EXTERNAL REVIEW DRAFT
United Srates f rin r l u in^
Environmental Protection c LflU-l, 1 n-4UD
Agency April, 1908
-EPA Research and
Development
DRINKING WATER CRITERIA DOCUMENT FOR
2.3.7.8-TETRACHLORODIBENZO-E-DIOXIN
Prepared for
OFFICE OF DRINKING WATER
Prepared by
Environmental Criteria and Assessment Office
Office of Health and Environmental Assessment
U.S. Environmental Protection Agency
Cincinnati, OH 45268
DRAFT: DO NOT CITE OR QUOTE
NOTICE
This document Is a preliminary draft. It has not been formally released
by the U.S. Environmental Protection Agency arid sfiould not* at this-stage be
construed to represent Agency policy. It Is being circulated for conments
on Its technical accuracy and policy Implications.
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DISCLAIMER
Mention of trade names or commercial products does not constitute
endorsement or recommendation for use.
11
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FOREWORD
Section 1412 (b)(3)(A) of the Safe Drinking Water Act, as amended In
1986, requires the Administrator of the Environmental Protection Agency to
publish maximum contaminant level goals (MCLGs) and promulgate National
Primary Drinking Water Regulations for each contaminant, which, In the
judgment of the Administrator, may have an adverse effect on public health
and which is known or anticipated to occur In public water systems. The
MCLG Is nonenforceable and is set at a level at which no known or antici-
pated adverse health effects in humans occur and which allows for an
adequate margin of safety. Factors considered in setting the MCLG include
health effects data and sources of exposure other than drinking water.
This document provides the health effects basis to be considered in
establishing the MCLG. To achieve this objective, data on pharmacokinetics,
human exposure, acute and chronic toxicity to animals and humans, epidemi-
ology and mechanisms of toxicity are evaluated. Specific emphasis is placed
on literature data providing dose-response Information. Thus, while the
literature search and evaluation performed i-n support of this document has
been comprehenslve, only the reports considered most pertinent In the deri-
vation of the MCLG are cited in the document. The comprehensive literature
data base in support of this document Includes Information published up to
1987; however, more recent data may have been added during the review
process.
When adequate health effects data exist, Health Advisory values for less
than lifetime exposures (1-day, 10-day and longer-term, -10% of an
individual's lifetime) are Included In this document. These values are not
used in setting the MCLG, but serve as Informal guidance to municipalities
and other organizations when emergency spills or contamination situations
occur.
Michael B. Cook
Director
Office of Drinking Water
111
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DOCUMENT DEVELOPMENT
Oefadas Mukerjee, Ph.D., Document Manager
Environmental Criteria and Assessment Office, Cincinnati
U.S. Environmental Protection Agency
Helen H. Ball, M.S., Project Officer
Environmental Criteria and Assessment Office, Cincinnati
U.S. Environmental Protection Agency
Authors
D1pak K. Basu, Ph.D.
Syracuse Research Corporation
Syracuse, New York
Denzll L. Tull1s, Ph.D.
Syracuse Research Corporation
Syracuse, New York
Scientific Reviewers
Larry D. Anderson, Ph.D.
Office of Drinking Water
U.S. Environmental Protection Agency
Washington, DC
Editorial Reviewers
Erma Durden, B.S.
Environmental Criteria and Assessment
Office, Cincinnati
U.S. Environmental Protection Agency
Judith Olsen, B.S.
Environmental Criteria and Assessment
Office, Cincinnati
U.S. Environmental Protection Agency
Document Preparation
Technical Support Services Staff: C
B. Zwayer, K. Davidson, J. Moore,
Office, Cincinnati
Cooper, P. Daunt, C. Fessler, K. Mann,
Environmental Criteria and Assessment
Special Note: Since this document was developed from the comprehensive
Information found 1n Ambient Water Quality Criteria for
2,3,7#8-Tetrachlorod1benzo~£-d1ox1n (EPA 440/5-84-007) and
Health Assessment Document for Polychlorlnated D1benzo-£-
dloxlns (EPA 600/8-84-014A), portions of this document were
extracted from these two documents.
W
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TABLE OF CONTENTS
Page
I. SUMMARY 1-1
II. PHYSICAL AND CHEMICAL PROPERTIES II-l
CHEMICAL STRUCTURE AND SYNONYMS II-l
PHYSICAL PROPERTIES II-l
STABILITY 11-4
SUMMARY 11-7
III. TOXICOKINETICS 111-1
ABSORPTION III-l
Absorption From the Gastrointestinal Tract III-l
DISTRIBUTION 111-6
METABOLISM 111-13
EXCRETION 111-17
SUMMARY III-23
IV. HUMAN EXPOSURE IV-1
EXPOSURE ESTIMATION IV-1
Drinking Water . IV-1
Diet IV-2
Air IV-4
SUMMARY IV-5
V. HEALTH EFFECTS IN ANIMALS V-l
EXPERIMENTAL ANIMALS V-l
Acute Toxicity V-l
Subchronlc Toxicity V-46
Chronic Toxicity V-53
TARGET ORGAN TOXICITY V-58
Hepatic Effects V-58
Immunological Effects V-62
Other Organ Systems V-64
OTHER EFFECTS V-66
Carcinogenicity V-66
Mutagenicity V-95
Teratogenicity and Reproductive Toxicity V-108
SUMMARY V-l 21
V
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TABLE OF CONTENTS (cont.)
Page
VI. HEALTH EFFECTS IN HUMANS VI-1
CLINICAL CASE STUDIES VI-1
EPIDEMIOLOGICAL STUDIES. VI-5
HIGH RISK SUBPOPULATIONS VI -61
SUMMARY VI -62
VII. MECHANISM OF TOXICITY VII-T
RECEPTOR-MEDIATED TOXICITY VII-1
2,3,7,8-TCDD: Segregation of Activity with
the Ah Locus VII-2
2,3,7,8-TCDD and Related Toxic Halogenated Aryl
Hydrocarbons: Structure-Activity Correlations . . . V11-4
METABOLISM VII-B
VITAMIN A DEPLETION VII-10
LIPID PEROXIDATION VII-10
ENDOCRINE IMBALANCE VII-11
SUMMARY VII -15
VIII. QUANTIFICATION OF TOXICOLOGICAL EFFECTS VIII-1
INTRODUCTION VIII-1
NONCARCINOGENIC EFFECTS VI11-6
1-Day HA VIII-7
10-Day HA VI11-10
Longer-Term Exposure V111 -10
QUANTIFICATION OF NONCARCINOGENIC EFFECTS VIII-H
Derivation of 1-Day HA V111-14
Derivation of 10-Day HA V111-15
Derivation of Longer-Term HA V111 -16
Assessment of Lifetime Exposure and Derivation
of a DWEL VI11-17
CARCINOGENIC EFFECTS VI11-19
QUANTIFICATION OF CARCINOGENIC EFFECTS VI11-23
EXISTING GUIDELINES, RECOMMENDATIONS AND STANDARDS . . . VIII-26
SPECIAL CONSIDERATIONS VI11-27
t
Synergistic Effects . VIII-27
High Risk Subpopulatlons VIII-28
SUMMARY - . VI11-28
IX. REFERENCES IX-1
v1
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LIST OF TABLES
No. T1tie Page
II-1 Solubility of 2,3,7,8-TCDD. II-2
II-2 Physical Parameters of 2,3,7,8-TCDD 11-3
111-1 Percentage of 2,3,7,8-TCDD 1n the Liver of Rats
24 Hours After Oral Administration of 0.5 mst of Various
Formulations Containing TCDD 111-3
II1-2 Tissue Distribution of 2,3,7,8-TCDD 111-7
111-3 Elimination of 2,3,7,8-TCDD 111-18
V-l Lethal Doses of 2,3,7,8-TCDD Following Acute Exposure ... V-2
V-2 Toxic Responses Following Exposure to 2,3,7,8-TCDD:
Species Differences ....... V-ll
V-3 Estimated Single Oral LD50 - 30 Values for PCDDs V-12
V-4 Immunological Effects of 2,3,7,8-TCDD 1n Animals V-29
V-5 Effects of Chronic Exposure to 2,3,7,8-TCDD In
Laboratory Rodents V-54
V-6 Carcinogenicity Bloassays of 2t3,7,8-TCDD
Administration by the Oral Route. . V-68
V-7 Carcinogenicity Bloassays of 2,3,7,8-TCDD
Administered by the Dermal Route V-72
V-8 2,3,7,8-TCDD Intake and Mortality in Male
Sprague-Dawley Rats V-74
V-9 Benign and Malignant Tumors 1n Rats Ingesting
2,3,7,8-TCDD . V-75
V-l0 Liver Tumors in Rats Ingesting 2,3,7,8-TCDD V-76
V-ll Tumors That Were Significantly Decreased 1n Rats
Following Exposure to 2,3,7,8-TCDD V-79
V-12 Tumor Incidence In Mice Treated with 2,4,5-TCPE
Contaminated with 2,3,7,0-TCDD. V-81
V-l3 Assessment of the Initiation and Promotion Activity
of 2,3,7,8-TCDD 1n Laboratory Animals . V-87
V-l4 The Results of Mutagenicity Assays for 2,3,7,8-TCDD
In Salmonella typh1mur1um . V-96
v11
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LIST OF TABLES (cont.)
No. Title Page
V-15 Studies on the Potential Teratogenic Effects of
2,3,7,8-TCDD Contaminated 2,4,5-T . . . V-109
V-16 Studies on the Potential Teratogenic and Reproductive
Effects of 2,3,7,8-TCDD V-113
VI-1 Distribution of Tumor Types In Two Case-Control
Studies of Soft-Tissue Sarcoma VI-23
VI-2 Exposure Frequencies In Two Case-Control Studies
of Soft-Tissue Sarcoma VI-24
VI-3 Relative Risks of Soft-Tissue Sarcoma In Relation
to Exposure to Phenoxyacetlc Acids and Chlorophenols
In Two Case-Control Studies VI-26
VI-4 Distribution of Histological Types of Soft-Tissue
Sarcomas. VI-31
VI-5 Midland County Soft and Connective Tissue Cancer
Deaths 1960-1981. .... VI-40
VI-6 Other Occupations (Mlnus/Forestry/Agrlculture). ...... VI-47
VI-7 Other Occupations (Mlnus/Forestry/Agrlculture/
Woodworkers) VI -48
VI-8 Analysis of Stomach Cancer Mortality 1n a Group of
West German Factory Workers Exposed to 2,3,7,8-TCDD .... VI-52
VI-9 Reanalysls of Stomach Cancer Mortality In a Group of
West German Factory Workers Exposed to 2,3,7,8-TCDD .... VI-55
VI—10 Stomach Cancer Mortality In Three Studies of Workers
Exposed to Phenoxyacetlc Acid Herbicides and/or
2,3,7,8-TCDD VI-57
VIII-1 Acute Toxicity of 2,3,7,8-TCD0. . . VI11-8
VIII—2 Effects of 4-13 Weeks Exposure to 2,3,7,8-TCDD VI11-11
VIII-3 Effects of Long-Term Oral Exposure to 2,3,7,8-TCDD. .... VIII-13
VIII-4 Carcinogenicity Bloassays of 2,3,7,8-TCDD by Oral
and Dermal Exposure VIII-21
VIII-5 Summary of Human Potency Estimates for 2,3,7,8-TCDD .... VIII-24
v 111
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LIST OF TABLES (cont.)
No. Title Page
VI11-6 Responses and Parameters of the Koclba Feeding Study. . . . V111-25
VI11-7 Summary of Noncarclnogenlc and Carcinogenic Effects
for 2,3,7 ,8-TCDD VII1-24
1 x
I
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LIST OF ABBREVIATIONS
ADI Acceptable dally Intake
AHH Aryl hydroxycarbon hydroxylase
bw Body weight
BCF BloconcentratIon factor
BromoPeCDD Bromopentachlorod1benzo-j>-d1ox1n
DCDD Dlchlorod1benzo-£-d1ox1n
DHBA Dimethylbenzanthracene
DMSG Dimethyl sulfoxide
DNA Deoxyribonucleic acid
EC/GC Electron capture/gas chromatography
ED50 Median effective dose
FEL Frank-effect level
GC/HS Gas chromatography/mass spectrometry
GC/SIH/MS Gas chromatography/sped f1c ion monitoring/mass spec-
trometry
GI Gastrointestinal
HPLC High performance liquid chromatography
HRGC High resolution gas chromatography
HRMS High resolution mass spectrometry
HxCDDs Hexachloro derivatives of dlbenzo-jj-dloxlns
1 .p. Intraperitoneal
LC50 Concentration lethal to 50% of recipients
LD50 Dose lethal to 50% of recipients
LOAEL Lowest-observed-adverse-effeet level
LRHS Low resolution mass spectrometry
3-MC 3-Methylcholanthrene
x
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LIST OF ABBREVIATIONS (cont.)
MFD Mixed function oxidase
NICI Negative Ion chemical Ionization
NOAEL No-observed-adverse-effeet level
NOEL No-observed-effect level
OCOD OctachlorInated d1benzo-£-d1ox1ns
PCDDs All polychlorlnated d1benzo-£-d1ox1ns
PCP Pentachlorophenol
PeCDDs Pentachloro derivatives of d1benzo-£-d1ox1ns
ppb Parts per billion
ppm Parts per million
ppt Parts per trillion
RBC Red blood cells
RNA Ribonucleic acid
SA Satel11te association
s.c. Subcutaneous
TCDDs Tetrachloro derivatives of d1benzo-£-d1ox1ns
TrICDD Tr1chlorod1benzo-f>-d1ox1n
2,4,5-T 2,4,5-TMchlorophenoxy acetic acid
TWA Time-weighted average
UV Ultraviolet
WCOT Wall-coated open tubular
x 1
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United Ste'es
Environmental Prot&cnon
Affe ncy
EPA Research and
Development
DRINKING WATER CRITERIA DOCUMENT FOR
2.3.7,8-TETRACHLORODIBENZO-£-DIOXIN
Prepared for
OFFICE OF DRINKING WATER
Prepared by
Environmental Criteria and Assessment Office
Office of Health and Environmental Assessment
U.S. Environmental Protection Agency
Cincinnati, OH 45268
DRAFT: DO NOT CITE OR QUOTE
NOTICE
This document Is a preliminary draft. It has not been formally released
by the U.S. Environmental Protection Agency and should not at this stage be
construed to represent Agency policy. It Is being circulated for comments
on Us technical accuracy and policy Implications.
¦HNAt—ORAf-V.
ECA0-C1N-40S
5ep4 efflbe f-.- - T9&1
h-PY t" \.
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I. SUMMARY
2,3,7,8-Tetrachlorod1benzo-E.-d1ox1n (2,3,7,8-TCDD) 1s one of the most
toxic and environmentally stable tricyclic aromatic compounds belonging to
*
chlorinated d1benzo-&-d1ox1ns. It 1s a contaminant formed 1n the production
of 2,4,5-trlchlorophenol. It Is also a contaminant of a few chlorinated
phenoxy acids (especially the herbicide 2,4,5-tMchlorophenoxy acetic acid
and sllvex) and hexachlorophene. 2,3,7,8-TCDD 1s considered relatively
stable toward heat, adds and alkalies. It begins to decompose at 500°C and
virtually complete decomposition occurs within 21 seconds at a temperature
of 800°C. It 1s very slightly soluble In water (0*2 ug/l). In aquatic
media, the compound Is expected to persist for a long time since It Is
likely to remain sorbed to sediments and biota.
2,3,7,8-TCDD Is readily absorbed by mammals following either oral or
dermal exposure. Because of Its relatively high lipid solubility, 2,3,7,8-
TCDD 1s rapidly distributed to tissues with a high lipid content. The liver
also represents a major site of accumulation 1n many species. Metabolism
occurs slowly, and the polar metabolites are excreted 1n the urine and
feces. 81Uary excretion of 2,3,7,8-TCDD metabolites also occurs.
Unmetabollzed 2,3,7,8-TCDD Is also excreted In the feces and 1n the milk of
lactatlng animals.
There are great differences' 1n species sensitivity to 2,3,7,8-TCDD,
with LDggS ranging from 0.6 vg/kg bw 1n the guinea pig to 5 mg/kg bw 1n
the hamster. In all species tested, thymic atrophy and severe weight loss
are characteristic of 2,3,7,8-TCDD poisoning, with death occurring several
01290
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09/17/84
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days to weeks following exposure. In rats, rabbits and mice, 2,3,7,8-TCDD
produces acute liver Injury which 1s not observed 1n either monkeys,
hamsters or guinea pigs. Suppression of the Immune system has been observed
1n mice, rats and guinea pigs. Clinical case studies and epidemiological
*
studies have Implicated 2,3,7,8-TCDD as the causative agent 1n the develop-
ment of chloracne, hyperplgmentatlon, altered liver function, porphyria
cutanea tarda and hirsutism In humans. In many respects, the type and
extent of effects observed are similar to those observed 1n nonhuman
primates, though there are differences In the dermal symptomology elicited.
Chloracne Is the major dermal finding 1n humans. In monkeys, common dermal
effects Include loss of hair, toenails and fingernails.
^ 2,3,7,8-TCDD has been demonstrated to be teratogenic 1n rats, mice and
rabbits, and fetid da1 1n monkeys. The major toxic signs and terata
observed were cleft palate In mice, edema, hemorrhage, and kidney anomalies
In rats and extra ribs 1n rabbits. Some epidemiological studies have Indi-
cated a possible teratogenic effect 1n humans, but the evidence is not suf-
ficient to reach a firm conclusion. -
In vivo and Jin vitro mutagenicity tests have produced Inconclusive evi-
dence as to the mutagenicity of 2,3,7,8-TCDD; however, a number of bloassays
have demonstrated this compound to be a potent animal carcinogen. Adenomas
or carcinomas of the thyroid, hepatocellular carcinomas, carcinomas of the
tongue and hard palate, and adenomas of the adrenal gland have all been
produced 1n rats and mice. Some evidence from human epidemiologic studies
associate exposure to herbicides contaminated with 2,3,7,8-TCDD with soft
tissue sarcomas and nonHodgklns lymphomas; however, the exposures to
2,3,7,8-TCDD were always compounded with exposures to herbicide chemicals.
01290 1-2 > 08/10/84
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These epidemiologic studies are consistent with the position that 2,3,7,8-.
TCDD 1s probably carcinogenic for humans. Because 2,3,7,8-TCDD Is usually
found In association with other materials (e.g., chlorophenols, phenoxy-
acetic acids, combustion products, etc.), it Is not presently possible to
evaluate the carcinogenicity of 2,3,7,8-TCDD by itself In humans.
A few possible mechanisms of toxicity have been proposed for 2,3,7,8-
TCDD. These Include receptor mediated toxicity, metabolism/disposition,
vitamin A depletion, increased lipid peroxidation and endocrine Imbalance.
All of the proposed mechanisms may account for some of the toxic effects of
2,3,7,8-TCDD and/or the species differences in sensitivity to the toxin.
However, further research is needed on the mechanism(s) for toxicity of
2,3,7,8-TCDD. Six metabolites have been identified in dogs exposed to
2,3,7,8-TCDD. The major metabolite is 1,3,7,8-tetrachloro-2-hydroxyd1benzo-
j>-d1ox1n.
A 1-day HA of l.OxlO"3 yg/fi, for a 10 kg child was calculated from a
single-dose oral LOAEL In guinea pigs, the species most sensitive to the
toxicity of this compound. A 10-day HA was calculated by dividing the 1-day
HA by 10. The resulting 10-day HA was l.OxlO"4 yg/il for a 10 kg child.
A longer-term HA of lxlO'5 yg/l for a 10 kg child and 3.5xl0~5
vg/9, for 70 kg adult were estimated from a LOAEL derived In a
3-generatlon reproductive study In rats (Murray et a!., 1979) along with the
rationale developed by the EPA. A DWEL of 3.5X10"5 yg/fi. has been
derived for lifetime exposure for an adult. However, the carcinogenicity
risk assessment indicates lower HAs for lifetime exposure to 2,3,7,8-TCDD.
Based on a linearized multistage model, and dose-response data for the
Incidence of tumors of the liver, lung, hard palate or nasal turbinates In
01290 1-3 * 09/23/87
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female rats, the concentrations of 2,3,7,8- TCDD in drinking water that
would result In Increased lifetime cancer risks of l(T4f 10~5, and
1Q"6 were estimated to be 2.2xl0~5, 2.2xl0~€ and 2.2xl0~7 vg/l,
respectively.
01290
1-4
09/23/87
i
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II. PHYSICAL AMD CHEMICAL PROPERTIES
Chemical Structure and Synonyws
2,3,7,8-Tetrachlorod1benzo-|j-d1ox1n (2,3,7,8-TCOD)
CI
CAS Registry Number: 1746-01-6
Chem. Abst. Name: 2,3,7,8-tetrachlorod1benzo[b,e](l,4)-d1ox1n
RTECS Number: HP35000
Synonyms: Dloxln; TCDBD; TCDO; 2,3,7,8-tetrachlorod1benzod1ox1n, 2,3,7,8-
tetrachlorodlbenzo-1,4-d1ox1n (IARC, 1977).
Physical Properties
2,3,7,8-TCOD has a molecular formula of C^H^CI^O^ and a roo1ecu*
lar weight of 321.9. In the pure form, 1t exists as colorless needles with
a melting point of 303-305°C (Cruwnett and Stehl, 1973). In a chloroform
solution, the maximum absorption of 2,3.7,8-TCDD occurs at 310 nm, with a
molar absorption coefficient of 5562 (NRCC, 1981). The available solubility
data (Table II-l) Indicates that 2,3,7,8-TCOD 1s a highly lipophilic
substance.
Values for other physical properties of 2,3,7,B-TC00 that are available
1n the literature searched are given 1n Table I1-2.
01300 II-l 09/18/84
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TABLE II-l
Solubility of 2,3,7,8-TCDD*
Solvent
Solubility (ppm)
Water
2 x 10"*
Lard oil
44
Benzene
570
o-01chlorobenzene
1400
Chloroform
370
Acetone
110
n-Octanol
50
Methanol
10
'Source: Adapted from Crummet and Stehl, 1973
01300
11-2
08/10/84
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*
TABLE II-2
Physical Parameters of 2,3t7#8-TCDD
Parameter Value Reference
Vapor pressure
1.7
X
10"*
NRCC, 1981
(mm of Hg)
Octanol/water
1.4
X
106
NRCC, 1981
partition coefficient
6.9
X
10*
Habey et al., 1981
1.9
X
10'
U.S. EPA, 1984a
Sorption partition
9.9
X
10s
NRCC, 1981
coefficient (Koc)
3.3
X
106
Habey et al., 1981
01300
II-3
09/1B/84
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Stability
2,3,7,8-TCDD Is considered relatively stable toward heat, acids and
alkalies (Albro, 1979). It begins to decompose at a temperature of 500°C
with virtually complete degradation at 800°C within 21 seconds (Stehl et
i
al., 1973). Garrma radiation degrades 2,3,7,8-TCDD (FanelH et al., 1978).
The four transformation processes (photoreactlon, biotransformation,
hydrolysis and radical oxidation) that control the fate of a chemical In
aquatic media do not appreciably transform 2,3,7,8-TCDD (Matsumura et al.t
1983). In organic solvents, 2,3,7,8-TCDD undergoes reductive photodechlorl-
natlon at wavelengths <320 nm (Crosby et al.f 1971; Llbertl et al.# 1978).
In aqueous solution, hydroxylatlve dechlorination has not been seen.
PUmmer et al. (1973) reported that a 2,3,7,8-TCDD suspension In distilled
water remained unchanged when Irradiated with a sunlamp for an unspecified
time. In contrast, 2,3,7,8-TCDD 1n methanol solution, or a benzene solution
of 2,3,7,8-TCDD In water In the presence of a surfactant, underwent substan-
tial photodegradatlon under sunlamp or sunlight Irradiation (PUmmer et al.,
1973; Crosby et al., 1971). The surfactant, 1-hexyldecylpyMdlnlum chlo-
ride, sensitized the photodecomposltlon of 2,3,7,8-TCDD In aqueous solution
(Botre et al., 1978). In order to explain the longer half-Hfe of 2,3,7,8-
TCDD (1.6 year vs. 1 year) 1n a model laboratory ecosystem than 1n an out-
door pond, Matsumura et al. (1983) and Tsushlmoto et al. (1982) speculated
that photolysis 1s the most likely cause. In the outdoor environment, where
the Intensity of sunlight Is higher compared with the laboratory experiments
(100 w fluorescent lamp over 40 cm of water surface), algae-mediated photo-
sensitization of 2,3,7,8-TCDO was speculated to have caused some photodecom-
posltlon of this compound. From the available Information, It 1s difficult
01300 I1-4 > 04/05/84
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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) 1n surface waters, photolysis may be a significant fate process.
2,3,7,8-TCDD exhibits relatively strong resistance to microbial
blodegradatlon. Only 5 of -100 microbial strains that have the ability to
degrade persistent pesticides show slight ability to degrade 2,3,7,8-TCDD
(U.S. EPA, 1980). 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, Tsushlmoto et al. (1982)
and Matsumura et al. (1983) estimated the apparent half-life of 2,3,7,8-
TCDD to be -1 year. Although blodegradatlon may have been responsible for
part of the degradation, other Investigators (Huetter and Ph1l1pp1, 1982)
have reported the virtually complete lack of b1odegradabH1ty of
2,3,7,8-TCDD.
The blodegradatlon half-Hfe of 2,3,7,8-TCDD can be estimated from the
u
theoretical rate constant values based on relative rates of transformation
reported 1n the literature or on structure-activity analogy values given by
Habey et al. (1981). Assuming the biotransformation rate constant of
lxlO"10 ml cell"1 hr"1 (Mabey et al., 1981) and the concentration of
microorganisms capable of degrading 2,3,7,8-TCDD as 5xl05 cell ml"1
(Burns et al., 1981), the half-Hfe of blodegradatlon Is estimated to be
>1 year.
01300
11-5
04/05/84
k.
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Although several Investigators Implicated volatilization as one of the
major reasons for the observed disappearance of 2,3,7,8-TCDD from aqueous
I
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 1n aquatic media
(Matsumura et al., 1983). A transport model to estimate 2,3,7,8-TCDD
volatilization from a cooling pond on an Industrial site based on measured
concentrations 1n the pond bottom sediment and pond surface area led to an
estimated rate of 15-16 mg/year (Thlbodeaux, 1983). Using the formulas of
L1ss and Slater (1974), a vapor pressure value of -10"6 torr (0.1 m Pa)
and a solubility value of 6.2xlO~10 mole/t, NRCC (1981) calculated the
volatilization half-Hfe 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-Hfe 1s
directly proportional to water depth and Inversely proportional to mass
transfer coefficient (Thlbodeaux, 1979). The limitations of the L1ss-Slater
theory to predict the rate of volatilization have been discussed 1n the NRCC
(1981) document. The L1ss-Slater model does not consider terrestrial
matrices (suspended solids, sediments, biota, etc.) normally encountered In
natural surface water. Employing a computerized EXAMS model for two stan-
dardized aquatic ecosystems and the Input parameters for 2,3,6,8-TCDD, as
discussed 1n NRCC (1981), volatilization has been estimated to account for
100% of the fraction lost and blodegradatlon has been calculated to be OX.
The volatilization half-Hfe for 2,3,7,8-TCDD has been estimated to be 5.5
and 12 years from pond and lake water, respectively. However, 1t should be
remembered that these are estimated values and experimental confirmation of
these values Is not available.
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Data from microcosm experiments Indicate that 2,3,7,8-TCDD Is highly
sorbed to sediments and biota (Isensee and Jones, 1975; Ward and Matsumura,
1970). More than 30% of 2,3,7,8-TCDD 1n an aquatic medium may be present 1n
the adsorbed state (Ward and Matsumura, 1978; Matsumura et aK, 1983). This
*
1s not surprising considering the low water solubility and the high octanol/
water partition coefficient. In fact, the equation of KaMckhoff et al.
(1979) predicts a sorption partition coefficient value of 104 for 2,3,7,8-
TCOD 1n sediments containing 2% organic carbon (NRCC, 1981).
Summary
2,3,7,8-TCDD has a molecular formula of C_.H_C1.0_ and a molecu-
12 4 4 2
lar weight of 321.9. In pure form, 1t exists as colorless needles with a
melting point of 303-305°C (Crunmett and Stehl, 1973) and 1s relatively
reslstent to degradation by heat, acids and alkalies. It Is very slightly
soluble In water (0.2 ppb) and somewhat soluble In organic solvents
(Crunmett and Stehl, 1973). The compound has a low vapor pressure
(1.7xl0~6 rmi of Hg) and a high octanol/water partition coefficient (NRCC,
1981; Habey et al.t 1981).
From the available information, 1t 1s difficult to predict the photo-
lytic fate of 2,3,7,8-TCDD 1n aquatic media. In the absence of hydrogen
donating substrates, photolysis does not appear to be a significant process
(Pllrrmer et al., 1973; Crosby et al., 1971). 2,3,7,8-TCDD exhibits rela-
tively strong resistance to microbial degradation (U.S. EPA, 1980; Huetter
and Ph1l1pp1, 1982). The eslmated blodegradatlon half-life of 2,3,7,8-TCDD
1s >1 year (Mabey et al., 1981; Burns et al., 1981). Hydrolysis and radical
oxidation do not appear to be significant processes for 2,3,7,8-TCDD 1n
aquatic media (NRCC, 1981).
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Little quantitative Information regarding the volatilization of 2,3,7,8-
TCDD from aquatic media 1s available. Theoretical modeling of 2,3,7,8-TCDD
(EXAMS model) provides a volatilization half-life for 2,3,7,8-TCDD of 5.5
and 12 years from pond and lake water, respectively (NRCC, 1981). However,
no* experimental confirmation of these values Is available. Experimental
data show that this compound Is likely to remain sorbed to sediments and
biota In aquatic media (Isensee and Jones, 1975; Ward and Matsumura, 1978).
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III. TOXICOKINETICS
The toxicokinetics of 2,3,7,8-TCDD has been Investigated In a number of
laboratory animals, and there are several recent reviews on this subject
(Neal et al., 1982; Gaslewlcz et al., 1983a; Olson et al.# 1983). This
sentlon will examine our cyrrent understanding of the absorption, distribu-
tion, metabolism and excretion of 2,3,7,8-TCDD 1n various manmallan species.
Absorption
The dermal and gastrointestinal absorption of 2,3,7,8-TCDD have been
Investigated 1n several species. No studies are available on the toxico-
kinetics of 2,3,7,8-TCDD through the Inhalation route of exposure.
Absorption From the Gastrointestinal Tract. Experimentally, 2,3,7,8-
TCDD Is generally administered 1n the diet or by gavage 1n an oil vehicle.
In Sprague-Dawley rats given a single oral dose of 1.0 vg [1*CJ2,3,7,8-
TCOD/kg bw, absorption from the Intestinal tract was estimated at -83% (Rose
et al., 1976). With repeated oral dosing at 1.0 yg/kg/day (5 days/week x
7 weeks), absorption was observed to be approximately that observed for the
single oral dose. With a much larger single oral dose, 50 pg/kg bw, -70%
of the dose was absorbed by Sprague-Dawley rats (Piper et al., 1973). In
these studies, the chemical was administered 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 acetonercorn oil was
absorbed (Nolan et al.# 1979). The gastrointestinal absorption of
2,3,7,8-TCDD was also examined in the hamster, the species most resistant to
the acute toxicity of this toxin (Olson et al., 1980a). Olson et al.
(1980a) administered hamsters a single, sublethal, oral dose of
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[1,6-*H]-2,3,7,8-TCDD In olive oil (650 vg/kg) and reported that 74% of
the dose was absorbed. When 2,3,7,8-TCDD was administered to rats In the
diet at 7 or 20 ppb (0.5 or 1.4 yg/kg/day) for 42 days, 50-60% of the
consumed dose was absorbed (Fries and Harrow, 1975). These findings
*
Indicate that over a wide range of doses and under these experimental condi-
tions, 2,3»7f8-TCDD 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-TCDO was administered in the diet or
through an oil vehicle.
The Influence of dose and vehicle or adsorbent on gastrointestinal
absorption has been Investigated In 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-TCDO
administered In 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 In Table II1-1. Admin-
istration of 2,3,7,8-TCDO In an aqueous suspension of soil resulted In a
decrease In the hepatic levels of 2,3V7V8-TCDD as compared with hepatic
levels resulting from administration of 2,3,7,8-TCDD In 50% ethanol. The
extent of the decrease was directly proportional to the length of time the
2,3,7,8-TCDO had been 1n contact with the soil. When 2,3,7,8-TCDD was mixed
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TABLE III-l
Percentage of 2,3,7,8-TCDO 1n the Liver of Rats 24 Hours After Oral
Administration of 0.5 mt of Various Formulations Containing TCDD*
Formulation TCOO Dose No. of Percentage of Dose
{ng) Animals 1n the Liver
50% Ethanol
14.7
7
36.7 ~
1.2
Aqueous suspension of soil
(37%, w/w) that had been
In contact with TCOO for:
10-15 hours
8 days
12.7, 22.9
21.2, 22.7
17
10
24.1 *
16.0 +
4.8
2.2
Aqueous suspension of
activated carbon
(25%, w/w)
14.7
6
<0.07
•Source: Adapted from Polger and Schlatter, 1980
01310 II1-3 08/10/84
I
-------
1n an aqueous suspension of activated carbon, absorption was almost totally
eliminated (<0.07% of the dose 1n hepatic tissues).
Ph1l1pp1 et al. (1981) and Hutter and PhlUppI (1982) have shown that
radiolabeled 2*3,7,B-TC00 becomes progressively more resistant with time to
extraction from soil. Similarly, the feeding of fly ash, which contains
PCOOs, to rats in the diet for 19 days resulted 1n considerably lower
hepatic levels of PCOOs than did the feeding of an extract of the fly ash at
comparable dietary concentrations of PCOOs (van den Berg et al., 1983). The
PCOOs were tentatively Identified as 2,3,7,8-TCOO, 1,2,3,7,8-PeCOO,
1,2,3,6,7,8-HxCOD and 1,2,3,7,8,9-HxCOO and the difference In hepatic levels
noted between fly ash-treated and extract-treated rats was greater for the
more highly chlorinated Isomers than 1t was for 2,3,7,8-TCOO. These results
Indicate the Importance of the formulation or vehicle containing the
toxln(s) on the relative bioavailability of 2,3,7,8-TCDD, PeCOO and HxCDOs
following oral exposure.
Information on the absorption of 2,3,7,8-TCDD through the skin Is found
only In a study by Polger and Schlatter (1980). The authors administered 26
ng 2,3,7,8-TCOO In 50 yl 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 1n 50% ethanol (In the
same study), the amount absorbed from a dermal application can be estimated
at ~40% 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
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In 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) in the rabbit ear assay.
Application of 2,3,7,8-TCDD In a soil/water paste decreased hepatic 2,3,7,8-
TCDD to -2% of the administered dose and increased the amount required to
produce skin lesions to 2-3 yg in rats and rabbits, respectively. Appli-
cation 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.
Since 2,3,7,8-TCDD 1n the environment is likely to be absorbed to soil,
McConnel et al. (1984) and Lucler et al. (1986) compared the absorption of
2,3,7,8-TCDD from contaminated soil to that from 2,3,7,8-TCDD administered
in corn oil. As Indicated by biological effects and the amount of
2,3,7,8-TCDD in the liver, the absorption from soil was -50% less than from
corn oil. Umbrelt et al. (1986a) showed that 2,3,7,8-TCDD contaminated soil
was less toxic than an equivalent amount of 2,3,7,8-TCDD, suggesting that
binding to soil had an influence on bioavailability. Although these data
Indicate that substantial absorption occurs from contaminated soil, soil
type and duration of contact, as suggested from the data which demonstrated
decreased extraction efficiency with Increasing contact time between soil
and 2,3,7,8-TCDD (PhllUpi et al., 1981; Huetter and Phlllipi, 1982), may
substantially affect the absorption of 2,3,7,8-TCDD from soils obtained from
different contaminated sites.
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Poiger and Schlatter (1986) Investigated the absorption of 2,3,7,8-TCDD
in a 42 year old man after ingestion of 105 ng 3H-2,3,7,8-TCDD in 6 ma.
corn oil and found that greater than 87% of the oral dose was absorbed from
the intestine. Following absorption, the half-life for elimination was
estimated to be 2,120 days.
Distribution
The tissue distribution of 2,3,7,8-TCDD in a number of species is summa-
rized in Table III-2. From these data It is 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. Host 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.
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-TCDD and/or Its metabolites was through the feces. With repeated
oral doses, the 14C activity was also excreted primarily through the
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TABLE 111-2
Tissue Distribution of 2,3,7,8-TCDD
Species Route of Tissues with the Highest Concentration References
Administration of 2,3,7,8-TCDD
Rat
oral
11 ver
Fries and Harrow, 1975
Rat
oral
1 iver
>
fat
Rose et al., 1976
Rat
oral
11 ver
>
fat
Piper et al., 1973
Rat
oral
1 Iver
>
fat
Koclba et al., 1978a,b
Rat
oral
1 Iver
>
fat
Allen et al., 1975
Rat
l.p.
1 Iver
>
fat
Van Hlller et al., 1976
House
oral
1 Iver
>
fat
>
kidney > lung
Manara et al., 1982
Mouse
l.p.
1 Iver
>
fat
>
kidney > lung > spleen
Hanara et al., 1982
Rhesus monkey
l.p.
fat >
skin >
liver > adrenal = thymus
Van Hlller et al., 1976
Golden Syrian
hamster
l.p. or oral
1 Iver
>
fat
Olson et al., 1980a
Guinea pig
oral
fat >
1 iver
>
adrenals > thymus > skin
Nolan et al., 1979
Guinea pig
l.p.
fat >
11 ver
>
skin > adrenals
Gaslewlcz and Neal, 1979
-------
Feces, but significant amounts were found in 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 12.5% in the urine (Rose et al., 1976).
Fries and Marrow (1975) have also reported evidence of sex differences in
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 in the liver,
while 70% of the total body residue of female rats was located In this organ.
Studies performed by Van Miller et al. (1976) on rhesus monkeys and rats
using single i.p. doses of tritlated 2,3,7,8-TCDD (400 yg/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 i.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 pg 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. administratis, 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 In 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).
Koclba et al. (1978a,b) found that female rats maintained on a daily
dietary 2,3,7,8-TCDD Intake of 0.1 yg/kg/day for 2 years had an average
01310 111-8 04/08/88
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2,3,7,8-TCDD content of 8100 ppt in fat and 24,000 ppt in the liver. Rats
given 0.01 pg/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 liver-.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 In
the guinea pig appears to be similar to the monkey, with the highest concen-
tration of the toxin being found In adipose tissue (Gaslewlcz 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-TCDD for the
hepatic microsomal fraction; however, the significance of these differences
remains In doubt. For example, the hepatotoxlclty 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 fetotoxlc in
the rat (see Teratogenicity 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 [14C]2,3,7,8-TCDD was Investigated by Moore et al. (1976). They
found low concentrations of 2,3,7,8-TCDD in the fetus at gestation days 14,
01310
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04/08/88
»
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18 and 21. The radioactlvity appeared to be evenly distributed throughout
the fetus on days 14 and 18; however, Increased levels of radioactivity were
detected in fetal liver on day 21. Nau and Bass (1981) (more recently
reported by Nau et a 1 -, 1982) Investigated the fetal uptake of 2,3,7,8-lCDD
in NMRI mice following oral, l.p. or s.c. administration of the compound at
dose levels of 5, 12.5 or 25 yg/kg in DMSOicorn 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 yg/kg. At 25
ug/kg, higher maternal and fetal tissue levels were obtained with s.c.
administration, and much higher levels were obtained with 1.p. administra-
tion, than were obtained with oral administration. Embryonic 2,3f7,B-TCDD
concentrations were maximal on gestational days 9 and 10; however, low
levels were found In the embryo and fetus between gestational days 11 and
18. This sharp decrease in 2,3,7,8-TCDD concentration coincides with
placentatlon. 2,3,7,8-TCDO concentrations in the placenta were an order of
magnitude greater than in the fetus itself. The affinity of fetal liver for
2,3,7,8-TCOD was relatively low, as compared to maternal liver; however,
2,3,7,8-TCDD levels In fetal livers were 2-4 times higher than the levels in
other fetal organs. An attempt was made to correlate 2,3,7,8-TCOD levels in
the fetuses with the observed incidence of cleft palate, but no clear rela-
tionship was observed.
Autoradiographic studies of tissue localization following 1 .v. adminis-
tratlon of [14C]2,3,7,8-TCDD in 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).
In pregnant mice, the concentration of radioactivity In the fetuses was
01310 111-10 04/08/88
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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-TCDD
in humans. Facchetti et al. (1980) reported tissue concentration of
2,3,7,8-TCDD at levels of 1-2 ng/g in Hver 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 adenocarcinoma (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 A1r Force officers with known heavy exposure to Agent
Orange during disposal operations, and 10 control veterans with no known
herbicide exposure. In the first group, 10 of the 20 had measurable levels
of 2,3,7,8-TCDD (5 with 5-7 ppt, 3 with 9-13 ppt 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-TCDD levels and these did not exceed 3 ppt. In the 10
control veterans, 4 had 2,3,7,8-TCDD levels between 7 and H ppt. Levels of
01310 111-11 04/08/88
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2,3,7,8-TCDD in adipose tissue did not appear to be associated in this study
with ill health or any particular symptom. However, it was considered that
information on background levels of 2,3,7,8-TCDD in adipose tissue was too
limited to draw any firm conclusions.
Following the analytical techniques described by Albro et al. (1985),
Ryan et al. (1985) analysed seventy-two autopsy samples of human adipose
tissue from North America and observed the presence of PCDDs and PCDFs at
low ppt levels.
Distribution of 2,3,7,8-TCDD has been reported In the general population
by Nygren et al. (1986); In Vietnam and veterans by Schecter et al. (1986,
1987); in mothers milk by Nygren et al. (1986), Rappe et al. (1984), and
Fuerst et al. (1987).
Although monitoring of adipose tissue may provide some qualitative
indication that exposure has occurred, there are no good correlations
available between adipose tissue levels and the extent of exposure. In
addition, the background level of 2,3,7,8-TCDD In the adipose tissue of
individuals with no known history of exposure to 2,3,7,8-TCDD generally are
In the range of 5-18 ppt. This would suggest an ubiquitous exposure to
2,3,7,8-TCDD, which makes It difficult to assess the contribution to body
burden from any particular small additional exposure. Similar lack of
correlation between estimated exposure to 2,3,7,8-TCDD and sera levels of
2,3,7,8-TCDD were reported In a preliminary report in the MMWR (1987) which
compared Vietnam veterans with military histories Indicating exposure to
herbicides containing 2,3,7,8-TCDD and non-V1etnam veterans with presumably
no unusual exposure to 2,3,7,8-TCDD. At least In these preliminary results
01310 111-12 h 04/12/88
-------
there was no difference in the range of 2>3,7,8-TCDD levels (1-9 ppt based
on lipid weight) or the median 2,3,7,8-TCDD level (3.9 ppt for the presum-
ably exposed group and 3.8 ppt for the non-exposed group). Biological
monitoring, such as monitoring levels in breast milk, only provides possible
qualitative Indications of exposure. With commonly available analytical
techniques, 2,3,7,8-TCDD is not detected In body fluids, such as blood or
urine, although a recent method with ppq (parts per quadrillion) sensitivity
has detected 2,3,7,8-TCDD in human serum (Patterson et al.f 1987b).
Metabolism
Vinopal and Casida (1973) found no evidence of water soluble metabolites
of 2,3,7,8-TCDD following Incubation with mammalian liver microsomes or l.p.
Injection Into mice. In the same experiment, only unmetabollzed 2,3,7,8-
TCDO 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 1.p. Injections suggested that
2,3,7,8-TCDD was not readily metabolized. Metabolites of 2,3,7,8-TCDD have
been detected in 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 (Poiger
et al., 1982a).
Poiger and Schlatter (1979), Ramsey et al. (1979) and Ramsey et al.
(1982) demonstrated biliary _ excretion of several metabolites of
[14C]2,3,7,8-TCDD by rats after repeated oral dosing. The metabolites
were tentatively 1 dent 1 fled as glucuronldes of hydroxy lated 2,3,7(8-TCDD.
The amounts of metabolites found were small, Indicating that 2,3,7,8-TCDD Is
only slowly metabolized In the liver. Previous work by Piper et al. (1973)
01310
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using single oral doses of 2,3,7,8-TCDD 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 14C activity
in the rat livers appeared to be present as unchanged 2,3,7,8-TCDD, a
significant amount of radioactivity found in the feces appeared to come from
substances other than 2,3,7,8-TCDD; the excretion of 14C in the urine also
indicated that metabolism had occurred.
Polger et al. (1982a) investigated the toxicity 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 in 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 toxicity of 2,3,7,8-TCDD to
guinea pigs was at least 100 times higher than was the acute toxicity of its
rnetabol 1 tes.
More recently, Olson et al. (1983) reported that all of the radioactiv-
ity 1n urine and bile from 1
-------
is dependent upon metabolism of the toxin. Although urine and bile appear
to be free of unmetabol ized TCDD, data from the hamster and rat Indicate
that from 10 to 40% of the 2,3,7,8-TCDD-der i ved radioactivity In feces
represents unchanged 2,3,7,8-1CDD (Olson et al.p 1983; Olson and Blttner,
1983). The daily presence of unchanged 2,3,7,8-TCDD In feces and its
absence in bile suggests that direct Intestinal elimination may be the
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 j_n 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 its elimination
or relative persistence in a given animal.
Several metabolites of 2,3,7,8-TCDD have recently been Identified.
Sawahata et al. (1982) investigated the j_n vitro metabolism of 2,3,7,8-TCDD
In Isolated rat hepatocytes. The major product was deconjugated with
[3-glucuronldase, derivatlzed with dlazomethane, and separated Into two com-
pounds by high performance liquid chromatography (HPLC). These metabolites
were subsequently identified as 1-hydroxy-2,3,7,8-TCDD and B-hydroxy-2,3,7-
tr1chlorodibenzo-£-d1ox1n. Poiger et al. (1982a) identified six metabolites
in the bile of dogs that were given a lethal dose of [3H]2,3,7,8-TCDD. The
major metabol1te was 1, 3f7,8-tetrachloro-2-hydroxyd1benzo-£-dioxin; 3,7,8-
tr1chloro-3-hydroxyd1benzo-£-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 trichloro-
hydroxyd1benzo-£-d1ox1ns and the third was apparently a chlorinated
2-hydroxyd1phenyl ether.
01310 111-15 04/08/88
-------
Data on the metabolism of 2,3,7,8-TCDD suggests that reactive epoxide
intermediates may be formed. Poland and Glover (1979) have Investigated the
iQ 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 amino acids in protein, 12 pmol 2,3,7,8-TCDD/mole of
nucleotide in rRNA, and 6 pmol of 2,3,7,8-TCDD/mole of nucleotide in DNA.
This corresponds to one 2,3,7,8-TCDD-DNA adduct/35 cells. Poland and Glover
(1979) suggest that It is unlikely that 2,3,7,8-TCDD-1nduced oncogenesis is
through a mechanism of covalent binding to DNA and somatic mutation.
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 toxicity of 2,3,7,8-TCDD.
Isolated rat hepatocytes In suspension have been used as an 1n_ 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-TCDD metabolism in
rat hepatocytes correlates directly with drug Induced changes in hepatic
cytochrome P-450 monooxygenase activity, suggesting that 2,3,7,8-TCDD Is
metabolized by this enzyme (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 its own
rate of metabolism. Beatty et al (1978) also found a correlation between
hepatic mixed-function oxidase (MFO) activity and the toxicity of 2,3,7,8-
TCDD In 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 LD^q.
