75 men, each
matched to study group members by age and date of entry Into employment, and
selected at random from a 11st of over 10,000 persons who had been Included
In 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.
In an update of this earlier study, Thelss et al. (1982) continued the
follow-up of his cohort through 1979 by adding 2 additional years of follow-
up and apparently reducing the size of his cohort from 75 to 74. Altogether
21 deaths (4 more than from the earlier study) occurred vs. 18 and 19 deaths
In 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
in 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 Rhinehessin-Palatlnate, 18
expected deaths based on 1970-1975 mortality statistics of Ludwlgshafen, and
20 expected deaths based upon 1971-1974 mortality statistics of the Federal
C-151
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Republic of Germany. Just as In the earlier study, the three stomach car-
cinomas noted earlier appear to be significantly elevated Irregardless of
which external comparison group 1s used (Table 20).
On the other hand, one stomach cancer appeared In 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<.016) and then after a lapse of 15 years (2 observed, 0.23
expected, P<.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, H Is Insensitive to the detection of a significantly elevated risk
for most causes of cancer, especially STS and lymphomas. Although, stomach
cancer is elevated significantly, it is based only upon three deaths and
since one stomach cancer death has been noted 1n an Internal control group
1n the updated version, it appears that this finding has been weakened some-
what. Furthermore, as was pointed out earlier, trends 1n stomach cancer
mortality during the 1950's, 1960's and 1970's could make the comparison of
stomach cancer mortality with expected deaths less valid based upon
1970-1975 rates.
In summary, the evidence that phenoxyacetlc 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 is based on only three
stomach cancer deaths. Further follow-up of these and similar cohorts is
warranted, but firm conclusions cannot be made on the basis of the available
data.
C-152
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TABLE 20
Reanalysls of Stomach Cancer Mortality 1n a Group
of West German Factory Workers Exposed to 2,3,7,8-TCDD*
Source for Stomach Cancer Deaths Relative Significance
Expected Deaths Risk Level
Observed Expected
Federal Republic of .on
Germany 1971-1974 3 0.7 4.3 0.
Rhlnehessin-
Palatlnate A _ n
1970-1975 3 0.64 4.7 0.
Ludwigs-Shafen
1970-1975 3 0.61 4.9 0.024
*Source: Thelss et al., 1982
C-153
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Four additional cohort studies have reported results that do not show
Increased stomach cancer mortality rates In groups of workers exposed to
phenoxyacetlc adds and/or 2,3,7,8-TCOD. These are studies of 2.4,5-T pro-
duction workers In Midland, Michigan (Ott et al., 1980), Finnish phenoxy-
acetlc add herbicide applicators (R11h1mak1 et al., 1978), the NHro study
1n which workers were exposed to 2,3,7,8-TCDO (Zack and Susklnd, 1980) and
trlchlorophenol manufacturing workers (Cook et al., 1980a).
As previously mentioned, the NHro study Included a single death from
STS and a weakly suggestive Increase In lymphatic and hematopoletlc system
cancer mortality. The Midland study Included only one cancer death, a tumor
1n the respiratory system. In the Finnish study, hlstologlc Information on
tumor types was not provided; however, there were no deaths from lymphoma.
The results pertinent to stomach cancer mortality In the three studies
are shown in Table 21. Neither the Midland study nor the NHro study con-
tradicts the findings of the Swedish and West German Investigations pre-
viously 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 Tables 14 and 19).
This Indicates that the relative risk estimates from the Midland and
NHro studies, even though equal to zero, are nevertheless not significantly
different from the estimates of 6.1, given the sample sizes, follow-up per-
iods, age distribution and comparison group rates.
C-154
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TABLE 21
Stomach Cancer Mortality In Three Studies of Workers Exposed
to Phenoxyacetlc Add Herbicides and/or 2,3,7,8-TCDD
Stomach Cancer Deaths Relative
Risk
Observed Expected
0 0.14a 0
5 6.9a«b 0.7
0 0.5& 0
95% Confidence
Interval
0-26.3
0.2-1.7
0-7.4
Reference
Ott et al., 1980
R11h1mak1 et al.,
1978
Zack and Susklnd,
1980
aEst1mated from total cancer expected deaths (see footnote In text).
^Entire follow-up period without regard for minimum time for cancer Induc-
tion (Ott et al., 1980 used a 10-year minimum Induction period).
C-155
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In addition, the smallest relative risk In the Midland study (a =
0.05,
-------
Finnish study set the minimum period of herbicide exposure for membership In
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 In the data from the Finnish
study which the authors note but find difficult to explain. In particular,
no cancer deaths occurred during the latter part of the study period among
Forestry Authority workers (1 of 4 groups Included In the cohort), even
though 9.0 deaths were expected. This finding strongly suggests some defi-
ciency In follow-up or In the source records from which vital status was
determined.
In summary, four cohort studies of workers exposed to phenoxyacetlc acid
herbicides and/or 2,3,7,8-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 in
stomach cancer risk. The available report of this study of Finnish herbi-
cide applicators contains methodologic questions that require clarification.
Summary of Epi^minloaical Studies. The net result of adding together
the number of workers exposed to phenoxy acids and/or chlorophenols from all
case studies was an unusually high number of STSs, considering the rarity of
the disease. It is suggestive of an association of cancer with exposure to
phenoxy acids and/or chlorophenols, and consequently, with the Impurities
found in these herbicides, including 2,3,7,8-TCDD.
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 dloxin contaminants, however. In fact, in one study,
C-157
-------
the risk was found to extend to phenoxy acids free of dloxln Impurities. In
that study, the risk Increases to 17 when phenoxy acids known to contain
dloxln impurities (polychloMnated dlbenzodloxlns and dlbenzofurans) are
considered. The extent of observer bias and recall bias Introduced Into
these studies by the employment of an undesirable methodology (self-adminis-
tered questionnaires) Is probably not of sufficient magnitude to have pro-
duced the highly significant risks found 1n the studies. However, the pos-
sibility exists that these biases could have played a role in the determina-
tion of these risks, and consequently the data must be considered limited
for the cardnogenidty of phenoxy add herbicides and/or chlorophenols in
the absence of confirmatory studies.
Later studies that did not reveal a significant excess risk of STS have
severe limitations with their methodologies. These problems make these
latter studies Inadequate to evaluate the risk of STSs from exposure to
phenoxy acids and/or chlorophenols and, consequently, 2,3,7,8-TCDD.
Therefore, the Swedish case-control studies provide limited evidence for
the carclnogenlcity of phenoxy acids and/or chlorophenols In humans. How-
ever, the evidence for the human carcinogenicity for 2,3,7,8-TCDD based on
the epldemiologic studies is only suggestive due to the difficulty of evalu-
ating the risk of 2,3,7,8-TCDD exposure in the presence of the confounding
effects of phenoxy adds and/or chlorophenol.