01310
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Olson and Bittner (1983) reported that the rate of 2,3,7,8-TCDD metabo-
lite formation j_n vitro was higher in hepatocytes from the hamster than in
hepatocytes from the rat. Qualitative evaluation of j_n vivo and u\ vitro
metabolites by HPLC also suggested significant interspecies variability.
The authors suggested that such differences in metabolism may partially
explain the differences In toxicity 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
on a semilogarIthrnlc plot, Indicating that elimination Is a single, first
order process. Hlles 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 111-3 summarizes results on the elimination
of 2,3,7,8-TCDD-derIved radioactivity, following a single exposure to
3H- or [14C]-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 toxicity 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 excreted radioactivity) (Olson et al., 1980b; Gas1ew1cz et al.,
1983a). The high levels found In the urine of Infant monkeys were probably
01310 111-17 04/08/88
-------
TABLF III-3
Elimination of 2,3,7,8-TCDD
Relative % of TCDD-Derlved
Single Treatment Half-Life for Radioactivity
Species (route) Elimination Reference
(days) Feces Urine
Guinea pig
2
(l.p.)
30.2
t 5.8
94.0
6.0
Gaslewlcz and Neal, 1979
Guinea pig
1.
45 (oral)
22 -
43
NT
NT
Nolan et al., 1979
Rat
1.
.0 (oral)
31 i
6
>99
<1
Rose et al., 1976
Rat
'50
(oral)
17.4
i 5.6
80.0
20.0
Piper et al., 1973
Rat
50
(oral)
21.3
± 2.9
95.5
4.5
Allen et al., 1975
Rat
400
(i.p.)
NT
91.0
9.0
Van Miller et al., 1976
Monkey
(adult)
400
(l.p.)
NT
78.0
22.0
Van Miller et al., 1976
Monkey
(Infant)
400
H.p.)
NT
39.0
61.0
Van Miller et al.. 1976
Mouse
C57B1/65
DBA/2J
B6D2F!/J*
10
10
10
(l.p.)
(1-p.)
(l.p.)
11.0
24.4
12.6
v 1.2
* 1.0
0.8
72.0
54.0
72.0
28.0
46.0
28.0
Gaslewlcz et al., 1983a,b
Gaslewlcz et al., 1983a,b
Gaslewlcz et al., 1983a,b
Hamster
650
(1-P-)
10.8
± 2.4
59.0
41.0
Olson et al., 1980a
Hants ter
650
(oral)
15.0
± 2.5
NT
Nl
Olson et al., 1980a
*0ffspr1ng of C57B1/6J and DBA/2J which are heterozygous at the Ah locus
NT = Not tested
-------
due to the incomplete 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 radioactivity) was recovered In the expired air.
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 yg/kg/day Monday
through Friday for 7 weeks, or a single dose of 1.0 yg/kg. In the single-
dose study, no 14C was excreted In 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 In the bodies of rats In -13 weeks. The rate constant
defining the approach to steady-state concentrations was Independent of the
dosage of 2,3,7,8-TCDD over the range studied. This Is consistent with the
observations of Fries and Marrow (1975) who found that the total retention
In the bodies of rats was proportional to total Intake. When rats were
maintained on a diet containing either 7 or 20 ppb 2,3,7,8-TCDD, the amount
of 2,3,7,8-TCDD retained In 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 III-3 suggest some Interspecies differences in the
half-life for elimination (t 1/2) of 2,3,7,8-TCDD. In the hamster, the
01310
111*19
04/08/88
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least sensitive species to the acute toxicity of 2,3.7,8-TCDD, a mean t 1/2
of 10.8 days was observed (Olson et al.# 1980a,b), and in the guinea pig,
the most sensitive species to the acute toxicity of 2,3,7,8-TCDD, the mean
t 1/2 was 30.2 days (Gasiewicz and Neal, 1979). The observed interspecies
differences in the t 1/2 of 2,3,7,8-TCDD may in part be related to the
relative sensitivity of a given species to the acute toxicity of
2.3,7,8-TCDD.
The intraspecies differences 1n the t 1/2 of 2,3,7,8-TCDD in three mouse
strains may be due to the finding that the DBA/2J strain possesses -2-fold
greater adipose tissue stores than the C57B1/6J and B6D2F^/J strains
(Gasiewicz et al.t 1983b). The sequestering of the lipophilic toxin in
adipose tissue stores of the DBA/2J mouse may contribute to the greater
persistence of 2,3,7,8-TCDD In this strain.
In all of the rat studies shown In Table III-3, urinary and fecal elimi-
nation were monitored for a period of only 20-22 days, and from these data
1t was assumed that elimination followed a single component, first order
kinetic model. Recently, Olson and BUtner (1983) examined the elimination
of 2,3,7,8-TCDD-derlved radioactivity In rats over a 35-day period following
a single l.p. exposure at 1 pg 3H-2,3,7,B-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 In the rhesus monkey indi-
cates that 2,3,7,8-TCDD may be exceptionally persistent 1n adipose tissue.
01310
III-20
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McNulty et a 1. (1982) estimated the apparent half-life of 2,3,7,8-TCDD 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 a 1., 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 unmetabolized 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). Unmetabolized 2,3,7,8-TCDD thus appears to enter the intestinal
lumen by some route other than bile (direct Intestinal elimination) for a
number of days following treatment. Studies In lac tat 1ng rats have also
found that unchanged 2,3,7,8-TCDD may be excreted In the milk of lactating
animals (Moore et al.f 1976; Lucler et al., 1975). Lactation, direct Intes-
tinal elimination, and perhaps sebum may serve as routes for excretion of
2,3,7,8-TCDD, which are not dependent upon metabolism of the toxin. These
data suggest that the j_n v1vo half-life for elimination of 2,3,7,8- TCDD may
not directly reflect the rate of 2,3,7,8-TCDD metabolism In a given animal
(Neal et al., 1982).
Due to the lipophilic nature' of milk, secretion of milk can provide a
relatively efficient mechanism for decreasing the body burden of
2,3,7,8-TCDD in females. As discussed by Graham et al. (1986), this
elimination of 2,3,7,8-TCDD through mother's milk can result In large
exposures of the Infant. Since both milk and the fatty tissues of fish are
01310 111-21 - 04/12/88
-------
essentially providing an oily vehicle, it would be likely that these sources
would provide 2,3,7,8-TCDD in a form that is readily bioavallable.
Several investigators have recently quantified the levels of 2,3,7,8-
TCDD In human milk samples. Many of the milk samples were pooled (Jensen,
1987). Rappe et al. (1984) reported levels of 1-3 ppt 2,3,7,8-TCDD in milk
fat from five volunteers In W. Germany and in a later report Rappe et al.
( 1985) reported an average level of 0.6 ppt 2,3,7,8-TCDD in milk fat from
four volunteers in northern Sweden. Furst et al. (1986) reported an average
level of 9.7 ppt 2,3,7,8-TCDD in milk fat from 3 Individuals in the Nether-
lands and <1.0 ppt 2,3.7,8-TCDD in milk fat from 2 individuals in Yugo-
slavia. Nygren et al. (1986) reported average levels of 2,3,7,8-TCDD In
human milk samples from 4 subjects 1n Sweden to be 0.6 pg/g in milk fat, in
5 subjects from W. Germany to be 1.9 pg/g in milk fat, and In 4 subjects
from Vietnam to be <0.5 pg/g In milk fat.
High levels of 2,3,7,8- TCDD have been reported In the milk of mothers
exposed to high levels of 2,3,7,8-TCDD in the environment. Reggiani et al.
(1980) reported levels between 2.3 and 28.0 ppt 2,3,7,8-TCDD in whole milk
from mothers in Seveso. Baughman (1976) reported levels between 40.0 and
50.0 ppt 2,3,7,8-TCDD In whole milk form mothers In South Vietnam. Schecter
et al. (1987), also, found high ppt levels of 2,3,7,8-TCDD in human milk
samples from Vietnam. These authors found the samples taken In 1985 from
Vietnameese mothers were comparable to the level of 2,3,7,8-TCDD presently
found In North American human milk samples (5 ppt).
01310
111-22
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Summary
The toxicokinetics of 2,3,7,8-TCDD has been investigated in a number of
laboratory animals; the reader is referred to recent reviews (Neal et al.,
1902; Gasiewicz et al.t 1983a; Olson et al., 1983) for Indepth discussions
of this subject.
Because 2,3,7,8-TCDD is a strongly lipophilic compound (Crummett and
Stehl, 1973), gavage treatment with single or repeated doses of the compound
in oil has resulted in absorption of -50% of the dose administered to guinea
pigs (Nolan et al., 1979), -70-83% of the dose administered to rats (Rose et
al., 1976; Piper et al., 1973) or to hamsters (Olson et al.f 1980a).
Dietary administration of comparable dose-ranges to rats resulted in some-
what reduced GI absorption (-50-60% of administered dose was absorbed)
(Fries and Marrow, 1975).
Using hepatic concentration of 2,3,7,8-TCDD as a endpoint, Poiger and
Schlatter (1980) demonstrated a linear relationship In rats between the
magnitude of an oral dose of 2,3,7,8-TCDD and absorption, up to a dose of
~1.0 yg. By treating rats with aqueous suspensions of soil treated with
2,3,7,8-TCDD these researchers (Poiger and Schlatter, 1980) were able to
show a decrease In GI absorption of 2,3,7,8-TCDD directly proportional to
the length of time the compound had been In contact with soil before the
suspension was made and the rats were treated. Mixing 2,3,7,8-TCDD with a
aqueous suspension of activated charcoal essentially eliminated absorption
as measured by hepatic concentrations of 2,3,7,8-TCDD (Poiger and Schlatter,
1980). That adsorbant materials may reduce the GI absorption of 2,3,7,8-
01310
111-23
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TCDO was observed by Van den Berg et a 1. ( 1983), who demonstrated that
absorption of PCDDs was less from fly ash (naturally containing PCDDs) than
from comparable doses of PCDDs from extracts of fly ash.
Percutaneous absorption of 2,3,7,8-TCDD has been estimated in rats to be
-40% of the absorption of an equivalent dose orally administered (Poiger and
Schlatter, 1980). Dermal application of the compound in vaseline, poly-
ethylene glycol or soil/water paste substantially reduced dermal- absorp-
tion. Application of the compound in activated carbon/water paste virtually
eliminated absorption.
Tissue distribution following oral or i.p. administration of 2,3,7,8-
TCDD to rats appears to be preferentially to the liver and adipose tissue
(Fries and Marrow, 1975; Rose et al., 1976; Van Miller et al.f 1976; Kociba
et al., 1978a). Other tissues showed substantially lower concentrations of
2,3,7,8-TCDD. Soon after treatment the liver may have concentrations =3
(Kociba et al.# 1978a) to 5 (Rose et al., 1976) times that in adipose tis-
sue. It was suggested that male rats accumulate 2,3,7,8-TCDD in the liver
more efficiently than female rats (Fries and Marrow, 1975). Tissue distri-
bution in mice (Manara et al., 1982) and hamsters (Olson et al., 1980a)
seems to be similar to that in rats.
Monkeys, however, appear to accumulate 2,3,7,8-TCDD preferentially in
adipose tissue > liver (Van Miller et al., 1976; McNulty et al., 1982). Two
years after a single oral dose to a monkey, the fat contained 100 ppt and
the liver 15 ppt 2,3,7,8-TCDD (McNulty et al., 1982). Prolonged tissue
01310
III-24
04/12/88
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retention of the compound was thus demonstrated. Tissue distribution In
guinea pigs appears similar to that in monkeys (Gasiewicz and Neal, 1979;
Nolan et al., 1979) in that tissue levels in fat exceed those in the liver.
Evidence that 2,3,7,8-TCDD accumulates 1n the adipose tissue of exposed
humans was presented by Young et al. (1983) who reported levels of 3-99 ppt
in the adipose tissue of armed forces veterans claiming health problems
related to Agent Orange.
Distribution of 2,3,7,8-TCDD to the fetus has been studied in rats
(Moore et al., 1976) and mice (Nau and Bass, 1981; Nau et al., 1982).
Levels of 2,3,7,8-TCDD were low in rat fetuses on gestation days 14 and 18
and appeared to be evenly distributed in all fetal tissues. At gestation
day 21, the fetal liver showed a marked affinity for 2,3,7,8-TCDD (Moore et
al.# 1976). 2,3,7,8-TCDD was distributed to the fetuses of mice following
oral, l.p. or s.c. administration (Nau et al., 1982). Maximum fetal concen-
trations occurred on gestation days 9 and 10; lower fetal concentrations
were observed on gestation days 11-18, coincident with placentatlon. The
fetal liver had less affinity for the compound that did the maternal liver.
In an early metabolism study, Vlnopal and Casida {1973) reported that _in
vivo or j_n vi tro studies with mice showed that polar metabolites of
2,3,7,8-TCDD were not produced by this species. In rats, however, hydroxy-
lation and conjugation with glucuronide and sulfate have been demonstrated
(Poiger and Schlatter, 1979; Olson et al., 1983; Polger et al., 1982a. Glu-
curonide conjugates tended to predominate In the bile (Polger and Schlatter,
1979) and sulfate conjugates were located in the urine (Olson et al., 1983).
01310 III-25 . 04/12/88
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Poiger and Schlatter (1979) stated that liver metabolism of 2,3.7,8-TCDD
proceeds slowly In the liver. Neal et al. (1982) demonstrated that the rate
of hepatic metabolism was enhanced by activated cytochrome P-450 mono-
oxygenase. It was suggested that metabolism of 2,3,7,8-TCDD proceeds by the
formation of reactive epoxide Intermediates (Poland and Glover, 1979). That
dechlorination also occurs was demonstrated by Olson et al. (1983) and
Sawahata et al. (1982) who Identified trl- and d1chlorodibenzo-£-d1oxins as
metabolites In j_n vitro rat hepatocyte systems. From the bile of dogs, six
major metabolites have been identified (Poiger et al.p 1982a); hydroxylated
conjugates of tetra-, tri- and d1chlorodibenzo-£-diox1n predominated.
When the excretion data are subjected to a semi-logarithmic plot, a
straight line results, suggesting that elimination of 2,3,7,8-TCDD 1s a
first-order phenomenon, particularly for rats. Excretion In the guinea pig
may be a zero-order process (Gasiewicz and Neal, 1979). The half-life for
body elimination varied considerably with ranges of -10-15 days In the
hamster (Olson et al.t 1980a), the species least sensitive to the toxic
effects of 2,3,7,8-TCDD, to estimates of -11-24 days In the mouse (Gasiewicz
et al., 1983a,b), -17-31 days In the rat (Rose et al., 1976; Piper et al.,
1973; Allen et al., 1975) and -22-30 days 1n the guinea pig (Gasiewicz and
Neal, 1979; Nolan et al., 1979). One strain of mice, DBA/23, had a half-
life for elimination (-24 days) about twice as high as other strains tested
by Gasiewicz et al. (1983a,b). These authors also noted that this strain of
mice accumulated 2,3,7,8-TCDD In adipose tissue more strongly than other
strains and that this phenomenon probably resulted In slowed body elimina-
tion. Half-Hves for body elimination of 2,3,7,8-TCDD have not been calcu-
01310
III-26
04/12/88
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lated for the monkey, but it was suggested that the tendency of this species
to accumulate 2,3,7,8-TCDD in body fat may well result In slowed body elimi-
nation (Van Miller et al., 1976).
Recently, Olson and Bittner (1983) examined the elimination of 2,3,7,8-
TGOD in rats over a longer period than the studies previously summarized and
determined that a biphasic elimination occurred. They suggested a half-life
of -7 days for the Initial rapid phase and a half-life of -75 days for the
slower phase, probably related to release from stores of body fat. McNulty
et al. (1982) estimated the half-life for elimination from the fat of mon-
keys to be -1 year.
The fecal route seems to be the major pathway for the elimination of
2,3,7,8-TCDD-derlved radioactivity from rats (Rose et al.t 1976; Piper et
al., 1973; Allen et al.# 1975; Van Miller et al., 1976), guinea pigs
(Gaslewicz and Neal, 1979) and mice (Gaslewlcz et al., 1983a,b). Urinary
excretion played less of a role In these species, accounting for <1-28% of
total excreted radioactivity while fecal excretion accounted for 72->99% of
the eliminated radioactivity. Urinary excretion accounted for a more sub-
stantial proportion of body elimination in hamsters (41% compared to 59% by
feces) (Olson et al.t 1980a) and that strain of mice (DBA/2J) which prefer-
entially accumulated 2,3,7,8-TCD0 in body fat (Gaslewlcz et al., 1983a,b).
The failure to detect metabolites of 2,3,7,8-TCDD In liver and fat
(Olson et al., 1983) Indicates that elimination of the metabolites occurs
rapidly and that the rate of elimination Is governed primarily by the rate
of hepatic metabolism.
01310
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IV. HUMAN EXPOSURE
Text to be provided by the Office of Drinking Water.
t
01320
IV-1
03/16/84
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V. HEALTH EFFECTS IN ANIMALS
Experimental Animals
Acute Toxicity.
Lethal Effects — There have been studies 1n a variety of species
i
defining the doses necessary to cause death after acute exposure to 2,3,7,8-
TCDD. A summary of the single dose LD^ data for 2,3,7,8-TCDD Is present-
ed In Table V-l. The dose that results In death varies extensively with
species, with the male guinea pig the most sensitive species tested (LD^q
of 0.6 v9/kg) (Schwetz et al.t 1973), and the male hamster the least
sensitive species tested (LD^ of 5051 yg/kg) (Henck et al., 1901). The
rat and monkey appear to be the second most sensitive species, with LD^s
between 22 and 70 pg/kg (Schwetz et al., 1973; HcConnell et al., 1978a),
while other species tested (rabbit and mouse) had LD^s between 114 and
283 wg/kg (Schwetz et al., 1973; McConnell et al., 1978b; Vos et al.,
1974). Schwetz et al. (1973) found male rats more sensitive to 2,3,7,8-
TCDD, while Beatty et al. (1978) found adult female and weanling male rats
more sensitive than adult male rats (see Table V-l). In C57B1/10 mice,
Smith et al. (1981) reported adult males to be far more sensitive to the
acute toxicity of 2,3,7,8-TCDD than adult females. Thus, data on sex
differences In sensitivity to the acute toxicity of 2,3,7,8-TCDD are con-
flicting and may depend on the species examined.
Harris et al. (1973) studied the toxic effects of 2,3,7,8-TCDD 1n rats,
mice and guinea pigs with regaM to single or multiple exposures. Similar
effects were observed after a single exposure to 2,3,7,8-TCDD as were
observed when multiple exposures totaled the same dose as received In the
single exposure. As Illustrated most clearly 1n rats, a single dose of
00110
V-l
09/18/84
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TABLE V-l
Lethal Doses of 2,3,7,8-TCDO Following Acute Exposure
Species/Strain Sex/No./Group
Route/
Vehicle
Dose Tested
(vg/kg)
Duration of
Observation
LD50
(pg/kg)
Coiments
Reference
Guinea pigs/
Hartley
Guinea pigs/
Hartley
Guinea pigs/
Hartley
Guinea pigs/
Hartley
Guinea pigs/
Hartley
Rats/
Sherman
Rats/
Sherman
Rats/Sprague-
Dawley
Rats/Sprague-
Dawley
N/NR
fl/NR
M/9
F/6
F/6
N/5-10
F/NR
B/6
F/6
gavage/corn
oil-acetone
(9:1)
gavage/corn
oil -acetone
<9:11
gavage/
corn oil
gavage/
corn oil
gavage/
methyl
cellulose
gavage/corn
oil-acetone
19:1)
gavage/corn
oil-acetone
19:1)
1.p./olive
oil
1.p./olive
oil
NR
NR
NR
0.1
0.5
2.5
12.5
20.0
0.1
0.5
2.5
12.5
20.0
6
16
32
63
NR
NR
NR
2-8 weeks
2-8 weeks
30 days
42 days
12 days
2-8 weeks
2-8 weeks
20 days
20 days
0.6
(0.4-0.9)*
2.1
(1.5-3)*
-- 2.5
(1.2-5.4. 95%
confidence)
19
(15-23. 95%
confidence)
22
45
(30-66)*
60
25
Tine to death was 5-34 days, the
2,3,7,8-TCDO was 91% pure
Time to death was 9-42 days, the
2,3,7,8-TCDD was 99% pure
Median time to death was 17-20 days,
marked weight loss, thymus atrophy.
Intestinal hemorrhage, no porphyria
and only mild liver Injury
Time to first death was 32 days In
the 2.5 yg/kg group, with 50%
mortality by day 42
Tim to first death was 12 days In
the 20.0 pg/kg group, with 67%
mortality by day 42
Time to death was 9-27 days, the
2,3,7,8-TCDO was 91% pure
Time to death was 13-43 days, the
2,3,7,8-TCDO was 91% pure
LD50 Ug/kg, mean ~ SE) adult
male, 60.2 ~ 7.8; weanling male,
25.2 ~ 1.4
Adult female had a mean ~_ SE of
24.6 ~ 2.0 vg/kg
Schwetz et al.,
1973
Schwetz et al.,
1973
HcConnell et al.,
1978b
Sllkworth et al.,
1982
Sllkworth et al.,
1982
Schwetz et al..
1973
Schwetz et al.,
1973
Beatty et al., 1978
Beatty et al., 1978
-------
TABLE V-l (cont.)
Species/Strain
Sex/No./firoup
Route/
Vehicle
Dose Tested
(ng/kg)
Duration of
Observation
L05O
(vg/kg)
Comnents "*
Reference
Monkey/rhesus
r/3
gavage/
corn oil
0
70
350
>35 days
<70
Weight loss, edema, severe thymus
atrophy, loss of hair, mild liver
damage
RcConnell et al.,
1978a
K1ce/C57B1
fl/14
gavage/corn
oil-acetone
0:1)
0
100
ISO
200 .
60 days
114
Time to death In the high dose group
was 15-20 days, bw loss, edema In
25% of treated animals, severe
thymic and spleen atrophy, hemor-
rhage In the region of the eye and
small Intestine, liver necrosis in
the centrllobular region
Vos et al., 1974
«1ce/C57Bl
N/9
gavage/
com oil
NR
30 days
283.7
Median time to death was 22-25
days, dose-related bw loss, thymic
atrophy. Increased liver weight
and porphyria, gross and historic
liver alterations, subcutaneous
edema. Intestinal hemorrhage
HcConnell et al.,
1978b
Mce/C57B1/10
H/5
gauge/
arachls oil
85
107
135
170
213
45 days
146
95* confidence limits of 111-211
pg/kg. Host deaths occurred from
22-26 days after dosing. Signs
of porphyria, edema, hemorrhage.
Smith et al., 1981
«1ce/C57Bl/10
r/s
gauge/
arachls oil
05
107
135
170
213
269
33B
426
536
45 days
>450
1 of 4 animals died at dose of
426 |ig/kg
Smith et al., 1981
Mce/C57B1/6J
B/NR
1.p./olive
oil
NR
30 days
132
BGD2Fi/J mice are the offspring
of C57B1/6J and DBA/2J.
6as1ew1cz et al.,
19B3a,b
«1ce/0BA/2J
K/NR
1.p./olive
oil
NR
30 days
620
at the Ah locus.
fiaslewlcz et al.,
1983a,b
H1ce/B6D2F|/J
H/NR
1.p./olive
oil
NR
30 days
300
No comnent
Gaslewlci et al.,
1983a,b
-------
TABLE V-l (conl.)
Specles/Stratn
Sex/No./Group
Route/
Vehicle
Dose Tested
(Mg/kg)
Duration of
Observation
L050
(wg/kg)
Comnenls ¦
Reference
Rabbits/
New Zealand
Hfcf/NR
gavage/corn
ol1-acetone
(9:M
NR
2-8 weeks
115
(30-345)*
Time to death was 6-39 days, the
2,3,7.8-TCD0 was 91% pure
Schwetz et al.,
1973
Rabbits/
New Zealand
HfcF/5
l.p./
corn oil
32
63
126
252
500
4 weeks
NR
Time to death was 6-23 days,
2-3 animals/group died In all
but the low exposure group
Schwetz el al.,
1973
Rabbits/
New Zealand
Hfcf/NR
dermal/
acetone
31.6
63
126
252
500
3 weeks
275
(142-531)*
Time to death was 12-22 days
Schwelz et al..
1973
Hamster/
golden Syrian
H/6
gavage/corn
oil-acetone
(91)
0
300
600
1000
3000
6000
55 days
5051
(3076-10,467.
95% confidence)
Time to death was 26-43 days, the
liver and thymus appeared to be the
primary target organs, only 1 death
occurred In the 300 and 3000 pg/kg
group
Henck et al., 1981
Hamster/
golden Syrian
Hfcf/5-6
l.p./
olive oil
0
500
1000
2000
3000
50 days
>3000
Significant, dose-related decrease
In thymus weight starting at
500 tig/kg. only 2 deaths occurred
out of 11 hamsters In the 3000 pg/kg
group.
Olson et al., 19B0b
Hamster/
golden Syrian
H/5
gavage/
olive oil
500
1000
2000
3000
50 days
1157
Death generally occurred between
24 and 45 days, decrease In bw above
2000 pg/kg, proliferative Ileitis
with mild to severe lnflamnatlon
Olson et al., 1960b
Dogs/beagle
n/2
gavage/corn
oil-acetone
(9:1)
3000
2-0 weeks
NA
All animals died
Schwetz et al.,
1973
Dogs/beagle
F/2
gavage/corn
oil-acetone
(9:1)
30
100
2-B weeks
NA
All animals survived
Schwetz et a 1.,
1973
'The number In parentheses appears to Indicate the range of lethal doses; however, the article did not specify what these numbers represented.
I.p. « Intraperitoneal; NR ¦= Not reported; NA - Not applicable
-------
25 yg/kg, 6 weekly doses of 5 yg/kg, or 30 dally doses of 1 yg/kg were
all the threshold dose for observing a decrease In body weight. Other
endpolnts, Including lethality, decrease In thymus weight, and a no effect
level for body weight change In rats, mice and guinea pigs 1n general
appeared likewise to require a specific threshold level regardless of
whether this level was achieved through a single exposure or a small number
of multiple exposures.
Although 2,3,7,8-TCDO has over a 10*-fold difference In toxicity
depending upon the species tested, some of the signs of lethal toxicity were
the same regardless of species. One of the most characteristic observations
after acute lethal exposure to 2,3,7,8-TCDO was the protracted time between
exposure and death (see Table V-l). In determining the 10^ In the least
sensitive animal, the hamster, the test animals died between 24 and 45 days
after a single acute exposure (Olson et al., 1980b), and similar observa-
tions were made 1n all other species, tested Including the most sensitive
species, the guinea pig, 1n which animals died up to 42 days after treatment
(Schwetz et al., 1973).
During this extended period between treatment and death the animals had
poor weight gain or loss of weight and appeared to be "wasting away." In
female Wlstar rats Intubated with 2,3,7,B-TCDD at a dose of 100 yg/kg, the
weight loss was blphaslc (Courtney et al., 1978). The Initial weight loss
occurred rapidly during the first 7-10 days after treatment and was asso-
ciated with decreased food and water consumption. This Initial phase of
weight loss was reversed with the resumption of normal food Intake for 4 or
5 days, only to be followed by a second, more gradual, decline In food and
00110 V-5 08/11/84
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water Intake and weight until death. Providing animals with an adequately
nutritious liquid diet by Intubation did not appreciably alter the pattern
of weight loss nor affect survival. In contrast, Gas1ew1cz et al. (1980)
observed that providing rats with total parenteral nutrition would prevent
some of the weight loss Induced by 2,3,7,8-TCOO; however, there was no
protection from the lethal effects of 2,3,7,8-TCDO. In yet another study,
Seefeld and Peterson (1983) suggest that a reduction 1n food Intake caused
by 2,3,7,8-TCDO 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-TCDO-
treated partners (25 or 50 yg/kg) until day 10 after treatment. At 20-35
days after treatment, the body weight of the two groups began to diverge,
with the pair-fed control group having body weights that were 20-30 g higher
than the corresponding 2,3,7,8-TCDO groups. The mortality 1n the 25 and 50
yg/kg groups was 33 and 75%, respectively, while In the corresponding
pair-fed groups the mortality was 0 and 15%. The authors proposed a hypoth-
esis that 2,3,7,8-TCDD lowers a regulated level or "set-point" for body
weight control In the rat. The ensuing change 1n food Intake was thought to
occur secondarily to the change 1n set-point.
Also, severe thymic atrophy 1s universally observed In all species given
lethal doses of 2,3,7,8-TCDD, and since weight loss and thymic atrophy are
both associated with malnutrition, van Logten et al. (1981) Investigated the
effects of dietary protein on 'the toxicity of 2,3,7,8-TCOO. Groups of
female Fischer 344 rats administered 2,3,7,8-TCOO (20 yg/kg) and main-
tained on low (3.5%), normal (26%) or high (55%) protein diets maintained
approximately the same amount of weight (-0.2+3, 7+6 and 7+3 g for each
00110
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dietary group, respectively) during the subsequent 10-day period. The
weight gain 1n treated animals was 10-10 g less than that 1n the respective
control rats. Dietary protein also had no effect on preventing or enhancing
the 2,3,7,8-TCDO Induced thymic atrophy. Although weight loss and thymic
t
atrophy were present In most species tested, there were other symptoms which
were characteristic of toxicity 1n only some species.
In the guinea pig. besides thymic atrophy, no gross changes were
observed 1n Internal organs after a lethal oral or 1.p. dose of 2,3,7,8-TCDD
(Grelg et al., 1973, Gupta et al.t 1973). Hemorrhages were observed 1n a
number of organs Including the adrenal gland, urinary bladder, 61 tract and
mesenteric lymph nodes; however, these were considered unremarkable changes
by 6upta et al. (1973). Histologic examination confirmed the gross observa-
tions with atrophy and lymphoid cell depletion 1n the thymus, spleen and
lymph nodes, and hemorrhages observed 1n many organs. In addition, marked
hyperplasia of the urinary bladder was observed. Of particular Interest was
the absence of severe toxic effects on the liver. Gross observation under
UV light Indicated no excess of porphyrin, while histologic examinations
revealed diffuse single cell necrosis. Identical observations were made by
HcConnell et al. (1978b) 1n guinea pigs administered lethal doses of
2,3,7,8-TCDD, with the additional observation that the sternal bone marrow
was hypocellular In all types of blood-forming cells.
Turner and Collins (1983) described some histologic changes In the liver
of guinea pigs treated with 2,3,7,8-TCDD. Groups consisting of 4-6 female
Hartley guinea pigs were treated with 2,3,7,8-TCDD at doses of 0.0, 0.1,
0.S, 2.5, 12.5 or 20 pg/kg, and 1 male guinea pig each was treated with a
00110
Y-7
08/11/84
-------
dose of 0.1 or 0.5 yg/kg. The 2,3,7,8-TCDD was administered by gavage as
an aqueous suspension 1n 0.75% methyl cellulose and surviving animals were
killed 42 days after treatment. A second group of guinea pigs (6 males and
6 females/dose) were administered soot generated from a fire 1n a trans-
t
former cooled by polychlorlnated blphenyls and chlorinated benzenes (1, 10,
100 and 500 mg/kg). The histologic observations as described were applied
1n general to both treatment groups and there was no apparent relationship
between dose and response. The authors reported that, compared to controls,
all experimental groups showed liver alterations but that qualitative dif-
ferences among the dosage groups were not detectable by light microscopy.
At the light microscope levelf hepatocellular hypertrophy, steatosis, focal
necrosis, cytoplasmic degeneration and addophl 11c hyalln-Uke cytoplasmic
Inclusion bodies were observed. Even though there was no dose-response
relationship for these liver lesions, the doses spanned a range that result-
ed In the lowest dose being nonlethal (none of the 4 female guinea pigs died
during the study), while In the high dose group 4 of 6 animals died before
42 days post-treatment. The LD^ for female guinea pigs was determined 1n
this study, and reported by SUkworth et al. (1982), to be 2.5 or 19 yg/kg
bw depending on whether the compound was administered by gavage In corn oil
or 1n aqueous methyl cellulose.
The greatest difference at necropsy 1n the gross and histologic effects
1n rats and mice of exposure to lethal doses of 2,3,7,8-TCDD was pathologic
alterations In the liver, as compared with guinea pigs. An early report by
Buu-Ho1 et al. (1972) described alterations In the architecture of the liver
of rats within 5 days of receiving a low dose of 2,3,7,8-TCDD (10 yg/kg by
l.p. Injection). At higher oral doses of 100 or 50 yg/kg, which killed 43
00110
09/18/84
-------
and 1% of the animals, respectively, Gupta et al. ( 1973) also observed
marked distortion of liver architecture In rats; however, only mild regener-
ative changes of the liver were observed at the sublethal dose of 5 yg/kg
administered weekly for 6 weeks. Liver toxicity appeared to develop slowly
In \he rat with no change In liver function, as indicated by plasma protein
and bilirubin levels, or alkaline phosphatase, glutamlc-oxalacetlc trans-
aminase (GOT) and glutamic-pyruvic transaminase (GPT) activity being detect-
ed 3 days after Intubation with 2,3,7,8-TCDD at a dose of 200 yg/kg (Grelg
et al., 1973). Bilirubin levels were, however, markedly elevated from 0.33
ug/100 ml 1n control animals to 10.97 yg/100 ml 1n treated animals
21 days after exposure (the other parameters were not measured at this time,
although plasma protein was slightly but significantly decreased when
determined 9 days posUtreatment). As 1n rats, the livers of mice exposed
to lethal levels of 2,3,7,8-TCDD had signs of necrotic changes (Vos et al.,
1974); however, Jones and Grieg (1975) reported that the centrllobular
necrosis, bile duct proliferation and lipid accumulation were more extreme
In mice than In rats. Examination of mouse livers using long wave UV light
showed fluorescence suggestive of excess porphyrin accumulation (HcConnell
et al., 1976b). Although excess porphyrins may be present 1n the livers
from 2,3,7,8-TCOO-exposed rats, fluorescence 1s not usually observed.
Besides effects on the liver, 2,3,7,8-TCDD exposure produced other toxic
effects In rats and mice that were not observed or were observed to a lesser
extent 1n guinea pigs. In rats that died from 2,3,7,8-TCDD exposure, there
were extensive hemorrhages of the heart, liver, brain, adrenal gland and 61
tract along with ulcers and necrosis of the glandular stomach, and 1n
females, atrophy of the uterus (Gupta et al., 1973). In mice, facial edema
00110
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was severe and the testicles of males appeared degenerated with necrotic
spermatocytes and spermatozoa present (McConnell et al., 1978b; Vos et al.,
1974). Death in mice was frequently attributed to terminal hermorrhages
(Vos et al., 1974).
t i
In monkeys exposed to lethal levals of 2,3,7,8-TCDD, HcConnell et al.
(1978a) reported clinical and histologic signs of toxicity, some of which
were similar to those already described for other species. Severe thymic
atrophy and edema occurred in treated animals, as well as extensive weight
loss that could account for up to 38% of the body mass. As 1n guinea pigs,
liver Injury appeared to be mild; however, Increased serum GOT and aldolase
activity and decreased albumin levels Indicative of liver pathology occurred
near the time of death. As observed 1n mice, the bone marrow of monkeys was
hypocellular. In addition to the above signs of toxicity, which were
observed in other species as well, monkeys had progressive loss of hair,
toenails and fingernails, with associated dermatitis consisting of the
development of a crusty texture to the skin, squamous metaplasia of sebace-
ous glands and gastric mucosal dysplasia. As with most other species, a
specific cause of death could not be determined for monkeys. Poland and
Knutson (1982) have summarized the toxic response of various species to
2,3,7,8-TCOD {Table V-2).
There was very little Information on the lethal effects of PCDD
congeners other than 2,3,7,8-TCDD. McConnell et al. (1978b) determined the
LDgg for nine congeners of PCDO following a single treatment by gavage In
mice and guinea pigs. A comparison of the LD^q expressed as v^ol/kg
body weight 1s presented 1n Table V-3. The limited data suggest that con-
geners containing chlorine In the 2,3,7,8 positions were more biologically
00110 V-10 09/18/84
-------
TABLE V-2
Toxic Responses Following Exposure to 2,3,7,8-TCDD: Species Differences3
Monkey Guinea Cow^ Rat House Rabbit'' Chicken15 Hamster
Pig
Hyperplasia and/or metaplasia
Gastric mucus
++c
0
+
0
0
0
Intestinal mucosa
~
+ +
Urinary tract
++
0
0
B1le duct and/or gall bladder
+ 4-
0
4-
++
0
Lung: focal alveolar
+ +
Skin
+ +
0
*d
0
0
~ +
0
Hypoplasia, Atrophy or Necrosis
Thymus
~
~
+
+
+
+
Bone marrow
+
+
Testicle
+
*
+
~
~
Other
Liver lesions
~
+
+ +
+
~ 1
+
4-
Porphyria
0
0
+
+ ~
+
0
Edema
+
0
0
~
+
References: Monkey (McConnell et al., 1978b; Norback and Allen, 1973; Allen et al.t 1977); guinea pig
(McConnell et al., 1978b; HcConnell, 1980; Moore et al., 1979; Turner and Collins, 1983); cow
(McConnell, 1980); rat (McConnell, 1980; Koclba et al., 1978a,b, 1979); mouse (Schwetz et
al., 1973; McConnell et al., 1978b; 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.
^Skln lesions 1n cattle are observed, but they differ from the skin lesions observed 1n other species.
Adapted from Poland and Knutson, 1982
-------
TABLE V-3
Estimated Single Oral LD50-30 Values for PCD0sa
t
Chlor1nat1on of PCDOs
Guinea P1gs
Mice
(pmol/kg)b
(pmol/kg)tl
2.8
>1180
NR
2.3,7
120.41
>10
2.3.7,0
0.006
0.88
1.2.3,7,8
0.009
0.94
1.2.4.7,8
3.15
>14
1.2,3,4,7,8
0.185
2.11
1.2,3,6.7,8
0.178-0.255c
3.19
1,2,3,7,8,9
0.153-0.255c
>3.67
1,2,3,4,6,7,8
>1.400
NR
aSource: Adapted from HcConnel1 et al.f 1978b
bSpearman-Karber method
cEst1mated range due to variability In replicates
NR = Not reported
00110
V-12
09/18/84
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active than congeners deficient 1n a chlorine from any one of these
positions. It also appears that addition of one or more chlorines to
2(3P7,8-TCDD results 1n a decrease In lethality. Although the congeners
vary 1n effective dose between mice and guinea pigs, the relative order of
toridty of these congeners did not change. Also, similar effects of
toxicity were observed for all congeners as described above for 2,3,7,8-TCDD
when the comparison was made within a single species.
Effects on the Liver — The histological and ultrastructural changes
1n the liver Induced by oral exposure to 2,3,7,B-TCDD have been reported by
Fowler et al. (1973), Jones and Butler (1974) and Jones (1975). Fowler et
al. (1973) treated groups of 30 male rats with a single dose of 2,3,7,8-TCOO
at 0.0, 5 and 25 yg/kg by gavage. The animals were killed 1n groups of 5
on days 1, 3, 6f 9, 16 and 28 after treatment and the livers were prepared
for histologic examination. The major ultrastructural change observed was a
dose-related Increase 1n the smooth and rough endoplasmic reticulum (ER) 1n
cells near the bile canallcull. The Initial Increases appeared at day 3,
with the maximal response occurring on days 6 and 9. By day 16 the smooth
ER was nearly absent from the parenchymal cells, although large amounts of
rough ER were still present. By day 28 the cells had returned to normal
appearance. These changes 1n liver cells following 2,3,7,8-TCDO treatment
would be consistent with the Induction of protein and RNA synthesis.
Transmission electron microscopic observations revealed that single l.p.
administration of 20 yg/kg of 2,3,7,8-TCOO In Sprague-Dawley male rats
produces necrotizing hepatic lesions which become progressively worse up to
the 16th week postexposure followed by gradual Improvement of the condition
and disappearance of the lesions (Weber et al., 1983).
00110
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09/18/84
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At higher doses of 200 wg/kg, Jones and Butler (1974) observed necro-
sis and proliferative changes In the liver of rats to be the predominant
lesions. After treatment by gavage, groups of 4 male and 4 female rats were
killed and examined on a weekly basis for 10 weeks. By the first week,
t
degenerating cells were observed near the central vein and these lesions
progressed to areas of focal necrosis by the sixth week. Superimposed on
the necrotic changes were hyperplasia of the viable cells with multinucle-
ated cells common by the ninth week. At week 10 central vein fibrosis and
scattered necrosis remained. Fine structure observed after this large dose
of 2,3,7,8-TCOD also revealed Increases 1n smooth ER; however, the most
striking effect was degeneration of the plasma membrane with the resulting
fusion of parenchymal cells. In a study of similar design, Jones (1975)
followed the distribution with time after treatment of membrane associated
ATPase activity by hlstochemlcal techniques. At 3 days after treatment, the
first changes In ATPase patterns were observed, with loss of activity along
the canalicular borders and some increased activity In the sinusoids. The
mldzonal and periportal zones had normal activity at this time. The loss of
ATPase activity persisted for 34-42 days, and paralleled the histologic
lesions described previously (Jones and Butler, 1974). In rats that
survived treatment, the ATPase activity was back to normal by 9 months.
Peterson et al. (1979a) further studied the effect of 2,3,7,8-TCDD at
lower doses on hepatocyte plasma membrane ATPase activity. Liver surface
membranes (LSM) Isolated from male Holtzman rats 2, 10, 20 or 40 days after
Intubation with 2,3,7,8-TCDO at 0.0, 10 or 25 >jg/kg were used for determi-
nation of Na+, K+-ATPase and Mg++-ATPase activity. The activity of
Na+, and K+-ATPase was depressed to the same extent for both doses of
00110
V-14
09/18/84
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2,3,7,8-TCDD from day 2-40 after treatment, while a similar depression of
the Mg++-ATPase activity was observed only 1n the high dose group. In the
low-dose group, there was a decrease In Mg++-ATPase at 20 days, but
recovery to normal levels occurred by 40 days post-treatment. It was demon-
i
strated that the effect of 2,3,7,8-TCDD on ATPase activity was not the
result of 2,3,7,8-TCDO Induced food deprivation and Iri vltro studies
Indicated that the loss of activity was not due to the direct Interference
of 2,3,7,8-TCDD with the enzyme. Quantitative changes (both Increases and
decreases) have been reported for the protein composition of plasma
membranes Isolated and analyzed by electrophoresis from Sprague-Dawley rats
10 days after an l.p. Injection of 2,3,7,8-TCDD, Indicating that exposure
was actually affecting membrane components (Brewster et al., 1982).
Peterson et al. (1979a) did observe a positive correlation between the
levels of LSM ATPase activity and both In vivo cumulative biliary excretion
of ouabain and bile flow (pi/mln/g liver). Using perfused liver, however,
Peterson et al. (1979b) reported a segregation between LSM ATPase activity
and biliary excretion of ouabain when 2,3,7,8-TCDD rats were exposed to the
protective agents pregnenolone-!6a-carbon1tr1le or spironolactone. It was
concluded that LSM ATPase did not directly participate In ouabain transport.
Additional studies have described the effect of 2,3,7,8-TCDD on the
biliary excretion of a variety of xenoblotlcs. Early studies by Hwang
(1973) Investigated 2,3,7,8-TCDD Inhibition of biliary excretion In male CD
rats given a single dose of 2,3,7,8-TCDD at 25 or 5 pg/kg by gavage.
Animals were examined for Indocyanlne green (ICG) excretion 1, 7 and 16 days
after treatment. Unlike Peterson et al. (1979a), Hwang (1973) observed an
00110
V-15
09/18/84
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Inverse relationship between 2,3,7,8-TCDD exposure and bile flow, with
maximum bile flow observed 1n the 25 wg/kg dose group at 16 days. Even
with this Increased bile flow, however, the cumulative biliary excretion of
ICG was decreased In a dose-dependent manner with the greatest depression
i
observed 7 and 16 days after the exposure to 2,3,7,8-TCDD. The levels of
ICS In the plasma and liver was higher In treated animals than 1n control
animals, while the concentration in the bile was lower, reflecting the
decrease 1n total excretion of ICG.
Yang and Peterson (1977) compared the effect of 2,3,7,8-TCDD on the
biliary excretion of the organic neutral compound, ouabain, with that of the
organic anions phenol-3,6-dibromophthaieln (DBSP) and sulfobromophthaleln
(BSP) in male Holtzman rats. Animals were Intubated with 2,3,7,8-TCOD at
doses of 10 or 25 yg/kg and excretion was evaluated periodically between
2-4 days postexposure. The biliary excretion of ouabain was depressed in a
dose-related manner starting on the second day post-treatment, with maximum
depression developing between 10 and 20 days, and some recovery observed by
day 40. Decreases in bile flow followed a pattern similar to that observed
for ouabain. The pattern of biliary excretion was different for DBSP and
BSP in which only a transient small decrease was observed 10 days after
exposure in the high dose group. In the low dose animals there was actually
an increase at days 10 and 25 in the excretion of the anions. The results
obtained for DBSP and BSP differ sharply from those for the organic neutral
ouabain or those reported by Hwang (1973) for the organic anion ICG, in
which a dose-related decrease in biliary excretion was observed. The
authors concluded that the effects of 2,3,7,8-TCDD on the multiple pathways
Involved in biliary excretion depend on the specific compound being studied.
00110
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09/18/84
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In the guinea pig and rhesus monkey, which do not develop significant
liver pathology after exposure to 2,3,7,8-TCDD, there was also little change
In ICG blood clearance rates, while 1n the rabbit, which develops 2,3,7,8-
TCDD-lnduced liver damage similar to the rat, there was reduced blood clear-
!
ance of ICG (Seefeld et al.» 1979, I960). In the rabbit, there were In-
creases In serum sorbitol dehydrogenase and GPT activity as further Indica-
tions of 2,3,7,8-TCDD-produced liver damage. In the monkey, which received
2,3,7,8-TCDD by gavage at doses of 5, 25 or 75 vg/kg, there was an Initial
slight Increase In the blood clearance of ICG at 2 days post-treatment,
followed 1n the two higher dose groups by a dramatic decrease a few days
before death. Although some serum enzymes (sorbitol dehydrogenase and GPT)
Indicative of liver damage were elevated, the hlstopathology of the liver
was within normal limits. It appears that major effects on biliary excre-
tion occur only 1n species that are sensitive to the hepatotoxlc effects of
2,3,7,8-TCDD.
Other gross signs of. the hepatotoxlc effects of 2,3,7,8-TCDD observed in
some species Included fatty degeneration and porphyria. Early observations
by Cunningham and Williams (1972) descrlbed'a decrease In In vivo (1 hour
pulse) Incorporation of ®H sodium acetate Into liver lipids after exposure
of male Wlstar rats to 2,3,7,8-TCDD. The rats (12-16 animals) were treated
with 2,3,7,8-TCDD at a dose of 10 pg/kg followed In either 3 or 7 days by
the assessment of lipid synthesis. At 3 days Incorporation decreased from
258 to 98 dpm/mg lipid 1n the'control and treated animals, respectively.
There was an approximately similar decrease observed 7 days postexposure.
00110
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When Individual classes of lipids were examined, there was a decrease In the
synthesis of triglycerides, dlglycerldes and phospholipids. Although
Cunningham and Williams (1972) observed that 2,3,7,8-TCDD decreased lipid
synthesis, Albro et al. (1978) reported an Increase 1n total lipids 1n the
livers of rats 13 days after treatment with 2,3,7,8-TCDD at a lethal dose of
50 pg/kg. For Individual classes of lipids there was an Increase In free
fatty acids and cholesterol esters, while no change occurred 1n the content
of phospholipids, free cholesterol or triglycerides. The fatty changes 1n
the liver were confirmed by ultrastructural examination of liver specimens.