Substantially weaker evidence exists Incriminating 2,4,5-T and/or
2,3,7,8-TCDD as the cause of malignant lymphoma and stomach cancer in humans.
Studies in Animals
When outbred Swiss mice were given weekly doses of 2,3,7,8-TCDD by
gavage, an increase in liver tumors was observed (Toth et al., 1979).
C-158
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Animals receiving 0.007 yg/kg/week for 1 year showed an elevated tumor
Incidence over vehicle-treated mice; 1n the 0.7 vg/kg/week group the
Increase was statistically significant (P<0.005). Mortality was sufficient-
ly high at 7.0 vg/kg/week as to Interfere with cardnogenldty evaluation.
D1G1ovann1 et al. (1977) reported a mouse skin painting study. The
authors Indicated that 2,3,7,8-TCDD was a weak Initiator on the skin.
A bloassay of 2,3,7,8-TCDD for possible cardnogenldty was conducted by
the Illinois Institute of Technology Research, Chicago, Illinois, on a con-
tract with the NCI Cardnogenesls Testing Program by dermal administration
of the test material In Swiss-Webster mice for 104 weeks (U.S. DHHS,
1980a). Thirty male and female Swiss-Webster mice were dermally treated
with an acetone suspension of 2,3,7,8-TCDD for 3 days/week for 104 weeks.
Similar groups were pretreated with 1 application of 50 vg dlmethylbenz-
anthracene (DMBA) In 0.1 mi acetone 1 week before 2,3,7,8-TCDD administra-
tion began. Female mice received 0.005 vg 2,3,7,8-TCDD/appl1cat1on, and
the male mice received 0.001 yg 2,3,7.8-TCDD. As vehicle controls, 45
mice of each sex received 0.1 mil acetone 3 times/week. Thirty animals of
each sex were used as untreated controls (Tables 22 and 23).
Throughout the bloassay, mean body weights of the male or female groups
of mice administered 2,3,7,8-TCDD, or 2,3,7,8-TCDD following DMBA, were
essentially the same as those of the corresponding vehicle-control group.
Mean body weights of dosed and vehicle-control groups of the females were
less than those of the untreated control group throughout the study, and for
the males were less than mean body weights of untreated controls during the
first 80 weeks.
In female mice, the Incidence of flbrosarcoma 1n the Integumentary
system in groups dosed with 2,3,7,8-TCDD and 2,3,7,8-TCDD following DMBA was
significantly higher than that 1n the corresponding controls (see Table 22).
C-159
-------
TABLE 22
iw °f,Pr1mary Tumors 1" female Swiss-Webster Mice by
Dermal Application of 2,3,7,8-TCDD or 2,3.7,8-TCOD Following DMBAa
Tissue:
Types of Neoplastic
Growth
Integumentary System:
Flbrosarcoma
Lung:
Alveolar/Bronchiolar
Adenoma
Alveolar/Bronchiolar
Carcinoma
Alveolar/Bronchiolar
Carcinoma or Adenoma
Hematopoietic System:
Lymphoma
All Sites:
Hemangioma
Hemangioma or
Hemangiosarcoma
Vehicle
Control
4/41 (10%)
5/41 (12%)
9/41 (22%)
2/41 (5%)
3/41 (7%)
2,3,7,8-TCDD
1/25 (4%)
1/25 (4%)
2/25 (8%)
14/41 (34%) 10/27 (37%)
0/27 (0%)
0/27 (0%)
2,3,7,8-TCDD
plus DMBA
2/41 (5%) 8/27 (30%)b 8/29 (28%)C
3/28 (11%)
3/28 (11%)
6/28 (21%)
8/29 (28%)
1/29 (3%)
1/29 (3%)
aSource: U.S. DHHS, 1980a
DP<0.007
CP<0.010
C-160
-------
TABLE 23
Incidence of Primary Tumors 1n Male Swiss-Webster Mice by
Dermal Application of 2,3,7,8-TCDD or 2,3,7,8-TCDD Following DMBA*
Tissue:
Types of Neoplastlc
Growth
Integumentary System:
Flbrosarcoma
Lung:
Alveolar/Bronchlolar
Adenoma
Alveolar/Bronchlolar
Carcinoma
Alveolar/Bronchlolar
Carcinoma or Adenoma
Hematopo1et1c System:
Lymphoma or Leukemia
All Sites:
Hemanglosarcoma
Vehicle 2,3,7,8-TCDD 2,3,7,8-TCDD
Control plus DMBA
3/42 (7%) 6/28 (21%) 6/30 (20%)
6/41 (15%) 1/28 (4%) 5/29 (17%)
1/41 (2%) 1/28 (4%) 2/29 (7%)
7/41 (17%) 2/28 (7%) 6/29 (21%)
4/42 (10%) 2/28 (7%) 5/30 (17%)
1/42 (2%) 4/28 (14%) 0/30 (0%)
*Source: U.S. DHHS, 1980a
C-161
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It was concluded that, under the conditions of this bloassay, 2,3,7.8-
TCDD applied to the skin was carcinogenic for female Swiss-Webster mice,
Inducing Increased Incidences of Mbrosarcoma 1n the Integumentary system
(U.S. DHHS, 1980a).
Van Miller et al. (1977a,b) administered 0, 1, 5, 50, 1000 and 5000 ppt
of 2,3,7,8-TCOD 1n the diet of male Sprague-Dawley rats. Higher concentra-
tions (50, 500 and 1000 ppb) were also administered, but all of the rats fed
at those three highest concentrations died early 1n the test. After 65
weeks, all surviving animals underwent laparotomles, and all tumors were
blopsled. The rats were kept on the 2,3,7,8-TCDD diet for a total of 78
weeks and then placed on the control diet. After a total of 95 weeks all
surviving animals were sacrificed and necropsled. The results of the study
are summarized 1n Table 24.
Although the study by Van Miller et al. (1977a,b) demonstrated the
occurrence of neoplasms upon 2,3,7,8-TCDD exposure, the study has a number
of shortcomings. The protocol was unusual and only a small number of
animals were used. The occurrence of tumors did not follow a clear-cut
dose-response relationship. Furthermore, the complete absence of tumors In
the controls 1s a highly unusual finding.
Kodba et al. (1978) reported a more extensive carclnogenlcHy study on
2,3,7,8-TCDD. Groups of 100 Sprague-Dawley rats (Spartan substraln, 50
males and 50 females/group) were maintained for up to 2 years on diets
supplying 0.1, 0.01 or 0.001 yg 2,3,7,8-TCDD/kg/day. Ine control group
consisted of 86 males and 85 females. The terminal necrospy examination was
conducted at the end of 2 years of treatment. Females redevlng 0.1 vg
2,3,7,8-TCDD/kg/day experienced a greater mortality than controls during the
second half of the study. Extensive clinical chemistry data were reported
as part of the study.
C-162
-------
TABLE 24
Summary of Neoplastlc Changes After TCOO 1n Rats3
o
I
CO
Concentration of
2,3,7,8-TCDD 1n
Diet (ppt)
0
1
5
50
500
Approximate
Dally Dose
yg/kg
0.0
0.00004
0.0001
0.0014
0.014
No. of Animals
with Neoplasmsb
0
0
5
No. of
Neoplasms
0
0
6
Diagnosis
NA
NA
1 ear duct carcinoma
1 lymphocytlc leukemia
1 adenocarclnoma (kidney)
1 malignant hlstlocytoma (peritoneal)
1 anglosarcoma (skin)
1 Leydlg cell adenoma
1 flbrosarcoma (muscle)
1 squamous cell tumor (skin)
1 astrocytoma (brain)
1 fibroma (muscle)
1 carcinoma (skin)
1 adenocarclnoma (kidney)
1 scleroslng semlnoma (testes)
-------
TABLE 24 (cont.)
Concentration of
2,3,7,8-TCDD 1n
Diet (ppt)
Approximate
Dally Dose
No. of Animals
with Neoplasms'1
No. of
Neoplasms
Diagnosis
o
1000
5000
0.057
0.29
10
1 cholanglocardnoma (liver)
1 anglosarcoma (skin)
1 glloblastoma (brain)
2 malignant hlstlocytoma (peritoneal)
4 squamous cell tumors (lung)
4 neoplastlc nodules (liver)
2 cholanglocarclnomas (Hver)
aSource: Van MUler et al., 1977a
&10 animals per group
NA = Not applicable
-------
A portion of the data for the Mstopathologlc lesions found 1s sum-
marized 1n Table 25. The only lesions that are listed are those which were
statistically different from control levels for at least one dose and In one
sex. The following neoplastlc lesions were found to be Increased above con-
trol levels (P<0.05):
Hepatocellular hyperplastlc (neo- - females only
plastic) nodules
Hepatocellular carcinomas - females only
Stratified squamous cell carcinoma - males and
of palate or nasal turbinae females
Keratlnlzlng squamous carcinoma of - females only
the lung
Stratified squamous carcinoma of the - males only
tongue
Adrenal cortical ademona - males only
Dr. Robert Squire, pathologist at the Johns Hopkins University Medical
School and consultant to the U.S EPA Carcinogen Assessment Group (CAG),
evaluated the hlstopathologlc slides from 2-year rat feeding studies on
2,3,7,8-TCDD by Kociba et al. (1978). Squire and his associates examined
all livers, tongues, hard palates and nasal turbinates, and lungs available
from the 2,3,7,8-TCDD study. His histopathological findings, as well as
Kociba's histopathological evaluations, are summarized in Tables 26 and 27.
Although there are some differences between the diagnoses of Kociba and
Squire, the conclusions about the target organ for cancer induction and the
dose levels at which Induction occurred are the same whether Squire's or
Kociba's diagnoses are considered.
A bioassay of 2,3,7,8-TCDD for possible carcinogenldty was conducted by
administering the test material by gavage to Osborne-Mendel rats and
B6C3F mice for 104 weeks (U.S. DHHS, 1980b). Fifty rats and mice of each
C-165
-------
TABLE 25
Summary of Neoplastlc Lesions Produced by 2,3,7,8-TCDO
In Sprague-Dawley Rats, Spartan Substraln that are Statistically
Significant 1n at Least One Sexa
Males
Dose vg/kg/day
Number of Animals
Hepatocellular hyper-
plastic nodule
Hepatocellular
carcinomas
Stratified squamous
carcinoma-palate
(Including nasal
turblnate tumors)
Lung-kerat1n1zing
squamous carcinoma
Benign tumor-uterus
Subcutaneous fibroma/
f 1broadenoma/l1poma
Benign mammary neo-
plasm
Mammary carcinoma
Stratified squamous
carcinoma-tongue
Pituitary adenoma
Adnar adenoma
pancreas
Adenoma-adrenal
cortex
Pheochromocytoma
0
85
6
2
0
0
-
10
0
0
0
26
14
0
28
0.001
50
0
0
0
0
-
lb
0
0
1
6
7
0
6
0.01
50
2
0
0
0
-
5
1
0
1
11
5
2
10
0.1
50
3
1
4b
1
-
6
2
2
3b
13
2b
5b
4b
0
86
8
1
0
0
28
1
73
8
1
43
0
9
7
Females
0.001
50
3
0
0
0
12
1
35
4
0
18
1
6
2
0.01
50
18b
2
f-
1
0
11
o
36
4
0
13
0
2
1
0.1
50
23b
lib
1 1
4b
~
7b
7b
o
24b
0
2
12b
1
5
3
Source: Koclba et al., 1978
Significantly different (p<0.05) from control by the Fisher exact text
C-166
-------
TABLE 26
2 3 7 8-TCDD Oral Rat Study by Dr. Koclba, with Dr. Squire's Review (8/15/80)
Female Sprague-Dawley Rats - Spartan Substraln (2 years)
Tissues and Diagnoses*
Lung
squamous cell
0
(control)
S K
0/86 0/86
Dose Levels (pq/kq/day)
0.001 0.01
S K S K
0/50 0/50 0/49 0/49
S
8/47
(P<10~3)
0.1
K
7/49
(P<10~3
)
o
I
cr>
Nasal turblnate/
hard palate
squamous cell
carcinomas
Liver
0/54 1/54 0/30 0/30
1/27
1/27
5/22 5/24
(P<10~2) (P<10 2)
Neopidsnc riuuuiei/
hepatocellular 16/86 9/86 8/50 3/50
carcinomas
Total combined (1,2, 16/86 9/86 8/50 3/50
or above) (each
animal had at least
one Lumor above)
27/50
(P<10~4)
27/50
18/50
(P<10~3)
18/50
(P<10~3)
33/47
(P<10~8)
34/47
(P<10~B)
34/48
34/49
'Where result 1s significantly different than the appropriate control, probability (P) of Incorrectly
rejecting the null hypothesis 1s approximated 1n parentheses.