At a sublethal dose of 10 pg/kg there was a different pattern of lipid
accumulation, with triglycerides and fatty adds Increased and cholesterol
esters decreased. The changes In the lipid profile of the liver was
attributed to 2,3,7,8-TCDD Induced mobilization of body fat, a decrease In
lysosomal acid lipase (74%-decline In this enzyme 10 days after a 50 pg/kg
dose of 2,3,7,8-TCDO) and an Increase In lipid peroxidation as Indicated by
a sharp Increase In the production of llpofusdn pigments.
Porphyria was Initially characterized quantitatively In mice by
Goldstein et al. (1978). Groups of 12 male C57B1 mice received 4 weekly
Intubations of 2,3,7,8-TCDD at doses of 0.0, 1, 5 or 25 pg/kg, or a single
dose of 150 pg/kg followed 21-25 days after treatment by analysis of the
liver for porphyrins. Porphyrin levels were unchanged except In the 25 and
150 pg/kg groups where the levels were Increased 2000- and 4000-fold,
respectively. The difference In responsiveness to the development of por-
phyria was studied by Smith et al. (1981) In C57B1 mice which were sensitive
to, and DBA/2 mice which were Insensitive to, the toxicity of 2,3,7,8-TCDD.
00110
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Male and female C57B1 mice had a dose-related Increase 1n hepatic porphyrins
1n the two high dose groups 3 weeks after a single exposure to 2,3,7,8-TCDD
at 0.0, 5, 15, 50 or 75 yg/kg, while only minimal nondose-related changes
1n hepatic porphyrin were observed In DBA/2 mice exposed to up to 1200
vg/kg. In the sensitive C57B1 mice there was only a small difference In
hepatic porphyrin between the sexes even though males were >3 times as sen-
sitive to the toxic effects of 2,3,7,8-TCDD than females (see Table V-1).
Results similar to those above were reported for urinary porphyrin levels In
male C57B1 and DBA/2 mice given 6 weekly doses of 2,3,7,8-TCDO at 25 yg/kg
(Jones and Sweeney, 1980). In the sensitive strain, the Initial elevation
of porphyrin occurred In the second week.
In rats. Increased urinary porphyrin was observed only after subchronlc
exposure to 2,3,7,8-TCDD (Cantonl et al., 1981). Female CD rats were orally
administered a weekly dose of 2,3,7,8-TCOD at levels of 0.01, 0.1 and 1.0
yg/kg for 45 weeks. The Initial Increase was observed In the high dose
group at 3 months, and In the other two groups at 4 months, after the start
of exposure. Not only did the absolute amount of porphyrin Increase, but
the relative distribution also changed to compounds containing more carboxyl
groups. Only in the high dose group did the livers, at the terminal
necropsy, show signs of excess porphyrin under examination by UV light.
In attempts to understand the mechanism of 2»3,7,B-TC0D Induced por-
phyria, the effects of 2,3,7,£-TCDD on the enzymes involved 1n the synthesis
and catabollsm of porphyrin have been studied. Goldstein et al. (1978)
showerl that ^-aminolevulinic acid synthetase, a rate-limiting enzyme 1n
porphyrin synthesis, was slightly Increased (2-fold) 1n male C57B1 mice
00110
V-l 9
09/18/84
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given 4 weekly doses of 2,3,7,8-TCOD at 25 yg/kg. This dose of 2,3,7,8-
TCOD Increased liver porphyrin levels 2000-fold. Catabollsm of porphyrin by
uroporphyrinogen decarboxylase (UO) also appeared to be decreased 1n
2,3,7,8-TCDD treated mice. Smith et al. (1981) reported a decrease 1n UO
ictlvlty from -25-7 nmole,s/hr/g liver In male and female C57B1 mice 3 weeks
after a single oral exposure to 2,3,7,8-TCDD at a dose of 75 pg/kg. No
effect of 2,3,7,8-TCDD on UO activity was observed In DBA/2 mice which were
insensitive to the induction of porphyria. . A time course of changes In UO
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 pg/kg. In this study the UD activity declined -48% In C57B1 mice
and only 4% In OBA/2 mice. Other factors besides the Increase In {-amino-
levulinic acid synthetase and the decrease 1n UD activity may also partici-
pate in the dramatic Increase In liver porphyrin 1n mice associated with
exposure to near lethal doses of 2,3,7,8-TCDD.
As a result of the protracted time observed between exposure to 2,3,7,8-
TCDD and the development of toxic effects, as well as the reported terato-
genic and carcinogenic potential of 2,3,7,8-TCDO, Investigations have been
conducted to determine the Influence of 2,3,7,8-TCDD on ONA synthesis in the
liver. Grelg et al. (1974) measured the In. vlvo incorporation of 3H-thy-
mldlne (1 hour pulse) Into liver DNA of male and female Porten strain rats
after a single exposure to 2,3,7,8-TCDD at doses of 10 and 200 pg/kg.
00110
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09/18/84
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When the 2,3,7,8-TCDD was given either 0, 24 or 72 hours before a 3/4
partial hepatectomy there was only a slight, but not significant, decrease
In thymidine Incorporation observed when DNA synthesis was measured 24 hours
after the operation.
f
Although 2,3,7,8-TCOD had no effect on in vivo ONA synthesis, similar
studies by Conway and Hatsumura (1975) and Olckens et al. (1981) demonstrat-
ed an Increase In thymidine Incorporation when determined Iji vUro. Conway
and Matsumura (1975) administered male Sprague-Dawley rats 2,3,7,8-TCDD at a
dose of 5 tig/kg followed In 10 days by removal of the liver and the
vltro determination of DNA synthesis 1n liver slices. Incorporation of
thymidine into the nuclei increased from 29 cpm/mg 1n control animals to 45
cpm/mg 1n treated animals, A similar near doubling of ONA synthesis was
observed by Dickens et al. (1981); however, when ONA synthesis was stimulat-
ed by a 1/3 partial hepatectomy, thymidine Incorporation Into liver slices
was Increased 10-fold 1n rats treated 5 days earlier with 2,3,7,8-TCDD as
compared with hepatectomlzed controls. The onset of ONA synthesis after
partial hepatectomy (-20 hours) was the same 1n both 2,3,7,8-TCDD treated
and control animals; however, the treated animals had a more rapid and
extensive Increase 1n DNA synthesis between 20 and 32 hours after the
partial hepatectomy. The rates of DNA synthesis were again the same 1n both
groups 35 hours after the operation. It was shown by hydroxyurea Inhibition
that the DNA synthesis 1n both the treated and control animals was predomi-
nantly semlconservatlve. Further studies are needed to determine the reason
for the difference observed between in vitro and in vivo measurements of DNA
synthesis In the liver after exposure to 2,3,7f8-TCD0.
00110
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Extensive hepatic necrosis In the rabbit may be responsible for death 1n
this species (Poland and Knutson, 1982).
Besides the effects on the liver of 2,3,7,8-TCDD exposure described
above, 1t 1s known tha't 2,3,7,8-TCOD 1s a potent Inducer of microsomal
enzymes. These studies will be discussed 1n the Enzyme Induction by TCDD
Section, which describes the ability of this xenoblotlc to induce microsomal
enzymes In a number of tissues and organs.
Effects on Other Organ Systems ~ The most noticeable feature of
2,3,7,8-TCDO toxicity Is the loss of body weight and the apparent "wasting
away" until death. Since decreased food consumption may not totally account
for these findings, the effect of 2,3,7,8-TCDD on Intestinal absorption has
been studied. Madge (1977) assessed the ability of the intestine to absorb
D-glucose, D-galactose, L-arg1n1ne and L-h1st1d1ne using the everted intes-
tinal sac technique In CD-I mice exposed to 2,3,7,8-TCDD. In measurements
made 7 days after treatment with doses of 0.0, 10, 25, 75, 150, 200 or 300
yg/kg, D-glucose was absorbed to a lesser degree at all doses than In
control animals. The two low doses produced a dose-related decrease 1n
absorption; however, at doses of >75 yg/kg the decrease was uniform. At a
dose of 150 yg/kg, decreased absorption of D-glucose was slight 3 days
after treatment, became maximally decreased by 7 days, and this depressed
level was maintained for 28 days, at which time the study was terminated.
Providing D-mannose to the Incubation mixture as an energy supply Increased
the absorption of D-glucose to control levels; however, the amount of
D-glucose on the serosal side was still lower than control levels. This
suggested that intestinal utilization of D-glucose was taking place and
might account for some of the observed malabsorption. Treatment with
00110 V-22 08/11/84
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2,3,7,8-TCOO had no effect on the absorption of the other compounds Investi-
gated. In a similar experiment In Sprague-Oawley rats, Ball and Chhabra
(1981) also observed malabsorption of D-glucose, In this study, however,
absorption of leucine was also decreased. The decrease 1n leucine absorp-
>
tlon took longer to manifest Itself, with a significant decrease only
observed 2 weeks after treatment with 2,3,7,8-TCDD.
In contrast to the results observed for 0-glucose, intestinal Iron
transport was shown to be elevated by exposure to 2,3,7,8-TCDO. Manls and
K1m (1979a) examined the effect of prior treatment of male Sprague-Oawley
rats on the 30-mlnute transport of "Fe out of a duodenal loop created by
Ugatlng a section of the Intestine 1_n situ. At single 2,3,7,8-TCOD doses
of between 22 and 84 yg/kg there was Increased serosal transfer of s,fe
measured 48 hours after treatment. At doses >42 pg/kg the Increase was
-100%. The time after treatment at which serosol transfer was greatest was
1 day, with rapid decline 1n stimulation to near the levels of controls
observed on days 2-7. There was also an apparent effect of route of
administration, with gavage treatment being more effective In Inducing Iron
transport than 1.p. Injection. In similar experiments calcium transport was
decreased, and galactose and proline transport were unaffected by prior
exposure to 2,3,7,8-TCDO, Hanls and Kim (1979b) had Identical results when
the everted Intestinal sac was used to assess Iron transport. It was
Interesting to note that only duodenal sacs were stimulated, with no effect
of 2,3,7,8-TCDO exposure observed 1n the adjacent distal segment of the
Intestine. Increased Iron transport was also observed by Manls and Kim
(1979a) in an unidentified strain of mice. Increased Iron transport may be
one of the earliest effects of 2,3,7,8-TCDO; however, at present the toxico-
logic relevance of this transient disturbance 1n Iron transport Is unknown.
00110 V-23 09/18/84
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One of the common gross observations of 2,3,7,8-TCDD toxicity Is severe
edema, suggestive of a breakdown 1n salt and water homeostasis. These
observations prompted Investigations to determine the effect of 2,3,7,8-TCDO
on the function of the kidney. Pegg et al. (1976) measured renal function
»
ill v^tro using renal cortical slices obtained from male Sprague-Dawley rats
3 and 7 days after Intubation with 2,3,7,8-TCDD at doses of 10 or 25
vg/kg. {These results were also described by Hook et al.» 1977). Anion
and cation transport were measured by the respective accumulation of
p-am1noh1ppur1c add and N-methylnicotinamide Into the cortical slices.
Anion accumulation was lower in the high dose group, while cation transport
was lower at both dose levels tested. The decrease 1n anion transport was
confirmed 1n an Iji vivo study. Ammonlogenesls and gluconeogenesls were not
affected 1n 2,3,7,8-TCDD treated rats, even when the animals were made
addotlc, Indicative of no effect on the kidneys1 ability to maintain add
base balance. Also, sodium reabsorptlon was shown 1_n vivo to be within
normal range. Since decreases In cation and anion transport were the only
effects observed, and since these compounds are transported by a different
mechanism, the authors concluded that the effects of 2,3,7,8-TCDD were
merely a general decrease 1n kidney functon reflecting the poor condition of
the treated animals (animals 1n all treated groups had decreased weight
gain), and not a cause of debilitation.
Although kidney function was only minimally affected by exposure to
2,3,7,8-TCDD, Grelg et al. (1974) demonstrated that pre-exposure to 2,3,7,8-
TCDO could reduce the ability of the rat kidney to respond to stimuli of DNA
sythesls. Folate-stlmulated DNA synthesis measured In vivo 1n Porten strain
rats was decreased between 67 and 25% In animals receiving 2,3,7,6-TCDO at a
00110
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dose of 10 yg/kg on day 0-9 before administration of folic add. No
significant difference 1n folate-stlmulated ONA synthesis was observed 1f
2,3,7,8-TCDD was given 23 hours after folic acid. The lack of effectiveness
of administering 2,3,7,8-TCOD shortly after treatment with folic acid
!
suggested that 2,3,7,8-TC00 did not directly Interact with cellular ONA, nor
Inhibit the protein synthesis necessary to support folate-stlmulated DNA
synthesis. Similar Inhibitory effects of 2,3,7,8-TCDD were observed when
lead acetate was used to stimulate kidney DNA synthesis. The mechanism by
which 2,3,7,8-TCDD prevents the kidney from responding to proliferative
stimuli Is not known, although 1t was demonstrated that another agent
capable of Inducing microsomal enzymes, 3-methylcholanthrene (3-HC), had
similar effects on the kidney.
Additionally a number a hematologic and clinical chemistry changes have
been observed In the blood of laboratory animals after exposure to 2,3,7,8-
TCDD. Many of these changes, as described by Zlnkl et al. (1973), reflect
damage to previously described organ systems. In female CD rats given 30
dally doses of 2,3,7,8-TCDD at levels of 0.1, 1.0 or 10 yg/kg, the clini-
cal chemistry of the serum reflected liver damage. In the high dose group,
serum GOT and serum GPT were elevated starting 13-17 days after Initial
treatment. There was a marginal change 1n GPT 1n the m1d-dose group and
lactic dehydrogenase (LDH) 1n the high group, but the Increases were only
transitory. Serum cholesterol was Increased In the high dose animals start-
*
1ng at day 10, with a transitory Increase again observed 1n the m1d-dose
group. Conversely, there was a decrease 1n serum protein from day 24 on In
the high-dose animals. Along with these clinical chemistry changes Indica-
tive of Hver damage, the only other major effect observed 1n the blood was
00110
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08/11/84
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thrombocytopenia. The decrease 1n platelet count was detected early, by day
3, 1n the 10 and 1 yg/kg groups, while In the 0.1 vg/kg group a signifi-
cant decrease was not observed until day 17. Thrombocytopenia was also
observed In female guinea pigs after 8 weekly oral doses of 2,3,7,8-TCDD at
t
0.2 yg/kg, and 1n mice (administered a single dose of 1.0, 10 or 50
wg/kg). In guinea pigs lymphopenia was also observed. Other hematologic
changes were attributed to hemoconcentratlon.
In a more extensive Investigation of 2,3,7,8-TCDD-1nduced hyper!1p1dem1a
1n male Sprague-Oawley rats, Poll et al. (1980) treated animals with a
single 1.p. Injection of 2t3,7,B-TC0D at 2 doses of 2.5, 5, 10 and 20
ng/kg. At day 21 after treatment there was a dose-related Increase In
total plasma cholesterol and high density lipoprotein cholesterol, while no
change was observed In triglycerides or very low and low density lipopro-
teins (VLDL and LDL, respectively). At a dose of 20 yg/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 In the apoprotein
of HDL from 2,3,7,8-TCDD rats and control rats were Indicative of new
apoprotein synthesis. Although the Increases 1n HDL cholesterol may be In
response to eliminating excess lipids, the exact function has not been
clearly shown. There Is some evidence from studies of workers exposed to
2,3,7,8-TCDD that there were reduced levels of blood HDL cholesterol and
raised total cholesterol as compared with a matched control group (Walker
and Martin, 1979),
00110
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In contrast to rats, male Hartley strain guinea pigs given a single 1.p.
Injection of 2,3,7,8-TCOO at a dose of 2 pg/kg had Increased hyperllpld-
emla characterized by Increases In VLOL and LDL (Swift et al., 1981). In
animals sacrificed 7 days after exposure to 2,3,7,8-TCDD, there was an
increase in total serum lipid, cholesterol esters, triglycerides and
phospholipids, when comparison was made with pair-fed, weight-paired or ad
libitum fed control groups. Serum-free fatty acids were not changed quanti-
tatively; however, some qualitative changes occurred, reflecting an Increase
1n the types of fatty acids that were abundant In the adipose tissue of
guinea pigs. Anaylsls of lipoproteins revealed a 19-fold Increase 1n VLDL
and a 4-foid Increase In LOL, with no change observed in the levels of HOL.
*
The VLOL was also qualitatively different 1n the 2,3,7,8-TCQD treated
animals, containing less cholesterol ester and an altered C apoprotein. The
Importance of these qualitative changes Is unclear. The hyperllpldemla may
result from the 2,3,7,8-TCDD mobilization of free fatty acids, which are
then used in the synthesis of VLDL and are subsequently formed Into IDL.
The relationship of the changes 1n serum lipid levels to the mechanism of
2,3,7,8-TCOO toxicity needs further study.
Elovaara et al. (1977) observed some changes 1n blochemlcals of the
brain of male Wlstar and heterozygous Gunn rats given a single Intubation of
2,3,7,8-TCOO at a dose of 20 pg/kg. At 7 days post-treatment, there was a
small but significant decrease as compared with vehicle treated control
animals 1n both the protein and RNA content of the Wlstar rats, while levels
of acid proteinase and DT-dlaphorase (an enzyme Induced by 2,3,7,8-TCDD In
the liver) had a small but significant Increase In the heterozygous Gunn
rats. There were no significant changes observed In homozygous rats given
00110
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2,3,7,8-TCDO at 20 yg/kg. The authors noted that acid proteinase may
participate 1n chemically Induced degeneration of the brain.
Inraunological Effects -- During acute toxicity studies with 2,3,7,8-
t
TCDD, thymic atrophy was: noted as a consistent effect In all species that
have been Investigated. This finding suggested that 2,3,7,8-TCDD may alter
the Iramine response, and Initiated Immunotoxiclty studies 1n exposed
animals. In guinea pigs treated with 8 weekly oral doses of 2,3,7,8-TCDD
(0, 0.008, 0.04, 0.2 or 1.0 pg/kg bw), body weight, spleen weight and
thymus weight were depressed, adrenal weight was Increased and leukocyte and
lymphocyte counts were elevated (Vos et al., 1973). Upon histological
examination, 2,3,7,8-TCDD-exposed rats had a severe depletion of lymphocytes
from the thymic cortex (Vos and Moore, 1974). Hematological changes were
noted In rats exposed to 10 and 14 dally doses of 10 pg/kg 2,3,7,8-TCOO
(Welssberg and Zlnkl, 1973). Increased red blood cell count, decreased
platelet count. Increased neutrophil count and Increased packed cell volumes
were reported 1n 2,3,7,8-TCOD-exposed rats. .A summary of the data available
on the Immunotoxlc effects of 2,3,7,8-TCDO 1n animals Is presented In Table
V-4. A review of 1imunotox1c1ty and Immunosuppression was reported by Vos
(1977).
Vos et al. (1973) Investigated the humoral and cell-mediated Immune
response 1n Hartley guinea pigs, CO rats and B6D2F^ mice. The humoral
Immune response was tested 'In 2,3,7,8-TCOD-treated hamsters by Injecting
tetanus toxoid (subcutaneously) Into the footpad and later testing for the
concentration of tetanus antitoxin from the serum by an Immunodiffusion
technique. Cell-mediated Immunity was tested by injecting Mycobacterium
00110
V-28
08/11/84
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TABLE V-4
Immunological Effects of 2,3,7,B-TCDD In Animals
o
o
Species/
Strain
Sex Exposure Route
Dose(s)
Duration of Exposure
Hlnlmura
Effective Dose
Parameter
Ef f ec t
Reference
H1ce/B6D2Fj M gavage
Hlce/C57Bl/6 F.N maternally
administered
(gavage)
Hlce/
C57Bl/6Jfh
gavage
0, 0.2, 1.0, 5.0, 4 weeks
25.0 |ig/kg bw/week
0. 1.0, 2.0, 5.0,
25.0 vg/kg
0, 0.5, 1, 5. 10.
20 wg/kg bw/week
4 or 6 weeks (3 or 5
administrations)
4 weeks
NA
5.0 iig/kg bw/week
5.0 wg/kg bw/week
1.0 |ig/kg bw/week
25.0 wg/kg bw/week
2.0 iig/kg bw/week
1.0 wg/kg bw/week
bw
thymus weight
graft-versus-
host response
thymus weight
PHA response
skin graft
rejection
Salmonel la
Infection
no change Vos et al..
decreased 1973
decreased
decreased
decreased
prolonged
Increased
mortality
and
decreased
time to
death
Vos and
Hoore, 1974
Thlgpen
el al.. 1975
i
ro
i£>
Htce/Swtss
H1ce/B6C3F
1
gavage
In vitro
(spleen cells)
0. 1.5. 5. 15.
50 wg/kg bw/week
4 weeks
0.5. 5.0, 50 ng/ml 5-60 seconds
1.5 |i9/kg bw/week
50 tig/ml
endotoxin |E. coll)
susceptlbl11ty
protein, DNA, and
RNA synthesis
Increased
mortalIty
decreased
Vos et al.,
197Ba
Luster et al.,
1979a,b
Hlce/Swlss-
Uebster
-Mlce/CO
f.H maternally
administered
(diet)
N gavage
0. I. 2.5, 5. 10.
20 ppb (dietary)
10 weeks (pre-gestatIon
and 3 weeks post-
parturltlon)
0, 0.01, 0.1, 1.0, up to B weeks
10.0 iiQ^kg bw/week
2.5 ppb
2.5 ppb
5 ppb
1 ppb
NA
0.01 wg/kg bw/week
1.0 wQ/k9 bw/wcek
antigenic RBC
reaction
thymic cortex
contact sensitivity
to DNfB
endotoxin
(Salmonella)
susceptibility
Listeria Infection
serum Immunoglobln
level
serum Inmunoglobln
level
decreased
atrophy
decreased
Increased
mortality
no change
Increased
decreased
Thomas and
Hlnsdl11. 1979
Sharma and
Gehrlng, 1979
o
id
Nice/CD
In vitro
10~«-10~» N
single
10^ H
lymphocyte blasto-
genlc transforma-
t Ion
Increased Sharma and
Gehrlng, 1979
CD
-p»
-------
TABLE V-4 (cont.)
Species/
Strain
Sex
Exposure Route
Dose(s)
Duration of Exposure
Minimum
Effective Dose
Parameter
Effect
Reference
Hlce/Swlss-
Mebster
F
oral (diet)
0. 10. 100 ppb
5 weeks (or more)
10 ppb
10 ppb
10 ppb
10 ppb
10 ppb
tetanus response
antigenic RBC
response
sensitization to
DNFB
resistance to
Salmonella
resistance In
Lister la
decreased
decreased
decreased
Increased
mortality
Increased
mortality
Htnsdl11,
et al.. 1900
Mice/
CS7B1/6J
M
Kp.
0. 1. 2. 6.
30 pg/kg bw
single Injection
1 pg/kg
macrophage and
natura 1 kl1ler
cell activity
macrophage and
natural killer
cell number
antibody
production
-no change
decreased
decreased
Hantovanl
el al.. 19B0
HIcemCSF!
M.F
maternally
administered
0. 1.0, 5.0,
1S.0 pg/kg bw/day
4 days during gestation
and lactation
1.0 pg/kg bw/day
1.0 ng/kg bw/day
S.O pg/kg bw/day
L. monocytogenes
susceptibility
PTB6-tumor suscep-
tibility
bone marrow hypo-
cellular Ity
Increased
Increased
Increased
Luster et al
1980
H1ce/C57Bl/6
N
l.p.
0. 0.4. 4.0.
40 pg/kg bu/ueek
4 weeks
4.0 pg/kg bw/week
0.4 pg/kg bw/week
thymus atrophy
cytotoxic T-cell
response
Increased
decreased
Clark et al.
1981
M1ce/C57Bl/6
N
I.p.
0. 0.004. 0.04,
0.4 pg/kg bw/week
4 weeks
0.004 pg/kg bw/week
In vitro genera-
tion of cytotoxic
T-cells
decreased
Clark et al.,
1981
Rat/CD
F
oral
0. 0.2. 1.0,
S.O |ig/kg bw/week
6 weeks
S.O pg/kg bw/week
S.O pg/kg bw/week
NA
bw
thymus weight
tuberculin hyper-
sensitivity
decreased
decreased
no change
Vos et al..
1973
Rat/CD
F
oral
0, 10 wg/kg bw/day
10. 14 days
10 pg/kg bw/day
10 pg/kg bw/day
10 pg/kg bw/day
erythrocyte count
platelet count
neutrophil count
Increased
decreased
Increased
Helssberg am
Zlnkl. 19/3
-------
TABLE V 4 (cont.)
Species/
Strain
Sex
Exposure Route
Dose(s)
Ouratton of Exposure
Minimum
Effective Dose
Parameter
Effect
Reference
Rat/F-344
F.N
maternally
0. 1.0, S.O wg/kg
4 or 6 weeks (3 or 5
1.0 pg/kg bw/dose
bw and thymus
decreased
Vos and
administered
bw/dose
administrations)
weight
Hoore. 1974
S.O |ig/kg bw/dose
spleen weight
decreased
S.O wg/kg bw/dose
PHA response
decreased
S.O iig/kg bw/dose
graf t-versus-host
decreased
response
S.O tig/kg bw/dose
skin graft
prolonged
rejection
NA
pseudorables
no change
virus infection
Rat/FIscher
F.H
maternally
NR
4-6 weeks (during ges-
NR
Con A and PHA
decreased
Hoore and
administered
tation and neonatally)
response
Faith, 1916
(NR)
NR
oxatolone skin
decreased
hypersensitivity
Rat/FIscher-
F.H
maternally
0, 5 pg/kg bw/dose
3 or 4 applications
S pg/kg bw/dose
antibody production
no effect
Faith and
Wlstar
administered
during gestation and
to bovine ganrna
Luster. 1979
(NR)
neonatally
globulin
S |ig/kg bw/dose
PHA and Con A
decreased
response
S pg/kg bw/dose
thymus and bw
decreased
until 13S
days
Rat/Sprague-
N
l.v.
0, 1 pg/kg bw
single Injection
1 pg/kg bw
thymic RNA
decreased
Kur1 et al.,
Dawley
synthesis
1982
thymic RNA
decreased
polymerase
activity
Guinea pig/
F
gavage
0. 0.008, 0.04,
8 weeks
0.04 |ig/kg bw/week
bw
decreased
Vos et al..
Hartley
0.?, 1.0 pg/kg bw
0.04 |ig/kg bw/week
thymus weight
decreased
1973
0.04 iig/kg bw/week
tuberculin hyper-
decreased
sensitivity
0.2 iig/kg bw/week
tetanus antitoxin
decreased
H . male; F = female; l.p. = Intraperitoneal l.v. ~ Intravenous; PHA * Phytohemagglutlnln; Con A =¦ Conconavalln A; RBC - red blood cell; DNFB - 2,4-dlnltro,
1-fluorobemene; NA = Not applicable; NR - Not reported
-------
tuberculosis (subcutaneously) Into guinea pigs on day 35 of 2,3,7,8-TCOD
treatment (during a schedule of 8 weekly doses). Intradermal tuberculin
hypersensitivity was determined by measurements of skin thickening on days
47 and 54. Decreased skin hypersensitivity was noted In hamsters treated
!
with 0.04 g 2,3,7,8-TCDD/kg and higher doses. Decreased tetanus antitoxin
levels were evident In guinea pigs treated with 0.2 yg 2,3,7,8-TCDD/kg,
but not at lower dose levels. Vos et al. (1973) also tested the cell-
mediated Immunity In rats exposed to 2,3,7,8-TCOD (0, 0.2, 1.0 or 5.0
yg/kg, once weekly for 6 weeks). H. tuberculosis was Injected Into rats
by day 28 of the treatment period, followed by Intradermal hypersensitivity
testing on day 42. No changes In the thickness of skin were noted In
2,3,7,8-TCDD-treated rats when compared with controls.
Mice were used to test the effect of 2,3,7,8-TCOD on cell-mediated
Immunity by use of the 'graft-versus-host" experiment (Vos et al., 1973).
In this test, spleen cells from 2,3,7,8-TCDD-exposed mice (0, 0.2, 1.0 or
5.0 yg/kg once weekly for 4 weeks) of the C57B1/6 strain were Injected
Into the right footpad of a hybrid recipient mouse (C5781/6 x 0BA-2). Donor
cells possessing sufficient activity will respond to the DBA-2 antigen on
the host cells, resulting In the enlargement of the popliteal lymph node.
Host cells are tolerant of the donor cells since both have C5781/6 antigens.
In this test Vos et al. (1973) noted a significant (p<0-01) dose-related
decrease 1n the activity of 2,3,7,8-TCDD-treated spleen cells (as measured
by the degree of popliteal lymph node enlargement on the site of the spleen
cell Injection). Lymph node enlargement was significantly less (p<0.01} 1n
hybrid recipient mice receiving spleen cells from mice treated with 5 yg
2,3,7,8-TCOD/kg/week than from donor cells of untreated mice.
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Studies continued In an attempt to Identify the mechanism of 2,3,7,8-
TCDO-lnduced Immunodeficiency. Rats (f-344) exposed pre- and postnatally by
maternal dosing (1 or 5 gg 2,3,7,8-TC00/kg administered to dams on days 11
and 18 of gestation and 0, 7 and 14 postnatally) had prolonged times until
t
graft rejection, decreased spleen cell graft-versus-host activity and
decreased binding response to phytohemagglutlnin (PHA) (Vos and Moore, 1974;
Moore and Vos, 1974). Response to conconavalln A (Con A), a humoral Immune
response, was actually Increased.
Since thymus-deMved lymphocytes (T-cells) play a central role In
cell-mediated Immunity and host defense mechanisms, Interest turned to these
areas of Immunology. The effect of 2,3,7,8-TCOO on host resistance to
Infection, a vital measure of Immune response, was tested by Thlgpen et al.
(1975) In male pathogen-free mice (C57B1/6Jfh). 2,3,7,8-TCDD was adminis-
tered to mice at 0.5, 1, 5, 10 or 20 vg/kg once weekly for 4 weeks
followed by Inoculation with Salmonella bern 2 days after the final 2,3,7,8-
TCOD administration. Mortality rates and "time until Infection" were used
to determine the Immunological effect of 2,3,7,8-TCOO. A significant
(p<0.05) Increase 1n mortality and decrease 1n time of infection were noted
1n groups treated with 1 yg/kg or higher doses of 2,3,7,8-TCOO when
compared with controls. 2,3,7,8-TCOO at 0.5 vg/kg did not alter these
parameters and was regarded as a no effect level. The Immune-resistance of
mice to S. bern Is therefore reduced by treatment with 1 yg 2,3,7,8-
TCDO/kg/week (for 4 weeks).
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Pretreatment with 2,3,7,8-TCDD greatly enhances the susceptibility of
mice to E. coll endotoxin (Vos et al., 1978a). Injection of 250 yg of
endotoxin to mice pretreated with 0, 1.5, 5 and 15 yg 2,3,7,8-TCDOAg
resulted In 0/5, 1/5, 6/6 and 6/6 deaths, respectively. H1ce pretreated
with 15 and 50 yg 2,3,7,8-TCDD/kg and Injected with 10 yg of endotoxin
had 1/4 and 2/4 deaths, respectively. Mice treated with lower doses of
2,3,7,8-TCDD were not susceptible to this quantity of endotoxin. Increased
mortality (2/6) In a control group was noted only when 500 yg of endotoxin
was administered, while 10 yg of endotoxin was sufficient to cause similar
mortality (2/5) In mice treated with 50 yg 2.3,7,8-TCD0/kg.
The Immunocompetence of 5-week-old offspring of Swiss-Webster mice fed
diets containing 1, 2.5, 5, 10 or 20 ppb 2,3,7,8-TCDD was tested by several
means (Thomas and Hlnsdlll, 1979). The number of cells reactive to anti-
genic RBC, differential white blood cell counts, organ weights, hlstopathol-
ogles, hypersensltlvlty to 2,4-d1n1tro-l-fluorobenzene (ONFB) and the
resistance to E. coll llpopolysaccharlde (LPS), Listeria monocytogenes and
Salmonella typhlmurlum LPS were all measured for mice exposed to different
levels of 2,3,7,8-TCDD. Adult female mice were exposed to 2,3,7,8-TCDD for
4 weeks before mating, throughout gestation and for 3 weeks postparturltlon.
Young mice being tested for Immunotoxldty were therefore exposed to
2,3,7,8-TCDD only In. utero and through lactation. The typical decrease In
thymus weight was noted 1n mice exposed to 2.5 and 5.0 ppb but was not
evident In the 1.0 ppb group. 'A decrease In the number of plaque-forming
cells (PFC) reactive to sheep RBCs was significantly reduced In the 2.5 and
5.0 ppb 2,3,7,8-TCDD-exposed groups. (Because of the poor survival of young
1n the 10 and 20 ppb 2,3,7,8-TCDD-exposed groups, results and comparisons
00110
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were usually reported For the three lower dose groups). The humoral content
of antl-RCD antibodies, however, was not lower 1n 2,3,7,8-TCOD-exposed
groups when compared with controls. A decrease In the skin hypersensitivity
to DNF8 following sensitization was noted In all 2,3,7,8-TCDD-treated groups
t
(only the 5 ppb group wks statistically reduced from controls). 2,3,7,8-
TCOD caused an Increased susceptibility (Increased mortality level) to S.
typhlmurlum 1n a dose-related fashion. The response to £. coll LPS and L.
monocytogenes was not different from controls. 2,3,7,8-TCDO exposure did
not alter the response of lymphocytes (Band T-cells) In, vltro to Con A, nor
was mltogen-1nduced lymphocyte proliferation affected (Thomas and Hlnsdlll,
1979).
Similar findings were reported In F1scher/W1star rats exposed to
2,3,7,8-TCDD during gestation (18th day) and neonatally, or neonatally alone
(on days 0, 7 and 14) (Faith and Luster, 1979), Dams were treated with 5
g/kg 2,3,7,8-TCDD on each dose day. Typically, body weight and thymic
weights were decreased 1n progeny, which lasted until 135 days of age. The
thymic- and splenic-cell response to PHA and Con A was decreased 1n all
2,3,7,8-TCDD-treated animals and did not return to normal until day 270.
Delayed hypersensitive reaction was also suppressed until 270 days of age.
The production of antibodies to bovine gamma globulin, which requires
T-helper cell function, was not affected by 2,3,7,8-TCDO exposure during rat
development (Faith and Luster, 1979).
00110
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*
-------
Neonatal B6C3F^ 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 ^g/kg 2,3,7,8-TCOD, were studied for Immunotoxlc effects and
host susceptibility (Luster et al., 1980). At the 15.0 pg 2,3,7,8-TCOD/kg
Jose level, 70% of the -neonates died with overt toxic effects (decreased
body weight, liver weight, spleen weight and thymus weight). Bone marrow
hypocellularlty and depressed macrophages-granulocyte progenltor cells and
pleurlpotent stem cells were associated with 2,3,7,8-TCDD exposure at the
5.0 and 15.0 yg/kg dose levels. Hematological changes, such as decreased
RBC count, hematocrit and hemoglobin, and lymphocyte count showed a dose-
related response. Host susceptibility to L. monocytogenes and PYB6-tumor
cells was tested 1n the 2,3,7,8-TCOD-exposed neonates. Death occurred 1n 73
and 40* of the L. monocytogenes Inoculated (1.2x10* viable organisms) mice
1n the 5.0 and 1.0 yg/kg dose groups, respectively, compared with 28% of
controls. Tumor development occurred in 44, 60 and 22% of the neonates
Inoculated with 5x10* tumor cells from the 5.0 pg 2,3,7,8-TCDD/kg, 1.0
vg 2,3,7,8-TCDD/kg and control groups, respectively.
H1nsd1l1 et al. (1980) reported that 2,3,7,8-TCOO administered In the
diet of Swiss-Webster mice at 100 ppb for 5 weeks caused a marked suppres-
sion of total serum protein, gamma globulin and albumin, but an Increase 1n
B-globul1ns. At 10 ppb In the diet, 2,3,7,8-TCOO caused decreased Immune
response to tetanus toxoid, sheep RBC, S. typhlmurlum and L. monocytogenes,
and lowered contact sensitivity to 0NFB. This study also suggested that
although young animals are more susceptible to 2,3,7,8-TC00, older animals
are still Immunosuppressed and exposure in utero and neonatally Is not more
crucial than In other periods. Vos and Moore (1974) had previously reported
00110
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-------
that 1-month-old mice were more sensitive to 2,3,7,8-TCOO than were 4-month-
o1d mice (C57B1/6). Decreased body weight and thymus weight and spleen cell
response to PHA were evident at lower doses 1n 1-month-old mice than In
4-month-old mice.
?
The effect of single 1.p. doses of 2,3,7,8-TCDD (1, 2, 6 and 30 ng/kg)
on peritoneal macrophage and splenic natural killer cell function in mice
(C57B1/6J) was studied by Mantovanl et al. (1980) and Vecchl et al. (1980).
2,3,7,8-TCOD treatment at all dose levels did not decrease the cytostatic
and cytocldal activity of macrophages or natural killer cells on a per cell
basis. The total number of macrophages and splenic natural killer cells
recovered from 2,3,7,8-TCOO-treated animals, however, was reduced when
compared with untreated controls. Marked hypocellularlty noted 1n the bone
marrow of 2,3,7,6-TCDD-treated mice may account for the decrease In periph-
eral cell counts (McConnell et al,, 1978b). The lack of macrophages and
natural killer cells was suggested as being Instrumental In the decreased
resistance to Infection common to 2,3,7,8-TCDD-exposed animals (Mantovanl et
al., 1980). Although 2,3,7,8-TCDD was a strong Immunosuppressant, animals
given a lethal dose of 2,3,7,8-TCD0 did not appear to die from Infections,
nor did a germ-free environment protect them from death (Gre1g et al., 1973).
The actual mechanism of 2,3,7,8-TCDD Imnunotoxlclty 1s unknown but
several Investigators have tested various hypotheses. Vos et al. (1973,
1978a,b) attempted to address the Indirect causes for decreased thymic
growth and altered T-lymphocyte activity following 2,3,7,8-TCDD treatment.
00110
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Vos et al. (1973) measured serum Cortisol and cortlcosteron levels 1n guinea
pigs exposed to 2,3,7,8-TCDD to evaluate the possible Indirect Irmiunosup-
presslon by these hormones. There was, however, no significant difference
In the level of these hormones between treated and control animals.
Fndlrect Immunosuppression of this type was unlikely. Later studies (Vos et
al.f 1978atb) investigated the role of thymic hormones (thymosin) on the
atrophy of the thymus during 2,3,7,8-TCDD treatment. Thymosin administered
In conjunction with 2,3,7,8-TCDD did not protect mice from the typical
2,3,7,8-TCDO-lnduced Inmunotoxlc alterations. Thymus weight was maintained
but not Increased by thymosin, and thymus-deMved cells continued to show
decreased responsiveness to mitogens (PHA, Con A). Thus, It Is unlikely
that 2,3,7,8-TCDD affects the supply or synthesis of thymic hormones which
could lead to the observed Immunosuppression.
van Logten et al. (1980) Investigated the possible Influence of the
adrenal gland, hypophysis and pituitary, and growth hormone on thymic
atrophy and Immunosuppression following 2,3,7,8-TCDD exposure 1n female
F-344 rats. Adrenalectomy and exogenous growth hormone had no preventative
action on thymic Involution. Hypophysectomlzed rats showed advanced thymic
atrophy.
Sharma and Gehrlng (1979) noted that 2,3,7,8-TCDD caused stimulation of
lymphocyte transformation to blast form cells (mltotlcally active precurs-
ors) when no mitogens were present 1n the culture system. This represents a
phenomenon similar to actual antigenic challenge. At low doses (0.01 and
0.1 pg 2,3,7,8-TCDD/kg/week for up to 8 weeks), serum Immunoglobulin
levels were elevated In male CD-I mice. Larger doses of 2,3,7,8-TCDD (1.0
00110
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and 10 pg/kg/week) resulted 1n a decrease 1n the serum Immunoglobulin
level. It was suggested that 2,3,7,8-TCDD may elicit an antigenic response
either by combining with a body protein or by causing cellular or biochemi-
cal damage that releases antigenic proteins. Sharma and Gehrlng (1979) also
t
noted that thymic atrophy was observed after 2 and 4 weeks of treatment but
not after 8 weeks. There may be a recovery of thymic tissue, either by
Immune tolerance or Immune unresponsiveness as a sort of adaptation to
2,3,7,8-TCOO-exposure and Its possible antigenic complex.
Luster et al. (1979a,b) reported that 2,3,7,8-TCOD affects the Immune
system directly by altering lymphocyte function. The function of T-helper
cells was not altered, since no change In response to bovine gamma globulin
(requires T-helper cell cooperation) was noted In W1star/F1scher and Fischer
rats exposed to 2,3,7,8-TCDD. In. vitro, 2,3,7,8-TCDO (100 ng/rna.) sup-
pressed DNA, RNA and protein synthesis 1n splenic lymphoid cells from
86C3F^ (Luster et al., 1979a). 2,3,7,8-TCOD, however, did not decrease
the binding of 3H-Con A to lymphocytes, Indicating that these receptors
are not blocked by 2,3,7,8-TCDO. T-lymphocytes were more susceptible to
2,3,7,8-TCOO, measured by specific mitogen binding assays, than B-lympho-
cytes. These authors (Luster et al.f 1979a) suggested that 2,3,7,8-TCDO may
bind directly to the lymphocyte cell membrane and alter Its function. Faith
and Luster (1979) reported that lymphocytes from the spleen, thymus, bone
marrow and lymph nodes of Fischer rats exposed to 2,3,7,8-TCOO showed
abnormal homing patterns within' the body. 2,3,7,8-TCDO exposure apparently
altered the cell surface markers so that spleen lymphocytes were taken up by
the thymus of recipient rats. These authors (Faith and Luster, 1979)
suggested that 2,3,7,8-TCDO may change cellular metabolism, which alters the
00110
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-------
cell membrane constituents or may Insert directly Into the membrane. Kurl
et al. (1982) reported that 2,3,7,8-TCDD causes changes 1n thymic transcrip-
tion and RNA synthesis that may lead to cell surface changes. Cell surface
changes could presumably result In altered antigen recognition and cell-to-
i
cell recognition, causing Inruinosuppresslon and thymic atrophy.
Clark et al. (1981) reported that 2,3,7,8-TCDD treatment (0.4, 4.0, 40
yg/kg weekly for 4 weeks by 1.p. Injection) caused functional Impairment
of cytotoxic T-cells 1n C5781/6 male mice. The authors felt that this
response was particularly sensitive to 2,3,7,8-TCDD treatment and hypothe-
sized that 2,3,7,8-TC0D directly inhibits the function of these cells.
Contrary to the hypothesis tested by these authors and that held by Luster
et al. (1979a,b), 2,3,7,8-TCOD treatment Impaired the generation of cyto-
toxic T-cells by the spleen (at doses as low as 0.004 yg/kg when detected
1_n vitro) but did not appear directly toxic to the cytotoxic T-cells. At
present, the mechanism of Immunosuppression caused by 2,3,7,8-TCDD 1s
unknown and the theories available are speculative. In a later study,
however, Clark et al. (1983) reported that a 10- to 100-fold greater dose of
2,3,7,8-TCDD was required to suppress cytotoxic T-cells 1n DBA/2 mice as
compared with C5681/6 mice. This Indicates that susceptibility to 2,3,7,8-
TCD0 1mmunotox1c1ty segregates with the Ah locus which Is consistent with a
receptor mediated mechanism. , The receptor mediated mechanism was further
supported by the susceptibility of the C5781/6 x DBA/2J hybrid mouse to
2,3,7,8-TCDO suppression of the cytotoxic T-cells which is again consistent
with the dominant Inheritance of Ah (Nagarkattl et al., 1984).
00110
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Few reports are available In which the Immunological effects of 2,3,7,8-
TCOD exposure were studied In humans. Regglanl (1960) reported that the
Immunocapablll ty of 17 people, ranging 1n age from 3-60 years, who had been
exposed to 2,3,7,8-TCOO, was normal In all cases. In a survey of 41 workers
^exposed to 2,3,7,8-TCDD, Ward (1982) measured 1mmunoglob1n G, A, M, D and E,
as well as lymphocytes, T-cells, B-cells, PHA response and blood cell
counts. These determinations were made 10 years after workers had developed
2,3,7,8-TCDtMnduced chloracne. In this group of workers, there was a
significant Increase In the proportion of cases with reduced IgO and IgN.
It was suggested that the 2,3,7,8-TCOO-exposed group had a reduced Immune
capability and a deficiency In T-cell and 8-cell cooperation. The Immuno-
toxlclty of 2,3,7,8-TCOD In humans cannot be properly assessed because of
the paucity of data recorded soon after exposure. The most prominent
effects In animals (I.e., humoral responses) were not measured In humans.
Enzyme Induction by TCOD —
In Cell Cultures. Although 2,3,7,8-TCDD has a very low toxicity to
cells In culture (Beatty et al., 1975; Bradlaw et al., 1976; Knutson and
Poland, 1980; Yang et al., 1983), It Is an extremely potent enzyme Inducer
In these systems (Kourl et al., 1974; N1wa et al., 1975; Bradlaw et al.,
1976; Malik and Owens, 1977 ; Malik et al., 1979; Bradlaw et al., 1980).
This enzyme induction Is so sensitive that 1t has been proposed as a bio-
assay for detecting planar polychlorlnated organic compounds (Bradlaw et
al., 1975, Bradlaw and Casterllne, 1979; N1wa et al., 1975).
00110
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KouM et al. (1974) found that 2,3,7,8-TCDD Induced aromatic hydrocarbon
hydroxylase (AHH) activity In cultured human lymphocytes to the same extent
as 3-MC; however, the concentration of 2,3,7,8-TCDD necessary for maximal
enzyme Induction was 40-60 times less than that of 3-MC. N1va et al. (197S)
Compared AHH Induction by 2,3,7,8-TCDD among cell cultures (H-4-II-E, VERGf
HTC, LB82, MA, Hepa-1, TRL2, ERl-2, NRKE and Chang). ED^ values ranged
from 0.12 nM In the Hepa-1 cell line to >100 nM 1n the VER0 and HTC cell
lines. 2,3,7,8-TCDO did not Induce AHH activity In LB82 cells. The respon-
siveness of AHH Induction to 2,3,7,8-TCDD was 250-900 times greater than to
3-MC. In addition, cell cultures derived from C57B1/6N mice were 16 times
as sensitive to 2,3,7,8-TCDD as cell cultures derived from DBA/2N mice. The
responsiveness of cell cultures t6 enzyme Induction by 2,3,7,8-TCDD 1s thus
similar to the effects seen in vivo. The Inductive effect of 2,3,7,8-TCOD
was blocked by actlnomycln 0 and cyclohex1rn1de, Implying that induction
Involved the sythesls of new mRNA and protein. Enzyme Induction by
2,3,7,8-TCDD, therefore, Involves an Initial RNA synthesis and continuous
protein synthesis (Malik and Owens, 1977; Malik et al., 1979).
In all of these studies, there was no correlation between cytotoxicity
and enzyme Induction. This Implies that, despite the correlation In. vivo.
there may be no direct connection betweeen enzyme Induction and the toxicity
of 2,3,7,8-TCDO.
In Mice and Rats. The effects of 2,3,7,8-TCDD on enzyme activity
In rats and mice have been Investigated extensively. 2,3,7,8-TCDO has been
found to alter many enzyme activities 1n a wide variety of organ systems
(vide Infra). This alteration primarily results In Increased enzyme
activity, although 2,3,7,8-TCDD has been observed to Inhibit some enzymes.