S = Dr. Squire's hlstopathologlc analysis; K = Dr. Kodba's h1stopatholog1c analysis
-------
TAUI h 71
2,3,7,8-TCDD Oral Rat Study by Dr. Kodba, with Dr. Squire's Review (8/15/80)
Male Sprague-Dawley Rats - Spartan Substraln (2 years)
o
I
cr
oc
Tissues and Diagnoses* 0
(control )
S K
Nasal turblnate/
hard palate
squamotis cell
carcinomas 0/55 0/51
Tongue
squamous cell
carcinomas 0/77 0/76
Total - 1 or 2 above 0/77 0/76
(each rat had at
least one tumor
above)
Dose Levels (uq/kg/day)
0.001 0.01 0.1
S K S K S K
1/34 1/34 0/26 0/27 6/30 4/30
(P<10~a)
2/44 1/49 1/49 1/49 3/44 3/42
(P<0.05) (P<10~3)
2/44 2/49 1/49 1/49 9/44 7/42
(P<10~4)
*Where result 1s significantly different than the appropriate control, probability (P) of Incorrectly
rejecting the null hypothesis 1s approximated 1n parentheses.
S = Dr. Squire's hlstopathologlc analysis; K = Dr. Kodba's hlstopathologlc analysis
-------
sex were administered 2,3,7,8-TCDD suspended 1n a vehicle of 9:1 corn
o1l:acetone 2 days/week for 104 weeks at doses of 0.01, 0.05 or 0.5
vg/kg/week for rats and male mice and 0.04, 0.2 or 2.0 vg/kg/week for
female mice. Seventy-five rats and 75 mice of each sex served as vehicle
controls. One untreated control group containing 25 rats and 25 mice of
each sex was present 1n the 2,3,7,8-TCDD treatment room, and one untreated
control group containing 25 rats and 25 mice of each sex was present In the
vehicle control room. All surviving animals were killed at 105-107 weeks.
In rats, a dose-related depression 1n mean body weight gain became
evident In the males after week 55 of the bloassay and In the females after
week 45. In mice, the mean body weight gain In the dosed groups was compar-
able with that of the vehicle-control groups, but H was lower than that of
the untreated controls.
In male rats, Increased Incidences of folllcular-cell adenoma or car-
cinomas In the thyroid were dose related and were significantly higher In
the low-, mid- and high-dose groups (P=0.001 for high dose) than In the
vehicles controls (Table 28). A significant Increase in the subcutaneous
tissue fibroma was found for the high-dose group (P=0.048).
In female rats, the Incidences of folllcular-cell adenomas of the
thyroid and hepatocellular carcinomas or neoplastlc nodules of the Hver
were dose-related, and the Incidence of hepatocellular carcinomas 1n the
high-dose group was significantly higher (P=0.001) than that In the vehicle
controls (Table 29), as were the Incidences of subcutaneous tissue flbro-
sarcoma (P=0.023) and adrenal cortical adenoma (P=0.039).
In both male and female mice, Incidences of hepatocellular adenomas or
carcinomas were dose related and the Incidences 1n the high-dose groups were
higher (P=0.001 male, P=0.002 female) than those 1n the corresponding
vehicle controls (Tables 30 and 31).
C-169
-------
TABLE 28
Incidence of Primary Tumors In Male Osborne-Mendel Rats
(2,3,7,8-TCDD Administered by Gavage)a«b
Tissue:
Types of Neoplastlc
Growth
Subcutaneous Tissue:
Fibroma or Flbrosarcoma
Fibroma
Flbrosarcoma
Circulatory System:
Hemangloma or
Hemanglosarcoma
Hemanglosarcoma
Liver:
Neoplastlc Nodule or
Hepatocellular Carcinoma
Neoplastlc nodule
Pituitary:
Adenoma or Chromophobe
Adenoma
Adenoma
Adrenal :
Cortical Adenoma
Pheochromcytoma
Thyroid:
Folllcular Cell
Adenoma or Carcinoma
Folllcular Cell Adenoma
C-Cell Adenoma
C-Cell Adenoma or
Carcinoma
Parathyroid:
Adenoma
Pancreatic Islets:
Adenoma
Vehicle
Control
12/75(16)
3/75(4)
9/75(12)
7/75(9)
4/75(5)
0/74(0)
0/74(0)
2/61(3)
0/61(0)
6/72(8)
5/72(7)
1/69(1)
1/69(1)
2/69(3)
2/69(3)
0/20(0)
2/70(3)
Dose (vq/kq/uk)C
0.01
4/50(8)
1/50(2)
3/50(6)
3/50(6)
3/50(6)
0/50(0)
0/50(0)
1/43(2)
1/43(2)
9/50(18)
0/50(0)
5/48(10)e
5/48(10)
2/48(4)
2/48(4)
2/41(5)
2/49(4)
0.05
5/50(10)
3/50(6)
3/50(6)
1/50(2)
0/50(0)
0/50(0)
0/50(0)
3/43(7)
2/43(5)
12/49(24)
1/49(2)
8/50(16)f
6/50(12)
4/50(8)
5/50(10)
1/40(3)
3/48(6)
0.5
10/50(20)
7/50(14)d
3/50(6)
4/50(8)
4/50(8)
3/50(6)
3/50(6)
3/40(8)
3/40(8)
9/49(18)
1/49(2)
11/50(22)9
10/50(20)
4/50(8)
4/50(8)
1/36(3)
1/50(2)
C-170
-------
TABLE 28 (cont.)
Tissue:
Types of Neoplastlc
Growth
Mammary Gland:
Adenocardnoma
Flbroadenoma
Dose (uq/ka/wk)c
Vehicle
Control
0/75(0)
5/75(7)
0.01
0/50(0)
0/50(0)
0.05
3/50(6)
1/50(2)
0.5
1/50(2)
0/50(0)
aSource: U.S. DHHS, 1980b
bValues In parentheses Indicate percent response.
CP = Values calculated using the Fisher Exact test
dP = 0.048
eP » 0.042
fP = 0.004
9P < 0.001
C-171
-------
TABLE 29
Incidence of Primary Tumors In Female Osborne-Hendel Rats
(2,3,7,8-TCDD Administered by Gavage)a-b
Tissue:
Types of Neoplastlc
Growth
Subcutaneous Tissue:
Fibroma or Flbrosarcoma
Fibroma
Flbrosarcoma
Liver:
Neoplastlc Nodule or
Hepatocellular Carcinoma
Neoplastlc nodule
Pituitary:
Adenoma
Chromophobe Adenoma
Adrenal :
Cortical Adenoma or
Adenoma
Cortical Adenoma or
Carcinoma or Adenoma
Thyroid:
FolUcular Cell Adenoma
FolUcular Cell Adenoma
or Carcinoma
C-Cell Adenoma
C-Cell Adenoma or
Carcinoma
Mammary Gland:
Adenocardnoma
Flbroadenoma
Brain:
Astrocytoma
Vehicle
Control
4/75(5)
4/75(5)
0/75(0)
5/75(7)
5/75(7)
1/66(2)
5/66(8)
11/73(15)
11/73(15)
3/73(4)
5/73(7)
7/73(10)
7/73(10)
3/75(4)
27/75(36)
0/75(0)
Dose (ua/kcr/wk)c
0.01
2/50(4)
0/50(0)
2/50(4)
1/49(2)
1/49(2)
5/47(11)
0/47(0)
8/49(16)
9/49(18)
2/45(4)
2/45(4)
1/45(2)
3/45(7)
3/50(6)
20/50(40)
3/47(6)
0.05
3/50(6)
0/50(0)
3/50(6)
3/50(6)
3/50(6)
2/44(5)
0/44(0)
4/49(8)
5/49(10)
1/49(2)
1/49(2)
8/49(26)
8/49(16)
2/50(4)
21/50(42)
0/49(0)
0.5
5/49(10)
1/49(2)
4/49(8)d
14/49(29)6
12/49(24)
3/43(7)
1/43(2)
14/46(30)f
14/46(30)
6/47(13)
6/47(13)
6/47(13)
6/47(13)
1/49(2)
17/49(35)
0/48(0)
Source: U.S. DHHS, 1980b
Values In parentheses Indicate percent response.