00110 V-42 08/11/84
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Hook et al. (1975a) reported that 2,3,7,8-TCDD supressed hepatic micro-
somal N-demethylatlon 1n male, but not female, rats; however, cytochrome
P-450 and benzpyrene hydroxylase activity were Increased. The suppression
of N-demethylase activity was undetectable for 73 days following a single
I
oral dose of 25 pg 2,3,7,8-TCOD/kg bw. The suppression of N-demethylase
activity was seen only In adult animals. In 10-day-old rats, 2,3,7,8-TCDO
had an Inductive effect on this activity.
The Inductive effects of 2,3,7,0-TCOO have been demonstrated to be organ
specific. Alt1o and Parkkl (1978) Investigated the effects of 2,3,7,8-TCDD
on the activities of AHH, ethoxycoumarln deethylase, cytochrome C reductase,
epoxide hydratase, LIDP glucuronosyltransferase, and glutathione S-transfer-
ase In the liver, kidney, lung, small Intestine and testes of male Wlstar
rats. Monooxygenase activity was stimulated 1n the liver, lung and kidney,
but not In any other tissue Investigated. UOP glucuronosyltransferase
activity Increased by a factor of 7 In the liver, by a factor of <2 1n the
kidney, and not at all In any other tissue. Epoxide hydratase and
glutathione S-transferase activities were not affected 1n any of the tissues
studied, although stimulation of hepatic glutathione S-transferase has been
reported by other Investigators (Manls and Apap, 1979). Enzyme Induction
has also been reported 1n rat mammary gland (Rlkans et al., 1979), mouse
testes (Mattlson and Thorgelrsson, 1978), and rat prostate gland (Lee and
Suzuki, 1980), but the rat adrenal gland 1s apparently Insensitive to
Inductive effects of 2,3,7,8-TCOD (Guenthner et al., 1979).
00110
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In the liver of rats and mice, 2,3,7,8-TCOO affects a wide range of
enzymatic activities, Including OT-dlaphorase (Beatty and Neal, 1976a,b),
bilirubin catabollsm (Kapltulnlk and Ostrow^ 1978), ornithine decarboxylase
(Potter et al., 1982), 7-ethoxycoumar1n O-demethylase (Greenlee and Poland,
»
1978), glutathione S-tran'sferase (Baars et al., 1978; Hanls and Apap, 1979),
aldehyde dehydrogenase (Llndahl et al., 1978; Deltrlch et al., 1977),
uroporphyrinogen decarboxylase (Jones and Sweeney, 1977), 5-am1nolevul1n1c
acid synthetase (Goldstein et al., 1982a; Woods, 1973), UDP-glucuronosyl
transferase (Harselos et al., 1978) and a number of microsomal oxidative
enzyme systems (vide Infra).
2,3,7,8-TCDD 1s four orders of magnitude more potent than 3-MC as an
inducer of hepatic AHH activity; however, the dose-response curve for the
two compounds are parallel and both produce the same maximal response
(Poland and Glover, 1974). Simultaneous administrations of maximally
Inducing doses of both compounds produced no greater response than either
alone and both produced a cytochrome with a shift In the absorption maximum
of the carbon monoxide difference spectrum from 450 to 448 nm. In a number
of studies, Increased AHH activity and cytochrome P-448 synthesis have been
separated (Chhabra et al., 1976); however, other researchers report an
apparent connection between cytochrome P-448 and AHH Induction (Kltchln and
Woods, 1977, 1978a,b). Thus, 2,3,7,8-TCDD not only stimulates AHH activity
by Inducing cytochrome P-450 formation, but may enhance AHH activity by
other mechanisms as well.
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In Rabbit. The response of the rabbit 1s quite different from that
observed In rats and mice (Hook et al., 1975a). The only changes In hepatic
enzyme activities observed were suppression of benzpyrene hydroxylase and
benzphetamlne N-demethylase. In the same study, blphenyl 4-hydroxylase was
induced 1n the lung and benzpyrene hydroxylase was Induced In the kidney.
In a similar study, a hepatotoxlc dose of 2,3,7,8-TCDD (30 yg/kg) failed
to alter prostaglandin synthetase activity In hepatic or renal tissue (Kohll
and Goldstein, 1981).
In a series of studies, Johnson and Muller-Eberhard (1977a,b,c,d),
Johnson et al. (1979), Norman et al. (1978), Hem et al. (1980) and Dees et
al. (1982) Isolated a series of cytochromes P-450 from rabbit liver micro-
somes. These cytochromes were Imnunologlcally distinct, functioned 1n dif-
ferent catalytic pathways, and responded differently to Induction by poly-
cycllc aromatic hydrocarbons. 2,3,7,8-TCDD was found to Induce two cyto-
chromes, designated as form 4 and form 6. Form 4 1s the major cytochrome
Induced 1n adult rabbit liver by 2,3,7,8-TCDO; however, form 6 Is the major
cytochrome Induced In newborn rabbit liver (Norman et al., 1978b), adult
rabbit lung, and adult rabbit kidney (Llem et al., 1980; Dees et al., 1982).
Other Species. The guinea pig, the species most sensitive to the
toxic effects of 2,3,7,8-TCDO, Is similar to the rabbit In Its response to
2,3,7,8-TCOO. Blphenyl 4-hydroxylase was Induced In the liver, lung and
kidney, blphenyl 2-hydroxylase suppressed 1n the liver, and benzpyrene
hydroxylase was Induced In the kidney (Hook et al,, 1975b). Testicular
microsomal cytochrome P-450 content was depressed following a single oral
dose of 1 yg/kgt reaching 52% of controls by 1 day and remaining at this
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level for 9 days (Tofilon et al., 1980). Testicular microsomal heme levels
and 4-amlnolevullnU acid synthetase activity were unaffected by this
treatment. Tn contrast to the rat, 2,3,7,8-TCOO did not Induce DT-dlaphor-
ase 1n brain, spleen, kidney, lung, heart or liver of male guinea pigs
^Beatty and Neal, 1978). -
Aryl hydrocarbon hydroxylase and ^-aminolevulinic add synthetase In
the chick embryo have been reported to be extremely sensitive to the
Inductive effects of 2,3,7,8-TCOO (Poland and Glover, 1973a,b)f with maximal
Induction occurring with 155 pmoles/egg. This Induction Is relatively long
lasting, with 10% of the maximum Induced activity present 5 days following a
single dose of 2,3t7#8-TC00. Structure-activity studies demonstrated a
perfect correspondence between the toxicity and Induction potency of a
series of d1benzo-£-d1ox1n congeners (Poland and Glover, 1973a),
Subchronlc Toxldty. Four laboratory studies described the systemic
toxic effects of subchronlc exposure to 2,3,7,8-TCOO in rodents. Also, one
semi-controlled study evaluated the toxic effects to rabbits after confine-
ment to an area containing soil contaminated with 2,3,7,8-TCOO. No Informa-
tion was found 1n the literature searched on the effects of subchronlc
exposure to 1,2,3,7,8-PeCOD, and only one preliminary study was available
describing the effects of subchronlc exposure to a mixture of two HxCODs In
rats and mice,
t
Kodba et al. (1976) exposed Sprague-Oawley rats to 2,3,7,8-TCDD for 13
weeks. The animals 1n groups of 12 males and 12 females received the com-
pound suspended 1n acetone-corn oil (1:9) by gavage 5 days/week at doses of
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0.0, 0.001, 0.01, 0.1 or 1,0 yg/kg bw. At the end of the treatment period
5 rats of each sex were killed for histopathologic examination, and the
remaining animals were continued for postexposure observation. This report
on gross, hematologic, clinical chemistry and histopathologic (on animals
*
terminated at the Interim kill or killed when moribund) observations was
prepared on data available 13 weeks after termination of treatment. Signs
of toxicity were observed only at the two higher dose levels, and female
rats appeared more sensitive to the toxic effects of 2,3,7,8-TCDO. During
the study there were five treatment-related deaths In the high dose group
females, with three occurring during treatment and two In the post-treatment
period. In male animals only two deaths occurred In the post-treatment
period In the high dose group. Both the male and female rats of the 0.1 and
1.0 gg/kg groups had depressed body weight; however, greater relative
depression of body weight was observed In the high dose females. Other
changes such as Increases In bilirubin concentrations, urinary coproporphy-
rln excretion, and changes 1n relative thymus or Hver-to-body weight ratio
occurred 1n the two high-dose female groups, but only In the 1.0 yg/kg
male group. Although male rats had significantly decreased hematologic
values (packed cell volume, RBC count and hemoglobin) In the two high-dose
groups, and these values were normal In all female rats, the authors pointed
out that these results may have been an artifact resulting from dehydration-
Induced hemoconcentratlon In the female rats. No specific data were
provided, however, to support this last conclusion.
f
After necropsy, gross examination revealed subcutaneous edema, a
decrease in the size of testes and uteri, and a decrease In the number of
corpora lutea. Histologic examination revealed Involution of the thymus,
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decreased number of thymocytes, and focal necrosis and pigment accumulation
In the liver. These observations were made only In the animals of the
high-dose group, with the exception of a slight decrease In the number of
thymocytes and mild microscopic distortion of the architecture of the 11verL
t
In the group fed 0.1 pg/kg. Although histologic evidence from animals
killed during the Interim sacrifice was consistent with the liver and thymus
being the primary target organs, In an animal that died during the study
there were signs of aortic thrombosis and adrenal hemorrhage, and In a
second animal there was severe anemia, suggesting possible Involvement of
the hematopoietic system near the time of death.
Liver toxicity was the only effect of treatment observed during histo-
logic examination of rats (Osborne-Mendel) and mice (86C3F^) administered
2,3,7,8-TCDO for 13 weeks In a preliminary subchronlc toxicity study
designed to define an acceptable dose for a chronic toxicity study (NTP
1980a). The animals 1n groups of 10 males and 10 females were administered
the compound 1n corn oil-acetone (9:1) twice a week at doses for rats of
0.0, 0.5, 1, 2, 4 and 8 pg/kg/week, and for mice at doses of 0.0, 1, 2, S,
10 and 20 pg/kg/week. Deaths occurred at the two high-dose levels In
rats, with 4 females In the 8 pg/kg/week and 1 In the 4 pg/kg/week group
dying, while only 2 male rats In the 4 pg/kg/week group died. Deaths were
accompanied by severe toxic hepatitis. Hepatic lesions were observed In all
other rats examined 1n groups administered 1-8 pg/kg/week; however, not
all animals In each group were submitted to necropsy. Normal liver
histology was observed 1n the 2 male rats examined from the low-dose groups
and only threshold toxic effects occurred In the low-dose female rats.
00110
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Similar effects of treatment were observed In mice, with a single death
occurring In each sex at the high-exposure level, along with reports of
hepatic lesions on histologic examination. In contrast to rats, female mice
were less sensitive to the hepatotoxlc effect of 2,3,7,8-TCDD than were the
*
male mice. Hepatic lesions were observed In all dose groups of male mice,
while the 1 and 2 yg/kg/week dose groups of female mice had normal livers.
Although the group sizes were small, making conclusions tenuous, It 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.
In a more extensive subchronlc study 1n rats. King and Roesler (1974)
followed the development of toxicity by a series of Interim sacrifices
during 28 weeks of exposure to 2,3,7,8-TCOO and a 12-week post-treatment
recovery period. Groups of 35 male and 35 female Sprague-Dawley rats were
Intubated twice weekly with 2,3,7,8-TCDD In corn oil-acetone (9:1) at cumu-
lative doses of 0.0, 0.1 and 1 yg/kg/week. No treatment-related deaths
occurred; however, 3 animals from each group of each sex were killed after
2, 4, 8 and 16 weeks, and 10 animals of each sex were killed after 28 weeks
of treatment. In addition, 3 rats of each sex were killed 4 and 12 weeks
after termination of exposure. Animals were monltoredj for gross changes
during the study and were examined for gross and histologic changes at
necropsy.
Besides a dose-related decrease 1n body weight gain In male rats and a
decrease In body weight gain In the high-dose female rats, the only effect
of exposure to 2,3,7,8-TCDD was histologic changes In the liver. Liver
pathology was normal In all treated groups up through the Interim kill at 16
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weeks. Fatty changes In the liver were considered the most Important obser-
vation. The fatty changes ranged from single large lipid droplets 1n a few
centrllobular hepatocytes to lipid droplets In all centrllobular hepatocytes
with extension Into the mldzonal hepatocytes. No clear dose-response
'pattern was observed In this study; however, It did appear that the severity
of fatty changes was greater In male rats. During the recovery period,
fatty changes progressively decreased 1n severity, but were still present In
some treated animals 12 weeks after cessation of exposure. Other histologic
changes observed 1n the liver predominantly In the animals killed at 28
weeks Included necrosis. Increased nuclear size, subtle distortion of liver
architecture, and hyperchromatlc nuclei. All of these lesions were consid-
ered to be slight or mild, and less toxlcologlcally relevant than the fatty
changes. The data suggested that the liver was the most sensitive organ to
the toxic effect of 2,3,7,8-TCDD, and although recovery occurred after
termination of treatment, the recovery process was slow.
The recovery time was also demonstrated to be long In a subchronlc study
by Goldstein et al. (1982b) of 2,3,7,8-TCDD Induced porphyria. Groups of 8
female Sprague-Oawley rats were given 2,3,7,8-TCDD 1n corn o1l-acetone (7:1)
weekly by gavage for 16 weeks at doses of 0.0, 0.01, 0.1 or 10.0 yg/kg/
week and killed 1 week after the last treatment. Additional groups of rats
received doses of 0.0 or 1.0 ^g/kg/week for 16 weeks and were allowed to
recover for 6 months. The high-dose level was lethal to all animals within
12 weeks, while the only other gross sign of toxicity was a decrease 1n body
weight gain In the group receiving 1.0 Mg/kg/week. After 16 weeks of
exposure to 2,3,7,8-TCDD, liver porphyrins were elevated -1000-fold In 7 of
8 animals receiving 1.0 pg/kg/week, but only 1 of 8 animals In the 0.1
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yg/kg/week group had elevated porphyrin levels. No ePFect was observed in
the low-dose animals. After a 6-month recovery period the porphyrin level
in animals exposed to 1 yg/kg/week was still 100-fold higher than values
in the control group. A similar pattern was observed for urinary excretion
of uroporphyrin. The rate limiting enzyme in heme synthesis, 6-am1nolevu-
linic acid synthetase, was also elevated at both the, time of termination of
treatment and at the end of the recovery period; however, other enzymes that
were Increased after 10 weeks of treatment, cytochrome P-450, AHH and glucu-
ronyl transferase, returned to near normal levels by 6 months. It was clear
that a 6-month recovery period from subchronlc exposure to 2,3,7,8-TCDD at a
dose of 1.0 ^g/kg/week was not sufficient for complete reversal of
2,3#7,8-TCDD-lnduced porphyria.
DeCaprlo et al. (1986) fed 2,3,7,8-TCDD In the diet for 90 days to male
and female Hartley guinea pigs and found NOELs of 0.61 and 0.68 ng/kg/day,
respectively. Decreased body weight gain, Increased relative liver weight,
decreased relative thymus weight and hepatocellular cytoplasmic Inclusion
bodies at 4.90 (males) and 4.86 (females) ng/kg/day and mortality and other
toxic effects at 26 (males) and 31 (females) ng/kg/day were also observed.
In addition to the above laboratory studies, Strlk and de Wit (1980)
attempted to Investigate the toxicologic effect on rabbits of exposure to a
natural environment that was contaminated with 2,3,7,8-TCDD. Groups of 20
female rabbits and 1 male rabbit .were housed for 5 months In pens, located
In five separate areas, on soil that had been contaminated with 2,3,7,8-
TCDD. The soil had been cleaned by replacement or cultivation before Initi-
ation of the study. The levels of 2,3,7,8-TCDD before cleaning were from
00110
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0.8-23.2 yg/m3; however, the levels of contamination after cleaning were
not determined. At ' the end of 5 months liver histology, including the
localization of porphyrin, was examined, and the levels of cytochrome P-45G
and P-420 were determined along with urinary levels of total porphyrin,
creatinine and D-glucaric-acid. All of the parameters examined were consid-
ered to be within the normal range. Since exposure data were not available,
the negative results of this study cannot be compared with the controlled
subchronic laboratory studies already described.
Information on the subchronic toxicity of HxCDD was provided in a pre-
liminary range-finding study for a chronic bioassay conducted by NTP (19Q0d)
on a 1-2 mixture of 1,2,3,6,7,8- and 1,2,3,7,8,9-HxCDD. Osborne-Mendel rats
and B6C3F.J mice in groups of 10 males and 10 females were administered the
HxCDD mixture in corn oil-acetone-(9:1) by gavage twice a week for 13 weeks.
The total weekly doses given rats were 0.0» 2.5, 5, 10, 50 and 100 yg/kg,
while mice received 0.0, 1.25, 2.5, 5t 10 and 50 yg/kg. At week 10 of the
study, the body weight In rats was decreased in a dose-related manner to a
maximum of -20% in the high-dose group. In mice, body weight was also
decreased 10-20% in the treated animals; however, there appeared to be no
correlation with dose. At the end of the study the animals were killed and
necropsies were performed on selected animals. In both species liver
pathology was observed, with threshold to moderate hepatotoxicity occurring
at doses of 5 and 10 yg/kg/week for male and female -rats, respectively,
and at 10 yg/kg/week for both sexe-s of mice. At higher exposures, splenic
hyperplasia and cortical atrophy of the thymus were also detected in rats.
In rats It was unclear whether the low-dose animals were free of any patho-
logic findings or none were subjected to necropsy. In mice it was stated
00110
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that no changes were observed in males exposed to 2,3,7,8-TCDD at 1.25
yg/kg/week or in females exposed to 1.25 or 2.5 yg/kg/week. Although
the data are limited, It appears that the same target organs are sensitive
to the toxic effects of both 2,3,7,8-TCDD and this mixture of HxCDD.
In addition, a second subchronic range finding study conducted by NIP
(1980c) evaluated the dermal toxicity of the above mixture of HxCDD. Groups
of 10 male and 10 female Swiss-Webster mice were treated by dermal applica-
tion 3 times/week for 13 weeks. The doses used were from 0.01-50 yg/
application with the test compound dissolved in acetone. There was 100%
mortality in the 25 and 50 yg/appl 1 catIon groups and 80% mortality in the
10 yg/appllcatlon group. On histologic examination, there were signs of
liver damage at the lowest dose tested In both sexes; however, the incidence
and degree of damage were not well correlated to the dose applied.
Chronic Toxicity> In rats and mice the toxic effects of chronic
exposure to 2,3,7,8-TCDD are summarized in Table V-5. These studies were
predominately designed to assess the carcinogenicity of 2,3,7,8-TCDD and the
observations of non-neoplast1c systemic toxicity are, therefore, limited.
Van Miller et al. (1977a,b) fed groups of 10 male Sprague-Dawley rats
diets containing 1, 5, 50, 500, 1000, 5000, 50,000, 500,000 or 1,000,000 ppt
2,3,7,8-TCDD (10~3 yg/kg diet) for 78 weeks In order to determine the
potential toxic and carcinogenic effects of 2,3,7,8-TCDD. The authors
estimated that these dietary levels corresponded to doses of 0.0003, 0.001,
0.01, 0.1, 0.4, 2.0, 24, 240 or 500 yg 2,3,7,8-TCDD/kg bw/week, respec-
tively. All animals that received diets containing >1000 ppt (>0.4 yg/kg
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TABLE V-S
Effects of Chronic
Exposure to 2.3,7,0-
•TC00 In Laboratory Rodents
Species/Strain
Sex/No.
Dose
Treatment Schedule
Duration of
Study
Parameters
Monitored
Effects of Treatment*
Reference
Rat/Sprague-
Dawley
male/10
0.0 ppt
NA
95 weeks
survival
40X survived until 95
weeks, the first death
occurred at week 68
Van Miller
et al., 1977a.b
IMle/10
1 ppt
contlnous In diet
for 7B weeks
95 weeks
survival
BOX survived until 95
weeks, the first death
occurred at week B6
iule/10
5 ppt
continuous In
for IB weeks
dlet
95 weeks
survival
60% survived until 95
weeks, the first death
occurred at week 33
male/10
iO ppt
continuous In
for 70 weeks
diet
95 weeks
survival
60K survived until 95
weeks, the first death
occurred at week 69
male/10
500 ppt
continuous In
for 7B weeks
diet
95 weeks
survival
5OX survived until 95
weeks, the first death
occurred at week 17
male/10
1000 and S000 ppt
continuous In
for 7B weeks
diet
95 weeks
survival
No animals survived until
95 weeks, the first death
occurred at week 31
male/10
50.000. 500,000
and 1,000,000 ppt
continuous In
for 78 weeks
diet
95 weeks
survival
No animals survived until
95 weeks, the first deaths
occurred at weeks 2 and 3
o
CD
S
CD
-------
1ABLE V-5 (cont.)
Species/Strain Sex/No.
Dose
Treatment Schedule
Duration of
Study
Parameters
Monitored
Effects of TreatmflJt*
Reference
Rat/Sprague-
Oawley
H and F/
50 and 50
-2193 ppt
(0.1 i>g/kg/day)
continuous In diet
for 2 years
2 years
H and F/
50 and 50
N and F/
50 and 50
-20B ppt
(0.01 ug/kg/day)
-22 ppt (0.001
i>g/kg/day)
continuous In diet
for 2 years
continuous In diet
for 2 years
2 years
2 years
extensive hlsto-
pathology, hema-
tology, urine
analyses, and
clinical chemistry
extensive hlsto-
pathology, hema-
tology, urine
analyses and
clinical chemistry
extensive htsto-
pathology, urine
analyses and
clinical chemistry
Cumulative mortality.
Increased (f);
Body weight gain,
decreased («.f>;
Red blood cell count,
decreased (H.F);
Packed cell volume,
decreased (H.F);
Hemoglobin,
decreased.(M,F);
Reticulocytes,
Increased (H.F);
White blood cell cotint,
decreased (F);
Serum glutamic pyruvic
transaminase. Increased
G-Glutamyl transferase.
Increased (F);
Alkaline phosphatase,
increased (F);
Urinary coproporphyria
Increased (F);
Urinary uroporphyrin.
Increased (F);
Urinary delta-amlno-
levullnlc add.
Increased hepatic
degeneratIon,
Increased (H.F)
Urinary coproporphyrln.
Increased (F);
Urinary uroprophyrln.
Increased (F);
Hepatic degeneration.
Increased (H.F)
No differences from
values obtained from
control animals
Kodba et al.
1976a, 1979
(F);
-------
TABLE V-5 (cont.)
Species/Strain Sex/No.
Oose
Treatment Schedule
Duration of
Study
Parameters
Monitored
Effects of Treatment*
Reference
Rat/Osborne-
Hendel
H1ce/B6C3F1
Nice/Swiss
H and F/ 0.0 pg/kg/week
75 and 75
N and F/ 0.5 pg/kg/week
50 and 50
H and F/ 0.05 pg/kg/week
50 and 50
N and F/ 0.01 pg/kg/week
50 and 50
H and F/
75 and 75
H and F/
50 and 50
n and F/
50 and 50
H and F/
50 and 50
H/38
H/44
H/44
H/43
0.0 pg/kg/week
0.5 pg/kg/week
-------
bw/weck) were dead by the end of the study (95 weeks). It appeared that
diets containing >1000 ppt (>0.4 yg/kg bw/week) 2,3,7,8-TCDD definitely
increased mortality. The three highest dietary levels (50,000, 500,000 and
1,000,000 ppt) were acutely toxic, causing death between the second and
Fourth weeks of the study, with severe liver necrosis, thymic atrophy and
cessation of growth. Conclusions could not be drawn concerning the effects
of lower doses (lower than 500 ppt) on survival because of the small sample
sires and the high mortality in the control group (6 of 10). In the groups
receiving <5000 ppt, weight gain was significantly depressed only in the
5000 ppt group. The only histopathologic changes reported in these groups
were neoplastic changes (see Carcinogenicity Section).
Koclba et al. (1978a, 1979) maintained male and female Sprague-Dawley
rats (50/sex/dose) on diets containing levels of 2,3,7,8-lCDD that resulted
in doses of 0.0Glt 0.01 or 0.1 yg/kg/day. Increased mortality was
observed in the high-dose females. In the groups receiving 0.01 or 0.1
yg/kg/day, treatment-related changes were observed in hematologic, clini-
cal chemistry and urinary analysis values. Urinary excretion rates of
coproporphyrIn and uroporphyrin were Increased in these groups. Histologic
examination revealed degenerative, necrotic and Inflammatory changes in the
liver. The NOEL In this study was 0.001 yg/kg bw/day.
Toxic hepatitis (lipidosis and hydropic degeneration of the cytoplasm of
the hepatocytes) was observed in.both sexes of Osborne-Mendel rats receiving
2,3,7,8-TCDD by gavage in corn oll:acetone (9:1) (0.25 or 0.025 yg/kg bw),
twice/week for 104 weeks (NTP, 1980a). No other nonneoplastic lesions were
observed, even though extensive histological examinations were performed.
This study demonstrated a N0AEL of 0.05 yg/kg bw/week for hepatitis.
00110 V-57 07/22/87
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In this same study, B6C3F -j mice were treated biweekly for 104 weeks
with 2,3,7,8-TCDD in corn oil:acetone (9:1). Males were given 0.0, 0.01,
0.05 or 0.5 yg/kg bw/week and females were given 0.0, 0.04, 0.2 or 2.0
yg/kg bw/week. Toxic hepatitis was observed in control and treated
groups, but appeared to be significantly elevated only in the high dose
groups (NTP, 1980a).
In another study, Toth et al. (1978, 1979) Intubated male Swiss mice
with 0.0, 0.007, 0.7 or 7.0 yg 2,3,7,8-TCDD/kg bw/week for 1 year.
Amyloidosis of the kidney, spleen and liver, and dermatitis were observed in
all three treatment groups. This suggests a L0AEL of 0.007 yg/kg bw/week
1n mice In this study, but a N0AEL was not established.
Some Information on the toxicity of chronic dermal exposure can be
obtained from the NTP (1980b) dermal carcinogenicity study In Swiss-Webster
mice. Thirty males were treated with 0.01 yg 2,3,7,8-TCDD/appHcation and
30 females were treated with 0.005 yg/appHcation, 3 times/week for 104
weeks. Vehicle-treated and untreated controls were included in the experi-
ment. Although no effects on body weight were observed, treated male mice
had a significant shortening of lifespan, and treated females showed non-
neoplastic hepatic lesions (hepatic cytomegaly and Inflammatory areas).
Target Organ Toxicity
Hepatic Effects. As previously described, when administered lethal
doses of 2,3,7 (8-TC0D, guinea pigs and monkeys have few or no histopatho-
logic changes In the liver, whereas rats and mice have extensive histopatho-
logic effects In this organ. Gupta et al. (1973) and Grieg et al. (1973)
00110
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observed marked distortion of liver architecture in rats given 50 or 100
ug 2,3.7,8-TCDD/kg, with marked necrosis of hepatocytes. The livers of
mice given lethal doses of 2,3,7,8-TCDD also had necrosis (Vos et a 1.,
1974). According to Jones and Grieg (1975), centrilobular necrosis, bile
duct proliferation and lipid accumulation were more severe in mice than in
rats. Livers of mice contained excess amounts of porphyrin (McConnell et
al., 1978a).
Light microscopic, ultras truetural and hlstochemical changes in the
livers of rats given 2,3,7,8-TCDD orally have been described by Fowler et
al. (1973), Jones and Butler (1974) and Jones (1975). Fowler et al. (1973)
administered 0.0f 5.0 or 25.0 yg 2,3,7,8-TCDD/kg bw by gavage to groups of
30 male rats. The rats were sacrificed 1, 3, 6, 9f 16 or 28 days after
treatment. Major ultrastructural changes occurred in the cells near the
bile canaliculi in both treatment groups. A dose-related increase in the
smooth and rough endoplasmic reticulum was observed starting on day 3,
reaching a maximum on days 6-9, and returned to normal by day 28.
Jones and Butler (1974) administered 200 >jg 2,3,7,8-TCDD/kg bw to
groups of 40 male and 40 female Porton rats and sacrificed 4 rats of each
sex/week for 10 weeks. The major lesions observed were necrosis and prolif-
erative changes In the liver. Degenerating cells were observed near the
central vein, progressing to areas of focal necrosis by week 6 and central
vein fibrosis with scattered necrosis by week 10. Additionally, hyperplasia
of the viable cells, with many multinucleated cells, was prevalent by
week 9. Jones (1975) gave 23 male Porton rats a single gavage dose of 200
vg 2t3,7,8-TCDD/kg in arachis oil, and 7 control rats received arachls oil
00110 V-59 07/22/87
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alone. Loss of ATPase activity along the canalicular borders and Increased
activity in the sinusoids in the livers of rats were observed 3 days follow-
ing treatment. The loss of ATPase activity persisted for at least 34 42
days, but returned to normal by 9 months.
Peterson et al. (1979a) investigated the effects of lower doses (10 or
25 yg/kg bw, oral) of 2,3,7,8-TCDD on AlPase activity in hepatocyte plasma
membranes. Liver surface membranes were isolated from male Holtzman rats
using a modified discontinuous sucrose gradient method on days 2, 10, 20 or
40 post-treatment. Both doses resulted in similar depression of Na/K-ATPase
on days 2-40; however, Mgf+-A7Pase was depressed to this extent only in
the high-dose groups. In the low-dose group, Mgff-ATPase activity was
decreased on day 20, but returned to normal levels by day 40 post-treatment.
2,3,7,8-TCDD did not Inhibit ATPase activity j_n vltro. nor did the decrease
in ATPase activity correlate with 2,3,7,8-TCDD Induced food deprivation.
There was, however, a correlation between liver surface membrane activity,
bile flow and biliary excretion of ouabain j_n vivo. In similar experiments,
Hwang (1973) reported that bile flow was stimulated, but Indocyanine green
excretion was inhibited, in male CD rats following treatment with 5 or 25
yg 2,3,7,8-TCDD/kg bw by gavage.
Peterson et al. (1979b) investigated the relationship between AlPase
activity and biliary excretion of ouabain In perfused liver from rats. They
discovered that liver surface membrane ATPase activity and ouabain excretion
could be segregated by cotreatment with the use of spironolactone, chemicals
that increase bile flow, or pregnenolone-!6-a-carbonitr lie, Indicating
that this ATPase activity was not directly Involved 1n ouabain transport.
00110 V-60 * 07/22/87
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Ihe ability of 2,3,7,8-TCDD to inhibit biliary excretion has been demon-
strated to correlate with the species sensitivity to the hepatotoxlc effects
of 2,3,7,8-TCDD (Seefeld et al., 1979, 1980). Yang et al. (1977) reported
that the effect of 2,3,7,8-TCDD on biliary transport is compound dependent.
In these studies, ouabain excretion, a neutral compound, and bile flow were
inhibited, while the excretion of the organic anions phenol-3,6-dibromo-
phthaleln and sulfobromophthaleln were essentially unchanged.
Cunningham and Williams (1972) detected a decrease in j_n vivo lipid
formation in the liver of male Wistar rats receiving 10 pg 2,3,7,8-lCDD/kg
bw, based on [aH]sod1um acetate incorporation. They determined that the
synthesis of triglycerides, dlglycerldes and phospholipids were all
depressed. Despite this Inhibition of lipid synthesis. Albro et al. (1978)
found an Increase in total hepatic lipids In rats 13 days following treat-
ment with 50 2,3,7,8-TCD0/kg bw« Levels of free fatty acids and
cholesterol esters were Increased while levels of phospholipids, free
cholesterol and triglycerides remained constant. When a sublethal dose, 10
vg/kg bw, was administered, triglycerides and fatty acids were Increased
and cholesterol esters were decreased. These changes were explained by
mobilization of body fat, decreased lysosomal acid lipase, and Increased
lipid peroxidation.
Goldstein et al. (1978) determined that 4 weekly doses of 25 yg
2,3,7,8-TCDD/kg bw or higher would result In Increased hepatic porphyrin
levels in male C57B1 mice. Smith et al. (1981) found that strain differ-
ences In sensitivity between C57B1 and DBA/2 mice given a single gavage dose
of 75 ug 2,3,7,8-TCDD/kg correlated with the ability of 2,3,7,8-TCDD to
00110
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induce increases in hepatic porphyrins; however, there was no correlation
between toxicity and increased porphyrins between sexes within strains.
Rats are less sensitive to 2,3,7,8-TCDD Induced porphyria, with increases in
porphyrin levels occurring only after subchronic exposure (>0.01 mg/kg/wcek
by gavage for 6 months) (Cantoni et al., 1981). The major factors involved
in 2,3,7,8-TCDD-lnduced porphyria have been identified as an Increase in
a-aminolevulinic acid synthetase (Goldstein et al,, 1978) and a decrease
in uroporphyrinogen decarboxylase activity (Smith et al.( 1981; Sweeney and
Jones, 1978).
A number of groups investigated the effect of 2,3,7,8-TCDD on hepatic
DNA synthesis. Grieg et al. ( 1974) measured the Incorporation of [^thy-
midine into the hepatic DNA of male and female Porten rats following partial
hepatectomy. No effect of 2,3,7,8-TCDD pretreatment (10 or 200 mg/kg bw)
was noted. In Hi vitro studies, however, Conway and Matsumura ( 1975) and
Dickens et al. (1981) demonstrated Increased [3 H]thymld1ne incorporation
into DNA in liver slices obtained from rats which had been pretreated with 5
vg 2,3,7(8-TCDD/kg bw. 2,3,7,8-TCDD is also a potent inducer of hepatic
microsomal enzymes (see Enzyme Induction In Chapter VII).
Immunological Effects, The consistent observation of thymic atrophy
in early acute toxicity studies suggested that 2,3,7,8-TCDD may alter the
Immune response and prompted investigations of immunotoxld ty, summarized in
Table V-4. The results suggest .that 2,3,7,8-TCDD interferes with the immu-
nologic capability of the animals tested. Vos et al. (1973) Investigated
the effect of orally administered 2,3,7,8-TCDD on the eel 1-mediated and
humoral Immune response of B6D2F^ mice, CD rats and Hartley guinea pigs.
00110 V-62 07/22/87
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Pretreatment with 0.04 2,3,7,8-TCDD/kg bw/week reduced the development
of skin hypersensitivity to Mycobacterium tuberculosis in guinea pigs; no
effect on this parameter was seen in rats. Tetanus antitoxin levels were
reduced in guinea pigs receiving 0.2 yg 2,3,7,8-TCDD/kg bw/week. 2,3,7,8-
TCOD has also been demonstrated to inhibit eel 1-mediated immunity in "graft-
versus-host" experiments utilizing spleen cells From mice which had received
4 weekly oral doses of 0, 0.2, 1.0 or 5.0 yg 2,3,7,8-TCDD/kg bw (Vos et
al., 1973).
Vos and Moore (1974) and Moore and Vos ( 1974) administered 1 or 5 yg
2,3,7,8-TCDD/kg bw to female F-344 rats on days 11 and 18 of gestation and
days 0, 7 and 14 postpartum. This treatment resulted in prolonged times
until graft rejection, decreased spleen cell graft-versus-host activity and
decreased binding response to phytohemogglutinln, but an increased humoral
Immune response to conconavalin A in the pups.
Thlgpen et al. (1975) determined the effect of 2,3,7,8-TCDD on host
resistance to injection by Salmonella bern In male SPF C57B1/&Jfh mice, an
effect largely mediated by thymus-derlved lymphocytes. Pretreatment with >1
yg/kg bw/week by gavage resulted in an increase in mortality and a
decrease in time until infection. The N0AEL was determined to be 0.5
yg/kg bw. Pretreatment with 2,3,7,8-TCDD has also been demonstrated to
enhance the susceptibility of mice to col 1 endotoxin (Vos et al.( 1978a).
Thomas and Hinsdill (1979) maintained female Swiss-Webster mice on diets
containing 1, 2.5, 5, 10 or 20 ppb 2,3,7,8-TCDD (lf 2.5, 5, 10 or 20 yg/kg
diet) from 4 weeks before mating until 3 weeks postpartum. Thymus weight
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was decreased in the pups from groups receiving >2.5 ppb, along with a
decreased number of plaque-forming cells reactive to sheep red blood cells
and an Increased susceptibility to S. typhlmurium. This treatment did not,
however, lower the humoral content of anti-RBC antibodies, affect the
response to E_. col 1 LPS or L monocytogenes, affect nitrogen-induced lympho-
cyte proliferation, or alter the response of B- and T-cells to conconcanva-
11 n A j_n vl tro. Similar results have been reported in F isher/Wistar rats
(Faith and Luster, 1979; Luster et al., 1980).
Hinsdill et al. (1380) found that feeding Swiss-Webster mice diets con-
taining 100 ppb (100 yg/kg diet) 2,3,7,8-TCDD for 5 weeks increased serum
[3-globul ins, but suppressed total serum protein, y-globulin and albumin.
A decreased immune response to tetanus toxoid, sheep red blood cells, S.
typhlmurium and L. monocytogenes and a lowered contact sensitivity to DNFB
were noted at dietary concentrations as low as 10 ppb (10 yg/kg diet).
Young animals were slightly more susceptible to the Immunosuppressive
effects of 2,3,7,8-TCDD than were older animals. Vos and Moore (1974) also
reported that 1-month-old mice are more sensitive to 2,3,7,8-TCOD than are
4-month-old mice.
Mantovanl et al. (1980) administered single l.p. doses of 1, 2, 6 or 30
yg 2,3,7,8-TCDO/kg bw to C57B1/6J mice. This treatment resulted in a
decreased total number of macrophages and splenic natural killer cells, but
did not affect the cytostatic or- cytocldal activity of the remaining cells.
McConnell et al. (1978a) suggested that the decrease in peripheral cell
counts may be the result of the hypocellularlty seen in the bone marrow of
2,3,7,8-TCDD-treated mice.
00110
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Other Organ Systems. One of the characteristic effects of 2,3,7.8-
TCDD is the loss of body weight. Because decreased food consumption does
not totally account for this finding, the potential effects of 2,3,7,8-TCDD
on intestinal absorption were of interest. Madge (1977) investigated the
effect of 2,3,7,8-TCDD on intestinal absorption in CD-I mice using the
everted intestinal sac technique. Treatment of the mice with single gavage
doses of 0, 10, 25, 75, 150, 200 or 300 yg 2,3,7,8-TCDD/kg bw decreased
the ]_n vitro Intestinal absorption of D-glucose, but did not affect absorp-
tion of D-galactose, L-argenine or L-histidine. These measurements were
made 7 days after treatment. In a similar study, Ball and Chhabra (1981)
reported that 2,3,7,8-TCDD reduced the absorption of D-glucose and leucine
in Sprague-Dawley rats.
Manis and Kim (1979a,b) found that 22-84 yg 2,3,7,8-TCDD stimulated
intestinal transport of iron In male Sprague-Dawley rats and in an unidenti-
fied strain oF mice. Gavage administration was more effective than l.p.
injection. This stimulation occurred in the duodenum, but the distal
segment of the Intestine was unaffected. In parallel experiments, calcium
transport was decreased and galactose and proline were unaffected by prior
exposure to 2,3,7,8-TCDD.
Pegg et al. (1976) and Hook et al. (1977) investigated the effect of 10
or 25 yg 2,3,7,8-TCDD on the U\ vitro function of renal cortical slices
obtained from male Sprague-Dawley rats 3 or 7 days following intubation.
Anion transport, as measured by accumulation of p-amlnohlppuric acid, was
decreased by N-methyl-nicotinamide accumulation, at both dose levels. In
the vjvo studies, sodium reabsorptlon was within the normal range and
00110
V-65
07/22/87
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ammonlogenesis and gluconeogenesis were unaffected, even when the rats were
made acldotic. The authors concluded that the observed decrease in kidney
function was a result of the generally poor condition of the treated animals
and not a direct effect of 2,3,7,8-TCDD.
Grieg et al. ( 1974) found that intraperitoneal administration of 10 yg
2,3,7,8-TCDD/kg bw inhibited the DNA synthesis stimulated by folate (250 mg
folic acid/kg) or lead acetate (40 mg Pb+ /kg) in the kidneys of male and
¦female Porton strain rats* This inhibition did not involve direct inter-
action with ONA or the inhibition of protein synthesis, but may Involve the
aromatic oxidases of liver microsome.
Zinkl et al. (1973) observed a number of hematologic clinical chemistry
changes in female CD rats given 30 daily doses of 0.1, 1.0 or 10 yg
2,3,7,8-TCDD/kg bw; however, they concluded that these changes simply
reflected damage to other organs, such as the liver, and hemoconcentration.
The only other major effect observed was thrombocytopenia, which was also
observed in mice and female guinea pigs. Lymphopenia was observed In guinea
pigs that received a single dose of 1, 10 or 50 yg/kg bw.
Poll et al. (1980) Investigated the induction of hyperlIpidemia in male
Sprague-Dawley rats following a single l.p. Injection of 2.5, 5, 10 or 20
yg 2,3,7,8-TCDD/kg bw. There was a dose-related Increase in total plasma
cholesterol and high density lipoprotein cholesterol by 21 days post-
treatment, but triglycerides and low and very low density lipoproteins were
unaffected. In contrast, hyperllpidemia In male Hartley guinea pigs given a
single l.p. Injection of 2 yg/kg bw was characterized by Increases in low
and very low density lipoproteins.
00110 V-66 07/22/87
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Other Effects
Carcinogenicity. 2,3,7,8-TCDD has been tested for carcinogenicity in
rats and mice by administering the compound in the diet and by gavage.
Also, the tumor Incidence In native mice inhabiting an area with heavy
exposure to the herbicide Agent Orange has been assessed and compared with
mice from an uncontaminated habitat. The results of these bloassays are
summarized in Table V-6. Along with studies using the oral route, 2,3,7,8-
TCDD has been tested for tumorlgenlclty by dermal application (Table V-7).
Using the skin two-stage tumorlgenlclty model, 2,3,7,8-TCDD has been tested
for promoting and Initiating activity as well as antlcarclnogenic activity.
Other model systems have been used to a more limited extent In studies of
the effect of 2,3,7,8-TCDD on the carcinogenic potential of chemical
carcinogens.
In a limited study, Van Miller {1977a,b) maintained small groups of male
Sprague-Dawley rats on diets containing 2,3,7,8-TCDD. The animals, in
groups of 10, were fed diets containing 0.0, 0.001, 0.005, 0.05, 0.5, 1.0,
5.0, 50, 500 or 1000 ppb (yg/kg diet) of 2,3,7,8-TCDD for 78 weeks. As
determined from the food consumption of two animals from each group, these
exposure levels corresponded to doses of 0.0, 0.0003, 0.001, 0.01, 0.1, 0.4,
2.0, 24, 240 and 500 >ig/kg/week, respectively. Animals exposed to >50 ppb
died before tumor development could occur. At week 65 of treatment, all
surviving animals were examined by laparotomy, and biopsy samples were
obtained from any gross tumors., Following termination of treatment, the
animals were observed for an additional 17 weeks before and until sacrific-
ing all surviving animals. At necropsy, performed on animals killed when
moribund, found dead, or killed at termination of the study, the animals
were examined for both gross and microscopic lesions.
00110 V-67 07/22/87
to.
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1A0LE V-6
Carcinogenicity Bloassays of 2,3,7,8-TCDD Administration by the Oral Route
Exposure
Route Species/Strain Sex Dose or Exposure
Duration Duration
of of Study
Treatment
Vehlcle
Tumor Type
Tumor
Incidence p Value
Reference
Gavage
rats/
Osborne-Hendel
Gavage
rats/
Osborne-Hendel
Gavage mlce/BbCSF)
H 0.0 pg/kg/ueek 104 weeks
0.01 pg/kg/week 104 weeks
0.0S pg/kg/week 104 weeks
0.5 ug/kg/week 104 weeks
F 0.0 pg/kg/week 104 weeks
0.01 pg/kg/week 104 weeks
0.0S pg/kg/week 104 weeks
0.5 pg/kg/week 104 weeks
M 0.0 |ig/kg/week 104 weeks
0.01 |ig/kg/week 104 weeks
0.0S Mg/kg/week 104 weeks
0.5 pg/kg/week 104 weeks
105 weeks corn oil-
acetone (9:1)
107 weeks corn oll-
acetone (9:1)
107 weeks corn oil-
acetone (9:1)
107 weeks corn oil-
acetone (9:1)
105 weeks corn oil-
acetone (9:1)
107 weeks corn oil-
acetone (9:1)
107 weeks corn olI-
acetone (9:1)
107 weeks corn oil-
acetone (9:1)
105 weeks corn oil-
acetone (9:1)
107 weeks corn oil-
acetone (9:1)
107 weeks corn oil-
acetone (9:1)
107 weeks corn oil-
acetone (9:1)
folllcular-cell adenomas of
the thyroid, carcinoma of
the thyroid
folllcular-cell adenomas of
the thyroid, carcinoma of
the thyroid
folllcular-cell adenomas of
the thyroid, carcinoma of
the thyroid
folllcular-cell adenomas of
the thyroid, carcinoma of
the thyroid
neoplastic nodule of the
liver, hepatocellular
carcinoma of the liver
neoplastic nodule of the
liver, hepatocellular
carcinoma of the liver
neoplastic nodule of the
liver, hepatocellular
carcinoma of the liver
neoplastic nodule of the
liver, hepatocellular
carcinoma of the liver
hepatocellular carcinoma
hepatocellular carcinoma
hepatocellular carcinoma
hepatocellular carcinoma
1/69 *0.006 NTP. 1980a
0/69
5/40 *0.042
0/48
6/50 *0.021
2/50
10/50 <0.001
1/50
5/75 <0.001 NTP. 1980a
0/75
1/49 NS
0/49
3/50 NS
0/50
12/49 *0.006
3/49
0/73 *0.002 MTP, 1980a
9/49 NS
8/49 NS
17/50 -0.002
-------
TABLE V-6 (cont.)
Exposure
Route
Species/Strain
Sex
Dose or Exposure
Duration
of
Treatment
OuratIon
of Study
Vehlcle
Tumor Type
Tumor
IncIdence
p Value
Reference
Gavage
m1ce/B6C3F,
F
0.0 pg/kg/week
104 weeks
105 weeks
corn oil-
acetone (9:1)
hepatocellular carcinoma,
folllcular-cell adenomas
of the thyroid
1/73
0/69
*0.006
t0.016
NTH, 1960a
0.04 pg/kg/week
104 weeks
107 weeks
corn oil-
acetone (9:1)
hepatocellular carcinoma,
folllcular-cell adenomas
of the thyroid
2/50
3/50
NS
NS
0.2 pg/kg/week
104 weeks
107 weeks
corn oil-
acetone (9:1)
hepatocellular carcinoma,
folllcular-cell adenomas
of the thyroid
2/46
1/4T
NS
NS
-
2.0 pg/kg/week
104 weeks
107 weeks
corn oil-
acetone (9:1)
hepatocellular carcinoma,
folllcular-cell adenomas
of the thyroid
6/47
5/46
>0.014
-0.009
Oral
rat/
Sprague-Oawley
H
0.0 ppb
0.001 ppb
78 weeks
78 weeks
95 weeks
95 weeks
in diet
In diet
all tumors3
all tumors'
0/10
0/10
NR
NR
Van Miller
et al.. 1977a,b
0.00S ppb
78 weeks
95 weeks
In diet
all tumors'
5/10
NR
0.05 ppb
78 weeks
95 weeks
In diet
all tumors'
3/10
NR
0.5 ppb
7B weeks
95 weeks
In diet
all tumors'
4/10
NR
1.0 ppb
78 weeks
95 weeks
In diet
all tumors'
4/10
NR
5.0 ppb
78 weeks
95 weeks
In diet
all tumors'
7/10
NR
Oral
rat/
Sprague-Oawley
H
0.0 wg/kg/day
105 weeks
105 weeks
In diet
squamous cell carcinoma of
the hard palate,
squamous cell carcinoma of
the tongue,
adenoma of the adrenal
cor lex
0/85
0/85
0/B5
NS
NS
NS
Koctba et al.,
1978a,b
0.001 ng/kg/day
105 weeks
105 weeks
In diet
squamous cell carcinoma of
the hard palate,
squamous cell carcinoma of
0/50
1/50
NS
NS
the tongue,
adenoma of the adrenal 0/50 NS
cortex
-------
TABLE V b (cont.)
Exposure Duration Duration
Route Species/Strain Sex Dose or Exposure of of Study
Treatment
Vehicle
Tumor Type
Tumor
Incidence p Value
Reference
Oral rat/ M 0.01 ug/kg/day 10S weeks 105 weeks In diet
Sprague-Dawley
0.1 pg/kg/day 10S weeks 105 weeks In diet
Oral
Oral
rat/ f
Sprague-Dawley
rat/
Sprague-Dawley
0.0 ng/kg/day
0.01 |ig/kg/day
F 0.1 |ig/kg/day
105 weeks 105 weeks In diet
0.001 pg/kg/day 105 weeks T05 weeks In diet
105 weeks 105 weeks In diet
105 weeks 105 weeks In diet
squamous cell carcinoma of 0/50 NS Koclba et al.
the hard palate, 197Ba.b
squamous cell carcinoma of 1/50 NS
the tongue,
adenoma of the adrenal 2/50 NS
cortex
squamous cell carcinoma of 4/50 <0.05
the hard palate,
squamous cell carcinoma of 3/50 . <0.05
the tongue,
adenoma of the adrenal 5/50 <0.05
cortex
hepatocellular carcinoma, 0/66 NS Koclba et al.
squamous cell carcinoma of 1978a,b
the tongue, 0/86 NS
squamous cell carcinoma of
the lung 0/86 NS
hepatocellular carcinoma, 0/50 NS
squamous cell carcinoma of
the tongue, 0/50 NS
squamous cell carcinoma of
the lung 0/50 NS
hepatocellular carcinoma, 2/50 NS
squamous cell carcinoma of
the tongue, 1/50 NS
squamous cell carcinoma of
the lung 0/50 NS
hepatocellular carcinoma, 11/49 <0.05 Koclba et al.
squamous cell carcinoma of 1978a,b
the tongue, 4/49 <0.05
squamous cell carcinoma of
the lung 7/49 <0.05
-------
TABLE V-6 (conl.)
Exposure
Route Species/Strain Sex Dose or Exposure
Duration (Juration
of of Study
Treatment
Vehlc le
Tumor Type
Tumor
Incidence p Value
Reference
Gavage mice/
Swlss/H/Rlop
Oral
mice/
Peromyscus
poltonotus
N 0.0 pg/kg/week 365 days
0.007 |tg/kg/week 365 days
0.7 iig/kg/week 365 days
7.0 Mg/kg/week 365 days
M 0.0012 ng/kg/day NA
0.0 |ig/kg/day NA
5BB days
649 days
633 days
424 days
NA
sunflower
ol 1
sunflower
oil
sunflower
oil
sunflower
ol 1
contaminated
soil
liver tumors''
liver tumors'*
liver tumors'*
liver tumors'*
liver
NA contaminated liver
soil
7/36 NS
13/44 NS
21/44 <0.01
13/43- NS
0/15 NS
0/15 NS
Toth et al..
1979
Cockerhara
et al.. 1980
aNo single target organ for cancer was outstanding.
''Includes hepatomas and hepatocellular carcinomas.
NR ¦ Not reported
NS = Not significant
-------
TABLE V-7
Carcinogenicity Bloassays of 2,3,7,8-TCDD Administered by the Dermal Route
Species
Sex
Dose''
Duration
of
Exposure
Target Organ
Tumor Type
Tumor
Incidence
Nice
M
0.01 yg/appl1cat1on
104 weeks
Integumentary
system
fibrosarcoma
6/28
0.0 ^/application
(vehicle control)
104 weeks
Integumentary
system
fibrosarcoma
3/42
0.0 ^/application
(untreated control)
NA
Integumentary
system
fibrosarcoma
0/20
F
0.005 uQ/appllcatlon
104 weeks
Integumentary
system
fibrosarcoma
0/20
0.0 iig/appllcatlon
(vehicle control)
104 weeks
Integumentary
system
fibrosarcoma
2/41
0.0 pg/appllcatlon
(untreated control)
NA
Integumentary
system
fibrosarcoma
1/27
aSource: NTP, 1980b
bThc compound was applied 3 times/week In 100 pi of acetone.
NA = Not applicable
-------
All animals In groups maintained on diets" containing 1-1000 ppb of
2,3,7,8-TCOD were dead by week 90 of treatment with the first deaths In
groups at the 1000 and 1 ppb levels observed at 2 weeks and 31 weeks of
treatment, respectively (Van Miller et al., 1977a,b). Animals exposed to
tf.001-0.5 ppb of 2,3,7,8-TCDO had similar food consumption (20 g/day) and
survival (4/10, 8/10, 6/10, 6/10, 5/10 survived In the 0, 0.001, 0.005, 0.05
and 0.5 ppb treatment groups, respectively) as control animals. All treated
and control animals had extensive senile degenerative changes In the
kidneys. Complete necropsies were done and samples of tissues were taken
for microscopic examination from the control groups and each treatment group.
Special staining methods were used as an aid 1n the diagnosis of
neoplasms. Various benign and malignant tumors were found In each treatment
group. No tumors were observed In the controls (Table V-8).
Statistically significant Increases of squamous cell tumors of the lungs
and neoplastic nodules of the liver were observed In rats Ingesting 5 ppb
TCDD (Tables V-9 and V-10). In addition, two animals In the 5 ppb dose
group and one animal In the 1 ppb dose group had liver cholanglocardnomas,
which are rare 1n Sprague-Dawley rats. These results provide evidence of a
carcinogenic effect.
The observation of no tumors of any kind In the controls 1s unusual for
Sprague-Dawley rats. In addition, the reporting of the study was not exten-
sive. These factors may tend to lessen the reliance which can be placed on
the positive results of this study. However, this study Is suggestive of a
carcinogenic response upon exposure to TC00 1n rats.
00110 V-73 09/18/84
-------
I
TABLE V-8
2,3,7,8-TCOD Intake and Mortality In Male Sprague-Dawley Ratsa
Dose^
Weekly Dose/Rat
Week of
Number
of Rats
(ppb)
{vg/kg bw)
First Oeath
Dead at
95th Week
0.0
68
6/10
(60%)
0.001
0.0003
86
2/10
(20%)
0.005
0.001
33
4/10
(*0%)
0.05
0.01
69
4/10
(40%)
0.5
0.1
17
5/10
(50%)
1
0.4
31
10/10
(100%)
5
2.0
31
10/10
(100%)
aSource:
Adpated
from Van M1 Her et
al., 1977a,b
bRats at
50, 500
and 1000 ppb dose
levels were al1
dead within 4 weeks.
00110
V-74
09/18/84
-------
TABLE V-9
Benign and Malignant Tumors In Rats Ingesting 2,3,7,8-TCD0a
Doseb
t
Benign
Malignant
Number of
Tumors
Number
With
of Rats
Tumors
0
0
0
0
0/10
(0X)C
1 ppt
0
0
0
0/10
(OX)
5 ppt
1
5
fed
5/10
(50X)e
50 ppt
2
1
3f
3/10
(30X)
500 ppt
2
2
49
4/10
(40X)h
1 ppb
0
4
51
4/10
(40X)
5 ppb
B
2
10J
7/10
(70XJ
3Source: Adapted from Van Miller et al.f 1977a,b
bRats at dose levels of 50, 500 and 1000 ppb were all dead within 4 weeks.
C4G male rats used as controls for another study, received at the same
time and kept under Identical conditions, did not have neoplasms when
kl1 led at 18 months,
^1 rat had ear duct carcinoma and lymphocytic leukemia
1 adenocarcinoma (kidney)
1 malignant histiocytoma (retroperitoneal)
1 angiosarcoma (sktn)
1 Leydlg cell adenoma (testis)
e3 rats died with aplastic anemia
f1 fibrosarcoma (muscle)
1 squamous cell tumor (skin)
1 astrocytoma (brain)
91 fibroma (striated muscle)
1 carcinoma (skin)
1 adenocarcinoma (kidney)
1 sclerosing seminoma (testis)
hl rat had a severe liver Infarction
^1 rat cholanglocarclnoma and malignant histiocytomas (retroperitoneal)
1 angiosarcoma (skin)
1 glioblastoma (brain)
1 malignant histiocytoma (retroperitoneal)
Jl rat had squamous cell tumor (lung) and neoplastic nodule (liver)
2 cholanglocardnomas and neoplastic nodules (liver)
3 squamous cell tumors (lung)
1 neoplastic nodule
00110
V-75
09/18/84
-------
I
TABLE V-10
Liver Tumors 1n Rats Ingesting 2,3,7,8-TC00a
Dose (ppb) Neoplastic Cholanglocardnomas Squamous Cell
Nodules Tumors of the Lungs
0 0/10 (OX) 0/10 (OX) 0/10
1 0/10 (OX) 1/10 (10X) 0/10
5 4/10 (40X) 2/10 (20X)b 4/10 (40X)
p=0.043 p=0.043
aSource: Adapted from Van Miller et al., 1977a,b
^The two animals had both neoplastic nodules of the liver and cholanglo-
cardnomas .
00110
V-76
09/18/84
-------
The second study employing oral administration to rats, performed by
Koclba et al. (1978a,b), was more extensive. Groups of 100 Sprague-Dawley
rats (50 males and 50 females) were exposed for 2 years to diets containing
-22, 208 and 2193 ppt of 99% pure 2,3,7,8-TCOD. The dietary levels were
Adjusted according to body weight 1n order to maintain 2,3,7,8-TCDD dose
levels of 0.001, 0.01 or 0.1 yg/kg/day. The control group consisted of
172 rats (86 males and 86 females) maintained on unadulterated diets. Dur-
ing the study, the animals were palpated periodically for gross tumors and
clinical chemistry analyses were performed on the blood and urine. Gross
and histopathologic examinations of major organs were conducted 1n animals
removed from the study before week 105, while extensive histopathologic
examinations of a large number of organs and tissues were conducted on
animals killed at the termination of the study.
Gross signs of 2,3,7,8-TCDD toxidty Included significantly increased
cumulative mortality In the high dose female rats, and lower body weights In
the high dose male and female and medium dose female rats (p values not
given). Hematologic values Including packed cell volume and hemoglobin
concentration were decreased (p<0.05) In both sexes at the 0.1 yg/kg/day
dose, and urinary coproporphyrin and uroporphryln were Increased (p<0.05) in
females receiving 0.01 and 0.1 pg/kg/day. Statistically significant
Increases In tumors at several sites occurred In both male and female rats
1n the high dose group and liver lesions, including hepatocellular neoplas-
tic nodules and lung lesions Including focal alveolar hyperplasia In mid-
dosed females. Tumors of the hard palate, tongue and adrenal cortex were
Increased in high-dosed males; tumors of the liver, tongue and lung were
Increased in high-dosed females. The most sensitive target organ appeared
01330
V-77
09/18/84
-------
to be the liver of female rats with hepatocellular carcinoma Incidences of
0/86, 0/50, 2/50 and 11/49 for the control, low, medium and high dose
groups, respectively. There was no Increase 1n the hepatocellular carcinoma
Incidence In male animals. Other tumors commonly observed In male and
tfemale rats were significantly (p<0.05) decreased In the treated animals
(Table V-ll). There was no Increase In the neoplastic lesions In low-dosed
males and females.
In a study to determine the carcinogenic potential of the herbicide
2,4,5-trlchlorophenoxyethanol (2,4,5-TCPE)f Toth et al. ( 1978, 1979) .tested
both this compound containing the known contaminant 2,3,7,8-TCQD and
2,3,7,8-TCDD alone by gavage 1n Swlss-H/Rlop mice. Groups of 45 male mice
were administered 2,3,7,8-TCDD dissolved 1n sunflower oil at dose levels of
0.007, 0.7 or 7.0 pg/kg bw once a week for 1 year. Other groups consist-
ing of 100 male and 100 female mice were administered 2,4,5-TCPE contami-
nated with measured amounts of 2,3,7,8-TCOD suspended In 0.5% carboxymethyl-
cellulose once a week for 1 year. Control groups administered each vehicle
alone were Included 1n the study for comparison. Following the treatment
period, the animals were observed until moribund or until spontaneous death.
At autopsy, animals were examined for tumors, and organs were obtained for
histopathologic examination.
The only tumors that were observed to occur at a significantly greater
Incidence 1n either the 2,3,7,8-TCDD or the 2,4,5-TCPE/2,3,7,8-TCDD treat-
ment groups compared with the appropriate vehicle control were tumors of the
liver. These tumors consisted of both benign hepatomas and hepatocellular
carcinomas. In the 2,3,7,8-TCDD treated mice, the Incidence of liver tumors
01330
V-78
09/18/84
-------
TABLE V-ll
Tumors That Were Significantly Decreased 1n Rats Following Exposure to 2,3,7,8-TCDDa
Tumor Incidence
Sex Tumor Type
0.0 tig/kg/day 0.1 pg/kg/day 0.01 ^g/kg/day 0.001 pg/kg/day
M
Acinar adenoma of the
pancreas
14/85
2/50b
5/50
7/50
H
Pheochromo'cytoma of
the adrenal
28/85
4/50b
10/50
6/50
H
Subcutaneous fibro-
adenoma/^ Ibroma/ lipoma
10/85
6/50
5/50
l/50b
F
Benign tumors of the
uterus
28/86
7/49b
11/50
12/50
F
Benign tumors of the
mammary gland
73/86
24/49b
36/50
35/50
F
Carcinoma of the
mammary gland
8/86
0/49b
4/50
4/50
F
Pituitary adenoma
43/86
12/49b
13/50
18/50
aSource: Koclba et al., 1978a
bThe tumor Incidence was significantly (p<0.05) different from the Incidence In this control group.
-------
was 7/38, 13/44, 21/44 and 13/43 for groups administered 0.0, 0.007, 0.7 and
7.0 yg/kg bw/week. The incidences of benign and malignant tumors were not
enumerated separately. Only the 0.7 yg/kg/week group had a statistically
significant (p
-------
TABLE V-12
Tumor Incidence In Nice Treated with 2.4.S-TCPE Contaminated with 2,3,7,B-TCDDa
TCPEb
Treatment
Number
Number of
Animals with
Tumors of:
Effective
of Tumor
Group
(mg/kg)
TCDD
Vehicle'
Sex
Number of
BearIng
Liver
Lung
Lymphomas
Other
Average
(wg/kg)
(mg/kg)
Nice
Nice
(X)
Organs
Lifespan
1
67.0
0.112
50
N
8B
69
42d (18)
50
7
16
595
(1.6 ppn)
F
83
7
(8)
52
15
25
652
2
70.0
0.007
50
98
78
57®
(SB)
IB
11
16
571
(0.1 ppm)
F
96
59
9
(9)
39
15
- 23
5B2
3
control
93
63
24
(26)
44
B
17
577
f
84
57
4
|5>
41
23
13
639
4
7.0
. 0.07
50
93
79
25
(27)
38
IB
22
641
(10 ppm)
F
96
60
10
(10)
38
19
19
589
5
7.0
0.0007
50
94
77
23
(24)
50
23
17
660
(0.1 ppm)
F
93
71
8
(9)
42
36
21
590
6
G.7
0.00007
50
97
78
24
(25)
51
20
17
643
(0.1 ppm)
F
94
64
5
(5)
38
22
21
S66
7
__
50
96
74
32
(33)
44
14
22
615
F
84
55
4
(5)
38
18
17
565
8
control
96
78
32
(33)
38
22
15
651
F
91
57
4
(«)
31
24
19
549
9
7.0
10
N
43
27
13
(30)
11
6
7
424
10
0.7
10
.M
44
36
21
(48)
18
12
4
633
11
0.007
10
N
44
39
13
(29)
27
10
6
649
12
--
10
N
38
27
7
(IB)
15
6
7
5BB
aSource: Toth et al., 1979
**TCPE • Trtchlorophenoxy ethanol
cCarboxymethyl cellulose In groups 1-8, sunflower oil In groups 9-12.
dp
-------
In the oral study, (NTP, 1 982a) groups of 50 male and 50 female rats
received 2,3,7,8-TCDD by gavage in corn oil:acetone (9:1) 2 days/week For
104 weeks. The weekly dose of 2,3,7,8-TCDD was 0.01, 0.05 or 0.5 yg/kg
bw. The male mice received identical oral doses of test compound on the
above schedule, while female mice received weekly doses of 0.04, 0.2 and 2.0
yg/kg bw for 104 weeks. In both species an additional 3-week observation
period followed treatment. Control groups for both rats and mice consisted
of 75 untreated animals and 75 vehicle-treated animals of each sex. At
death or termination of the study, gross and histopathologic examinations
were performed on all major organs and gross lesions.
In this chronic study the only overt sign of toxicity in rats was a
slightly lower body weight in the high dose male and female animals after 55
and 45 weeks of treatment, respectively. Survival among control and treated
groups was not significantly different. Histologic examination revealed
that toxic hepatitis was common in the high dosed animals, with the liver
developing lipidosis and hydropic degeneration of the hepatocytes, and pro-
liferation and fibrosis of the peripheral bile ducts. Histologic examina-
tion also revealed Increased Incidences of neoplastic lesions In both liver
and thyroid. The Incidence of hepatic neoplastic nodules or hepatocellular
carcinomas was 0/74, 0/50, 0/50 and 3/50 (all neoplastic nodules) for males,
and 5/75, 1/49, 3/50 and 15/49 for females In the control, low, medium and
high dose groups, respectively. The incidence of hepatic neoplastic nodules
in females was 5/75, 1/49, 3/50 and 12/49, respectively. In both males and
females, the incidence of these combined lesions had a positive dose-related
trend by the Cochran-Armitage test (p=0.0Q5 and p=0.001 for males and
females, respectively); however, the Incidence of these tumors was
01330
V-82
04/08/88
-------
significantly (p=0.001) higher than controls only 1n female rats In the high
dose group. The Incidence of hepatic neoplastic nodules In high-dose
females was statistically significantly elevated (p=0.006) as compared to
controls. In male rats, the Incidence of thyroid follicular cell adenomas
or carcinomas was 1/69, 5/48, 8/50 and 11/50 In the control, low, medium and
high dose groups, respectively, and these tumor Incidences showed a
significant dose-related trend along with the Incidences 1n the two higher
doses being significantly (p=0.021 and 0.001, respectively) Increased over
controls. Incidence of other tumors, Including thyroid folHcular-cell
adenomas, adrenal adenomas or carcinomas and subcutaneous adenomas 1n
females, subcutaneous flbromas and cortical adenomas showed a dose-related
trend or a significant Increase over controls at the low or medium dose
level, but did not meet the requirements for overall significance of 0.5
using the Bonferronl Inequality criteria. The only tumors considered by the
NTP to be related to exposure to 2,3,7,8-TCOO were the thyroid tumors In
male rats and the liver tumors 1n female rats.
As observed In the rats, administration of 2,3,7,8-TCDD by gavage
produced no overt signs of toxicity 1n mice with the only nonneoplastic
histopathologic effect being toxic hepatltls. Neoplastic lesions that
demonstrated both a significant dose-related trend and a greater Incidence
1n the high dose animals Included hepatocellular adenomas or carcinomas In
both male and female mice and thyroid follicular adenomas 1n female mice.
The Incidence of liver tumors was 8/73, 9/49, 8/49 and 17/50 in males and
1/73, 2/50, 2/48 and 6/47 In females, while the Incidence of thyroid tumors
1n females was 0/69, 3/50, 1/47 and 5/46 for the controls, low, medium and
high dose groups, respectively. The Incidence of histiocytic lymphomas and
01330
V-83
09/18/84.
-------
subcutaneous fibromas in female mice and alveolar/bronchiolar adenomas or
carcinomas of the lung in male mice were significant by either the Cochran-
Armitage test or the Fisher exact test but not both, and did not meet the
Bonferroni inequality criteria for overall significance. Under test condi-
tions It was concluded by the NIP (1982a) that 2,3,7,8-TCDD was carcinogenic
to both male and female B6C3F^ mice.
Cockerham.et al. (1980) performed a field study on beach mice, Pero-
myscus pollonotus, that inhabited an area which was heavily treated with the
herbicide 2,4,5-T of which 2,3,7,8-TCDD was a contaminant. Analysis of the
soil in the contaminated area revealed average 2,3,7,8-TCDD levels of 150
ppt at the surface. Measured levels of 2,3,7,8-TCDD in the liver of beach
mice from the contaminated area was determined to be 1300 ppt In males and
960 ppt in females. Detection of 2,3,7,8-TCDD in the liver Indicates that
the compound was absorbed; however, since seeds in the area did not contain
2,3,7,8-TCDD it was believed that the animals Ingested the compound from
contaminated dust while grooming. In the 10 male and 5 female animals cap-
tured in the contaminated area, there were no histopathologic differences,
including neoplastic lesions, observed in the liver as compared with 9 male
and 6 female beach mice captured In a noncontamlnated area. The only
observed difference In the two groups of mice was a statistically signifi-
cant (95% confidence) Increase In liver to body weight ratios. The authors
back-calculated from the 2,3,7,8-TCDD levels of the liver and estimated a
daily 2,3,7,8-TCDD dose of 0.0012 pg/kg bw. It was noted that this expo-
sure was much lower than the exposure used In laboratory studies to produce
tumors.
01330
V-84
04/08/88
kk
-------
2,3,7,8-TCDD (NTP, 1982b) has been tested in mice for tumorigenic poten-
tial by dermal application. This study was conducted under the NTP and the
description of the chemicals used was the same as previously presented in
the discussion of NTP (1982a).
Groups of 30 male and 30 female Swiss-Webster mice were treated with 100
\il of a solution of the test compound in acetone 3 times/week for 104
weeks. Groups of 45 animals were employed as vehicle controls and 2 groups
of 15 animals were used as untreated controls. The concentration of
2,3,7,8-TCDD used resulted In a dose of 0.01 pg/applIcatlon in male and
0.005 yg/applIcation in female mice. Subchronlc toxicity studies used to
define the dose levels for the chronic bloassay Indicated that all the doses
used resulted in some liver damage but no increase 1n mortality. In the
chronic study, animals were killed when moribund at the termination of the
study and examined for gross tumors. Microscopic examinations of all major
organs were also made.
In mice exposed dermally to 2,3,7,8-TCDD (NTP, 1982b), there was no
treatment-related difference In body weight of either sex between exposed
animals and control groups; however, male mice treated with 2,3,7,8-TCDD had
a significant shortening of lifespan (I.e., 10/30, 7/45 and 3/30 survived to
the end of the study In the untreated control, vehicle control and 2,3,7,8-
TCDD-treated groups, respectively). Nontumorigenlc hepatic lesions were
observed 1n treated female mice, but not In treated male mice. The only
tumors that were treatment related were Integumentary system fibrosarcomas,
with tumors developing on or near the site of application. The Incidence of
01330
V-85
04/08/88
-------
these tumors in male mice was 3/42 and 6/28» and 1n female mice the Inci-
dences were 2/41 and 8/27, respectively, for the vehicle control groups and
the treated animals. Only the tumor Incidence In female mice was statisti-
cally (p=0.007) greater than control values; however, life table analyses
1r*d1cated that the time to tumor was shorter 1n both the male and female
treated mice. The Incidence of tumors In untreated and vehicle control
groups was slmllar.
Using the mouse skin two-stage tumoMgenesis model, 2,3,7,8-TCOO has
been tested for 1n1tat1ng and promoting activity. In this model, an Initia-
tor 1s a chemical that 1s applied to the skin for a very limited time under-
a dose schedule which will not result In tumor formation during the'course
of the study. Treatment with the Initiator, however. Is considered to have
caused some Irreversible change 1n the treated cells since subsequent
repeated exposures to a promoting agent (a compound that does not produce
tumors at the concentration tested after chronic exposure) results in the
development of tumors. The results of these studies and similar studies
assessing promotion of hepatocarclnogenesls are summarized In Table V-13.
Also, In additional Investigations, the effect of previous or simultaneous
exposure to 2,3,7,8-TCDD on the tumor yield of known chemical carcinogens
has been studied.
OIGIovannl et al. (1977) tested 2,3,7,8-TCDD, which was 98.6% pure, for
1n1tat1ng activity on the skin of female CD-I mice. Groups of 30 animals
received an Initial single dermal dose of 2,3,7,8-TCOO of 2 yg/mouse
followed by twice weekly promotion with 5 yg of the known promoter 12-o-
tetra-decanocyl-phorbol-13-acetate {TPA) for 32 weeks. The level of
01330
V-86
09/18/84
-------
TABLF V 13
Assessment of the Initiation and Promotion Activity of 2,3,7,8-TCOD In Laboratory Animals
O
u
cj ¦—1— —
o
Number of % or Incidence
Species/Strain Sex An Ima Is/Group Jnltlator/Dose Promoter/Dose Tumor Type of Animals Reference
With Tumors
Hlce/CD-1
f
30
2.3.7,8-TCDD/
2 |ig/mouse
TPA/5 pg/applIcatlon.
2 times/week for 32 weeks
dermal
papilloma
14
DIGIovannl
et al.. 1977
F
30
DHBA/
2.S6 gg/mouse
TPA/5 pg/applIcatIon,
2 times/week for 32 weeks
dermal
papilloma
63
Nice/
Swiss-Webster
H
~5
none
none
dermal
papl1loma
3/42
NTP, 1982b
N
30
none
2,3,7.8-TCDD/O.OOl Mg/
application, 3 times/week
for 104 weeks
dermal
papilloma
6/28
N
30
DHBA/50 pg
(single
applIcatlon)
2,3,7,8-TCOD/O.OOl pg/
application. 3 times/week
for 104 weeks
dermal
papilloma
5/30
Nice/
Swiss-Webster
F
45
none
none
dermal
papilloma
2/41
F
30
none
2,3,7,8-TCDD/O.005 Mg/
application, 3 times/week
for 104 weeks
dermal
papilloma
8/27
F
30
DHBA/50 pg
(single
applIcatlon)
2.3.7.8-TCDD/0.005 Mg/
application, 3 times/week
for 104 weeks
dermal
papilloma
8/29
Hlce/CD-1
F
30
none
2.3.7,8-TCDD/0.1 Mg/
application, 2 times/week
for 30 weeks
dermal
papilloma
o.ox
Berry et al.,
1970, 1979
F
30
DMBA/200 nmol
(single
applIcatIon)
2.3.7.8-TCDD/0.1 vq/
application, 2 times/week
for 30 weeks
dermal
papl 1 Ionia
o.ox
F
30
DHBA/200 nmol
(single
applIcatlon)
TPA/2 pg/applIcatIon,
2 times/week for 30 weeks
dermal
papilloma
92X
CO
-------
TABLE V-13 (cont.)
Number of % or Ipcldence
Species/Strain Sex Antra Is/Group Initiator/Dose Promoter/Dose Tumor Type of Animals Reference
With Tumors
Mlce/HRS/J
(hr/t)
Mlce/HRS/J
(hr/hr)
Rats/
Charles River
20
20
20
20
20
20
20
20
4
4
0MBA/0.? ymol
(single
application)
none
DMBA/0.2 »mo1
(single
application)
DMBA/0.2 pinol
(single
application)
0MBA/0.2 vmo^
(single
application)
none
0MBA/0.2 nmol
(single
application
DMBA/0.2 i>mol
(single
application)
DEN/10 mg/kg
none
none
2,3,7,8-TCDO/blweekly
application of SO ng/
application for B weeks
followed by 20 ng/applt-
catlon for 17 weeks
2.3.7.8-TCDD/blweekly
application of SO ng/
application for B weeks
followed by 20 ng/
application for 17 weeks.
TPA/blweekly application
of 2 iig/mouse
none
2,3,7,8-TCDD/blweekly
application of SO ng/
application for B weeks
followed by 20 ng/appll-
catlon for 17 weeks
2.3,7.B-TCOO/blweekly
application of SO ng/
application for 8 weeks
followed by 20 ng/appll-
catlon for 17 weeks
TPA/b1weekly application
of 2 ^g/mouse
none
2.3,7,8-TCDO/O.14 „g/kg
twice a week for 28 weeks*
dermal papilloma 0/20
dermal papilloma 0/20
dermal papilloma 0/20
dermal papilloma 20/20
dermal papilloma 1/20
dermal papilloma 0/20
dermal papilloma 15/19
dermal papilloma -10%
hepatocellular 0/4
carcinoma
hepatocellular 0/4
carcinoma
Poland et
al.. 1982
Pltot et al
1980
-------
TABLE V-13 (cont.)
Species/Strain
Sex
Number of
Animals/Group
Initiator/Dose
Promoter/Dose
Tumor Type
% or Incidence
of Animals
Ulth Tumors
Reference
Rats/
Charles River
F
5
none
2.3.7.B-TCDD/1.4 pg/kg
twice a week for 20 weeks*
hepatocellular
carcinoma
0/5
Pltot et al.,
1900
f
S
DEN/10 nig/kg
2.3.7.8-TC0D/0.14 wg/kg
twice a week for 28 weeks*
hepatocellular
carcinoma
0/5
f
7
DEN/10 mg/kg
2.3.7.B-TC0D/1.4 „g/kg
twice a week for 2B weeks*
hepatocellular
carcinoma
5/7
*The 2,3,7,8-TCDD was Administered by subcutaneous Injection.
-------
2,3,7,8-lCDD exposure was chosen from the ED^ for induction of aryl
hydrocarbon hydroxylase activity, and this level resulted in the death of
1/3 of the. animals before the end of the study. In the group Initiated with
2,3,7,8- TCDD and promoted with TPAt there was a skin papilloma incidence of
14% with an average of 0.1 papi 1 lomas/mouse. This was in comparison with
animals initiated with dimethylbenzathracene (DMBA), a potent tumor
Initiator, and promoted with TPA where the papilloma Incidence was 63% with
2.2 papillomas/ mouse. The authors concluded that 2,3,7,8-TCDD was a weak
tumor initiator; however, no vehicle control groups or TPA-treated-only
control groups were included In the study for comparison.
The tumor promoting activity of 2,3,7,8-TCDD was Investigated, along
with the complete carcinogenicity in the NTP (1982b) bloassay. In the tumor
promotion study, groups of 30 mice of each sex were Initiated by a single
dermal application of 50 yg of the tumor Initiator (DMBA). A week after
the Initiation dose, the male mice received applications of 0.001 yg 3
times weekly and the female received 0.005 yg of 2,3,7,8-TCDD for 104
weeks. The skin tumor Incidence in both male and female mice exposed to
DMBA prior to 2,3,7,8-TCDD application or the same level of 2,3,7,8-TCDD
alone were nearly identical with respective tumor incidence In males of 5/30
and 6/28, and Incidences 1n females of 8/29 and 8/27. This NTP (1982b)
dermal bloassay found no statistically significant difference in the
incidence of tumors between the groups of animals treated with 2,3,7,8-TCDD
alone and those treated with' DMBA prior to 2,3,7,8-TCDD application.
Furthermore, the incidence of hemanglosarcoma was higher In male mice 1n
2,3,7,8-TCDD treated group than In the animals treated with DMBA prior to
2,3,7,8-TCDD. 30 female CD-I mice were Initiated with a single 200 nmol
application of DMBA followed by twice weekly applications of 0.1 yg of
01330 V-90 * 04/12/88
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2,3,7,8-TCDD for 30 weeks In a bioassay by Berry et al. (1978, 1979). At
the termination of promotion, there were no dermal papillomas In either the
DMBA plus 2,3,7,8-TCDD group or the 2,3,7,8-TCDD group. In an additional
group of mice initiated with DMBA followed by promotion twice a week with 2
yg of the known promoter (TPA) there was, however, a 92% incidence of skin
papillomas. The average number of tumors/mouse was 8.1. In the mouse skin
two-stage tumorigenesis model, 2,3,7,8-TCDD did not demonstrate tumor
promoting activity. Slaga and Nesnow (1985) observed that 2,3,7,8-TCDD
either had no promoting activity or very weak promoting activity in Senear
mice skin.
Poland et al. (1982) described studies which Indicate that genetic
differences in mice affect the tumor promoting capacity of 2,3,7,8-TCDD in
the skin two-stage tumorlgenesis model. Both 2,3,7,8-TCDD and TPA were
compared for tumor promoting activity In DBA-Initiated HRS/J mice that were
either heterozygous (hr/+) or homozygous (hr/hr) for the recessive "hair-
less" trait. Promotion with biweekly applications of 2 yg of TPA for 25
weeks resulted In papilloma Incidences of 100 and 70% in (hr/+) and (hr/hr)
mice, respectively. Promotion of DMBA-lnltlated (hr/f) mice with 2,3,7,8-
TCDD (50 ng/application for 8 weeks followed by 20 ng/application) did not
result in the formation of tumors, while promotion of (hr/hr) mice resulted
in both the same incidence and multiplicity of tumors as observed in TPA-
promoted mice. With either DMBA or N-nitrosoguanidine (MNNG)-initiated
(hr/hr) mice, the effective doser of 2,3,7,8-TCDD was -100-fold less than TPA
on molar basis. Histologic examination of the skin showed that TPA produced
both acute Inflammation and hyperplasia In (hr/+) and (hr/hr) mice, while
2,3,7,8-TCDD produced hyperplasia and hyperkeratosis only In (hr/hr) mice
01330
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with no inflammatory response. The lack of a 2 ,3,7,8-TCDD-induced inflamma-
tory response suggested to the authors that 2,3,7,8-TCDD-promoted skin
papillomas in (hr/hr) mice by a mechanism different from TPA.
Pi tot et al. (1980) investigated the ability of 2,3,7,8-TCDD to act as a
promoter for the hepatocarcinogen diethylnitrosamine (DEN). Female Charles-
River rats were given DEN at a single intragastric dose of 10 mg/kg during
the time of liver regeneration after a 70% partial hepatectomy. Following
this initiation with DEN, rats received twice weekly subcutaneous injections
of either 0.14 or 1.4 yg of 2,3,7,8-TCDD/kg bw For 28 weeks. Additional
groups of partially hepatectomized animals received only the initiation
treatment or the promotion treatment. No tumors were observed in the rats
given DEN or 2,3,7,8-TCDD alone, or 1n the animals exposed to DEN plus the
low dose of 2,3,7,8-TCDD. In the rats exposed to DEN plus the high dose of
2,3,7,8-TCDD, however f the hepatocellular carcinoma incidence was 5/7.
Similar high incidences, 8/10, of hepatocellular carcinomas were observed in
rats exposed to DEN followed by dietary administration of phenobarbltal, a
promoter of liver carcinogenesis. Foci of cells with altered enzyme pat-
terns indicative of preneoplastic lesions were Identified in rats initiated
with DEN and promoted with either dose level of 2,3,7,8-TCDD or phenobarbi-
tol. Although this study Is limited by the small size oF the experimental
groups, the authors concluded that 2,3,7,8-TCDD was a promoter for DEN-
initlated hepatocarcinogenesls In rats.
Abernathy et al. (1985) Investigated the promotion effects of
2,3,7,8-TCDD Vn vitro using C3H/10T1/2 cells Initiated with N-methyl-
N'-n1tro-N-n1trosoguan1d1ne. Maximal enhancement of focus formation
01330
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occurred at 40 pM 2,3,7,8-TCOD, a concentration 10,000-fold lower than the
optimal concentration of 12-o-tetradecanoylphorbo1-13-acetate.
Investigations have also been conducted on the effects of prior or
simultaneous treatment with 2,3,7,8-TCDD on the subsequent development of
skin tumors by chemical carcinogens. When 2,3,7,8-TCDD (0.1 yg) was
administered simultaneously with DMBA (200 nmol) to the backs of CD-I mice
in a single initiation dose, the skin papilloma incidence following promo-
tion with TPA was nearly the same as when DMBA alone was used as the initia-
tor (DiGlovanni et al., 1977). Although simultaneous exposure to 2,3,7,8-
TCDD and DMBA did not appreciably affect tumor yield, Berry et al. ( 1979 )
demonstrated a marked 93% decrease In the incidence of DMBA Initiated tumors
when CD-I mice were pretreated 3 days before DMBA Initiation with 1 yg/
mouse of 2,3,7,8-TCDD. The time of treatment with 2,3,7,8-TCDD In relation
to Initiation was shown to be critical 1n the antitumorIgenlc effect of
2,3,7,8-TCDD (Berry et al., 1979; Dlglovannl et al., 1979b, 1980). Maximum
tumor inhibition of between 86 and 95% occurred when pretreatment was
between 1 and 5 days before initiation. If pretreatment was 10 days before
DMBA initiation, the tumor yield was decreased by 78%, while 2,3,7,8-TCDD
treatment 5 minutes before or 1 day after DMBA Initiation had no effect on
tumor yield. There was some Indication of an Inverse relationship between
the pretreatment (3 days before DMBA Initiation) dose of 2,3,7,8-TCDD and
the Incidence of tumors. Doses of 2,3,7,8-TCDD of 0.0, 0.01, 0.1 and 2
yg/mouse resulted 1n decreases 1n tumor yields, respectively, of 0, 83, 92
and 96% (DiGlovanni et al., 1979b). Also under similar experimental condi-
tions Cohen et al. (1979) observed a 75% decrease 1n the Incidence of skin
tumors In Senear mice pretreated with 1 yg of 2,3,7,8-TCDD 3 days before
Initiation by DMBA. Kourl et al. (1978) reported that l.p. Injection of
01330 V-93 h 04/12/88
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1-100 mg 2,3,7,8-TC0D/kg preceded by MCA but not trioctanoin raised the
carcinogenic index in B6 but not D2 mice. The term "carcinogenic index" was
defined by Kouri et al. (1978) as percentage of tumor incidence 8 months
after treatment divided by the average latency in days multiplied by 100.
DiGiovanni et al. (1980) investigated the antitumorigenic effect of
2,3,7,8-TCDD in CD-I mice with chemical carcinogens other than DMBA. As
observed with DMBA, exposure to 2,3,7,8-TCDD 3 days before initiation with
either benzo(a)pyrene (BaP) or 3-MC resulted in a decrease in tumor yield as
compared with acetone pretreated animals, while pretreatment with 2,3,7,8-
TCDD 5 minutes before or 1 day after initiations was Ineffective in changing
the tumor yield. The maximum decrease in tumor production was 86 and 57%,
respectively, for BaP and 3-MC initiated mice. A different temporal rela-
tionship was observed in the ability of 2,3,7,8-TCDD to inhibit tumor forma-
tion by BaP-d1o1-epoxide as compared with the previously studied polyaroma-
tic hydrocarbons (PAH). When 2,3,7,8-TCDD was applied 3 days or 5 minutes
before, or 1 day after Initiation with BaP-dlol-epoxide, decreases in tumor
yield were 81.5, 49 and 39%, respectively. Examination of PAH metabolism in
the skin of mice treated with 2,3,7,8-TCDD showed a 21-fold Increase in aryl
hydrocarbon hydroxylase (AHH) activity 72 hours after treatment (DiGiovanni
et al., 1980). The j_n vitro metabolism of DMBA by dermal homogenates from
2,3,7,8-TCDD-treated mice indicated both qualitative and quantitative
changes 1n metabolism (I.e., changes In both rates of metabolism and rela-
tive types of metabolites formed) (Cohen et al., 1979 ; DiGiovanni et al.t
1979b; Berry et al.# 1979). The similarity In the time frame of AHH induc-
tion and the antitumorigenic effect of pretreatment with 2,3,7,8-TCDD sug-
gested that the antitumorigenic properties of 2,3,7,8-TCDD resulted from
01330
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2 , 3 ,7,8-1 COD-induced alteration in the metabolism of the initiating chemi-
cal. Although metabolic change was a possible mechanism for the inhibition
of DMBA, 3-MC and BaP Initiation, the ability of 2,3,7,8-TCDD to inhibit
tumor yield when administered 1 day after Initiation with BaP-diol-epoxide
(which does not require metabolic activation) Indicated to D1G1ovanni et al.
(1980) that more than one mechanism may participate in the anticarcinogenic
effect of 2,3,7,8-TCDD.
Mutagenicity. Short-term in vitro test systems have been developed to
assess the biologic, toxic and genotoxic effects of chemicals. These assays
have proven to be useful indicators of potential activity of diverse Indus-
trial chemicals, a broad range of drugs and xenobiotlcs, carcinogens and
crude environmental extracts. The most widely used short-term test system,
the Ames test for bacterial mutagenesis, employs several strains of Salmo-
nella typhlmurlum that are highly susceptible to the effects of mutagenic
chemicals. Despite the obvious utility of the Ames test and related short-
term assays, their predictive capabilities (i.e., the correlation between
bacterial mutagenicity and carcinogenicity) have not been fully assessed
(Bartsch et al., 1982).
Mutagenicity assays In microorganisms have been used to assess the geno-
toxic effects of 2,3,7,8-TCDD; however, the results of most of these assays
have indicated little potential for mutagenic effects (Table V~14).
Hussaln et al. (1972) exposed S. typhlmurlum histldlne-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
01330
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£ table v-m
u
o
The Results of Hutagenlctty Assays for 2,3,7,8-TCDO In Salmonella typhlnmrluii
Strains of Salmonella typhlmurluw
Type of Assay
S-9
TA98
TA1530
TAIS35
TA1537
TA1538
TA1532
TA1950
TA1975
TA1978
G4b
TA100
TA1S31
TA1534
Reference
Spot test
~/-
NT
NT
0
0
0
0
NT
NT
NT
NT
NT
NT
NT
NcCann, 197B
Plate
Incorporation
~/-
NT
NT
0
0
0
0
NT
NT
NT
NT
NT
NT
NT
NcCann, 1978
Plate
Incorporation*
~/-
0
0
0
0
0
0
0
0
0
0
0
NT
NT
Gilbert et al.. 1980
Fluctuation
test
~/-
0
0
0
D
0
0
0
0
0
0
0
NT
NT
61 lbert et al.. 1980
Spot test
-
NT
0
NT
NT
NT
~
NT
NT
NT
0
NT
OR
QK
Seller, 1973
Plate
Incorporation
*
0
NT
0
0
0
NT
NT
NT
NT
NT
0
NT
NT
6e1ger and Neal, 1981
Plate
Incorporation
-
NT
NT
NT
0
NT
NT
NT
NT
NT
NT
NT
NT
NT
Gelger and Neal, 1981
Suspension
assay
-
NT
0
NT
NT
NT
~
NT
NT
NT
NT
NT
NT
NT
Hussaln et al., 1972
Suspension
assay
*/-
0
NT
0
0
NT
, NT
NT
NT
NT
NT
0
NT
NT
Zelger, 1983
•The assay was performed under both aerobic and anaerobic conditions.
NT - Not tested; QR - Questionable response; 0 - Negative response; ~ - Positive response
o
cO
\
CD
N.
CD
-------
and 10 yg/mi. 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 yg/mi, which resulted in
a 0-50% decrease in survival; however, at 2,3,7,8-TCDD levels that resulted
in a 99% decrease in 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, Seiler (1973)
observed a positive mutagenic response in a spot test of 2,3,7,8-TCDD per-
formed in the absence of a metabolic activation system. However, the purity
of the sample studied was not provided. In tester strains G46 and TA1530,
the ratio of revertants/108 cells in the treated plates divided by spon-
taneous revertants/100 cells was <1. In strains TA1531 and TA1534, the
ratio was between 1 and 2, which was considered a "doubtful" mutagenic
response, while in strain TA1532, the ratio was >10. There was no mention
of the 2,3,7,8-TCDD levels tested in this assay. The positive controls,
diethylsulfate, 2-aminopurlne and 2-aminofluorene, produced ratios of 2 to
5, <1 and 5 to 10, respectively, in strain TA1532. In both the study by
Hussain et al. (1972) and the study by Seller (1973), 2,3,7,8-TCDD produced
a positive mutagenic response only in the S. typhlmurium strain 1A1532,
which is sensitive to frameshift mutagens.
Hussain et al. (1972) also performed a mutagenicity test of 2,3,7,8-TCDD
In two other microbial test systems. A positive response was observed in
Escherichia coli Sd-4 as indicated by a reversion to streptomycin independ-
ence. In this assay, cells were treated in suspension for 1 hour with
2,3,7,8-TCDD at 0.5-4 yg/ml. The greatest mutation frequency (256
mutants x 10"e, as compared with the control frequency of 2.2 mutants x
01330
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]Q~a) occurred at a dose level of 2 yg/ml. The absolute number of
colonies/plate was 7 for the control and 46 for the treated plate. The dose
of 2 yg/ms, 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,.
coli K-39 cells was examined. The vehicle control, DMSO, inhibited prophage
induction as compared with the untreated controls, while the most effective
dose level of 2,3,7,8-TCDD (0.5 yg/mi) resulted in an increased prophage
induction as compared with the vehicle control but not as compared with the
untreated controls. Hussain et al. (1972) concluded that 2,3,7,8-TCDD was
capable of causing increases in the reverse mutation rate in E_. coli Sd-4
and that 2,3,7,8-TCDD had a weak ability to induce prophage in E_. coli K-39
cells.
The studies that followed these two early reports of Hussain et al.
(1972) and Seiler (1973) failed to detect mutagenic activity of 2,3,7,8-TCDD
in S. typhlmurium. Wassom et al. (1978) cited a personal communication from
McCann (1978), which reported that 2,3,7,8-TCDD was inactive in both the
spot test and plate incorporation assay with S. typhimurium strains 1Al532»
TA1535, TA1537 and TA1538. Doses and other experimental protocols were not
mentioned except that the tests were performed both with and without metabo-
lic activation. Gilbert et al. (1980) reported that 2,3,7,8-TCDD gave
"substantially negative results" with S. typhlmurium strains TA98, TA100,
TA1530, TA1535, TA1537, TA1538, G46, TA1532, TA1950, TA1975 and TA1978.
Both the standard plate incorporation assay and the bacterial fluctuation
test were used, and both were performed with and without S-9 prepared from
the livers of Aroclor 1254 pretreated rats. In the plate incorporation
assay, the test compound was tested at 1-2000 yg/plate under both aerobic
and anaerobic conditions. Details were not provided for the fluctuation
01330 V-98 04/08/88
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assay. It is difficult to assess possible reasons for the conflicting
results between the earlier studies and these later mutagenicity assays,
since information on experimental conditions was limited in the negative
studies.
In an attempt to resolve the conflicting results and observe a mutagenic
response, Geiger and Neal (1981] tested 2,3,7,8-TCDD in the standard plate
incorporation assay using S-9 prepared From different sources. In order to
maximize the amount of compound tested, 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 yg/
plate to be tested, a dose that was shown to be non-toxic to the cells. The
S-9 used in these assays was prepared from the livers of Aroclor 1254 pre-
treated 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 concen-
trations of 0.2, 2, 5 or 20 yg/plate, and regardless of the source of the
S-9, there was no observed mutagenic response. In further attempts to
duplicate the previous positive results, Geiger and Neal (1981) tested the
same concentrations of 2,3,7,8-TCDD In strain TA1537, a more sensitive
direct descendent of strain TA1532, for mutagenic activity in the absence of
S-9. Again, no Increase In the number of revertants was observed. In
assays either with or without S-9, positive controls had predictable
increases 1n the number of revertant colonies. The authors concluded that
2,3,7,8-TCDD was not active under the conditions of this assay; however,
testing at higher concentrations may elicit a positive response. It was
also noted that many other polychlorlnated aromatic compounds are not muta-
genic in the Ames test, even though there 1s positive evidence of
carcinogenicity.