P = Values calculated using the Fisher Exact test
P = 0.0023; 6P = 0.001; fP = 0.039
C-172
-------
TABLE 30
Incidence of Primary Tumors 1n Female B6CF1 Mice
(2,3,7,8-TCOD Administered by Gavage)a«b
Tissue:
Types of Neoplastlc
Growth
Subcutaneous Tissue:
Flbrosarcoma
Lung:
Alveolar/Bronchlolar
Adenoma
Alveolar/Bronchlolar
Adenoma or Carcinoma
Hematopo1et1c System:
Lymphocytlc Lymphoma
H1st1ocyt1c Lymphoma
All Lymphoma
Lymphoma or Leukemia
Liver:
Hepatocellular Adenoma
or Carcinoma
Hepatocellular Adenoma
Hepatocellular Car-
cinoma
Pituitary:
Adenoma
Thyroid:
FolUcular-Cell Adenoma
Vehicle
Control
1/74{1)
2/74(3)
2/74(3)
5/74(7)
9/74(12)
18/74(24)
18/74(24)
3/73(4)
2/73(3)
1/73(1)
0/62(0)
0/69(0)
0.01
1/50(2)
3/49(6)
3/49(8)
6/50(12)
4/50(8)
11/50(22)
12/50(24)
6/50(12)
4/50(8)
2/50(4)
2/39(5)
3/50(6)
Dose (uQ/kq/wk)c
0.05
1/48(2)
4/48(8)
4/48(8)
4/48(8)
8/48(17)
13/48(27)
13/48(27)
6/48(13)
4/48(8)
2/48(4)
0/38(0)
1/47(2)
0.5
5/47(ll)d
1/46(2)
2/46(4)
6/47(13)
14/47(30)6
20/47 ( 43 )f
20/47(43)f
11/47(23)9
5/47(11)
6/47(13)
2/33(6)
5/46(11)"
^Source: U.S. DHHS, 1980b
bValues In parentheses Indicate percent response.
cp = Values calculated using the Fisher Exact test.
dp = 0.032; eP = 0.016; fP = 0.029; 9P = 0.002; hP
= 0.009
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TABLE 31
Incidence of Primary Tumors In Male B6CF1 Mice
(2,3,7,8-TCDD Administered by Gavage)a»b
Tissue:
Types of Neoplastlc
Growth
Subcutaneous Tissue:
Flbrosarcoma or Fibroma
Flbrosarcoma
Lung:
Alveolar/Bronchlolar
Adenoma or Carcinoma
Alveolar/Bronchlolar
Adenoma
Hematopo1et1c System:
H1st1ocyt1c Lymphoma
Lymphoma or Leukemia
Circulatory System:
Hemanglosarcoma
Liver:
Hepatocellular Adenoma
or Carcinoma
Hepatocellular Adenoma
Hepatocellular Car-
cinoma
Thyroid:
FolUcular-Cell Adenoma
Eye/Lacr1mal Glands:
Adenoma
Vehicle
Control
9/73(12)
8/73(11)
10/71(14)
7/71(10)
5/73(7)
8/73(11)
1/73(1)
15/73(21)
7/73(10)
8/73(11)
0/69(0)
0/73(0)
Dose (vq/lcq/ulf )C
0.01
6/49(12)
5/49(10)
2/48(4)
2/48(4)
0/49(0)
3/49(6)
2/49(4)
12/49(24)
3/49(6)
9/49(18)
3/48(6)
1/49(2)
0.05
5/49(10)
4/49(8)
4/48(8)
4/48(8)
3/49(6)
4/49(8)
1/49(2)
13/49(27)
5/49(10)
8/49(16)
0/48(0)
1/49(2)
0.5
3/50(6)
3/50(6)
13/50(26)
11/50(22)
0/50(0)
6/50(12)
3/50(6)
27/50(54)d
10/50(20)
17/50(34)
0/49(0)
3/50(6)
aSource: U.S. DHHS, 1980b
^Values 1n parentheses Indicate percent response.
CP = Values calculated using the Fisher Exact test.
dP<0.001
C-174
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In female mice, folllcular-cell adenomas In the thyroid and Mstocytlc
lymphomas In the hematopoletlc system occurred at dose-related Incidences,
and the Incidences were significantly higher In the high-dose groups than
those in vehicle controls. The high-dose group of females also showed a
significantly higher Incidence of subcutaneous fIbrosarcomas (P=0.032) and
lymphoma or leukemia (P=0.029) (see Table 31).
It was concluded that, under the conditions of this bloassay, 2,3,7,8-
TCDD was carcinogenic for Osborne-Mendel rats, Inducing significant dose-
related increased Incidences of folllcular-cell thyroid tumors In males and
liver tumors 1n females. 2,3,7,8-TCDD was also carcinogenic for B6C3F1
mice, Inducing significant dose-related Increased Incidences of liver tumors
1n males and females and of thyroid tumors In females (U.S. DHHS, 1980b).
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CRITERION FORMULATION
Existing Guidelines and Standards
The National Academy of Sciences Committee on Drinking Water and Health
(NAS, 1977} suggested an acceptable dally Intake (ADI) of 1(T« vg 2,3,7,8-
TCDD/kg/day. At that time. 2,3,7,8-TCDD was not considered to be a carcino-
gen, and the ADI was based on a 13-week feeding study 1n rats by Koclba et
al. (1976).
The FDA has Issued a health advisory stating that fish with residues of
2,3,7,8-TCDD >50 ppt should not be consumed, but fish with residues of <25
ppt pose no serious health concern (FDA, 1981, 1983; Cordle, 1981). Federal
legal limits for Great Lakes fish distributed in Interstate commerce were
deemed unnecessary because most samples analyzed by the FDA contained <25
ppt. Canada has established a 20 ppt concentration limit for 2,3,7,8-TCDD
In Lake Ontario commercial fish exported into the United States to comply
with the levels believed by FDA to be safe. No tolerances have been estab-
lished for 2,3,7,8-TCDD on food crops. A tolerance of 0.05 ppm hexachloro-
phene in or on cottonseeds (used as livestock feed), with a proviso that the
technical grade of hexachlorophene shall not contain >0.1 ppm 2,3,7,8-TCDD,
was published in 40 CFR 180.302.
The Ministry of Labour of Canada has set a tentative Ambient Air Quality
criterion for PCDDs of 30 pg/m3 (Harding, 1982).
Current Levels of Exposure
The extent of human exposure to 2,3,7,8-TCDD that can be directly
attributed to the water route cannot be readily determined. While 2,3,7,8-
TCDD does not appear to occur naturally in the environment, it can be pro-
duced with low efficiency from the combustion of 2,4,5-T-containing mate-
C-176
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rials (Stehl and Lamparskl. 1977); H may also be produced In a large vari-
ety of normal combustion processes (Anonymous, 1978; Bumb et al., 1980), but
It 1s not produced during all combustion processes (Klmble and Gross, 1980).
The Impact of these processes on human exposure Is unknown. The high affin-
ity of 2,3,7,8-TCDD for soils with significant organic content would seem to
reduce the likelihood of groundwater contamination; however, as the organic
content of soil declines the likelihood of groundwater contamination by
2,3,7,8-TCDD Increases.
Contaminated beef fat samples have been found to have concentrations as
high as 60 ppt of 2,3,7,8-TCDD In one sample (Ross, 1976). 2,3,7,8-TCDD
residues also have been detected In the edible portions of fish from the
Tlttabawassee, Grand and Saginaw Rivers, Lake Michigan and the Saglnaw Bay
in Michigan at concentrations ranging from 4-695 ppt (Harless and Lewis,
1980).
The reports of Incidents of 2,3,7,8-TCDD exposures In Industrial plants
and of accidents where 2,3,7,8-TCDD was more widely disseminated are useful
in identifying some of the effects of 2,3,7,8-TCDD exposure 1n man. Unfor-
tunately, the existing human data can only roughly estimate the extent and
duration of 2,3,7,8-TCDD exposure which produced the toxic symptoms.
Special Groups at Risk
The most obvious groups at risk are those employed In the manufacture of
chemicals 1n which 2,3,7,8-TCDD may occur as an unwanted by-product. The
spraying of herbicides containing traces of 2,3,7,8-TCDD has become less of
a problem because of restrictions on the use of such agents. Considering
the reproductive toxicity of 2,3,7,8-TCDD, women of child-bearing age, and
especially the fetus, are at high risk from exposures to 2,3,7,8-TCDD.
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Basis and Derivation of Criterion
2,3,7,8-TCDD 1s an unusually toxic compound with demonstrated acute.
subacute and chronic effects In animals and man. Acute or subchronlc expo-
sures to 2,3,7,8-TCDD can adversely affect the skin, the liver, the nervous
system and the Immune system.
2,3,7,8-TCDD displays an unusually high degree of reproductive toxlc-
Hy. It 1s teratogenVc, fetotoxlc and reduces fertility. In a 3-generatlon
reproductive study, Murray et al. (1979) reported a reduction 1n fertility
after dally dosing at 0.1 or 0.01 yg 2,3,7,8-TCDD/kg 1n the F and F
generations of Sprague-Dawley rats. Although Murray et al. (1979) con-
sidered the lowest dose tested, 0.001 yg/kg, to be a no-observed-effect
level (NOEL), a re-evaluation of these data by Nlsbet and Paxton (1982),
using different statistical methods, Indicated that there was a reduction in
the gestation Index, decreased fetal weight, Increased liver to body weight
ratio, and Increased Incidence of dilated renal pelvis at the 0.001 vg/kg
dose. The re-evaluated data would suggest that equivocal adverse effects
were seen at the lowest dose (0.001 vg/kg/day) and that this dose should,
therefore, represent a lowest-observed-adverse-effect level (LOAEL).
Schantz et al. (1979) found reductions in fertility and various other toxic
effects In rhesus monkeys fed a 50 ppt 2,3,7,8-TCDD diet for 20 months.
This corresponds to a calculated dally dose of 0.0015 vg 2,3,7,8-TCDD/kg/
day. These results suggest that monkeys may be somewhat more sensitive than
rats, since the effects in monkeys were more severe and not equivocal.
A tox1c1ty-based criterion has been calculated for comparison with the
cancer-based criterion In accordance with public comments. Since the data
from the limited study by Schantz et al. (1979) are supportive of the find-
Ings by Murray 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
C-178
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existence of lifetime animal studies and knowledge of effects In man as per
MAS (1977) guidelines, and then an additional 10 because a LOAEL Is used as
the basis of this C3lcul3t1on,* then the ADI would be:
ID-3 yg/kg/day (LOAEL) _
ADI ! — =1 x 10 * yg/kg/day.
100 x 10
Thus, the acceptable dally Intake for a 70 kg man would be 7.0x10'* yg
2,3,7,8-TCDD/day. Using a BCF of 5000 and assuming a dally consumption of
6.5 g of fish, the water concentration corresponding to this ADI would be:
7.0 x 10"5
water concentration = —— = 2-° x 10 "
However, this concentration may not be sufficiently protective of human
health since It does not take Into account the demonstrated carcinogenic
effects of 2,3,7,8-TCDD In animals and the probability that 2,3,7,8-TCDD 1s
a human carcinogen (see cancer-based criterion derivation).
The carcinogenic potential of 2,3,7.8-TCDD has been established by feed-
Ing studies In rodents. The results of the study by Van Miller et al.
(1977a,b) are summarized In Table 24, and the findings of the more extensive
study by Kodba et al. (1978) are summarized In Table 25. The Van Miller et
al. (1977a,b), the Toth et al. (1979) and recent NCI data (U.S. DHHS,
1980a,b) summarized 1n Tables 28, 29, 30, and 31, reinforce the findings of
Kodba et al. (1978) and establish that 2,3,7,8-TCDD Is an animal carcinogen
and 1s probably carcinogenic 1n humans.
'According to the methods published by EPA (45 FR 79353), an additional
uncertainty factor between 1 and 10 must be used because the calculation Is
based on a LOAEL. An uncertainty factor of 10 was chosen because of the
adverse effects seen 1n rhesus monkeys at 0.0015 yg/kg/day, despite the
equivocal nature of the effects In rats seen at the 0.001 yg/kg/day dose
level.
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Furthermore, the epldemlologlcal findings (Harden, 1977, 1979; U.S.