01330 V-99 04/08/88
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Mutagenic effects of 2,3,7,8-lCDD in yeast were observed by Bronzetti et
al. (1983). Positive results for reversion and gene conversion were
obtained j_n vitro and in the host-mediated assay. -The _i_n vitro experiments
yielded small dose-related increases in trpf convertants and ilv*"
revertants. An SI0 metabolic activation system was required. Exposure of
the yeast to 2,3,7,B-TCDD at the highest level tested (10 pg/mi) 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 pg of
2,3,7,8-TCDD/kg (Bronzetti et al., 1983). After 5, 10, 20 or 30 days, 0.2
ml of a yeast culture (4 x 10® 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 in 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. The positive results described
in this paper suggest that 2,3,7,8-TCDD is mutagenic in yeast, but more
definitive studies are needed before a firm conclusion can be drawn.
Hay (1982) has found that 2,3,7,8-TCDD dissolved in DMS0 transformed
baby hamster kidney cells (BHK) in vitro. The dloxin isomers 2,8-dichloro-
and 1,3,7-trichlorod1benzo-£-d1oxin also transformed BHK cells, but the
response was weak. The unchlorInated dibenzo-£-d1ox1n and the fully chlori-
nated octachlorodlbenzo-g-dloxln were both negative in the BHK assay (i.e.,
there was no cell transformation). More recently, Rogers et al. (1982)
01330
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reported that 2,3,7,3-TCQD induced mutations in the excess thymidine, thio-
guanine and methotrexate selective systems in L5178Y mouse lymphoma cells in
culture.
The National Toxicology Program (NTP) (Zeiger, 1983) provided data on
2.3,7,8-TCDD from four assay systems: the S. typhlmurium (strains TA98,
TA100, TA1535 and TA1537) hlstidine reversion assay, the sex-linked reces-
sive lethal test in Drosophila. and cytogenetic studies (sister chromatid
exchange and chromosome aberrations) in Chinese hamster ovary cells. Nega-
tive results were obtained in all of these assays. These studies cannot be
evaluated, however, because the procedures used to obtain the data were not
described.
iH v^tro reactions of 2,3,7,8-TCDD with bacteriophage QB RNA were eval-
uated by Kondorosi et al. ( 1973). Active RNA was purified from QB phage
followed by Incubation for 1 hour at 37°C with 0.0, 0.2, 2.0 or 4.0
yg/mfi. 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 .alkylating agents methyl, ethyl and isopropyl methane-sulfonate, and
diethyl pyrocarbonate, all of which inactivated QB RNA under the same exper-
imental conditions. 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.
In vivo binding of radiolabeled 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 [1,6-3H]2,3,7,8-TCDD 1,p. at a dose of
01330 V-101 04/08/88
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7.5 pg/kg. This dose corresponded to a tritium level of 0.87 mCi/kg. Ihe
animals were killed 12, 48 and 168 hours after treatment, or 24 hours after
treatment when the animals were pretreated with the enzyme inducers pheno-
barbital or unlabeled 2,3,7,8-TCDD. Following sacrifice, isolation of
macromolecules, and removal of free labeled 2,3,7,8-TCDD, the amount of
label bound to protein, RNA and DNA was determined. The greatest 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 radio-
activity 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 aber-
rations in the bone marrow of male rats were reported in an abstract by
Green and Moreland (1975). In the Initial experiment, no increase In
chromosomal aberration was observed after five daily gavage treatments at a
2,3,7,8-TCDD dose of 10 yg/kg. In the second portion of this study, rats
were exposed by a single Intraperitoneal injection of 2,3,7,8-TCDD at 5, 10
or 15 yg/kg or a single gavage treatment at 20 yg/kg. The animals at
the two highest exposure levels were killed 24 hours post-treatment, while
the remaining animals were killed 29 days post-treatment. Again, no
Increase In chromosomal aberrations was observed, except In the positive
control group exposed to trlethylenemelamlne.
01330
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In a later report, a small but significant increase in chromosomal
aberrations was observed in the bone marrow cells of male and female
Osborne-Mendel rats (Green et al., 1977). Bone marrow cells for cytogenetic
analysis were obtained from Osborne-Mendel rats used in 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 ug/kg for 13 weeks. Because It was not required for the
range-finding study, a control group was not included. Bone marrow cells
were analyzed for abnormalities and cells In mitosis In the animals that
survived to the end of the study (4-8 animals/group). The only significant
Increases in chromosomal aberrations in comparison with the low dose group
were in males at 2 and 4 yg/kg and females at 4 yg/kg. The greatest
incidence observed was 4.65% of the cells with chromosomal breaks 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 toxicity, makes the conclusion from this study that 2,3,7,8-
TCDD produced chromosomal breaks tenuous.
A similar weak response was observed by Loprieno et al. (1982) in male
and female CD-I mice which received an i.p. Injection of 2,3,7,8-TCDD at a
dose of 10 yg/kg. At 96 hours post-treatment, there was a significant
(p<0.01) Increase in bone marrow cells with gaps and chromatid aberrations.
When chromosomal aberrations were analyzed at 24 hours post-treatment, there
was no significant change In the Incidence of cells with aberrant chromo-
somes. The study was continued with a more extensive experiment using
CD-COBS female rats. The rats were treated weekly by gavage (vehicle
01330 V-103 04/08/88
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acetone-corn oil 1:6) at closes of 0, 0.01, 0.10 or 1.00 wg/kg for 45
weeks. Analysis of bone marrow cells for chromosomal aberrations 24 hours
after the last treatment failed to detect any significant increases.
A limited number of initial studies on the mutagenicity of 2,3,7,8-TCDD
in bacteria reported positive results in S. typhimurlum strain TA1532 in the
absence of a mammalian metabolic activation system (Hussain et al., 1972;
Seller, 1973). More recent attempts to repeat these results with strain
TA1532 or related strains have failed (Geiger and Neal, 1981; Nebert et al.,
1976; Gilbert et al., 1980; McCann, 1978). These authors have also reported
no increase in mutation rate when 2,3,7,8-TCDD was tested in the presence of
a mammalian metabolic activation system. In other j_n vitro assays, 2,3,7,8-
TCOD has produced a positive response in reversion to streptomycin independ-
ence in E_. coli Sd-4 cells and questionable positive response with prophage
induction in coli 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 j_n
vitro 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 system. In the E_. col 1 studies, the poor survival of
the cells or the interference of the vehicle solvent, DMS0, with the assay
makes the evaluation of the studies difficult. With the data available, it
is not possible to resolve the conflicting reports on the mutagenic poten-
tial of 2,3,7,8-TCDD.
Overall, the data indicate little potential for the interaction of
2,3,7,8-TCDD with nucleic adds or the ability of 2,3,7,8-TCDD to produce
chromosomal aberrat ions. Kondoros1 et al. (1973) demons trated that 2,3,7,8-
01330
V -104
04/14/88
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TCDD did not react with RNA j_n vitro in the absence of a metabolic activa-
tion system. I_n vi vo studies using radiolabeled 2,3,7,8-TCDD Indicated some
association of non-extractable label with RNA and DNA (Poland and Glover,
1979); however, the level of bound label was very low. Similar marginal
data were available on the clastogenic effect of 2,3,7.8-TCDD. Although two
lH vo studies in rats (Green and Moreland, 1 975; Loprieno et al.t 1982)
failed to demonstrate any treatment-related chromosomal aberration, a second
study by the same authors (Green et al.f 1977 ) using a longer exposure
period reported a small increase in the number of aberrations. A similar
small Increase was observed by Loprieno et al. (1982) following a single
i.p. injection of 2,3,7,8-TCDD in mice. In humans exposed to 2,3,7,8-TCDD
during the manufacture of 2,4,5-TCPE and Bumlnol, Czeizel and Kiraly (1976)
reported an increase In the number of chromosomal aberrations, while no
increase was detected in individuals exposed to 2,3,7,8-TCDD following an
industrial accident in Seveso, Italy (Reggiani, 1980; Mottura et al., 1981).
The studies of the clastogenic effect of 2,3,7,8-TCDD were presented with
little or no experimental detail to assist in evaluating the merits of the
reports. The data available are too limited to Indicate whether 2,3,7,8-
TCDD can interact with nucleic acids or produce chromosomal aberrations.
Conflicting evidence exists on the mutagenic and genotoxlc effects of
2,3,7,8-TCDD, and although there is evidence for the genotoxlc effect of
2,3,7,8-TCDD additional testing will be required to demonstrate this
activity with certainly (Giri, 1986).
The differences among the results reported could be due to several
factors, such as treatment protocols, solubility problems, purity of the
samples tested and the high toxicity of 2,3,7,8-TCDD. This chemical may be
01330
V-105
04/14/88
-------
a weak mutagen, but because it is very toxic, the dose range for detecting a
positive genetic effect may be very narrow. Therefore, additional experi-
mentation 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 in Drosophila.
Other mutagenicity studies involving humans are found in the Mutagenicity
Section of Chapter VI.
Teratogenicity and Reproductive Toxicity, A number of investigators
have studied the role of 2,3,7,8-TCDD contamination in 2,4,5-T-1nduced
teratogenicity (Table V-15). Neubert and Dlllmann (1972) investigated the
teratogenicity of purified 2,3,7,8-TCDD, purified 2,4,5-T and samples of
2,4,5-T containing 0.05+0.02 ppm (0.05 mg/kg sample) or an undetermined
amount of 2,3,7,8-TCDD. The ED^ for the production of cleft palate by
2,3,7,8-TCDD was determined to be 4.6 ug/kg bw.
Lamb et al. (1980) studied the effect of herbicides containing 2,3,7,8-
TCDD on the reproductive behavior of male C57B1/6 mice. 2,3,7,8-TCDD was
mixed with 2,4-D and 2,4,5-T to simulate Agent Orange and added to the diet.
At doses that resulted in hepatic and thymic toxicity, no significant
changes were noted In mating frequency, average fertility, percentage
implantation, number of resorption sites, percentage of fetal malformations,
survival of offspring or neonatal development.
Rats are apparently less sensitive than mice to the teratogenic effects
of 2,3,7,8-TCDD contaminated 2,4,5-T. Only skeletal anomalies (wavy ribs,
fused sternum) Indicative of fetal toxicity have been reported (Khera et
01330 V-106 04/14/88
-------
TABLE V—15
Studies on the Potential Teratogenic.Effects of 2.3.7.0-TCOD Contaminated 2.4.S-T
O-
Specles/
Strain
Vehicle
Form of
2.4.5-T
TCDD
Level
Oally
Oose
Treatment
Days
Observation
Day
Haternal
Response
fetal Response
Reference
Hlce/
NHRI
Rape-seed
oil
acid
<0.02 ppn
(Sample A)
Rape-seed
oil
Rape-seed
oil
Rape-seed
oil
Hlce/ NR
.NMRI
acid
acid
butyl
ester
acid
0.05*0.02
ppm
(Sample B)
NR
(Sample C)
NR
0.05t0.02
ppm
Hlce/ Corn oil: acid
CD-I acetone
(9:1)
8. 15. 30.
45. 60. 90,
and 120 mg/kg
6-15
30. 60 and 6-15
90 mg/kg
IB 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
90 mg/kg
12 and IT
mg/kg
20. 35. 60.
90 and 130
mg/kg
6t.15
6-15
18
IB
6-15
NR
<0.05 ppm 115 mg/kg
10-15
18
No toxic effects
but decreased
maternal weight
No toxic effects
Toxic effects
observed at 90
and 130 mg/kg
No significant
effect on weight
gain or liver-to-
body weight ratios
Significant Increases In
the Incidence of cleft
palates at doses above
30 mg/kg (see text for
additional details).
Significantly decreased
(p<0.005) fetal weight
at all dose levels.
Increases In the Incidence
of cleft palate at 60 and
90 mg/kg; significant decrease
In fetal weight (p<0.005)
at all dose levels
Increase In the Incidence
of cleft palate; significant
(p<0.005) decrease In fetal
weight
Significant decrease in
fetal weight but no effect
on mortality; Increase In
the frequency of cleft
palate similar to that seen
with acid (see text)
Neubert and Dlllmann.
1972
Increases In the percent-
age of resorptions and/or
dead fetuses at 90 and 130
mg/kg; Increases In the
Incidence of cleft palate
and retardation of skeletal
development at 35 mg/kg
and above
No effect on fetal mortal-
ity or fetal weight but
an Increase in the inci-
dence of cleft palate
Roll. 1971 (Taken
from EPA. 1979a)
Courtney. 1977
-------
TABLE V-1S (cont.)
Species/
Form of TCOD
Dally
Treatment
Observation
Maternal
Fetal Respoqse
Reference
Strain
Vehicle
2.4,5-T Level
Dose
Days
Day
Response
dice/
Honey:water
acid 30 ppm
21.5, 46.4
6-14
10
NR
Significant (p<0.01)
Cour tney et al.,
C57B1/6
(1:1) or
and 113 mg/kg
Increases In the Incidence
1970a,b
or Nice/
OHSO
of cleft palate In the high
AKR
dose group and cystic
kidney In both dose groups;
Increased fetal mortality
also observed In the high
dose group
Hlce/
Honey-.water
acid 30 ppm
113 mg/kg
6-15
19
Increase In
Significant (p<0.05)
Courtney et al..
AKR
(1:1)
llver-to-body
Increases In the Incidence
1970a.b
weight ratio
of cleft palate and fetal
mortality
Rats/
Gavage/
acid ¦ 0.S ppn
1. 3. 6. 12
6-15
20
No effect on body
A slight but statistically
Emerson et al.,
Sprague-
hydroxy-
or 24 mg/kg/
weight and no
significant (p<0.05)
1970, 1971 N.B.
Oawley
propyl-
day
observable signs
decrease in Implantations
This appears to
{groups
methyl-
of toxicity
and litter size In lowest
be a full publi-
of 25
cellulose
dose group only; no frank
cation of the
rats)
teratogenic effects based
abstract summary
on a detailed examination
by Thompson
of the control and 24 mg/kg
et al., 1971
dose group; the only effect
noted was an Increase In
the Incidence of 5th par-
i
tially ossified stemebrae
Rats/
Gavage/
add <0.5 mg/kg
25. 50. 100 '
6-15
22
Some maternal mor-
At 100 or 150 mg/kg.
Khera and HcKlnley,
Wlstar
aqueous
or 150 mg/kg/
*
tality and
decreased fetal weight,
1972; Khera et al..
gelatin or
day
decreased body
Increased fetal mortality
1971
corn oil
weight gain at
and an Increase In the
150 mg/kg; no
Incidence of skeletal
signs of toxicity
anomalies; no significant
at 100 mg/kg or
effect at the two lower
below
dose levels
Savage/
butyl <0.5 mg/kg
50 or 150
6-15
22
NR
No significant effect on
Khera and HcKlnley,
aqueous
ester
mg/kg/day
fetal mortality, fetal
1972; Khera
gelatin or
weight, or the Incidence
et al.. 1971
corn oil
of anomalies
-------
CJ TABLE Vf-15 (cont.)
CO
o
Species/ Form of TCOO Dally Treatment Observation Maternal
Strain Vehicle 2,4,5-T Level Oose Days Day Response
Fetal Response
Reference
o
kO
Rats/
Holtxman
Rats/CO
Rats/
Strain
not
speci-
fied
Gavage/
1:1 solu-
tion of
honey and
water
Gavage/
15X sucrose
solution
Gavage/
methocel
add 30 ppm
acid
0.S ppm
acid
0.S ppa
4.6. 10.0.
and 46.4
mg/kg/day
10.0. 21.5.
46.4 and
BO.O mg/kg/
day
50 mg/kg
10-15
20
6-15
20
6-15
NS
NR
Reduced maternal
weight gain at the
2 higher dose
levels (p<0.05)
and Increased
liver-to-body
weight ratio at
the highest dose
level (p<0.05)
No effect on mor-
tality or body
weight gain
Significant (p<0.01)
Increases In fetal mor-
tality at the 2 higher
dose levels; dose-related
Increases In the percentage
of abnormal fetuses per
litter; a high Incidence
of cystic kidneys In
treated groups
Increase In the Incidence
of kidney anomalies but
no Increase In cleft
palate
No significant effect on
fetal mortality or fetal
weight; a significant
(p<0.05) Increase In the
Incidence of delayed
ossification
Courtney et al.,
1970a.b
Courtney and Moore.
1971
Sparschu «t al..
1971b
o
s
CO
qo
Syrian
hamsters/
Heso
crlcetus
euratus
Gavage/
methocel
Gavage/
acetone,
corn oil,
and car-
boxymethyl
eellulose
In ratio
of 1:5.8:
10
acid 0.5 ppm
acid
<0.1-4.5
ppm
100 mg/kg
20. 40. BO
and 100
mg/kg
6-10
6-10
NS
14
Increased mor-
tality and
decreased body
weight gain
NS
Increase In the Incidence
of delayed ossification
and poorly ossified or
malaltgned sternabrae
(p<0.05)
Dose-related Increases In
fetal mortality, gastro-
intestinal hemorrhages,
and fetal abnormalities;
Sparschu et a I.,
1971b
Collins et al., 1971
NS ¦= Not specified; NR = Not reported
-------
a 1. , 1971; Khera and McKinley, 1 972 ). At doses <100 mg/kg bw/day of contam-
inated 2,4,5-T, the only skeletal effect appeared to be delayed ossification
(Emerson et al., 1970, 1971; Sparschu et al., 1971b).
The teratogenicity of purified samples of 2,3,7,8-TCDD is presented in
Table V-16. Courtney and Moore (1971) administered subcutaneous injections
of 1 or 3 yg of purified 2,3,7,8-TCDD/kg bw/day to CD-I, DBA/2J and
C57B1/6J mice on days 6-15 of gestation. This treatment had no effect on
fetal mortality; however, cleft palate and unspecified kidney anomalies were
found at all dose levels in all strains, with C57B1/6J being the most sensi-
tive strain.
Moore et al. (1973) administered oral doses of 2,3,7,8-TCDD (1 or 3
yg/kg/day) to C57B1/6 mice by gavage on days 10-13 of gestation. Cleft
palate and hydronephrosis were observed in both dose groups. These kidney
lesions were apparently reversible, since very few litters (1/14) that were
cross-nursed on control mothers had pups with kidney abnormalities. In
contrast, kidney lesions were found in 4 of 14 control litters that were
nursed on 2,3,7,8-TCDD treated mice.
Neubert and Dlllman (1972) and Neubert et al. (1973) administered daily
oral doses of 0.3, 3.0, 4.5 or 9.0 yg/kg bw to NMRI mice on days 6-15 of
gestation. Extensive resorption (6 of 9 litters completely resorbed) occur-
red in the high-dose group. Cleft palate, but not kidney abnormalities,
were observed at a dose of 3.0 yg/kg bw/day or higher, with 0.3 yg/kg
bw/day being the N0AEL In this study. A single dose of 45 yg/kg bw could
produce cleft palate If given before day 13 of gestation, with maximum
incidences occurring when the dose was given on day 8 or 11.
01330
V-110
04/14/88
-------
TABLE V-16
Studies on the Potential Teratogenic and Reproductive Effects of 2,3,7,B-TC00
-L
Specles/Straln Vehicle4
Compound Daily Oose Treatment Observation Maternal
Days Day Response
fetal Response
Reference
House/C0-1
mouse/0BA/2J
mouse/C57B1/6J
Mouse/C57BI/6
House/CD-I
Mouse/CF-1
Mouse/NMRI
Rat/CD
Rat/Sprague-
Oawley
OHSOb
Acetone:3
corn oil
(1:9)
0NS0b or
corn oil
corn olI1:
acetone
(96:2)
rape-seeda
oil
DHSOb
corn olla/
acetone
2,3.7,8-TCDD 1. 3 ug/kg
2.3.7.B-TCDD 1. 3 ,.g/kg
6-15
17c or IB Increased liver/ cleft palate, kid- Courtney and Moore,
body weight ratio ney anomalies'' 1971
2,3.7,8-TCDD
2.3.7.B-TCD0
10-13 or 1BC
10
2.3.7.B-TCDD 25. 50. 100, 7-16 18e
200. 400 „g/kg
0.001. 0.01,
0.1. 1.0.
3.0 wfl/kg
0.3, 3.0. 4.5.
9.0 i>g/kg
2.3.7.B-TC00 0. 0.5,
2.0 pg/kg
2,3.7,8-TCDD 0. 0.03.
0.125, 0.5,
2.0 and
8.0 |ig/kg
6-15
6-15
6-15,
9 and 10.
or 13 and
14
6-15
18c
IB
20c
20c
none reported
Increased liver/
body weight ratio
none reported
no effect observed
none reported
vaginal hemorrhage
at 2.0 and
B.O tig/kg
cleft palate, kid-
ney anomalies''
cleft palate,
hydronephrotlc
kidneys,
hydrocephalus,
open eyes,
edema,
petechlae
cleft palate,
dilated renal
pelvis
fetocldal at the
high dose, cleft
palate at doses
at or above
3 pg/kg
kidney malforma-
tions at both
dose levels
Intestinal
hemorrhage at
0.125 and
0.5 ng/fcg.
fetal death of
higher doses,
subcutaneous
edema
Moore et al., 1973
Courtney, 1976
Smith et al., 1976
Neubert and Dlllmann,
1972
Courtney and Moore,
1971
Sparschu et al.,
1971a
-------
TABLE V-16 (cont.)
Species/Strain
Vehicle3
Compound
Dally Dose
Treatment
Observation
Maternal
Fetal Response
Reference
-
Days
Day
Response
Rat/Ulstar
corn oil®/
2.3.7.8-TCDD
0.0. 0.12S.
6-15
22
maternal toxicity
Increased fetal
Khera and Ruddlck,
anlsole
0.25. 0.5.
observed at or
death observed at
1973
1. 2. «. 8.
above T ug/kg
or above 1 ug/kg.
16 ug/kg
subcutaneous edema
and hemorrhages In
the 0.25-2.0 iig/kg
groups
Rat/Sprague-
corn oil1/
2.3.7.8-TCOD
0.0. O.T25,
1-3
21
decrease In body
decreased fetal
Gtavlnl et al., 1982a
Dawley
acetone
0.5. 2.0 >ig/kg
weight gain In the
weight In the 0.5
(9:1)
high dose group
and 2 pg/kg group.
*
cystic kidneys and
dilated renal pel-
vis occured in the
2 wg/kg group
Rat/Sprague-
diet
2.3.7.8-TCDD
0.001. 0.01
throughout
postpartu-
low fertility at
low survival at
Hurray et al., 1979
Oawley
and 0.1
gestation
rltlon
0.01 and 0.1 |ig/kg.
0.01 and 0.1 tig/kg.
wg/kg'
decreased body
decreased body
weight at 0.01
weight at 0.01,
and 0.1 wg/kg.
slight dilated
dilated renal
renal pelvis at
pelvis
0.001 i>g/kg
Rabbit/
corn oil'/
2.3.7.8-TCOD
0.0. 0.1.
6-15
28
maternal toxicity
Increases In extra
Glavlnl et al.. 1982b
New Zealand
acetone
0.25, 0.5
at doses of 0.25
ribs and total soft
(9:1)
and i ug/kg
iig/kg and above
tissue anomalies
rHonkey/rhesus
diet
2,3,7,8-rTCOO
8.6 pg/kg/day
7 months
at term
6/8 conceived, nor-
3/8 normal births
Allen et al.. 1979
before
mal serum estradiol
and during
and progesterone
gestation
Monkey/rhesus
diet
2.3,7.8-TC00
55.7 pg/kg/day
7 months
at term
3/8 conceived, de-
1/B normal births
Allen et al.. 1979
before
creased serum estra-
¦
and during
diol and progester-
ges tat Ion
one
Administration was by gavage.
Administration was by subcutaneous Injection.
cflrst day of gestation designated day lero.
^Kidney anomalies were not specifically defined.
ef1rst day of gestation designated day one.
-------
Courtney (1976) compared the effectiveness of oral and subcutaneous
administration in CD-I mice. Subcutaneous administration was found to pro-
duce a greater teratogenic response at a lower dose than did oral
administration.
¦ Smith et al. (1976) determined the minimum effective oral dose (MED) for
producing teratogenic effects in CF-1 mice to be 1 yg 2,3t7t8-TCDD/kg
bw/day. The NOAEL in this study was 0.1 yg/kg bw/day.
Courtney and Moore (1971) determined the teratogenic potential of
subcutaneously injected 2,3,7,8-TCDD (0.5 or 2 yg/kg bw/day) In CD rats.
The compound was administered in dimethylsulfoxide on days 6-15, 9-10 or
13-14 of gestation. The only developmental anomalies observed were kidney
malformations in all dose groups. Six hemorrhagic gastrointestinal tracts
were found; however, this was considered a primary fetotoxic effect and not
a malformation.
Sparschu et al. (1971a) administered 0.03, 0.125, 0.5, 2.0 or 8.0 yg
2,3,7,8-TCDD/kg/day by gavage to Sprague-Dawley rats on days 6-15 of gesta-
tion. A number of dose-related fetotoxic effects were found, including
edema, increased numbers of resorptions, numbers of dead fetuses and Intes-
tinal hemorrhage. No teratogenic effects were reported In the surviving
fetuses.
Khera and Ruddlck (1973) Intubated groups of 7-15 pregnant W1star rats
with 0.125, 0.25, 0.5, 1, 2, 4, 8 or 16 yg 2,3f7t8-TCDD/kg bw/day on days
6-15 of gestation. Severe fetotoxic effects were observed at doses of 1
yg/kg bw/day or higher, with no live fetuses found In the groups exposed
01330 V-113 04/14/88
-------
to 4, 8 or 16 yg/kg bw/day. No anomalies were observed in the group
receiving 0.125 yg/kg bw/day. In the intermediate dose groups, 0.25-2.0
yg/kg bw/day, a number of anomalies, Including ~subcutaneous edema of the
head and neck and hemorrhages in the intestine, brain, and subcutaneous
tissue, were observed. When the dams were allowed to litter and wean the
pups, none of the pups in groups receiving >1 yg/kg bw/day survived until
weaning. Fostering pups from dams exposed to 1 yg/kg bw/day to control
dams did not appreciably increase survival (36/42 died).
Giavini et al. (1982a, 1983) administered 0, 0.125, 0.5 or 2.0 yg
2,3,7,8-TCDO/kg/day by gavage,. to Sprague-Dawley rats on days 1-3 days of
gestation or to female CRCD rats dally for 2 weeks before mating. In the
groups dosed on days 1-3 of gestation, fetal weight was significantly
decreased in the 0.5 and 2.0 yg/kg bw/day groups, but no statistically
significant Increases in malformations were noted. When adult female rats
were treated for 2 weeks before mating, an increased number of cystic kid-
neys and dilated renal pelvis were observed in the pups in the high dose
group. In these studies, 0.125 yg/kg bw/day was the NOEL for both mater-
nal toxicity and adverse effects on the fetus.
The reproductive effects of 2,3,7,8-TCDD were also studied in a 3-gener-
ation study using Sprague-Dawley rats (Murray et al., 1979). Throughout the
study, animals were continuously maintained on diets providing doses of 0,
0.001, 0.01 or 0.1 yg 2,3,7,8-TCDD/kg/day. The parental group (fQ) was
maintained for 90 days on the test diets prior to mating. The f rats
were mated twice, producing the filial generations (f^ and
Selected f,D and rats were mated at ~130 days of age to produce the
ID d
and f-j litters, respectively. In, later generations, the high dose
01330 V-114 04/14/88
-------
group (0.1 yg 2,3,7,8-TCQD/kg/day) was discontinued because few offspring
were produced in this group. At the intermediate dose (0.01 yg/kg/day),
2,3,7,8-TCDD caused lower body weight in exposed rats of both sexes (f ^
and f^)- At the low dose, no toxic effects were discerned.
Fertility was greatly reduced in the f generation exposed to 0.1 yg
2,3,7,8-TCDD/kg/day. At 0.01 yg 2,3,7,8-TCDD/kg/day, fertility was
significantly (P<0.05) reduced in the and f^ rats. Fertility in rats
(of any generation) exposed to 0.001 yg 2,3,7,8-TCDD/kg/day was not
different from that of control rats. Decreases in litter size were noted in
the group exposed to 0.1 yg/kg/day and the f^ and litters
exposed at 0.01 yg/kg/day. Statistically significant decreases in fetal
survival throughout gestation were noted in and f^ litters of the
0.01 yg 2,3,7,8-TCDD/kg/day exposed dams. At 0.001 yg 2,3,7,8-TCDD/kg/
day, a decreased gestational survival was reported for the f2 Utters, but
not for other generations. Decreased neonatal survival was noted among
f 1A and pups exPosed t° 0.01 yg 2,3,7,8-TCDD/kg/day, but not among
f 1B or ^ pups. Postnatal body weights of the f^ and f^ litters at
0.01 yg 2,3,7,8-TCDD/kg/day were significantly depressed. At the low dose
(0.001 yg 2,3,7,8-TCDD/kg/day), necropsy of 21-day-old pups revealed a
statistically significant (P<0.05) Increase In dilated renal pelvis in the
f.j generation. Subsequent generations at this dose level or any at the
Intermediate dose (0.01 yg 2,3,7,8-TCDD/kg/day) did not have a significant
Increase in this abnormality. .Significantly decreased thymus weight and
increased liver weight were reported In the f^ generation, but not in the
f.j 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
01330
V-115
04/14/88
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0.1 yg/kg/day impaired reproduction among rats, and NOAEls were associated
with 0.001 yg 2,3,7,8-TCDD/kg/day.
Nisbet and Paxton (1982) reevaluated the primary data of Murray et al.
(1979) using different statistical methods. From this revaluation it was
concluded that 2,3,7,8-TCDD significantly reduced the gestational index,
decreased fetal weight, and increased liver to body weight ratios and the
incidence of dilated renal pelvis in both lower dose groups. Nisbet and
Paxton (1982) concluded that the dose of 0.001 yg/kg/day was not a N0AEL
In this study. The FIFRA Scientific Advisory Panel has also reviewed the
data from this three generation study and concluded that the effects
observed at the 0.001 yg/kg dose were not consistent enough between the
different generations to consider them treatment-related (U.S. EPA, 1979b).
Although the panel considered the data suggestive of an embryotoxic effect,
they concluded that 0.001 yg/kg represented a NOEL. Subsequently, EPA did
further evaluation of Murray et al. (1979) data and arrived at a conclusion
that 0.001 yg/kg represents a L0AEL (U.S. EPA, 1984a).
It has been demonstated that both genetic susceptibility and concomitant
exposure to other compounds affect the developmental toxicity of
2,3,7,8-TCDD. Poland and Glover (1980) and Oencker and Pratt (1981)
demonstated genetic differences In the susceptibility of mice; only
responsive C57B1/6J mice developed the characteristic cleft palate and
hydroneophrotic kidneys after treatment. This indicates that developmental
toxicity, as many other toxicologic endpolnts of 2,3,7,8-TCDO, segregates
with the Ah locus. Additionally, it was demonstrated that simultaneous
exposure to 2,3,7,8-TCDD and specific polychlorInated b1 phenyls (Blrnbaum et
al., 1985), or the hormones hydrocortisone (Blrnbaum et al., 1986) or
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thyroxine and triiodothyroxine (Lamb et al., 1986) increases the sensitivity
of mice to the developmental effects of 2,3,7,8-TCDD, and an additive effect
was observed by Weber et al. (1985) for simultaneous exposure to
2,3,7,8-TCDD and 2,3,7,8-tetrachlorodibenzofurans (TCDF).
Berry et al. ( 1 976, 1 977) treated Sprague-Dawley rats on day 17 of
gestation with 0.2, 0.5, 2.5 or 6 \iq 2,3,7 ,8-TCDD/kg bw Intraperitoneal 1y.
Maximal induction of fetal hepatic aryl hydrocarbon hydroxylase (AHH) acti-
vity (measured by fluorometric methods) and N-hydroxylation of FAA (measured
by autoradiography) was observed at a dose of 2.5 pg/kg bw. At lower
doses these effects were not observed 1n either the fetus or the dams.
Electron microscopic examination revealed eellular necrosis, increased
glycogen and rough endoplasmic reticulum and swollen mitochondria in the
liver at dose levels that Induced AHH activity. Induction of epoxide hydra-
tase was also observed in the lungs (ratio 2,3,7f8-TCDD/control=2.85),
kidney (ratio 2,3,7,8-TCDD/control=l.06) and skin (ratio 2,3,7,8-TCDD/ con-
trol=2.62)-
Lucler and McDanlel (1979 ) Intubated CD rats with 0 or 3 yg 2,3,7,8-
TCOD/kg on days 5, 10 or 16 of gestation. They measured the activity of
fetal and newborn hepatic microsomal benzo[a]pyrene hydroxylase and p-nltro-
phenol glucuronlde formation on gestation day 21, postnatal day 8 and post-
natal day 21. The BaP hydroxylase activity of controls on gestation day 21,
treated rats on gestation day 21, controls on postnatal day 8, treated rats
on postnatal day 8, controls on postnatal day 21 and treated rats on post-
natal day 21 was 0.003, 0.032 (p<0.01), 0.214, 0.719 (p<0.01), 0.224 and
0.863 (p<0•01) nmol/mln/mg, respectively. The rate of p-nltrophenyl glu-
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curonlde formation at the above intervals was 13.4, 14.4, 28.4, 99.5
(p<0.01), 23.1 and 164.2 (p<0.01} nmol/min/mg, respectively.
Giavini et al. ( 1982b) administered 0.1, 0.25, 0.5 or 1 ug 2,3,7,8-
TCDD/kg bw/day to groups of 10-15 New Zealand rabbits by gavage on days 6-15
of gestation. Maternal toxicity was observed at doses of 0.25, 0.5 or 1.0
vg/kg bw/day. There were Increases in abortions and resorptions at doses
of 0.25 or 0.5 pg/kg bw/day, with no live fetuses found in the 1.0 pg/kg
bw/day dose groups. Extra ribs were found in all dose groups. Hydronephro-
sis was a common finding in all groups, but the increase in treated groups
over control values was not statistically significant. 2,3,7,8-TCDD also
induced fetal liver microsomal enzymes in New Zealand rabbits (Norman et
al., 1978). The BaP hydroxylase activity of the liver microsomal fraction
of adult males and newborn pups was determined following subcutaneous injec-
tion of 30 nmol/kg Into adult animals. Dams received treatment on the 24th
day of gestation. The benzo[a]pyrene hydroxylase activity of newborn con-
trols, adult controls, treated newborns and treated adults was 0.3, 1.8, 1.6
and 3.7 nmol/mg protein, respectively.
Allen et al. (1979) fed adult female rhesus monkeys on diets containing
50 or 500 ppt (50 or 500 ng/kg diet) 2,3,7,8-TCDD for 7 months. The ani-
mals consumed a total dose of 1.8 and 11.7 yg 2,3,7,8-TCDD, respectively.
The animals were bred at 7 months of treatment resulting in pregnancy in 6
of 8 females In the low-dose group and 3 of 8 in the high-dose group. They
were continued on treatment during pregnancy. In both groups, two-thirds of
the pregnancies ended In spontaneous abortions. There were no reported
malformations In the three surviving Infants. All of the controls (one
group of 8 and another of unspecified size) conceived and gave birth to
01330 V-118 > 04/14/88
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normal offpsring. McNulty (1978) reported a dose-related Increase in
spontaneous abortions In rhesus monkeys given oral doses of O.0r 0.2, 1 or 5
yg/kg bw 3 times/week for 3 weeks starting -20 days preconception. The
group sizes (2-4 animals) were too small for adequate statistical analysis.
Summary
There are wide variations in the species sensitivity to the acute toxi-
city of 2, 3,7,8-TCDD. LD^s ran9e ^rom 0.6 yg/kg bw for the male guinea
pig to >5000 pg/kg bw for the male hamster (Schwetz et al., 1973; Olson et
al., 1980b; Henck et al., 1981). The toxic manifestations seem to be the
same whether the compound Is given as a single oral dose or as a limited
number of multiple treatments, with death occurring from 5-45 days post-
treatment. Lethal exposures result in weight loss, often described as
"wasting away," and thymic atrophy. In some species, particularly rats and
mice, extensive liver damage is observed (Gupta et al.# 1973). In general,
no specific cause of death has been identified, although extensive hemor-
rhaging has been implicated in mice (Vos et al., 1974).
In rats and mice, single high doses produce liver necrosis (Jones and
Butler, 1974), while lower doses produce fatty changes and proliferation of
the endoplasmic reticulum (Fowler et al., 1973). Other effects seen in some
species Include induction of microsomal enzymes, degeneration of plasma
membranes with loss of ATPase activity, a decreased ability to excrete some
xenoblotics In the bile, porphyria, altered gastrotlntestlnal absorption of
some nutrients and decreased blood cellularlty.
2,3,7,8-TCDD 1s an Immunotoxln, predominately affecting eel 1-mediated
immunity. Hypersensitivity, adverse effects on the thymus and increased
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sensitivity to antigens have demonstrated the immunotoxic potential of
2,3,7,8-TCDD.
In rats and mice, the liver appears to be the most sensitive organ
following chronic or subchronic exposure. Hepatotoxicity develops following
a long induction period and the changes persist for long periods following
the termination of exposure (King and Roesler, 1974; Goldstein et al.,
1982b).
In the subchronic studies reviewed in this report, the NOAEL of 0.01
yg/kg bw/day (Kociba et al., 1976) and 0.07 ug/kg bw/day (NTP, 1980a)
have been reported for rats. A NOAEL of 0.29 yg/kg bw/day was evident for
female mice and a L0EL of 0.14 yg/kg bw/day for male mice in the NTP
(1980a) study. A NOAEL of 0.001 yg/kg bw/day, a NOAEL of 0.05 yg/kg
bw/dayt and a frank effect level (FEL) of 0.1 yg/kg bw/day have been
reported for rats following chronic exposure (Kociba et al.t 1978a,b, 1979;
NTP, 1980a). Toth et al. ( 1978, 1979) observed toxic effects in mice at
doses as low as 0.007 yg/kg bw/week.
In a preliminary study by Van Miller (1977a,b), 2,3,7,8-TCDD was tested
for carcinogenicity following oral administration to rats. Increases in the
Incidence of total tumors was observed In some groups; however, the group
sizes, -10 animals/group, were too small for an assessment of a treatment-
related response. In a second,, more extensive study by Kociba et al.
(1978a) a positive carcinogenic response was detected. In this study the
estimated intake of 2,3,7,8-TCDD from the diet was 0.0, 0.001, 0.01 and 0.1
01330
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yg/kg/day. In the high dose group, both male,.and female animals had sig-
nificant increases in site specific tumors. The target organs and tumor
types In male animals were squamous cell carcinomas of the tongue and hard
palate, and adenomas of the adrenal cortex; in female animals the target
organs and tumor types were hepatocellular carcinomas, squamous cell car-
cinomas of the tongue and of the lung. The data demonstrate that dietary
exposure to 2,3,7,8-TCDD at levels that produce a dally dose of 0.1 pg/kg
produce Increased tumor incidences in both male and female rats. Though the
increase was not significant, these tumor types were also found in lower
dose groups.
Under the National Toxicology Program, 2,3,7,8-TCOD was tested for
carcinogenicity In rats following administration by gavage (NTP, 1982a).
Both male and female animals were exposed to weekly doses of 0.0, 0.01, 0.05
and 5 pg/kg bw. The only tumors that appeared to be treatment-related
were follicular cell adenomas or carcinomas of the thyroid In male animals,
and neoplastic nodules or hepatocellular carcinomas of the liver In female
animals. The Incidence of these tumors was significantly greater than
control in the high dose groups and the Incidence of both tumors showed a
positive dose-related trend. Under the conditions of this assay, 2,3,7,8-
TCDD was concluded to be carcinogenic In both male and female rats.
Further studies In mice exposed by gavage have provided support for the
carcinogenicity of 2,3,7,8-TCDD. Toth et al. ( 1979 ) exposed male mice to
2,3,7,8-TCDD at doses of 0.0, 0.007, 0.7 and 7.0 yg/kg week 1n a study to
determine whether 2,4,5-TCPE, Its contaminant 2,3,7,8-TCDD, or both were
carcinogens. At the 0.7 yg/kg/week level there was a significantly
Increased incidence of liver tumors. Liver tumors were not significantly
01330
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increased in the high dose group; however, early mortality in this group
from high doses may have precluded observing late developing tumors. Simi-
larly increased incidences of liver tumors were observed in the NTP (1980a)
study in the high dose male mice exposed to 0.5 yg/kg/week and in the high
dose female mice exposed to 2 yg/kg/week of 2,3,7,8-TCDD by gavage.
Female mice also had an increased incidence of fol1icular-cel1 adenomas of
the thyroid. In both studies, 2,3,7,8-TCDD was carcinogenic to mice with
effective doses ranging between 0.5 and 2 yg/kg/day depending on sex and
the individual study.
The mouse skin two-stage tumoMgenlclty model has also been used to test
the carcinogenic potential of 2,3,7,8-TCDD. Following long-term dermal
application 3 times/week of 2,3,7,8-TCDO at levels of 0.01 and 0.005 yg/
application to male and female mice, respectively, there was an increased
Incidence of skin tumors only in female mice (NTP, 1982b). Along with the
Indication that 2t3,7t8-TCDD was a complete carcinogen in this system,
DIGIovanni et al. (1977) reported that 2,3,7,8-TCDD was also a tumor initia-
tor in mouse skin. The ability of 2,3,7,0-TCOD to Initiate, however, has
yet to be confirmed since appropriate vehicle and promotion-only control
groups were not Included. Attempts, to demonstrate tumor promoting activity
with 2,3,7,8-TCDD on mouse skin have produced negative results In some
assays (NTP, 1982b; Berry et al., 1978, 1979; Slaga and Nesnow, 1985);
however, Poland et ,al. (1982) reported that 2,3,7,8-TCDD was a tumor
promoter when tested on the skin, of mice homozygous for the "hairless" trait
but not in mice heterozygous for this recessive trait. P1tot et al. (1980)
also reported that 2,3,7,8-TCDD was a promoter for DEN-lnltiated hepatocar-
clnogenesis in rats following parenteral administration of the compounds.
On mouse skin, 2,3,7,8-TCDD was a complete carcinogen and possibly a tumor
01330
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initiator, while no tumor promoting activity could be attributed to
2,3,7,8-TCDD in the assays. In rat liver Initiated with DEN, 2,3,7,8-TCDD
was a tumor promoter.
In studies of the Interaction of 2,3,7,8-TCDD with other chemical carci-
nogens, Kouri et al. (1978) reported that 2,3,7,8-TCDD was a cocarcinogen
with 3-MC when administered by subcutaneous injection. In the mouse skin
bioassay, initiation with simultaneous administration of 2,3,7,8-TCDD and
DMBA, however, did not affect tumor yield (DiGiovanni et al., 1977). Simi-
larly, no effect was observed when 2,3,7,8-TCDD was administered either
immediately before (5 minutes) or 1 day after DMBA initiation (Berry et al.,
1979; DiGiovanni et al.# 1977, 1979a; Cohen et al., 1979). When treatment
with 2,3,7,8-TCDD occurred 1-10 days before DMBA Initiation, 2,3,7,8-TCDD
demonstrated a potent antlcarclnogenlc action. Although 1-5 days prior
exposure to 2,3,7,8-TCDD inhibited tumor Initiation by BaP, 3-MC and BaP-
dlol-epoxide, the tumor Initiating ability of the latter compound was also
inhibited when 2,3,7,8-TCDD exposure occurred either 5 minutes before or 1
day after Initiation (DiGiovanni et al., 1980). The Increased AHH activity
resulting from 2,3,7,8-TCDD exposure may account for the anticarclnogenic
activity by altering the metabolism of the Initiating compound; however,
DiGiovanni et al. (1980) suggest that the Inhibition of the initiating
activity of BaP-dlol-epoxide 1 day after Initiation Indicates that more than
one mechanism participates In the antlcarclnogenlc activity of 2,3,7,8-TCDD.
Early reports indicated that 2,3,7,8-TCDD was mutagenic in S. typhlmur-
Ium strain TA1532 (Hussaln et al., 1972; Seller, 1973); however, later
attempts to confirm these results have been unsuccessful (Geiger and Neal,
1981; Nebert et al., 1976; Gilbert et al., 1980; McCann, 1978). 2,3,7,8-
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TCOD has been reported to be mutagenic to coli in vitro (Hussain et al.t
1972) and to S. cerevisiae j_n vitro, and in the host-mediated assay
(Bronzetti et al.( 1980). Covalent interactions with nucleic acids are
minimal if they occur at all (Kondorosi et al., 1973; Poland and Glover,
1979). Only marginal effects have been observed on the incidence of chromo-
somal aberrations in vivo (Green and Moreland, 1975; Green et al., 1977).
2,3,7,8-TCDD has been demonstrated to be teratogenic in all strains of
mice tested. The most common malformations observed are cleft palate and
kidney anomalies; however, other malformations have been observed occasion-
ally. With a MED of 1 pg/kg/day, 2,3,7,8-TCDD is the most potent terato-
gen known. At higher doses, 2,3,7,8-TCDD has a marked fetotoxic effect, as
measured by decreased fetal weight and increased fetal toxicity. Hemor-
rhagic GI tract has been associated with 2,3,7,8-TCDD fetal toxicity.
In rats, it has also been consistently observed that 2,3,7,8-TCDD
produced teratogenic and fetotoxic responses in all strains tested. In this
species, the most common fetal anomalies observed were edema, hemorrhage' and
malformation of the kidney with effects observed at doses of >0.01
^g/kg/day. In addition, there Is some evidence that 2,3,7,8-TCDD can
Induce microsomal enzymes in the fetus exposed j_n utero, and this induction
1s accompanied by damage to the fine structure of the liver cell; however,
other reports Indicate that enzyme induction occurs only after birth follow-
ing exposure to 2,3,7,8-TCDD through the mother's milk. As 1n mice, hemor-
rhagic GI tracts have been observed In rat fetuses exposed j_n utero to
2,3,7,8-TCDD.
01330
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Rabbits and monkeys are also susceptible to the fetotoxic effects of
2,3,7,8-TCDD; however, the studies of these species have been too limited to
clearly demonstrate a teratogenic response or define a threshold dose for
fe totox 1 city.
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VI. HEALTH EFFECTS IN HUMANS
Clinical Case Studies
Acute exposure to 2,3,7,8-TCDD results In nausea and vomiting, headache,
and Irritation of the eyes, skin and respiratory tract. The Initial skin
^reaction Is a cutaneous reaction resembling a chemical burn, followed
several days to weeks after by chloracne (Taylor, 1979). Chloracne, the
typical human dermal reaction to 2,3,7,8-TCDD, Is a cutaneous eruption of
comedones, cysts, and In severe cases, pustules. These usually occur on the
face and shoulders as a result of squamous metaplasia of the dermal glands
{Crow, 1978; Passl et al.# 1981). Most of the documented acute exposures to
2,3,7,8-TCDD have been the result of chemical Industry accidents Involving
2,4,5-tr1chlorophenoxyacet1c acid, which 1s contaminated with 2,3,7,8-TCOD.
According to Holmstedt (1980), the first cases of chloracne associated
with exposure to dloxlns occurred following an explosion 1n a chemical plant
producing 2,4,5-T 1n 1949. Zack and Susklnd (1980) recorded nausea, head-
aches, fatigue, muscular aches and pains, and chloracne as the frequent com-
plaints among the 228 workers exposed. Chemical tests revealed elevated
lipid levels and prolonged prothrombin times. Residual chloracne, pe-
ripheral neuropathy, fatigue and severe aches and pains persisted for up to
2 years.
Holmstedt (1980) and May (1973) reviewed reports of three other Indus-
trial explosions: a BASF factory In Ludwlgshafen, Germany 1n 1953; the
Coalite and Chemicals plant In England 1n 1968; and a 2,4,5-T producing
factory In Amsterdam In 1963. Severe chloracne was the most common symptom
among the exposed workers. Nervous system and unspecified Internal organ
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damage were reported In the German workers. Clinical examinations were per-
formed on 14 of the -90 workers In the Coalite and Chemicals plant at the
time of the explosion. Eleven of the 14 had altered liver function, altered
hematological parameters or glucosurla.
fc
An accident at the ICHESA plant at Seveso, Italy, 1n 1976, resulted In
the exposure of at least 8655 workers and nearby residents when a reactor
vessel used 1n manufacturing 2,4,5-T exploded (Garattlnl, 1982). From this
population, there were a total of 447 reported cases of chloracne, along
with complaints of nausea, vomiting, headache* diarrhea, hyperhldrosls and
Irritation of the eyes (Taylor, 1979; GlanotM, 1977; Crow, 1981). Serious
cases of chloracne occurred 1n children within several weeks of the exposure.
The Lombardy Regional Authority has compiled extensive data regarding
the health effects of 2,3,7,8-TCOO on children and adults at Seveso
(Pocchlarl et al., 1979; BoeM, 1978; Chlapplno et al., 1978; Slrchla,
1978). Reduced peripheral nerve conduction velocities occurred In both
adults and children, with a correlation between the Incidence and the dis-
tance from the plant. Total serum complement activity, lymphocyte blasto-
genlc response and peripheral blood lymphocytes were elevated In children
exposed to the accident (Tognonl and Bonaccorsl, 1982). The limited number
of studies regarding the Immunological effects of 2,3,7,8-TCDD in adults
have not revealed any reduction In Imrminocapablllty (Regglanl, 1980; May,
1982).
Caramaschl et al. (1981) reported an Increase In the frequency of head-
aches, eye Irritation, gastrointestinal tract symptoms and abnormal t-GT»
01340
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serum GPT and aminolevulinic acid levels In children, living In the Seveso
area, who developed chloracne. Increased urinary glucarlc acid levels,
Indicative of Increased microsomal enzyme activity, were found In children 3
years after the accident (Ideo et al., 1982).
*
Six children dermally exposed to contaminated soil (30 ppm, 30 mg/kg
soil) In horse arenas In Eastern Missouri developed headaches, skin lesions
and polyarthralgla (Klmbrough et al., 1977 ). In the most severe case,
eplstaxls and lethargy were reported.
Numbness of the extremities, skin rashes and Irritation, liver dysfunc-
tion, weakness, loss of sex drive and psychological changes have been asso-
ciated with exposure to 2,3,7,8-TCDD and other dloxlns, which occur as.con-
taminants In Agent Orange, 1n veterans and residents of Vietnam. The rela-
tionship between exposure to 2,3,7,8-TCDD and the development of these symp-
toms 1s, as yet, unknown (Holden, 1979; Bogen, 1979).
Stevens (1981) estimated the cumulative minimum toxic dose of 2,3,7,8-
TCDO In man to be 0.1 yg/kg, based on analogy to 2,3,7,8-tetrachlorod1-
benzo-£-furan. Given the conditions prevailing 1n Vietnam, Stevens esti-
mated that 5 years of exposure to Agent Orange would be required to reach
this dose level.
Cytogenetic studies of lymphocytes from Individuals exposed to 2,3,7,8-
TCDO, usually by exposure to contaminated 2,4,5-T, have been performed. In
two of the studies Involving exposure during the Seveso Incident, no
Increase In the Incidence of chromosomal aberrations was observed as com-
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pared with nonexposed controls (Regglanl, I960; Hottura et ah, 1981). In a
third such study, changes suggestive of a mutation In functional nucleolar
organizing regions of lymphocyte chromosomes were observed (OILernla et ah,
1982), but this assay has yet to be validated. Workers exposed to the
i
herbicides 2,4,5-trlchWophenoxy-ethanol (2,4,5-TCPE) and Bumlnol, contami-
nated with 2,3,7,8-TCDD, were found to have an Increased Incidence (p<0.001)
of chromatld-type and unstable chromosomal aberrations In peripheral lympho-
cytes (Czelzel and Klraly, 1976). No Increased Incidences of chromosomal
aberrations were observed 1n soldiers exposed 10 years previously to Agent
Orange, as compared with unexposed subjects (Mulcahy, 1980). With the
exception of the Hulcahy (1980) study, the studies described In this para-
graph were performed within a few weeks of exposure. All studies Involved
exposure to other chemicals and exposure levels were not characterized.
Thirty-five cases of various types of cancer were reported In 570
Vietnam veterans. The cancers (Including 1 lung, 3 kidney and 2-3 In
testes) were attributed to 2,3,7,8-TCDD exposure during "Agent Orange" (a
mixture of 2,4-D, 2,4,5-T and 30-50 ppm 2,3,7,8-TCDD) sprayings In Vietnam
(Holden, 1979). These data are of little value because the 570 veterans
were not selected at random; Instead, they were selected because they com-
plained of symptoms related to Agent Orange exposure. According to the
study, there was no reference group with which to compare these statistics.
A few occurrences of soft tissue sarcoma have been reported among chemi-
cal Industry workers 1n the United States who were exposed to varying levels
of 2,4,5-T, 2,4,5-TP, chlorophenols and 2,3,7,8-TCDD contaminants (Cook et
ah, 1980; Moses and Sellkoff, 1981). Honchar and Halperln (1981) reported
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that 3 of 105 deaths among phenoxy acid workers reported by two chemical
companies were from soft tissue sarcoma. This represents a 2.9% mortality
rate due to this cancer while only 0.07% of the deaths among males In the
United States were due to soft tissue sarcoma. Cook (1981) reported a
I
fourth case of malignant fibrous histiocytoma 1n a phenoxy acid chemical
worker. For a similar Industrial setting, except that 2,3,7,8-TCDD levels
were <1 ppm, Ott et al. (1900) reported no Increase in the cases of cancer
among 204 chemical workers. This study reported on 22 deaths among the
workers, however, and >75% of the men worked for <12 months In a Job Involv-
ing some 2,3,7,8-TCDD exposure.
Epidemiological Studies
Poland et al. (1971) reported the results of a health survey conducted
among 73 workers who had been exposed to 2,4,5-T, d1- and trlchlorophenols,
dloxln contaminants and 2,4-0. Thirteen of the workers developed moderate
to severe cases of chloracne. Other complaints Included minimal active
acne, eye Irritation, hyperplgmentatlon, hirsutism and gastrointestinal
symptoms. These effects could not be attributed to exposure to any specific
compound. In a study by Walker and Martin (1979), chloracne and elevated
y-GT, triglyceride and cholesterol levels have been associated with occu-
pational exposure to 2,3,7,8-TCDD. Other studies have reported a correla-
tion between the extent of occupational exposure to 2,3,7,8-TCOD and the
development of chloracne (Ott et al., 1980; Cook et al.t 1980). Neuropath-
ies have been reported in workers Involved In the production of 2,4,5-sodlum
trlchlorophenoxyacetate and trlchlorophenoxyacetate butyl ester (Pazderova-
Vejlupkova et al., 1981) and phenoxy acid herbicide (Singer et al.f 1982).
01ox1n contaminants were suspected to be the causative agents in these cases.
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A positive association between 2.4,5-T exposures and Increases 1n birth
defects or abortions has been reported In human populations 1n Oregon [U.S.
EPA, 1979a), New Zealand (Hanlfy et al., 1981) and Australia (Field and
Kerr, 1979). A lack of any such association has been reported In human
*
¦populations 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 correla-
tion studies, and because of the uncertainties Inherent 1n this type of
epidemiologic Investigation, as well as the difficulties In distinguishing
the effects of 2,4,5-T from those of 2,3,7,8-TCDD contamination, none of the
reportedly positive associations unequivocally Identify either 2,4,5-T or
2,3,7,8-TCDO as the causative agent. Similarly, the reportedly negative
associations do not rule out 2,4,5-T or 2,3,7,8-TCDO as potential teratogens
or abortlfaclents In humans.
Based on a report of a high Incidence of abortions 1n a small group of
women living around Alsea, Oregon, who may have been exposed to the herbi-
cide 2,4,5-T from aerial spraying (Smith, 1979), the U.S. EPA (1979a) Ini-
tiated 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 if such seasonal variations were associated with 2,4,5-T
spray application.
Spontaneous Abortion Rate Index, as defined by the U.S. EPA, 1s "basic-
ally the ratio of the number of hospitalized spontaneous abortions to the
number of births corresponding to the spontaneous abortions, based on the
01340
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residence zip code of the women contributing to each event." Upon comple-
tion of the study, the U.S. EPA concluded that (1) the 1972-1977 Spontaneous
Abortion Rate [ndex for the study area was significantly higher than in the
Rural Control Area or the Urban area; (2) there was a statistically s1gn1f1-
I
cdnt seasonal cycle In the abortion Index In each of the areas with a period
of ~4 months. In particular there was an outstanding peak In the study area
In 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 U.S. EPA concluded how-
ever, "This analysis Is a correlational analysis, and correlation does not
necessarily mean causation."
H1lby et al. (1980), citing three critiques of the Alsea II study that
were not published In 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 1n 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 report
01340
VI - 7
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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,
t
In the only other report encountered on a population 1n the United
States, Nelson et al. (1979) noted a general Increase In the reported Inci-
dence 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 correlations
were noted, and the general Increase with time 1n the Incidence of facial
clefts was attributed to better reporting procedures; however, there does
not have to be a direct correspondence of malformations 1n human beings and
experimental animals.
Of the four reports available from New Zealand (Dept. of Health, New
Zealand, 1900; McQueen et al.f 1977 ; Hanlfy et al., 1981; Smith et al.,
1982a), the report by the Department of Health Is essentially anecdotal,
Involving two women who gave birth to malformed children (one with an atrial
septal defect and a malformation of the tricuspid valve of the heart and the
other with biliary atresia). In both cases, exposure to 2,4,5-T could not
be ruled out. Based on an analysis of spraying records, the time course of
the pregnancies and plant damage near the women's homes, however, the
Department of Health, New Zealand (1980) concluded that there was Insuffl-
01340
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08/10/84
-------
dent evidence to Implicate 2,4,5-T spraying as a causative factor. Even If
the spraying had been Implicated, a lack of Information on 2,3,7,8-TCDG
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.
The study by McQueen et al. ( 1977) 1 s not published 1n the open litera-
ture but 1s summarized by Kllby et al. (1900). 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 In human birth defects."1 No additional
detalIs are provided.
Hanlfy et al. (1981) performed an epidemiologic study In Northland, New
Zealand, 1n areas where spraying of 2,4,5-T was conducted by various com-
panies for a number of years. The rate of birth defects was obtained from
an examination of hospital records 1n 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 nonexposed population
since this occurred before 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
01340
VI-9
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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,
t
however, no statistically significant association where 1.0 was used as the
fractional removal rate.
Smith et al. (1982a) Investigated the outcome of pregnancy In families
of professional 2,4,5-T applicators and agricultural contractors in 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 03% of the agricultural contractors
responding to questions asking whether they used 2,4f5-T and Its temporal
relationship to reproductive histories regarding birth, miscarriages, still-
births and congenital defects. The relative risks of congenital defects and
miscarriages were 1.19 (0.58-2.45% confidence limits) and 0.89 (0.61-1.30%
confidence limits) for the wives of chemical sprayers as compared with the
wives of agricultural contractors. These data Indicate that exposure of
fathers and mothers (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 1s taken from
01340
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08/12/84
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M1lby et al. (1980): The report concluded that birth defects 1n a group of
babies born 1n the [Yarram] district 1n 1974 and 1976 could not be attrib-
uted to exposure to 2,4,5-T or 2,4-0." . Additional details that might be
useful 1n assessing the rationale for this statement are not provided In the
summary. The report by Field and Kerr (1979 ) plotted the Incidence of
neural-tube defects (anencephaly and meningomyelocele) In New South Wales,
Australia, over the years 1965-1975, and the previous years usage of 2,4,5-T
In all of Australia. The authors noted a decrease 1n the incidence of
neural-tube defects expected on the basis of the plotted line 1n 1975 and
1976, when Australia Instituted monitoring of 2,4,5-T to ensure a 2,3,7,8-
TC00 level <0.1 ppm. The data were not tested for significance, although
Field and Kerr (1979) indicate that they consider the epidemiological data
on neural-tube defects to be "relatively complete"; however, they do not
comment on the Increasing Incidence of neural-tube defects with time and
whether or not an Increase 1n the thoroughness of reporting neural-tube
defects could have contributed to the apparent correlation of 2,4,5-T
exposure with these defects, A visual replottlng of the data suggests that
the Incidence of cleft palate correlates better with 2,4,5-T usage than with
time. Nonetheless, the appropriateness of correlating. 2,4,5-T usage 1n all
of Australia with the Incidence of defects In one area of Australia 1s
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 Up, cleft palate, spina
bifida, anencephalus and cystic kidney disease In all of Hungary betweer
1976 and 1980 with 2,4,5-T use 1n 1975 in all of Hungary. Because Hungary
requires compulsory notification of malformations diagnosed from birth to
01340 VI-11 08/10/84
-------
age 1 year, because a relatively large percentage (55%) of the Hungarian
population lives 1n rural areas where 2,4,5-T exposure may be expected to be
greatest, and because annual use of 2,4,5-T In Hungary had risen from 46,000
ktji 1n 1969 to 1 ,200,000 kg 1n 1975, Thomas ( 1980) considered Hungary to be
"...probably the best country In 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-TCDO exposure, 2,3,7,8-TCOD Is also an Inadvertent contaminant of
2,4,5-trlchlorophenol (TCP). Chronic exposure to 2,3,7,8-TCDD may occur
during the manufacture of TCP and high level acute exposure to 2,3,7,8-TCOO
has occurred after an accident In July 1976, at the ICHESA TCP chemical
factory In Seveso, Italy (Bonaccorsl et al.t 1978). In this accident, the
reaction used to produce TCP became uncontrolled, producing conditions
favorable for 2,3,7,8-TCOO formation before venting the contents of the
chemical reactor Into the atmosphere. The resulting cloud of chemicals
settled over a heavily populated area. Although the amount of 2,3,7,8-TCOO
released was not known, the reported cases of chloracne, a symptom of acute
exposure to 2,3,7,8-TCOO, Indicated that exposure to 2,3,7,8-TCOO had
occurred. Some preliminary results are available from epidemiologic studies
of reproductive events 1n the Inhabitants of Seveso, and recently a study
has become available on the reproductive history of men employed In the
chemical manufacturing Industry with possible chronic exposure to 2,3,7,8-
TCOO (Townsend et al., 1982).
01340
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Epidemiologic 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 Karch 1976 and January 1978 have
fc
been reported for Inhabitants 1n the area around Seveso by Bonaccorsl et al.
(1978), Regglani (1980) and Bisantl 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 in the spontaneous
abortion rate. The incidence rates of malformations were also examined;
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-TCOD exposure In 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 before the accident as a result of
suspected under-reporting. The inadequate reporting even after the accident
was 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-TCDO-induced birth defects. In a recent review of the
progress of epidemiologic investigations of the Seveso accident, Tognoni and
Bonaccorsl (1982) Indicated that the data on spontaneous abortions and mal-
formation rates still needed verification and that these data were too pre-
liminary to allow for conclusions.
01340
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Townsend et al. (1982) Investigated the reproductive history of wives of
employees potentially exposed to 2,3,7,8-TCDO 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
fc
potential 2,3,7,8-TCDD exposure. Exposure estimates of low, moderate and
high were made by an Industrial hyglenlst primarily from Job description and
surface contamination data; however, the high potential exposure group was
reserved for process workers during 1963-1964 when changes In operations
resulted 1n 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,
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 In
the exposed category and 1785 conceptions 1n the control category (concep-
tion that occurred In the exposed group before availability of work records
Indicating potential exposure to 2,3,7,8-TCDD were placed In the control
group), there was no statistically significant Increase 1n spontaneous
abortions, stillbirths, Infant deaths or selected congenital malformations.
Sample sizes were too small to provide meaningful data 1f the populations
were subdivided by extent of exposure. It was suggested that many
confounding factors could account for these negative results, such as the
Inappropriate selection of ' the populations, unidentified covarlables and
Insufficient power; however, the authors maintained that these results were
consistent with animal data, which report that paternal exposure to
2,3,7,8-TCDO does not affect the conceptus.
01340 VI-14 08/10/84
-------
Poole (1983), 1n testimony before the House Committee on Science and
Technology, described a reanalysls of the primary data used by Townsend et
al. (1982). In this reanalysls, the rate of cleft palate and cleft Hp were
reported to be elevated by 1.9 (90* confidence Intervals of 1.0-3.6) In the
k
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 Oefect Monitoring Program of the Center
for Disease Control on the yearly rate of cleft palate alone or cleft Hp
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 100% In
the years 1971-1975, with the rate returning to expected from 1976-1981.
The observed Increase was only statistically significant 1f the rates for
cleft palate alone and cleft lip with or without cleft palate were combined;
however, H 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, In addition,
demonstrated that the same results occurred 1f the comparison was made with
other counties In Michigan as well as with the general population of the
United States. It was noted In this report that "runs1 of Increases In 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 If any common factors
may exist, such as exposure to chemicals contaminated with 2,3,7,8-TCDO.
01340
VI-15
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Several Investigators have suggested that 2,3,7,8-TCOD Is the causative
agent of excess liver carcinoma and soft-tissue sarcoma associated with
occupational or accidental exposure to phenoxyacetlc acid herbicides.
Direct evidence Is limited since the dloxlns usually occur In conjunction
with other chemicals and quantitative exposure data are not available.
Observations of an unusual occurrence of relatively rare soft-tissue
sarcomas were first made by Hardell (1977). Of some 87 patients seen from
1970-1976 at the Department of Oncology, University Hospital, Umea, Sweden,
seven Individuals with soft-tissue sarcomas were Identified. All seven had
had occupational exposure to phenoxy acids 10-20 years earlier. The tumors
were 2 leiomyosarcomas, 1 llposarcoma, 1 rhabdomyosarcoma, 1 myxofibrosar-
coma and 2 additional sarcomas of which the hlstopathology was uncertain,
but one was probably a neurofibrosarcoma and the other a rhabdomyosarcoma.
The clustering of this rare tumor type among these patients prompted the
author to suggest that epidemiological studies be done to determine If
exposure to phenoxy adds and the Impurities they contain are related to the
occurrence of soft-tissue sarcomas.
Zack and Susklnd (1980) reported a soft-tissue sarcoma death In a cohort
study of workers exposed to 2,3,7,8-TCDD In a trlchlorophenol process acci-
dent In Nltro, West Virginia. This tumor, a fibrous histiocytoma, was noted
by the author as a rare event. This study, referred to as the Nltro study,
1s discussed later.
Cook et al. (1980) 1n a cohort mortality study of 61 male employees of a
trlchlorophenol manufacturing area, who exhibited chloracne following a 1964
01340
VI-16
08/10/84
-------
exposure incident, noted Pour deaths by the end of his study period, one oP
which was due to a Pibrosarcoma. The authors did not seem to attribute any
special significance to this finding at the time.
Qtt et a "I. ( 1980) in a cohort mortality study of 204 employees exposed
to 2,4,5-T during its manufacture from 1950 to 1971, found no soft-tissue
sarcomas among 11 deaths that had occurred by 1976. One of these 11 deaths
was due to a malignant neoplasm.
In a review of the studies of Zack and Suskind (1980), Cook et al.
(1980), an unpublished study by Zack (in which a liposarcoma was found), and
a 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 0.07%
deaths due to soft tissue sarcoma expected In United States males 20-84
years old, (ICD 171, 8th Revision, 1975)* Indicating an unusual excess of
such tumors. The researchers underestimated the results because of the
possiblity that some soft-tissue sarcomas may have been coded to categories
other than ICD 171. Individually, none of these case studies reported a
significant excess of soft-tissue sarcomas. Cook (1981) found an addi-
tional malignant fibrous histiocytoma after a later review of the medical
records from his earlier cohort study. Cook, who was familiar with the
three earlier cases, noted that frank chloracne occurred previously in two
cases of the four having a diagnosis of malignant fibrous histiocytoma. A
third person diagnosed as having $ fibrosarcoma worked In a trichlorophenol
*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.
01340
VI-17
04/14/88
-------
(TCP) process area contaminated with 2 ,3,7f8-TCDD. This individual exhibit-
ed facial dermatitis but no diagnosis of chloracne. The fourth case (diag-
nosed as a liposarcoma) was an individual who had been employed earlier in a
plant producing 2,4,5-T. Cook (1981) noted that although chloracne was not
reported, it could not be discounted. He also noted that all four were
smokers and suggested that cigarette 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 Hardel 11 s seven cases exhibited chloracne before the appearance
of the soft-tissue sarcomas, and that in their subsequent case control
study, they found no difference In smoking habits between his cases and
controls.
Moses and Selikoff (1981) reported a fifth soft-tissue sarcoma in a
worker "employed at the Monsanto Chemical Company at a time when trichloro-
phenol and 2,4,5-T were being produced. He died of a retroperitoneal neuro-
genic sarcoma (malignant schwanoma) in 1980 at the age of 58. The employee,
prior to his death, in a detailed occupational history said that he believed
he was exposed to these chemicals while he was a truck driver, hauler and
maintenance worker, but that he did not work In the production of either
chemical. He was a nonsmoker and did not have a history of chloracne.
Johnson et al. (1981) treated a father and son with soft-tissue sarcomas
(the 33-year-old son was diagnosed as having a fIbrosarcomatous mesothe-
lioma, while the 53-year-old father had a liposarcoma). Both were exposed
01340
VI -18
04/14/88
*
-------
to halogenated phenol derivatives. The author noted that 2,4-dlchlorophenol
can be a precursor of 2,4-D and 2,4,5-T, The father had had prolonged expo-
sure before the diagnosis. The son supposedly had a shorter latency,
according to the author. In neither case was the follow-up time given.
I
Sarma and Jacops (1981) reported three cases of thoracic soft-tissue
sarcoma In Individuals who were presumably exposed to Agent Orange while
serving In Vietnam. The diagnoses were fibrous histiocytoma, mediastinal
fibrosarcoma, and a p!eural/d1aphragmat1c leiomyosarcoma. All three served
In areas where defoliants were used at the time. One was drenched with the
material 1n a single spraying.
Bishop and Jones (1981) found two cases of non-Hodgkln's lymphomas of
the scalp In a related clinical study of 158 employees of a pentachloro-
phenol manufacturing plant 1n Wales. Homologues of 2,3,7,8-TCDD occurred as
contaminants at up to 300 ppm at Intermediate manufacturing stages and 5 ppm
1n the final products. M1ld, moderate and severe cases of chloracne were
seen 1n many employees, Including the two men who subsequently developed
lymphomas. Both men worked In processes where exposure to other chemicals
occurred, Including exposure to aromatic hydrocarbons. The authors reported
that only 0.28 tumors of this type could be expected to occur 1n a group of
158 workers (ICO 200 and 202J, although the basis for the computation of
expected numbers Is not stated.
>
Olsson and Brandt (1981) noted that of 123 male patients seen at their
clinic Yn Sweden with a recent diagnosis of non-Hodgkln's lymphoma (NHL), 5
had cutaneous lesions as the only clinically detectable manifestation of
NHL. Four of them were reported to have had repeatedly sprayed large areas
01340 VI-19 08/10/84
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with phenoxy add herbicides. In the remaining 118 NHL patients, only seven
had a similar occupational exposure to phenoxy adds. The authors reported
this to be significant at p<0.001. Olsson and Brandt (1981) suggested that
a relationship exists between cutaneous presentation of NHL and occupational
fc
exposure to phenoxy adds, and believed their observations were similar to
those of Bishop and Jones (1981).
The total number of workers with these Illnesses who were exposed to
phenoxy acids or chlorophenols or both Is small, but considering the rarity
of this cancer, It 1s unusual that so many cases of soft-tissue sarcomas
have occurred. A Lancet editorial (Anonymous, 1982) calls this phenomenon
"disturbing."
Soft-tissue sarcomas (STS) constitute a collection of heterologous
lesions that Include both malignant and nonmallgnant tumors. Not all of
them have their origin In primordial mesenchymal cells. Some exceptions are
tumors of peripheral nerves, and neuroectodermal tumors that are classified
as STS, but are derived from nonmesenchymal cells. Classification, grading
and staging of STSs Is difficult because of the capacity of such cells to
differentiate Into many different tissues. Fairly precise hlstogenetlc
classification of such tumors 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 dif-
ferent classes of mesenchymal cells that develop Into the following six
well-defined tissue complexes: fibrous tissue, tendosynovlal tissue,
adipose tissue, muscle, vessels and bone. STSs can be Induced In any of
these tissue types (Hajdu, 1983). The classification of STSs for cause of
death coding In the ninth and latest revision of the International Classlfl-
01340 VI-20 08/10/84
-------
cation of Diseases (ICD, 1975) places STSs Into one of several categories.
But chiefly, they fall Into 'malignant neoplasms of connective and other
soft-tissue" (ICD 171). Lymphosarcomas, retroperitoneal sarcomas and extra
skeletal STSs of the bone are coded elsewhere. In some Instances, If site
ts mentioned, It Is coded to the site, e.g., leiomyosarcoma of the stomach
(ICD 151.9), neurofibroma of the chest wall (215.4).
Questions have been raised concerning the appropriateness of lumping
together malignant tumors of different sites and tumor types in order to
derive risk estimates. It may not be scientifically appropriate to do so
because an elevated risk cannot readily be ascribed to a particular site or
type as 1s usual with most carcinogenic chemicals and substances. Unfortu-
nately, with respect to STSs, tallies of deaths caused by STSs of particular
sites and types are not maintained separately by the vital statistics
offices because of their rarity, and therefore, 1t Is Impossible to derive
risk estimates for particular types at given sites. Altogether, -2000
deaths/year can be attributed to STSs in the United States, most of which
are coded to ICD category 171 for purposes of developing Incidence and mor-
tality rates for this composite cause. Within ICO 171, Individual types
that may be correlated with exposure cannot be Identified.
A separate problem that potentially could arise from assigning STSs to
multiple ICO codes 1s that Incidence and death rates from STSs may be
underestimated. Furthermore, risk estimates derived from dividing observed
cases (or deaths) by expected cases (or deaths) could be biased upward.
This could happen when observed STSs classified to ICD codes other than ICD
01340
VI-21
08/10/84
-------
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 or chlorophenols or
both, could tad to risk estimates that may be biased upward by the
inclusion of STSs in the observed category for risk es11ma11 on that should
be coded to categories other than 171.
Prompted by clinical observations over a 7-year period of malignant sar-
comas 1n seven men with previous occupational exposure to phenoxyacetlc add
herbicides (Harden, 1977), researchers at the Department of Oncology,
University Hospital, Umea, Sweden, Initiated case-control epidemiologic
studies (Cole, 1979) to test the hypothesis of an etlologlc association
(Hardell and Sandstrom, 1979). Cases were defined as male patients with
sarcomas of soft connective tissue, such as smooth muscle (leiomyosarcoma)
and fat (llposarcoma). The distribution of tumor types In the two studies
Is shown In Table VI-1. Sarcomas of tissues, such as bone and cartilage,
were excluded as cases. According to the authors, these tumors may have a
different etiology and there occurred a different age-d1str1but1on In
patients with these tumors as compared with that of STS (Hardell, 1983).
Two case-control studies were conducted, the first In northern Sweden
(referred to as Study A), and the second In the southern part of the country
(Study B). The exposures to the substances of primary Interest are shown in
Table VI-2. In the north (Study A), occupational exposure to phenoxyacetlc
acids took place In both forestry and agricultural work. In the south
(Study B), these exposures were predominantly agricultural. The phenoxy-
01340
VI-22
08/10/84
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TABLE VI-1
Distribution of Tumor Types 1n Two Case-Controls Studies
of Soft-Tissue Sarcoma
Percent
of Cases
Diagnosis
Tissue of Origin
Study Aa
(n-52)
Study Bb
(n.110)
Leiomyosarcoma
Smooth muscle
30
23
Fibrous histiocytoma
>
Subcutaneous connective
tissue
17
25
Llposarcoma
Fat tissue
14
6
Neurogenic sarcoma
Nerve tissue
10
4
Angiosarcoma
Blood vessels
8
2
Myxosarcoma
Primitive connective
tissue
6
8
Fibrosarcoma
Fibrous tissue
4
8
Other sarcomas
11
24
Total
100
100
aUnpubl1shed Information supplied by Hardell to
strom, 1979)
EPA (Hardell
and Sand-
b£r1lcsson et al., 1979,
1981
01340
VI-23
08/10/84
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I
TABLE VI-2
Exposure Frequencies In Two Case-Control Studies of Soft-Tissue Sarcorna
Percent Exposed
Substance(s) Study A Study B
Cases
(n=52)
Controls
(n=206)
Cases
(n=110)
Controls
(n-219)
Phenoxyacetlc acids only
23.1
6.3
12.7
2.3
Chlorophenols only
11.5
2.4
10.0
3.6
Both
1.9
0.5
0
0
Total
36.5
9.2
22.7
5.9
~Sources: Study A, Hardell
1979, 1981
and Sandstrom,
1979; Study
B, Eriksson
et al.,
01340
VI-24
08/10/84
-------
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
chlorinated dibenzodloxin impurities (Levin et al., 1976) occurred mostly in
sawmill work and paper pulp production. Very few persons reported exposure
both to phenoxyacetic acid and chlorophenols in these studies. Of the two
predominant phenoxyacetlc acids, only 2,4,5-T is 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 In relation to exposure to phenoxyacetic acid
herbicide other than 2,4,5-T (2,4-D, MCPA, mecoprop, dlchloroprop).
Relative risks in relation to the three major categories of exposure are
shown in Table VI-3.* Studies A and B Indicate a risk of developing STSs
among workers exposed to phenoxyacetlc adds only, chlorophenols only, or
phenoxyacetic acids and/or chlorophenols several times higher than among
persons not exposed to these chemicals. In each comparison, the relative
risk is high (p<0.005) and unlikely to have resulted by chance alone.
Since little is known of the etiology of STSs, the consideration of con-
founding factors In these studies was largely a hypothetical matter. The
authors prevented the effects of age, sex, and place of residence as
possible
*In the analyses considering phenoxyacetlc acids only and chlorophenols
only, persons exposed to the other categories of substances were excluded.
In Study A, the three persons exposed to both chlorophenols and phenoxy-
acetic adds were Included in all comparisons.
01340
VI — 25
04/14/88
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TABLE VI-3
Relative Risks of Soft-Tissue Sarcoma 1n Relation to Exposure to
Phenoxyacetlc Acids and Chlorophenols 1n Two Case-Control Studies3
Phenoxyacetlc Acids
Only
Study A
Study B
Chlorophenols
Only
Study A
Study B
Phenoxyaqetlc Acids
and/or
Chlorophenols
Study A
Study B
Relative r1skb 5.3 6.8 6.6 3.3 5.7 4.7
90% Confidence Interval^ 2.7-10.2 3.1-14.9 2.8-15.6 1.6-7.0 3.2-10.2 2.7-8.3
Significance leveld <0.001 <0.001 <0.001 <0.005 <0.001 <0.001
aSource: Study A, Hardell and Sandstrom, 1979; Study B, Eriksson et al.f 1979, 1981
^Unmatched odds ratio
cTest-based method of Mlettlnen, 1976
dchl square statistic, no continuity correction, one-tailed test
-------
confounding factors In the selection of controls.* Because of the high cor-
relation between exposure to the substances of Interest and employment In
agriculture and forestry, a possible alternative hypothesis could be
developed that some other unknown factor present 1n these occupations was
I
responsible for the elevated relative risks.
To test this hypothesis, 1t Is possible to calculate the relative risk
In relation to the phenoxyacetlc add exposure In Study 0, restricting the
analysis to workers within agriculture and forestry. The result 1s a rela-
tive risk of 6.1 (90* confidence Interval 2.4-15.4). This finding 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 In relation to phenoxyacetlc acid exposure.
Because exposure histories were obtained by means of questionnaires and
Interviews, the major potential source of bias 1n these studies stems from
the need to rely upon the personal recollection of cases and controls for
exposure histories. The published papers Indicate that the researchers paid
a great deal of attention to this potential problem and specific efforts
were made to avoid 1t during the conduct of the study.
In addition, the relative risk calculated by considering the agriculture
and forestry workers who did not report exposure to phenoxyacetlc acids or
~Controls were matched Individually to cases on the basis of these factors.
Unmatched analyses are presented 1n Table VI-3 for the sake of simplicity.
The matched-method relative risks for exposure to phenoxyacetlc acids
and/or chlorophenols were 6.2 (p<0.001) In Study A and 5.1 (p<0.001) 1n
Study B.
01340
V1-27
OB/10/84
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chlorophenols and comparing them with unexposed persons 1n other occupations
was 0.9 (90X confidence Interval 0.3-2.4) In Study 8* This suggests that
little recall bias was present (Axelson. 1980).
*In an update of their earlier study, Eriksson et al. (1981) obtained
Information on the effects of phenoxy acids 1n the absence of the Impurl-
t1es--polychlor1nated dlbenzodloxlns and dlbenzofurans. The risk ratio
given exposure to phenoxy adds free of polychlorInated dlbenzodloxlns and
dlbenzofurans equaled 4.2 based upon 7 out of 14 respondents who Indicated
exposure to phenoxy acid herbicides. When consideration was given to
persons exposed to only phenoxy acids that contain such Impurities, the
relative risk was 17.0. A description of the basis for the determination of
exposure or nonexposure to dloxlns Is not well presented In this study.
The authors concluded that exposure to phenoxy adds and chlorophenols
¦might constitute a risk factor 1n the development of soft-tissue sarcomas."
This risk relates not only to 2,4,5-tMchlorophenoxy acids containing dloxln
Impurities, but to other phenoxy acids as well. Some doubt was raised con-
cerning the possible m1sclass1f1cat1ons of Individuals who were exposed to
phenoxy adds free of polychlorlnated dlbenzodloxlns [I.e., 1n particular,
"dlchoroprop" 1n the Eriksson et al. (1981) study]. In a recent communica-
tion from Hardell (1983), Eriksson recalculated risk estimates after
reclassifying dlchoroprop-exposed cases and controls Into the category of
probable exposure to phenoxy acids contaminated with polychlorlnated dlben-
zodloxlns and removing them from the nonexposed category. The new estimates
were 4.0 based upon 5 out of 8 respondents who were exposed to phenoxy acids
free of contamination and 10.9 for those exposed to contaminated phenoxy
01340
VI-28
09/18/04
-------
acid. The 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 al. (1980), studied 61 males
Involved In a 1964 exposure Incident who had chloracne caused by absorption
of 2,3,7,8-TCDO. The skin lesions characterizing chloracne ranged from a
few comedones on the back of one employee (predating his entry Into the
process area where exposure could occur) to severe cysts and comedones over
the faces, scalps, ears, necks and backs of the remaining employees of the
group. Since the main route of exposure was not through the respiratory
tract, no measurements of dloxln 1n the air were provided by the author. On
the other hand, the author did divide the cohort of 61 males Into potent-
ially "high" vs. "low" exposure by place of work based upon dermal exposure,
although not stated. Vital status was traced from the data of the Incident
through 1978. Altogether only 4 deaths were observed by the end of the
follow-up vs. 7.8 expected. Of these, 3 were cancer deaths vs. 1.6
expected. The remaining death was hypersensitive heart disease vs. 3.8
expected. The histopathologic causes of death of the three cancer victims
were 1) fibrosarcoma, 2) glioma with metastases, and 3) adenocarcinoma. The
authors report that all three victims smoked a minimum of one pack of cigar-
ettes a day for "many years.* Hot enough Information is provided by the
authors to conclude that any of these four deaths were smoking related.
Site of tumor 1s not mentioned 1n the cancer deaths.
Cancer mortality was slightly elevated 1n this cohort. This study had
low sensitivity and lacked a sufficient latent period. This Increased
01340
VI-29
09/18/84
-------
mortality was not attributable to any particular cause and no deaths were
attributable to liver cancer. Additionally, the authors state that only one
of the -cancer deaths possessed "documented" evidence of chloracne, although
this appears to be at variance with the definition of the cohort, which was
reported by the authors to consist of males who reported to the medical
department with skin conditions subsequently 'diagnosed as chloracne." The
authors concluded that the latency period was sufficient to "allow the
Identification of a potent human carcinogen," since It "exceeded 14 years."
Orris (1981) noted that 1n the Hardell and Sandstrom (1979) study the
authors stated that the latent period for soft-tissue tumors may be as long
as 27 years and for many, over 14 years. In any case, Hueper and Conway
(1964) noted that the latent period for the chemical Induction of solid
malignant tumors In man exceeds 15 years and 1s probably <30 years.
Smith et al. (1982b) conducted an Initial case-control study of 102
males Identified from the New Zealand Cancer Registry as having STSs (ICO
171) between 1976 and 1980. For each case, three controls each with another
form of cancer were matched by age and year of registration. The selection
of cancer controls from the same registry was done to eliminate recall bias
or Interviewer bias or both. The distribution of histological types 1n the
cases Is given 1n Table VI-4. An Interview to elicit occupational history
Information was accomplished by telephone either to the next of kin or the
patient himself, If he was well enough, although the Information was not
used 1n this preliminary analysis.
Comparisons between cases and controls were accomplished by use of
occupational groupings according to the Standard Classification System of
01340
VI-30
08/10/84
-------
I
TABLE VI-4
Distribution of Histological Types of Soft-Tissue Sarcomas'*
Cell Type
Number of Cases
Percent
Fibrosarcoma
25
24
Llposarcoma
20
20
Rhabdomyosarcoma
9
9
Leiomyosarcoma
7
7
Malignant Histiocytoma
6
6
Other
22
21
Unspecified
13
13
Total
102
100
~Source: Smith et al.# 1982b
01340
VI-31
08/10/84
-------
New Zealand focusing on those occupational groups with a potential for
exposure to phenoxy herbicides and chlorophenols. Expected cases for each
major occupational classification were derived based upon the occupational
distribution of the controls. The authors found no unusual excess of cases
o\ STS 1n any major occupational category. In agriculture, forestry and
fishing, 14 cases were observed vs. 14.0 expected. In laborers, production
and transport workers, 35 cases were observed vs. 37.0 expected. A further
breakdown of these two broad categories Into finer subcategories within the
major occupational categories revealed no significant excesses. The study,
however, Is not useful In assessing the risk of STS from exposure to phenoxy
acids or chlorophenols for several reasons. First, as was pointed out by
the authors but subsequently dismissed by them as having not much of an
Influence, Is the possibility that movement from one major occupational
category to another over the time period Involved for latent conditions to
manifest themselves could Introduce a negative bias into any estimates of
relative risks. The latency for STS was suggested to be a minimum of 15
years (Hueper and Conway, 1964).
The finding of no switching from one occupational category to another
that was noted In the "first 20 Interviews" In which a change could be noted
1s not necessarily Indicative of fidelity to the same Job over long periods
1n all 408 cases and controls. Information Identifying a change may be
lacking In those cases and controls 1f In fact one did occur possibly for
several reasons, (separation of 'the earlier work history from the latter;
purging of earlier employment records, etc). Besides, the "first 20
Interviews" where a change could be noted Is not necessarily representative
of the entire cohort 1n any case.
01340
V1-32
09/18/84
%
-------
Furthermore, the authors do not know absolutely that any of their cases
and controls were exposed to phenoxy acids or chlorophenols, since appar-
ently no effort was made to confirm "potential" exposures. Only differences
in occupational classification were noted where "potential" cases or con-
trols could have had exposure to the dioxin-containing herbicides. It was
pointed out that the risk estimates noted do not "preclude" the possibility
that an association may be found in this study when the cases and controls
(or surviving kin) are interviewed for chemical spraying at a later time.
The authors themselves conclude that the preliminary study results "should
not be taken as substantial evidence against the hypothesis that phenoxy
herbicides and chlorophenols may cause human cancer" (Smith et al.r 1982b).
The distribution of tumor types differed considerably in the Hardell and
Eriksson et al. (1981) study compared to the,Smith et al. ( 1982b) study.
Leiomyosarcomas, malignant histocytomas, neurogenic sarcomas and myxosarcoma
seem to predominate in the Hardell and Eriksson (1981) study, whereas fibro-
sarcomas and llposarcomas appear prominently in the Smith et al. (1982b)
study. More attention should be devoted to the study of the distributions
of STS types in registry data everywhere in order to determine if such
variations in 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.
t
Smith et al (1983) conducted another case-control study of STSs in 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
01340 VI-33 04/14/88
-------
each. Controls were matched for year of registration and by date of birth
+2 years. Inquiries were made by the authors with the hospital consultant,
family doctor, and finally the next-of-kin or patient If alive. Telephone
Interviews were conducted by only one Interviewer who had no knowledge of
the* patients cancer history and were completed on 00 cases and 92 controls.
Because some 32 potential cases (14 Ineligible) and 20 controls were
excluded or lost from the study for various reasons, It raises a question
whether control of confounding by age and year of registration was main-
tained In the final group of 172 cases and control Included 1n the analysis.
Presumably the corresponding ¦matched" case or control to each of the 52
lost members of the total study group were not excluded. However, since the
span of registration was only 5 years, not much age confounding could occur.
Patients were classified as having had potential exposure to phenoxy-
acetlc acids 1f they had definite, probable or possible exposure to phenoxy-
acetlc acid through spraying or hand contact. The actual chemical was
Identified only 1n some Instances. The authors concluded in all remaining
situations that 1f the member sprayed "gorse" or "blackberries," this was
tantamount to potential exposure to phenoxyacetlc add. Smith et al. (1983)
calculated elevated but nonsignificant relative risks of exposure to phen-
oxyacetlc acid ranging from 1.3 1n those Individuals who were "probably
exposed" for a minimum of 5 days not In the previous 10 years before cancer
registration to 1.6 In Individuals "probably exposed" for a minimum of 1 day
not 1n the previous 5 years before cancer registration. When risk ratios
were calculated after stratifying by year of birth and whether or not the
patient or a relative was Interviewed, the rates Increased to 1.7 (from 1.6)
01340
V1-34
08/12/04
-------
1n the latter and 1.4 (from 1.3) in the former calculation, although still
nonsignificant. If the numbers would allow, 1t would be of Interest to
repeat the above calculations excluding only those with potential exposure
occurring only within the 15-year period Just before cancer registration.
The*small numbers that remain following the 15-year lapse precludes such an
analysis. Furthermore, the categories of exposure "probably or definitely"
exposed for >1 day or even 5 days raises a question whether any of the cases
or controls could really be said to have ever come 1n contact with enough
phenoxyacetlc add to justify such a designation. It could be that, 1n
fact, potentially exposed Individuals In New Zealand have had Uttle 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 In the last 5 years cannot be entirely
discounted. But then the authors state that 1f the length of exposure was 5
days or more prior to 10 years before cancer registration they would expect
an Increase; since they do not see an Increase, there Is no evidence of a
¦real causal link." One might ask whether this 1s a suitable criterion for
providing evidence of a causal association. Perhaps a more valid group for
study would be one where the potential exposure was considerably longer than
"5 days- and >15 years before initial cancer registration. As a subtle
justification for the finding of no significant risk 1n workers exposed In
phenoxy adds, the authors (Smith et al., 1983) allude to the fact that
there are currently 500 full-time workers registered 1n New Zealand who do
full time ground spraying and altogether some 2000 workers who were at some
time professionally Involved 1n phenoxyacetlc add herbicide spraying from
the air or ground with exposure *very much greater- than that of patients 1n
01340
V1-35
09/18/84
-------
this study. This kind of argument has appeal if these workers could be
shown to have had their exposure sufficiently far In the past that latency
considerations could be adequately addressed. However, the real question
again remains how much real exposure did those patients In the study have
10-15 years earlier, and In what numbers. The authors remark that It Is
surprising that they found no STS victims who had ever worked full-time 1n
phenoxyacetlc acid herbicide spraying. Perhaps they have not yet been
observed for a long enough period. However, as was pointed out by the
authors, the findings do not support the hypothesis that exposure to
phenoxyacetlc acid herbicides causes STS, But neither do they support a
negative finding without better documentation regarding actual exposure and
time of actual exposure. The author does not (Smith, 1963), however, state
that his documentation of exposure to 2,4,5-T (and 2,4-D) Is at least as
good as that In the Hardell study, and that although Hardell noted higher
relative risks of <30 days exposure. Smith (1983) did not. Hence the
paradox. Smith does admit the possibility that 2,3,7,8-TCOO contamination
might be lower 1n New Zealand as opposed to 2,3,7,8-TCDO contamination 1n
the Swedish studies. Although, there 1s no evidence for It. Smith (1983)
still maintains that his study shows that exposure to phenoxyacetlc acids
may not be associated with STS.
Pazderova-Vejlupkova et al. (1981) studied 80 workers Involved In the
production of 2,4,5-sodlum trlchlorophenoxyacetate and butylester of tr1-
chlorophenoxyacetlc acid who • subsequently became 111 from exposure to
2,3,7,8-TCOD during the period 1965-1968. Only 55 members of this group
were followed for 10 years. The remaining 25 either refused participation
or moved leaving no forwarding address. Host patients developed chloracne
while 11 developed porphyria cutanea tarda. Chief chemical signs were meta-
01340 VI-36 09/18/84
-------
bol1c disturbances, pathologically elevated lipids with abnormalities In the
lipoprotein spectrum, and "pathological" changes In glucose tolerance.
Other symptoms noted were biochemical deviations consistent with "a mild
liver lesion," light steatosis, periportal fibrosis or activation of Kupffer
I
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 bronchogenic carcinoma, 1 from cirrhosis, 1
atherosclerosis predpue cerebl and 2 1n auto accidents. No STSs or lympho-
mas were found. Since there was no comparison population with which to
estimate relative risk for cancer, the study must be classified at best as
clinical with respect to cancer. The six deaths that occurred during the
10-year observation period In the 55 cannot be construed to be associated
with exposure to the 2,4,5-T. Because of ttje small number of cases and the
short follow-up period, nothing can be said concerning the association of
exposure with cancer, especially specific types of cancer such as STS or
non-Hodgkln's lymphoma.
R11h1mak1 et al. (1982, 1983) studied a cohort of 1926 herbicide appli-
cators formed 1n 1972 from personnel records of four Finnish employers
(e.g., the Forestry Authority, Highway Authority, State Railways and a
state-owned electric power company). Chlorinated phenoxyaclds had been used
since the 1950s 1n Finland for spraying. They constituted 2:1 mixtures of
emulsified esters of 2,4-D and 2,4,5-T dissolved In water. Analyses from
old herbicide formulations dating back to the 1960s revealed that these
mixtures contained 0.1-0.9 mg of 2,3,7,8-TCDD/kg formulation).
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
01340 VI-37 09/18/84
-------
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
I
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
23% 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
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 and 82% (even after 15 years from Initial exposure) 1s probably a
consequence of the "healthy worker effect" 1n that only able-bodied and
healthy Individuals were selected Into the industry. The fact that the
cohort was assembled In 1972 from records of persons who were exposed as
early as 1955 (17 years prior) raises the likelihood that In 1972 a "survi-
vor" population remained (45 deaths before 1972 were eliminated from the
01340 VI—38 08/10/84
-------
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
I
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 1s needed 1n order to provide a stable assessment of the relation-
ship between exposure and cancer. The authors concluded that this study
will allow no assessment of STS because "the number of persons having a suf-
ficiently long latency period 1s too small.1 It was suggested that more
valid conclusions could be made only with the passage of time (RUhlmakl et
al., 1983).