EPA, 1980c; Harden et a!., 1980; Hardell and Sandstrom, 1979; EMcksson et
al., 1979; Thelss and Frentzel-Beyme, 1977; Jlrasek et al., 1973, 1974;
Pazderova et al., 1974; Axelson et al., 1980; Zack and Susklnd, 1980; Zack.
1980; Cook et al., 1980a) are consistent with the conclusion from animal
studies that 2,3,7,8-TCOO 1s a probable human carcinogen. In addition,
2,3,7,8-TCDO has been shown to be a potent liver cancer promoter (PHot et
al., 1980) and a cocardnogen (Kourl et al., 1978).
Under the Consent Decree In NRDC vs. Train, criteria are to state
"recommended maximum permissible concentrations (Including where appro-
priate, zero) consistent with the protection of aquatic organisms, human
health, and recreational activities." 2,3,7,8-TCDD 1s suspected of being a
human carcinogen. Because there 1s no recognized safe concentration for a
human carcinogen, the recommended concentration of 2,3,7,8-TCOO 1n water for
maximum protection of human health 1s zero.
Because attaining a zero concentration level may be 1nfeas1ble 1n some
cases, and In order to assist the Agency and states In the possible future
development of water quality regulations, the concentrations of 2,3,7,8-TCDD
corresponding to several Incremental lifetime cancer risk levels have been
estimated. A cancer risk level provides an estimate of the additional Inci-
dence of cancer that may be expected 1n an exposed population. A risk of
10~5, for example, Indicates a probability of one additional case of can-
cer for every 100,000 people exposed, a risk 10~6 Indicates one additional
case of cancer for every million people exposed, and so forth.
In the November 1980 Federal Register notice of availability of ambient
water quality criteria (45 FR 79318), the U.S. EPA presented a range of con-
centrations for carcinogens corresponding to cancer risks of 10~5, 10~6
C-180
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or 10~7, based on the upper 95% confidence level of calculated Incremental
risk. The criteria for 2,3,7,8-TCDD are shown In the following table:
Exposure Assumptions 9554 Upper-Limit Risk Levels and Corresponding 95%
(per day) Lower-Limit Criteria (1) for 2.3.7,8-TCDD
0 10~7
2 a of drinking water 0 1.3xlO"9
and consumption of 6.5 g
fish and shellfish. (2)
Consumption of fish and 0 1.4xlO~9
shellfish only
2 «. of drinking water 0 2.2xlO~8
10~fe 10~5
1.3xlO~8 1.3xlO~7
1.4xlO~8 1.4x10''
2.2xlO~7 2.2xlO"6
only
(1) The animal bloassay data used 1n these calculations are presented In
the Appendix of this document. These levels are calculated by applying
a linearized multistage model as discussed In the Human Health Method-
ology Appendices to the Federal Register notice concerning water qual-
ity criteria. Since the extrapolation model Is linear at low doses,
the additional lifetime risk is directly proportional to the water con-
centration. Therefore, water concentrations corresponding to other
risk levels can be derived by multiplying or dividing one of the risk
levels and corresponding water concentrations shown in the table by
factors such as 10, 100, 1000 and so forth.
(2) Approximately 94.2% of the 2,3,7,8-TCDD exposure results from the con-
sumption of aquatic organisms which exhibit an average bloconcentratlon
potential of 5000-fold. The remaining 5.8% of 2,3,7,8-TCDD exposure
results from drinking water. Correspondingly, if no contaminated
shellfish or fish are eaten, the water contamination level could be 17
times as high for the same risk level, or 2.2xlO"7 yg/S. for a
10"* upper-limit risk level, vs. 1.3xlO~8 yg/8. when contaminated
fish and water are consumed.
C-181
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Concentration levels were derived assuming a lifetime exposure to vari-
ous amounts of 2,3,7,8-TCDD, (1) occurring from the consumption of both
drinking water and aquatic life taken from waters containing the correspond-
ing 2,3,7,8-TCDD concentrations, (2) occurring solely from consumption of
aquatic life grown 1n waters containing the corresponding 2,3,7,8-TCDD con-
centrations and (3) occurring from the consumption of drinking water only.
Because data Indicating other sources of 2,3.7,8-TCDD exposure and their
contributions to total body burden are Inadequate for quantitative use, the
figures reflect the Incremental risks associated with the Indicated routes
only.
The above criteria, which have been calculated on the basis of health
effects data, are below the limit of detection for 2,3,7,8-TCDD 1n water by
current analytical methods. The detection limit presently 1s estimated to
be ~3xlO~5 Mg/a (Harless et a!., 1980). The detection limit should also
be considered when Issuing guidance based on these criteria.
Estimates by Others of Carcinogenic Potency and Criteria
The U.S. Food and Drug Administration concluded that an advisory level
of 25 ppt for Great Lakes fish contaminated with 2,3,7,8-TCDD does not pose
an unacceptable risk to public health (FDA, 1981). EPA has reviewed the
recent testimony before Congress of Dr. S.A. Miller (FDA, 1983), discussing
cancer risk associated with 1ngest1on of these fish. The FDA estimate of
the 95% upper-limit carcinogenic potency factor for 2,3,7,8-TCDD 1s q * =
1.75xl04 (mg/kg/day)"1, which Is less potent than EPA's estimate of
q-j* = 1.56xl05 (mg/kg/day)-i (see Appendix) by a factor of 9. Even
though both Agencies used the same data base (Kodba et al., 1978) and risk
extrapolation model, some subtle differences 1n methodology exist which
account for this factor of 9. The major part of this difference 1s a factor
C-182
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of 5.38 which EPA uses for rat-to-man extrapolation on the assumption that
dose per unit body surface area, rather than dose per unit body weight, 1s
an equivalent dose between species (45 FR 79351). Most of the remaining
factor of -1.7 Is due to the FDA's use of the Kodba hlstopathologlcal
diagnosis alone, without Including that of Squire, and EPA's adjustment of
Us calculations to compensate for the high early mortality observed 1n the
Kodba et al. (1978) study (see Appendix).
FDA and EPA also differ In their assessment of human exposure to
2,3,7,8-TCDD In fish, 1n keeping with their respective regulatory approach-
es. EPA calculates water quality criteria to protect a body of water as
though H were the direct source of 100% of a human population's average
dally Intake of water and/or freshwater and estuarlne fish or shellfish.
The concentration of a pollutant In the tissues of all such fish or shell-
fish Is further assumed to be determined by the water concentration and the
bloconcentratlon factor (BCF) of the pollutant. FDA, on the other hand,
premised Us exposure assessment on the assumption that only limited amounts
of fish having 2,3,7,8-TCDD levels at or near the advisory level will
actually be consumed. For example, FDA assumed that for this substance,
significant contamination problems were limited to bottom feeders such as
catfish and carp.* It also assumed that actual average residue levels 1n
the flesh of bottom-feeding species reaching the market would not exceed
one-third of the advisory level (I.e., ~8 ppt) and further, that for most
Individuals, 90?t of the fish consumed would be comprised of other species
showing no measurable contamination, or would be taken from uncontamlnated
*However available data Indicate that other species, especially trout and
salmon, taken from some areas of the Great Lakes may also have tissue
residues of 2,3,7,8-TCDD which exceed 25 ppt (see Table 1).
C-183
-------
areas. Under these assumptions, and using an upper 90 percentlle value for
freshwater fish consumption of 15.7 g/day, the FDA potency estimate yields
an upper-limit risk estimate of 2.86xl(T« for consumers of these fish. If
the same exposure assumptions were used with EPA's potency estimate a some-
what higher upper limit risk of 2.92x10"= would result.
The Center for Disease Control (CDC) has also calculated an upper-limit
potency value for 2,3,7,8-TCDD (Klmbrough et al., 1983). The CDC estimate
1s based on the Squire hlstopathologlcal results, and, like that of FDA,
extrapolates from rat to man on a basis of dose equivalence per unit body
weight. The CDC difference from both the EPA and FDA approaches Is that the
curve fit was done, not on administered dose, but on liver concentration at
terminal sacrifice. Also, like FDA, CDC did not adjust for high early
mortality. The final result 1s that the CDC 95% upper-limit potency value
estimate when converted back to administered dose Is q * = 3.6x10"
(mg/kg/day)-i which 1s more potent by a factor of 2 than that of FDA and
less potent by a factor of 4 than that of the EPA.
In January 1984 the three Agencies met to review the differences 1n car-
cinogenic potency estimation. The three Agencies agreed that they are using
virtually the same methodologies for potency estimation although there are
differences 1n some assumptions used. Further, there was agreement that
correction for mortality 1s appropriate, making the differences less between
the EPA estimate and the other estimates. Lastly, the Agencies agreed that
the remaining differences are within the range of uncertainty Inherent 1n
the risk assessment process.
*The difference between the EPA and FDA risk estimates results from the
difference 1n potency estimates, described above, and the use by FDA of an
average human body weight of 80 kg, versus 70 kg used by EPA
C-184
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Van Miller, J.P., 3.3. Lallch and 3.R. Allen. 1977a. Increased Incidence
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APPENDIX
Summary and Conclusions Regarding the Carcinogenicity of 2,3,7,8-Tetra-
chlorodibenzo-p-Dioxin (TCDD)*
2,3,7,8-TCDD is probably carcinogenic for humans on the basis of animal
carclnogenlclty studies which were positive in multiple species and organs.
Ep1dem1ological studies of workers exposed to chemicals contaminated with
2,3,7,8-TCDD such as 2,4,5-trichlorophenoxyacetlc acid and 2,4,5-trichloro-
phenol are consistent with the position that 2,3,7,8-TCDD is probably carci-
nogenic for humans; the available evidence indicates an excess incidence of
soft tissue sarcomas. Because 2,3,7,8-TCDD Is almost always found in
association with other materials (e.g., chlorophenols, combustion products,
etc.), it may never be possible to evaluate the cardnogenicity of
2,3,7,8-TCDD by itself in humans.
SUMMARY OF HUMAN POTENCY ESTIMATES BASED ON PERTINENT DATA
A summary of 95% upper-limit human carcinogenic potency estimates for
2,3,7,8-TCDD derived from the Kodba et al. (1978) and NCI (U.S. DHHS,
1980b) studies in rats and mice, with two pathologists1 findings for the
Kodba study, are given in Table 32. These potency estimates have been
calculated using the linearized multistage model by a previously described
methodology (45 FR 79350-79353). The largest of these potency factors
(q *) comes from data In an independent pathologist's (Dr. R. Squire)
review of the Kodba feeding study of female Sprague-Dawley rats. An
adjustment for high early mortality in the high dose groups led to a
slightly lower estimate. The mean of the two pathologists1 estimates after
mortality adjustment is:
i /? -
q * = [(1.51 x 10s) x (1.61 x 105)]" = 1.56 x 10s (mg/kg/day) *
*Th1s summary was prepared and approved by an expert peer review panel on
dioxlns convened by the U.S. EPA 1n Cincinnati on July 17, 1983.
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TABLE 32
Summary of Human Potency Estimates for 2,3,7,8-TCDD
Species Study Sex Pathologist Human Potency Estimate
q-j* 1n (mg/kg/day)"1
Rat
Rat
Rat
Mouse
Mouse
Kodba et al.a male Koclba
Squire
Koclba et al.a female Kodba
Unadjusted
Adjusted for
early mortality
Squire
Unadjusted
Adjusted for
early mortality
NCIC female NCI-rev1ewed
NCIC male NCI-rev1ewed
NCIC female NCI-reviewed
1.47
1.73
2.52
1.51
4.25
1.61
3.28
7.52
4.56
x 10*
x 10«
x 10s
x 10sb
x 10s
x 10sb
x 10*
x 10*
x 10"
aSource: Kodba et a!., 1978
bValues used to determine the geometric mean of 1.56xl05 (mg/kg/day)"1
cSource: U.S. DHHS, 1980fa
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These potency estimates were derived from the Kodba feeding study. The
responses and parameters of the Kodba feeding study In female rats are
given below. The number with tumors refers to the number of animals with at
least one of liver, lung, hard palate, and/or nasal turblnate tumors.
Adjustment for early mortality refers to eliminating those animals which
died during the first year of study. The first tumor appeared In the high
dose group during the thirteenth month.
Dose
(mg/kg/day)
0
0.001 x 10~3
0.01 x 10~3
0.1 x 10~3
No. with
Adjusted
Squire
16/85
8/48
27/48
34/40
Tumors/No. Examined
for Early Mortality
Kodba
9/85
3/48
18/48
34/40
le = 720 days Wn = 70 kg
Le = 720 days W0 = 0.450 kg
L = 720 days R = 5000 H/kg
With these parameters, the mean 95% upper-limit carcinogenic potency
factor for humans, q * Is 1.56xl05 {mg/kg/dayT1. For a 70 kg human
drinking 2 5, water/day and eating 6.5 g of contaminated fish and shell-
fish, the water concentration should be <1.3xlO"B yg/8. 1n order to keep
the upper-limit Individual lifetime cancer risk below 10~6, for example.
If fish and shellfish alone are consumed, the corresponding water concen-
tration for this level of risk should be <1.4xlO~8 yg/i, and 1f water
alone 1s consumed, the corresponding water concentration should be
<2.2xlO"7
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