Recently, the Michigan Department of Public Health (1983b), conducted an
ecological study of soft and connective tissue cancer mortality rates In
Midland and other selected Michigan counties. They found that mortality
rates for this cause were 3.8-4.0 times the national average for the periods
1960-1959 and 1970-1978, respectively, for white females In Midland. These
estimates are based upon 5 deaths and 7 deaths, respectively, and are listed
1n Table VI-5. 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 1s 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 for the period
01340 VI-39 08/10/84
-------
TABLE VI-5
Midland County Soft and Connective Tissue Cancer Deaths 1960-1981*
Identification Type of Malignancy
Year of
Death
Sex
Age
Type
Primary Site
Metastases
Month and Year
Diagnosed
1961
F
24
Hemanglosarcoma
Face
Skull and upper lobe
of lung
5-58
1963
F
75
Llposarcoma
Right gluteal
Unknown
Unknown
1964
F
51
Leiomyosarcoma
Uterus
Widespread
11-63
1968
F
37
Llposarcoma
Spine
Lungs, pelvis
1-66
1969
F
45
Fibrosarcoma
Leiomyosarcoma
Right thigh
Uterus
Lung, liver
Adrenal gland and skin
10-68
1970
F
59
Kaposi sarcoma
Right leg
Lymph nodes
8-68
1970
F
56
Fibrosarcoma
Leiomyosarcoma
Right thigh
Abdominal wall
Spine
Lung
1960
1967
1974
F
1
Rhabdomyosarcoma
Inguinal area
Unknown
8-73
1976
F
77
Llposarcoma
Right thigh
Buttock, lung, rib,
lymph nodes
12-74
1978
F
64
Leiomyosarcoma
Left knee
Liver, lymph nodes.
7-70
lung, bone
-------
TABLE VI-5 (cont.)
Identification Type of Malignancy
Year of
Death
Sex
Age
Type
Primary Site
Metastases
Month and Year
Diagnosed
1978
F
26
Rhabdomyosarcoma
Rectum
Lung, neck, Inguinal
region
6 76
1978
F
88
Fibrosarcoma
Right cheek
Facial area
6-78
1979
F
27
Leiomyosarcoma
Left thigh
Lung
3-78
1962
M
63
Rhabdomyosarcoma
Left lower leg
Lung and right outer
chest wall
8-61
1967
M
77
Mesothelioma
Lung
Lung, peritoneum and
diaphragm
6-67
1967
M
20
Rhabdomyosarcoma
Pharynx
Periorbital area and
liver
1-67
1969
M
32
Llposarcoma
Left arm
Perineum and buttock
6-64
1971
M
76
Leiomyosarcoma
Smal 1
Intestine
Liver
10-69
1972
M
89
Leiomyosarcoma
Retro-
perl tonal
region
Hepatic system
7-72
1976
M
53
Fibrosarcoma
Per 1tloneum
Lung, liver
3-75
~Source: Michigan Department of Public Health, 1983b
-------
1970-1979. Midland County 1s the home of a major chemical company that pro-
duced phenoxyacetlc acid herbicides until recently. The authors stated 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 epidemiological evidence for STS from expo-
sure to 2,4,5-T-contalnlng herbicides, the United Kingdom Ministry of Agri-
culture, Fisheries and Food (1983) concluded that there was no evidence to
reconnend 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 (1981) as being biased, and 1t
makes no reference to the later validity study by Hardell (1981) of his own
work utilizing colon cancer controls. In this report Hardell effectively
answered these early criticisms that were reiterated by the British In their
report. At the same time, the British report appears to put undue emphasis
on nonposltlve studies that do not demonstrate a risk, although most of them
have methodological limitations (e.g., low power, Insufficient latency and
Inappropriate study method). In short, the British review appears to be
overly optimistic about the safety of 2,4,5-T herbicides.
In summary, the associations reported In the two Swedish soft-tissue
sarcoma studies are great enough to make It unlikely that they have resulted
01340
VI-42
09/18/64
-------
entirely from random variation bias or confounding, even though the pos-
sibility cannot be excluded. These studies provide a strong suggestion that
phenoxyacetlc acid herbicides, chlorophenols or their Impurities are
carcinogenic 1n humans.
I
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 phenoxyacetlc acid,
chlorophenols, and other organic compounds (Harden et al.p 1980, 1981).
Approximately 33% of the cases In this study were patients with Hodgkln's
disease; the remainder of the cases were non-Hodgk1n's lymphomas.
This study employed essentially the same methods and produced results
comparable with those of the STS studies: statistically significant 5-fold
to 6-fold relative risks In relation to phenoxyacetlc adds and chloro-
phenols. In addition, an elevated relative risk was found In connection
with exposure to organic solvents, such as benzene, trlchloroethylene and
styrene. In the published report, the methods and results were Incompletely
documented, especially the possibility of confounding by exposure to the
organic solvents.
In the update of the earlier 1980 study, Hardell et al. (1981), utiliz-
ing the same basic data source, found that 36.IX 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
01340
VI-43
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risk of lymphoma from phenoxy acids alone was 4.8 (95% C.I. 2.9-8.1). On
the other hand, if exposures to phenoxy acids are excluded and consideration
is given to just chlorophenols (which includes combined exposure to phenoxy
acids and chl orophenol s), then the relative risk equaled 4.3 (95% C.I.
2.7-6.9). The authors further subdivided this group into "low-grade'1 vs.
"high-grade" exposures to chlorophenols. A continuous exposure of not more
than 1 week or repeated intermittent exposures totaling not more than 1
month was classified as low-grade. The relative risk for high-grade expo-
sure was 8.4 (95% C.I. 4.2-16.9), while that for low-grade exposure equaled
2.9 (95% C.I. 1.6-5.2). If exposure to organic solvents is examined, given
that cases and controls exposed to only phenoxy acids or chlorophenols or
both were excluded except for combined exposure to organic solventst it is
found that highgrade 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 authors
noted that exposure to phenoxy acids and high-grade organic solvents (expo-
sure 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 or
phenoxy acids constitutes a risk factor for malignant lymphoma."
This latter study is still subject to the same methodological criticisms
to which the earlier study was subjected. Chief among those 1s the pos-
sibility of observational 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 on the risk
estimate, although the authors assure the reader that they did not (Hardell
et al., 1981).
01340
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Other research has tentatively suggested that lumberjacks may be at
increased risk of lymphoma (Edling and Granstam, 1979). The Nitro study
(Zack and Suskind, 1980) found three deaths from cancers of the lymphatic
and hematopoietic system, against only 0.88 expected (p=0.06, one-tailed
Poi sson test).
The lymphoma case-control study (Hardell et al., 1980, 1981) is con-
sistent with the two STS studies discussed. On the other hand, the
consistency could also reflect an as-yet unidentified consistent flaw in all
these studies.
The two Swedish case control studies on STSs and a later case control
study of malignant lymphoma (Hardell et al., 1981) were subjected to a
validity analysis with respect to the assessment of exposure by Hardell and
Eriksson (1981). To answer the question raised regarding the recall of
occupation In a forestry/agriculture job, secondary to the recall of expo-
sure to phenoxy acids or chlorophenols or both, 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 1n an agriculture/
forestry job but not exclusively, and the remainder who never worked in a
forestry/agriculture job. The study found that the risk ratio was still 8.2
for STS in exclusively agriculture/forestry workers who were exposed to
phenoxy acids compared with workers found In other occupations having no
apparent exposure to phenoxy acid's or chlorophenols. Even when comparing
phenoxy acid or chlorophenol exposed agricultural/forestry workers exclu-
sively with nonexposed agricultural/forestry workers, the risk ratio was
still 7.1. This argument seems to effectively answer questions regarding
recall of occupation secondary to exposure.
01340
VI -45
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On the other hand, the relative risk remains 5 A when comparing phenoxy
acid or chlor.ophenol exposed workers exclusively In occupations other than
agriculture/forestry with nonexposed workers in those same occupations, thus
suggesting the presence of either recall bias or still another occupation
with potential exposure to phenoxy acids or chlorophenols (Table VI-6).
When woodworkers are separated out (possible exposure to chlorophenols
in treatment of wood) the risk ratio becomes 9.7 (Table VI-7). These data
suggest the presence of some recall bias.
Another focus of this study (Hardell and Eriksson, 1981) was to deter-
mine 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 question-
naires only with the combined information from both the phone Interviews and
questionnaires. The study found no substantial differences in the frequency
of reporting exposure.
Still a third consideration of possible bias involves recall of exposure
to phenoxy acids or chlorophenols because of subject knowledge of having
cancer In the cases.versus no knowledge of cancer in the referent popula-
tion. The study chose as a referent group for the 52 STS cases (Hardell and
Sandstrom, 1979) and the 169 malignant lymphomas (Hardell et al., 1981) a
group of 154 colon cancer cases from the same population source and compared
their exposure to phenoxy acids or chlorophenols by broad age groupings and
by rural versus urban residence.
01340
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I
TABLE VI-6
Other Occupations (Minus Forestry/Agriculture)*
Group Phenoxy Ac Ids/ChiorophenoIs Non-exposed
Cases 11 68
Referents 5 167
RR = 5.4 X' = 11.01 (p<0.01
*Source: Adapted from Hardell and EMkkson, 1981
RR = relative risk
01340
VI-47
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I
TABLE VI-7
Other Occupations (Minus Forestry/Agrlculture/Woodworkers)*
Group Phenoxy Adds/Chlorophenols Non-exposed
Cases 4 66
Referents 1 160
RR = 9.7 Xa = 5.98 (p<0.05)
*Source: Adapted from Hardell and EMkkson, 1981
RR = relative risk
01340
V1-48
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Utilizing a HanteUHaenszel rate ratio, the study found the risk of
exposure to phenoxy acids remaining significantly high at 5.5 and to chloro-
phenols 5.4 In the STS cases compared with the colon cancer controls. Simi-
larly, with the malignant lymphomas, the Identically derived risk ratios
Remain significantly high at 4,5 with respect to phenoxy acids or
chlorophenol exposure 1n the cases; hence, the study concludes that, no
¦substantial observational bias" exists. If the study Is assuming that
recall bias was and 1s 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 It does not rule out the
possibility that recall bias can still be present In their data for other
reasons. Hardell and Eriksson (1981) refer to an intense "debate about
phenoxy acids and their presumptive risk" 1n Sweden at the time the colon
cancer study was conducted. But, there 1s no reason to think that colon
cancer victims would assume their disease was brought about from exposure to
dloxln containing chemicals 1f no connection was suggested.
It seems plausible that STS and 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.
01340 VI-49 09/18/84
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Therefore, although this study by Harden and Eriksson (1981) may
explain any biases introduced from secondary recall of occupation, observa-
tional bias introduced from the telephone interviewer and recall bias based
on subject knowledge of cancer, it does not adequately answer questions of
recall bias introduced through the acquired awareness on the part of the
victim of STS or non-Hodgkin1s lymphoma that his condition may have been
caused by exposure to dioxin containing herbicides.
Lynge ( 1985 ) compared the incidence of soft tissue sarcomas in two
phenoxy herbicide manufacturing plants in Denmark. An increased incidence
of soft tissue sarcomas was observed only in workers from the plant that had
a history of producing 2f4,5-T. Other studies that have reported an
elevated risk of soft tissue sarcomas, as supposedly associated with
exposure to 2,3,7,8-TCDD, include Cantor (1982), Milham (1982), Kogan and
Clapp ( 1985), Fett et al. (1984), Puntoni et al. ( 1986), and Merlo and
Puntoni (1986). ,
Studies of two of the oldest cohorts of workers known to have been
exposed to phenoxyacetic acid herbicides or 2,3,7,8-TCDD or both report
stomach cancer mortality rates significantly higher than expected. The
results in 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 herbicide
exposures: those primarily exposed to phenoxyacetic acids (2,4-D and
2,4,5-T) only, to amitrole (aminotrlazole) only, and to both types of
herbicides. After a 10-year latency was achieved, 3 stomach cancer deaths
were observed vs. 0.71 expected (p
-------
alone, but two were assigned to phenoxy acids alone while the remaining
stomach cancer death occurred in a worker exposed to both amitrol and
phenoxy acids in combination. The excess was more pronounced (3 observed
vs. 0.57 expected, p<0.05) among those with early exposure (1957 -1961) to
phenoxy acids or amitrol or both. If persons who were exposed to just
amitrol alone are excluded, thus leaving individuals exposed to phenoxy acid
alone and amitrol in combination, the excess is enhanced further (3 observed
vs. 0.41 expected, p<0.01).
Axelson et al. (1980) also note an excess in total "tumors" after 10
years latency as well (15 observed vs. 6.87 expected, p
-------
The results, shown in lable VI-8, 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. All three
stomach cancer deaths in the Ludwigshafen 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 relative risk estimate by
reducing the number of expected deaths.
Secondly, national and regional mortality rates from the 1970s were used
to generate expected deaths to compare with observed mortality over a much
longer period (1953-1977). Strong temporal trends in stomach cancer mortal-
ity in West Germany during the late 1950s and 1960s would make these
expected figures too Targe.
The researchers also used an Internal control group that does not raise
the second concern discussed above. This group consisted of 75 men, each
matched to study group members by age and date of entry Into employment, and
selected at random from a 11st of over 10,000 persons who had been included
In previous cohort studies by the same Investigators. No stomach cancer
deaths occurred In this control group during the follow-up period. Thus,
use of the Internal control groups also indicates an excess of stomach can-
cers In 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-
01340 VI-52 04/08/88
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TABLE VI-8
Analysis of Stomach Cancer Mortality in a Group of
West
German Factory Workers Exposed
to 2,3,7,8-TCDD*
Source for
Stomach Cancer Deaths
Relative Significance
Expected Deaths
Risk Level
Observed Expected
Federal Republic
of Germany
1971-1974
3 0.559
5.4 0.02
Rhlnehessen-
Palatinate
1972-1975
3 0.495
6.1 0.01
*Source: Adapted from Theiss and Frentzel-Beyme, 1977
01340
VI -53
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!
I
-------
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-Palatinate, 18
expected deaths based on 1970-1975 mortality statistics of Ludwigshafen, and
20 expected deaths based upon 1971-1974 mortality statistics of the Federal
Republic of Germany. Just as in the earlier study, the three stomach car-
cinomas noted earlier appear to be significantly elevated regardless of
which external comparison group is used (Table VI-9).
On the other hand, one stomach cancer appeared in the randomized inter-
nal control group. None appeared In 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 Rhinehessin-Palatinate, 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, it 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, 1t is based only upon three deaths and
since one stomach cancer death has been noted in an internal control group
01340
VI -54
04/08/88
*
-------
TABLE VI-9
Reanalysls of Stomach Cancer Mortality in a Group
of West German Factory Workers Exposed to 2,3,7,8-TCDD*
Source for Stomach Cancer Deaths Relative Significance
Expected Deaths Risk Level
Observed Expected
Federal Republic of
Germany 1971-1974 3 0.7 4.3 0.034
Rhinehessln-
Palatinate
1970-1975 3 0.64 4.7 0.027
Lti'dwigsshafen
1970-1975 3 . 0.61 4.9 0.024
*Source: Adapted from Theiss et a 1., 1982
01340
r VI - 5 5
04/08/88
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in the updated version, it appears that this finding has been weakened some-
what. Furthermore, as was pointed out earlier, trends in stomach cancer
mortality during the 1950s, 1960s and 1970s could make the comparison of
stomach cancer mortality with expected deaths less valid based upon
1970-1975 rates.
In summary, the evidence that phenoxyace'tic acids or 2,3,7,8-TCDD or
both 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 yet be made.
Four additional cohort studies have reported results that do not show
increased stomach cancer mortality rates in groups of workers exposed to
phenoxyacetic acids or 2,3,7,8-TCQD or both. These are studies of 2,4,5-T
production workers in Midland, Michigan (Ott et al.t 1980), Finnish phenoxy-
acetic acid herbicide applicators (Riihimaki et al., 1978), the Nitro study
in which workers were exposed to 2,3,7,8-TCDD (Zack and Suskind, 1980) and
trichlorophenol manufacturing workers (Cook et al., 1980).
As previously mentioned, the Nitro study included a single death from
STS and a weakly suggestive increase in lymphatic and hematopoietic system
cancer mortality. The Midland ,study included only one cancer death, a tumor
in the respiratory system. In the Finnish study, histologic information on
tumor types was not provided; however, there were no deaths from lymphoma.
The results pertinent to stomach cancer mortality In the three studies
are shown in Table VI-10. Neither the Midland study nor the Nitro study
01340 VI-56 04/08/88
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TABLE VI-10
Stomach Cancer Mortality 1n Three Studies of Workers Exposed
to Phenoxyacetic Acid Herbicides and/or 2,3,7,8-TCDD
Stomach
Observed
Cancer Deaths
Expected
Relative
Risk
95% Confidence
Interval
Reference
0
O.Ha
0
0-26.3
Ott et al., 1980
5
6.9a-b
0.7
0.2-1.7
Ri ihimaki et al.,
1978
0
0.5b
0
0-7.4
Zack and Suskind,
1980
Estimated 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).
01340
VI -57
04/08/B8
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contradicts the findings of the Swedish and West German investigations pre-
viously discussed. This can be shown in two ways. First, the upper 95%
confidence limits for the relative risk estimates from these two "negative"
studies exceed even the highest point estimates of relative risk (6.1) from
the two "positive" studies (see Table VI-8).
This indicates that the relative risk estimates from the Midland and
Nitro studies, even though equal to zero, are nevertheless not significantly
different from the estimates of 6.1, given the sample sizes, follow-up per-
iods, age distribution and comparison group rates.
In addition, the smallest detectable relative risk in the Midland study
(a = 0.05, cp = 0.2 one-tailed Poisson test) was 21.4 (3 observed deaths,
0.14 expected).* Similarly, the smallest detectable relative risk in the
Nitro study (a = 0.05,
-------
Midland studies had very low probabilities of detecting the ~6-fold in-
creases in risk suggested by the Swedish and West German investigations.
Statistically, the study of Finnish herbicide applicators is incon-
sistent with the results of the Swedish and West German cohort studies. The
smallest reasonably detectable relative risk (a = 0.05, 1 spraying
season).
There are also certain inconsistencies in the data from the Finnish
study that the authors note but find difficult to explain. In particular,
no cancer deaths occurred during the latter part of the study period among,
Forestry Authority workers (1 of 4 groups included in the cohort), even
though 9.0 deaths were expected. This finding strongly suggests some defi-
ciency in follow-up or in the source records from which vital status was
determined.
*The expected stomach cancer deaths were estimated in the same manner as for
the Midland study* A proportion of 20% of all cancer deaths was applied
because Finnish male mortality rates are known to be very high.
01340
VI-59
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In summary, four cohort studies of workers exposed to phenoxyacetic acid
herbicides or 2,3,7,8-TCDD or both do not report increased risks of stomach
cancer. 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.
By adding together the number of workers exposed to phenoxy acids or
chlorophenols or both from all case studies, an unusually high number of
STSs is shown, considering the rarity of the disease. This excess is
suggestive of an association of cancer with exposure to phenoxy acids or
chlorophenols or both, 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 or chlorophenols or both. They do not
pinpoint the risk to the dioxin contaminants, however. In fact, in one
study, the risk was found to extend to phenoxy acids free of dioxin impur-
ities. In that study, the risk increases to 17 when phenoxy acids known to
contain dioxin impurities (polychlorlnated dibenzodloxins and dibenzofurans)
are considered. The extent of possible observer bias and recall bias intro-
duced into these studies by using self-administered questionnaires is not of
sufficient magnitude to have produced the highly significant risks found in
the studies.
Later studies did not reveal a significant excess risk of STS. However,
methodology problems make these latter studies limited with respect to
01340
VI-60
04/08/88
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evaluating the risk of STSs from exposure to phenoxy acids or chloro-
phenols or both and, consequently, 2,3,7,8-TCDO.
The Swedish case-control studies provide limited evidence for the
carcinogenicity of phenoxy acids or chlorophenols or both In humans.
However, with respect to the dioxin impurities contained therein, the
evidence for the human carcinogenicity for 2,3,7,8-TCDD based on the epi-
demiologic studies is only suggestive because of the difficulty of
evaluating the risk of 2,3,7,8-TCDD exposure in the presence of the con-
founding effects of phenoxy acids and/or chlorophenol.
There Is less evidence incriminating 2,4,5-T or 2,3,7,8-TCDD or both as
the cause of malignant lymphoma and stomach cancer in humans.
Mutagenic^tv Studies
Czeizel and Kiraly (1976) reported an increased incidence (p<0.001) of
chromatid-type and unstable chromosome aberrations in the peripheral lympho-
cytes of workers exposed to the herbicides 2,4,5-trichlorophenoxyethanol
(2,4,5-TCPE) and Bumlnol. The 2,3,7,8-TCDD levels in the final product were
<0.1 mg/kg; however, the exposure levels for Individual workers were not
available.
Mulcahy (1980) reported no increased incidences of chromosomal aberra-
tions in the lymphocytes of 15 soldiers exposed to Agent Orange. The expo-
sure was for 6-15 months and all subjects complained of symptoms, Including
skin eruptions, which they associated with Agent Orange. The analyses were
performed with lymphocytes obtained -10 years after the last exposure, and
comparisons were made with eight subjects who had no history of exposure to
01340
VI -61
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2 , 3,7,8-TCDD. Neither sister chromatid exchange nor structural aberrations
including both gaps and breaks were increased. The authors note that the
long time between exposure and analysis may have accounted for the negative
results.
Also, both Reggiani (1980) and Mottura et al. (1981) have studied inhab-
itants in Seveso, Italy, exposed to 2,3,7,8-TCDO from an accident in a
trichlorophenol manufacturing plant. Reggiani (1980) examined 4 adults and
13 children (3-13 years) for chromosomal aberrations within 2 weeks of the
accident. These 17 individuals were examined to support claims of and
determine extent of injury. Although burn-like skin lesions in these 17
individuals indicated chemical exposure, no increase In chromosomal aberra-
tions was detected. The methods of performing the analyses and the actual
number of aberrations detected were not described. Similar negative results
were reported in an abstract by Mottura et al. (T981). In this study, sub-
jects were chosen from the area of heavy contamination following the acci-
dent (acute high level exposure), from the working population of the plant
(chronic low level exposure) and a nonexposed control population. The num-
ber of subjects in each group was not provided. The specimens were examined
by three Independent laboratories and no laboratory reported an increase in
chromosomal aberrations, although there was a significant difference in the
reported scores between laboratories. There 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 chromoso-
mal aberrations.
Dilernia et al. (1982) conducted additional studies on lymphocytes
prepared in 1976 and 1979 from eight persons considered acutely exposed to
01340 VI-62 * 04/14/88
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2,3,7,8-TCDD in the Seveso accident, eight ICMESA factory workers (consider-
ed chronically exposed), and 14 control subjects (eight had chromosome prep-
arations made in 1976 and six in 1979 ). Cells were examined for average
number of SAs (evidence for functional ribosomal genes), both on a cell
basis and for the large acrocentric chromosomes (D group chromosomes).
There was no change in the frequency of SAs on a per cell basis in any of
the groups as compared to control values, nor In D group chromosomes from
acutely exposed subjects examined immediately after the accident. There
was, however, a decrease in the average frequency of SAs in 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, DILernla et a "I. (1982)
observed a similar decrease In SAs after exposure of lymphocytes to
x-irradlation. It was concluded that the decrease In SAs may have resulted
from mutagenic damage to functional nucleolar organizing regions.
High Risk Subpopulatlons
Little information was found In the available literature to indicate
that specific human subpopulatlons may be unusually susceptible to the toxic
effects of 2,3,7,8-TCDD. Limited evidence from the Seveso area and Eastern
Missouri indicates that children may be more sensitive to the immunological
effects of 2,3,7,8-TCDD. Tognonl and Bonaccorsi (1982) found elevated
peripheral blood lymphocytes, lymphocyte blastogenlc response, and serum
complement activity In exposed children; however, no immunological effects
were detected in adults (Regglani, 1980; May, 1982). Among adults and
children exposed to contaminated soil In horse arenas in Eastern Missouri,
the only adverse health effects reported were in children (Klmbrough et al.,
1977). Children playing In the arena were probably In more Intimate contact
01340 VI-63 04/14/88
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with the contam-inated soil, and thus subject to higher exposures, than were
the adults.
Summary
By adding together the number of workers exposed to phenoxy acids and/or
chlorophenols from all case studies, an unusually high number of STSs is
shown, considering the rarity of the disease. This excess is suggestive of
an association of cancer with exposure to phenoxy acids and/or chloro-
phenols, 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
pinpoint the risk to the dloxln contaminants, however. In Fact, in one
study, the risk was found to extend to phentfxy acids free of dloxin
impurities. In that study, the risk Increases to 17 when phenoxy acids
known to contain dloxln Impurities (polychlorinated dibenzodioxins and
dibenzofurans) are considered. The extent of possible observer bias and
recall bias Introduced into these studies by using self-administered
questionnaires is not of sufficient magnitude to have produced the highly
significant risks found in the studies.
Later studies did not reveal a slgnficant excess of risk of SIS.
However, methodology problems make these latter studies limited with respect
to evaluating the risk of STSs from exposure to phenoxy acids and/or
chlorophenols and, consequently, 2,3,7,8-TCDD. Epidemiological studies on
more recently exposed populations, where dloxln exposure can be more accur-
ately assessed, and subsequent studies, Including a higher number of deaths,
01340
VI-64
04/14/88
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on populations previously studied will increase the pool of data on dioxin
and help evaluate Us carcinogenic risk in humans.
Either acute or chronic exposure to 2,3,7,8-TCDD may result In chlor-
acne, altered liver function, hematological pathologies, porphyria cutanea
tarda, hyperpigmentation, hirsutism and neural degeneration in the extremit-
ies. Stevens (1981) has estimated that the minimum cumulative toxic dose of
2,3,7,8-TCDD in humans 1s 0.1 yg/kg.
The toxic effects of exposure to 2,3,7,8-TCDD may persist for many
years, though some effects seem to be reversible in some cases. Even though
2,3,7,8-TCDD has been found to be fetotoxic and/or teratogenic in all animal
species tested (see the Teratogenicity and Reproductive Toxicity Section in
Chapter V), epidemiological studies have failed to demonstrate a convincing
connection between 2,3,7,8-TCDD exposure and spontaneous abortions or
malformations in humans. These studies are difficult to interpret, since
quantitative exposure data are not available. Some evidence of cyto-
genetic damage has been reported In humans exposed to chemicals contaminated
with 2,3,7,8-TCDD, but negative results have also been reported; exposures
were not quantitated; and the other chemicals cannot be ruled out as causa-
tive agents.
01340
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VII. MECHANISM OF TOXICITY
A number of studies have attempted to determine the mechanism of toxic-
ity of 2,3,7,8-TCOO. The ultimate purpose Is to provide a better estimate
of man's relative sensitivity to 2,3,7,8-TCDQ and other compounds having a
similar mode of action. Specifically, these studies may be able to explain
the reason for the marked Interspecies differences In 2,3,7,8-TCOO toxicity
and, thus, help determine If humans possess factors that are associated with
sensitivity to 2,3,7,8-TCDD toxicity.
Receptor-Mediated Toxicity
Pharmacogenetlc studies have played an Important role In understanding
the biologic and toxic effects of drugs and xenoblotlcs. Nebert and
coworkers have shown that carcinogenic polycycllc aromatic hydrocarbons
{PAHs) Induce the cytochrome P-450-dependent monooxygenase AHH 1n certain
responsive strains of mice (e.g., C57B1/6J, BALBc, C3HF/He] whereas this PAH
Induction activity 1s minimal or nonexistent 1n nonresponslve strains
(DBA/2J) (Nebert, 1979, 1982; Nebert and Glelen, 1972; Nebert and Jensen,
1979; Nebert et al., 1972, 1981, 1983). The gene complex responsible for
the Induction of AHH and several other enzymes has been designated the Ah
locus which comprises regulatory, structural and possible temporal genes.
Extensive studies on genetically Inbred responsive and nonresponslve mice
(and their backcrosses) Indicate that these differences are related to the
Ah regulatory gene and Its 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.
01350
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Since the carcinogenic and toxic effects of PAHs are dependent on their
oxidative metabolism to reactive electrophlllc forms, It 1s not surprising
that the Ah receptor plays an Important role In mediating their toxicity and
carcinogenicity (Kourl, 1976; Kourl et al., 1974; 8ened1ct et al.t 1973;
*
Sham et al., 1979; Thomas et al., 1973; Legraverend et al., 1980; Duran-
Reynolds et al., 1978; Robinson et al.t 1975; Mattlson and Thorgelrsson,
1979). Responsive mice are more susceptible to the toxic (Inflammation,
fetotoxIcUy, primordial oocyte depletion) and carcinogenic effects of PAH
at organs/tissues 1n direct contact with the applied chemical; In contrast,
nonresponslve mice are more susceptible to the tumorlgenlc effects of PAHs
at tissue/organ sites remote from the initial site of exposure to the PAHs.
These differences In susceptibility are due to several factors including
AHH-medlated toxlcatlon and detoxlcatlon.
%
2.3.7.8-TCPO: Segregation of Activity with the Ah Locus. Genetic
studies also support the role of the Ah receptor In mediating the toxic and
biologic effects of 2,3,7,8-TCOO. Initial studies by Poland and coworkers
(Poland et al., 1974, 1983; Poland and Glover, 1975; Nebert et al., 1975)
demonstrated that the microsomal AHH-1nduc1ng activity of 2,3,7,8-TCDD and
3-MC 1n several genetically Inbred mice strains were similar. Like 3-MC and
related PAHs, 2,3,7,8-TCOD Induced AHH 1n several responsive mouse strains
(I.e., C57B1/6J). In contrast to 3-MC, 2,3,7,B-TCDD Induced microsomal AHH
1n the 08A/2J nonresponslve mice; however, the E0^o for this biologic
response was significantly higher than values reported for the responsive
mice. In genetic crosses between responsive C5781/6 and nonresponslve DBA/2
mice it was also shown for both 3-MC and 2,3,7,8-TCDD that the trait of
responsiveness Is Inherited In a simple autosomal dominant mode (Poland and
01350 VI1-2 09/18/84
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Knutson, 1982). It has been suggested that the observed differences 1n the
activities of 3-MC and 2,3,7,8-TCDD are related to their relative. Ah
receptor affinities (Poland and Knutson, 1982) and the pharmacokinetic and
metabolic factors which would more rapidly diminish the ¦available" concen-
t
tratlons of 3-MC caused by metabolism and excretion.
Several studies with 2,3,7,8-TCOD In genetlcally Inbred mice support the
receptor mediated hypothesis. The Induction of UDP-glucuranosyl transfer-
ase, DT dlaphorase, 4-am1nolevul1n1c ac1dt glutathlone-S-transferase 8,
T-aldehyde dehydrogenase and cholIneklnase by 2,3,7,8-TCDD or 3-MC 1n
genetically Inbred mice have also been shown to segregate with the Ah locus
(Beatty and Neal, 1976b; Owens, 1977; Klrsch et al., 1975; Dietrich et al.,
1978; I s hi da te et al., 1980; Poland and Glover, 1973a). Toxicology studies
with genetically-Inbred mice confirm the role of the Ah locus In mediating
several toxic effects Including porphyria, Immunotoxlclty (a wasting syn-
drome), thymic atrophy and cleft palate formation (Jones and Sweeney, 1980;
Poland and Glover, 1980; Courtney and Moore, 1971; Vecchl et al., 1980,
1983). Poland et al. (1982) have also linked the tumor-promoting activity
of 2,3,7,8-TCDD 1n hairless mice to the cytosollc receptor. In vitro
studies with XB cells 1n culture also support the role of receptor 1n
mediating a dose-related cell keratlnlzatlon by 2,3,7,8-TCDO which resembles
some of the characteristics of chloracne (Knutson and Poland, 1980). This
cell line Is also responsive to ANH Induction and contains a cytosollc
receptor binding protein. Although the murine Ah receptor has not been
characterized, several studies confirm that a protein with high affinity for
3-MC and 2,3,7,8-TCDD Is present In low concentrations In the hepatic
(-30-50 fmolar) and extrahepatlc tissues of responsive C57B1/6J mice
01350
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(Greenlee and Poland, 1979; Okey et al., 1979, 1900; Poland et al., 1976;
Mason and Okey, 1982; Gaslewlcz and Neal, 1982; Okey and Vella, 1982; Okey,
1983; Nebert et al., 1983). In responsive C57B1/6J mice and Sprague-Dawley
rats, but not 1n nonresponslve 0BA/2J mice, the Ah receptor can be Induced
by pretreatment with phenobarbltal which Is the only known agent at present
that has been demonstrated to affect tissue concentrations of the receptor
(Okey and Vella, 1984). Although the Ah receptor has not been detected 1n
the cytosol of D8A/2J mice, after the administration of radiolabeled
2,3,7,8-TCDD to these mice, some of the radlolabel Is detected In the nuclei
of the nonresponslve mice. Moreover, the sedimentation characteristics of
the [3H]-2,3,7,8-TCDD:nuclear protein complex In 0BA/2J mice are similar
to those observed with the bound Ah cytosollc receptor protein In C57B1/6J
mice using a sucrose density gradient centrlfugatlon separation technique
(Okey, 1983). Several reports have also demonstrated that the cytosollc Ah
receptor protein migrates Into the nucleus of the cell only after binding
with 2,3,7,8-TCDD (Greenlee and Poland, 1979; Okey et al., 1979, 1980) and
this parallels the observations noted for the Interactions between steroids
and their receptor proteins.
2.3.7.8-TCOD and Related Toxic Halogenated Arvl Hydrocarbons:
Structure-Actlvltv Correlations. The evidence for a receptor mediated
mechanism of action for 2,3,7,8-TCDD 1s supported by data reported for the
effects of other halogenated aryl hydrocarbons In genetically Inbred mice
and other diverse animal species. A number of reviews and comparative
studies (Allen et al., 1979; Allen and Norback, 1977; Klmbrough, 1974;
Klmbrough et al.,- 1978; McConnell and Moore, 1979; Taylor, 1979) clearly
Indicate that the toxic halogenated mixtures and Individual compounds
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{Including the PCDDs, PCDFs, PC B s and PBBs) elicit similar toxic and
biologic responses which Include 1) a wasting syndrome which 1s manifested
by a progressive weight loss and decreased food consumption by the treated
animals; 2) skin disorders including acneform eruptions or chloracne,
fc
alopecia, edema, hyperkeratosis, and hypertrophy of the Meibomian glands;
3) lymphoid involution and atrophy; 4) porphyria (resembling porphyria
cutanea tarda); 5) endocrine and reproductive disorders; 6) modulation of
chemical carcinogenesis; and 7) the induction of numerous enzymes including
the cytochrome P-448 (or P-450c) dependent monooxygenases. It is apparent
that the effects of these compounds are not manifested in all the animal
species tested. McConnell and Moore (1979) summarized the pathologic
findings observed in several animal species after pretreatment with PCDDs,
PCDFs, PCBs and PBBs and these data illustrate the different species and
organ/tissue susceptibilities to these compounds. It is also evident that
for most of these effects, all the toxic halogenated aromatics elicit
similar effects 1n these species which also contain the cytosollc receptor
protein (Carlstedt-Duke, 1979; Carlstedt-Ouke et al., 1979, 1981; Okey,
1983; Okey and Vella, 1982; Mason and Okey, 1982). These observations
support a common mechanism of action for all the toxic halogenated aryl
hydrocarbons (Poland and Knutson, 1982; Safe et al., 1982; HcConnell and
Moore, 1979).
Several reports have demonstrated the effects of structure on the activ-
ity of PCDDs. The most active member of this group is substituted in the
lateral 2, 3, 7 and 8 positions; activity is decreased with 1) decreasing
lateral substituents, and 2) increasing CI substitution. Moreover, for
several PCDDs, there is an excellent correlation between the toxicity of
01350
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Individual PCOO congeners in guinea pigs and mice (HcConnell et al., 1978b)
and their AHH Induction potencies in chick embryos and rat hepatoma H-4-II-E
cells in culture and their binding affinities for the C57B1/6J mouse hepatic
cytosollc receptor protein (Poland et al., 1976, 1979; Bradlaw et al.t 1980;
Bradlaw and Casterllne, 1979). Comparable structure-activity correlations
have been reported for the PCDFs 1n which the most active compound, 2,3,7,8-
TCDF, 1s an approximate isostereomer of 2,3,7,8-TCDD (Poland et a 1., 1979;
Poland and Knutson, 1982). Moreover, like the PCDDs, there was an excellent
correlation between the toxicity of several Individual PCDFs (Yoshlhara et
al., 1981), their AHH induction potencies In rat H-4-II-E hepatoma cells and
binding affinities to male Wlstar rat hepatic cytosollc receptor protein
(Bandlera et al., 1983).
The most active PCB congeners, 3,4,4',5-tetra-, 3,3',4,4'-tetra-,
3,3',4,4',5-penta- and 3,3*,4,4',5P5'-hexachlorobiphenyl, are substituted at
both para and at two or more meta positions. The four coplanar PCBs Induce
rat hepatic microsomal AHH and cytochromes P-450a, P-450c and P-450d and
resemble 3-HC and 2,3,7,B-TCDD In their mode of Induction of the cytochrome
P-450 Isozymes (34) (Parkinson et al., 1980a,b, 1983; Safe et al., 1982;
Sawyer and Safe, 1982; Poland and Glover, 1977; Goldstein et al., 1977).
Like Aroclor 1254, all the monoortho and at least eight diortho-chloro
analogs of the coplanar PCBs exhibited a "mixed-type" Induction pattern and
Induced microsomal AHH, DMAP N-demethylase and cytochromes P-450a to P-450e
{Parkinson et al., 1980a,c, 1983). Quant1tative structure-activlty rela-
tionships (QSARs) within this series of PCBs were determined by comparing
their AHH Induction potencies (ECjq) In rat hepatoma H-4-II-E cells and
their binding affinities (ED^q) for the 2,3,7,8-TCDD rat cytosollc recep-
tor protein (Sawyer and Safe, 1982; Bandlera et al., 1983). The results
01350 VI1-6 09/10/84
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showed that there was an excellent correlation between AHH Induction
potencies and receptor binding avidities of these compounds and the order or
activity was coplanar PCBs (3,3',4,4'-tetra-, 3,3',4,4',5-penta- and
,4,4*,5,5'-hexachlorob1phenyls) > 3,4,4*,5-tetrachlor obi phenyl > mono-
ortho coplanar PC8s > dlortho coplanar PCBs. It was also apparent that the
relative toxicities of this group of PCBs paralleled their biological
potencies (Blocca et al.# 1981; Yoshlhara et al,» 1979; Marks et al., 1981;
McKlnney et al., 1976; Yamamoto et al.f 1976; Ax and Hansen, 1975; KurokV
and Hasuda, 1977).
The coplanar and monoortho coplanar PCBs also exhibit differential
effects In the Inbred C57B1/6J and DBA/2J mice. These compounds Induce AHH
and cause thymic atrophy In the former "responsive" mice whereas at compar-
able or higher doses none of these effects are observed in the nonresponslve
0BA/2J mice (Parkinson et al., 1982; Robertson et al., 1984). The results
obtained for structurally diverse PCDDs, PCBs and PCDFs clearly support the
role of the receptor protein In Initiating the broad spectrum of biologic
and toxic effects elicited by these chemicals. Bandlera et al. (1983) have
demonstrated that the 2,3,7,8-TCDD receptor protein 1s not only susceptible
to halogen substitution patterns but also the structure of the substltuent.
The cytosol receptor binding avidities and AHH Induction potencies In rat
hepatoma H-4-II-E cells for several 4,-X-2,3,4>5-tetrachlorob1pheny1s were
remarkably dependent on the structure of the X substltuent. The binding
data for 13 different substltuents was subjected to multiparameter regres-
sion analysis to correlate binding avidities with the physical chemical
characteristics of the critical lateral X substltuents. The equation
log (1) = 1.53
-------
showed that Ugand binding was dependent on substltuent electronegativity
{c), llpophlllclty (1) and hydrogen binding (H8) with a correlation
coefficient (r) equal to 0.978 for 13 different substltuents.
The receptor mediated hypothesis for the mechanism of action of 2,3,7,8-
TCDD still requires further confirmation and numerous problems must be
clarified. For example:
1. Several cell culture lines which appear to have the Ah recep-
tor are highly resistant to the toxicity of 2,3,7,8-TCDD; the
nonresponslve HTC and responsive H-4-II-E cell lines (I.e.,
for AHH InduclblHty by 2,3,7,8-TCDD) do not possess cytosollc
receptor; however, the nonresponslve HTC cells possess more
nuclear receptor binding protein than the responsive H-4-II-E
cells (Okey, 1903; Okey et al., 1980).
2. Hepatic cytosollc receptor levels In rats (Wlstar and Sprague-
Dawley), C5781/6J mice, hamsters and guinea p 1 gs are compar-
able (Gaslevlcz et al., 1983b); however, their susceptibility
to the biologic and toxic effects of 2,3,7,8-TCOD are highly
variable: guinea pigs are highly susceptible to the lethal
effects of 2,3,7,8-TCDD (LD50 = 1-2 yg/kg) whereas the
susceptibility of the other species follows the order rat >
C57B1/6J mice > DBA/2J mice > hamster (Neal et al., 1982).
Metabolism
The metabolism of 2,3,7,8-TCDD has been examined in the guinea pig, rat,
mouse and hamster. Urine and bile from 1*C-TCOD-treated animals were
found to be free of unmetabollzed 2,3,7,8-TCOO, demonstrating that metabol-
ism was required for elimination through these routes (Olson et al., 1983).
The direct Intestinal elimination of unchanged 2,3,7,8-TCDD 1n feces
suggests, however, that some 'routes of excretion may not be dependent on
prior metabolism of the toxin (Olson et al., 1983). Thus, 1t 1s not
possible to directly correlate the half-life for elimination of 2,3,7,8-TCDD
with Us In v1 vo rate of metabolism In a given species. The relative per-
sistence of 2,3,7,8-TCDD In a given species may be related to the in vivo
01350
VI1-8
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rate of 2,3,7,8-TCDD metabolism, excretion of the toxin not dependent upon
metabolism (direct Intestinal elimination, lactation, sebum), and the rela-
tive tissue distribution of 2,3,7,8-TCDD, particularly to adipose stores.
C^ual 1tative and quant1tat1ve dlfferences 1n the metabolIsm and d1sposl11 on
of 2,3,7,8-TCDD have been observed between various species, and these may 1n
part be related to the remarkable Interspecies differences 1n sensitivity to
2,3,7,8-TCDD toxicity (Olson et al.t 1983).
Polger et al. (1982a) suggest that 2,3,7,8-TCDD metabolism represents
detoxification, since they observed relatively little toxicity In guinea
pigs given extracts of dog bile containing 2,3,7,8-TCDD metabolites. How-
ever, a recent study proposes that metabolites of 2,3,7,8-TCDO may inhibit
uroporphyrinogen decarboxylase activity and lead to 2,3,7,8-TCDD-induced
porphyria (DeVerneull et al., 1983). Current data on the structural Identi-
fication of 2,3,7,8-TCDD metabolites suggest that reactive epoxide Inter-
mediates may be formed during metabolism (Polger et al., 1982b; Sawahata et
al., 1982). Poland and Glover (1979) reported that the maximum possible in
vivo covalent binding of 1,6-aH-2,3,7,8-TCDD derived radloactlvi ty to
hepatic DNA was 4 orders of magnitude less than the levels of binding
observed with other chemical carcinogens. The study did find much higher
levels of 2,3,7,8-TCDD derived radioactivity bound to hepatic protein of the
rat. No data is available, however, on the degree 2,3,7,8-TCDD derived
radioactivity 1s bound to tissues of various species of laboratory animals,
which have demonstrated remarkable variability 1n sensitivity to 2,3,7,8-
TCOD. While biliary excretion products may represent detoxified, polar
metabolites of 2,3,7,8-TCOD, it remains to be shown whether unexcreted
reactive metabolites Initiate some of the toxic responses associated with
exposure to this toxin.
01350 VII-9 h 08/10/84-
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Vitamin A Depletion
Many of the toxic effects of 2,3,7,8-TCDD resemble the effects of
vitamin A deficiency, such as epithelial lesions, keratosis and Immuno-
suppression. The administration of a single oral dose of 0.1, 1.0 or 10
»g 2,3,7,8-TCDD/kg bw produces a dose-related decrease 1n the hepatic
storage of retlnol In Sprague-Dawley rats (Thunburg et a 1.. 1979, 1930).
The authors suggested, but did not demonstrate, that the low storage of
retlnol In the 2,3,7,0- TCOD-treated animals 1s the result of an Increased
turnover of retlnol. These results suggest that an Induced vitamin A defi-
ciency may be responsible for some* but not all, of the toxic effects pro-
duced by 2,3,7,8-TCDD. At the highest dose of 2,3,7,8-TCDD, dietary retlnol
supplements could not fully compensate for the 2,3,7,8-TCDD-produced
decrease In hepatic retlnol content.
Lipid Peroxidation
Increased lipid peroxidation has been suggested as a possible mechanism
of 2,3,7,8-TC0D-1nduced toxicity (Sweeney and Jones, 1983). This hypothesis
is based on the following limited pieces of evidence. First, Iron defi-
ciency Inhibits In. vitro lipid peroxidation (Bus and Gibson, 1979; Sweeney
et al., 1979) and reduces the hepatotoxlc effects of 2,3,7,8-TCDD (Sweeney
et al., 1979). Secondly, llpofusdn pigments, by-products of lipid peroxi-
dation, are Increased 1n the heart muscle of rats treated with 2,3,7,8-TCDD
(Albro et al., 1978). Thirdly, Sweeney and Jones (1983) reported that
administration of the antioxidant butylated hydroxyanlsole (BHA) at a level
of 0.75% 1n the diet provided some protection from 2,3,7,8-TCDD-1nduced
prophyrla and neutral lipid accumulation. At this dose level of BHA, 4 of
the 6 mice (sex not specified) tested were protected; however, at a lower
01350
VII-10
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dose (0.25%), all animals were protected from these toxic effects. No
beneficial effects were observed when the antioxidant vitamin E (0.01%) was
Included 1n the diet.
* Recently, Stohs et al. (1983) obtained direct evidence that 2,3,7,8-TCDD
accelerates lipid peroxidation In Sprague-Oawley rats. Groups of 4-0 female
rats were treated for 3 days with 2,3,7,8-TCDD at doses of 0, 10, 20 or 40
ug/kg by gavage (In a corn oil vehicle). At days 1, 6 and 11 after the
last treatment the animals were sacrificed and lipid peroxidation was deter-
mined in Isolated liver microsomes by the reaction of formed malondlaldehyde
with thlobarbltur1c acid. At all sacrifice periods, Increased lipid peroxi-
dation was observed and the Increase was dose-related. The maximal Increase
detected on day 6 after the last treatment was 5- to 6-fold greater than In
the controls. In addition, these workers measured lipid peroxidation In
vivo by the determination of conjugated dlenes 1n rats receiving 2,3,7,8-
TCD0 at 40 yg/kg. Using this latter method, similar Increases In lipid
peroxidation were detected, although the maximal Increase of 2.35-fold was
observed at day 1 postexposure rather than day 6. The authors suggested
that the in vivo formation of reactive free radicals during lipid peroxida-
tion could account for the nonspecific nature of 2,3,7,8-TCDD toxicity.
Endocrine Imbalance ^
Some of the toxic response to 2,3,7,8-TCDD, Including hirsutism and
diminishing libido. Indicate that 2,3,7,8-TCDD may produce some of Us
toxicity through endocrine disturbances (Oliver, 1975). Nlenstedt et al.
(1979) reported that a single oral dose of 20 pg 2,3,7,8-TCDD/kg bw
significantly reduced testosterone catabollsm. Catabollsm of exogenous
estrogen In ovarlectomlzed rats 1s also decreased by 2,3,7,8-TCDD pre-
01350 VI1-11 > 09/18/84
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treatment (Shlverlck and Huther, 1982). In this study, there was a 57%
Increase 1n serum estrone concentrations following administration of 10 mg
estrone/100 g by/day for 4 days to either control or 2,3,7,8-TCOD pretreated
ovaMectomlzed rats. No differences were observed 1n the Increase In
*
uterine wet weight following estrone administration 1n control and 2,3,7,8-
TCDD pretreated rats. Thus, the uterotrophlc response was not altered by
any 2,3,7,8-TCDD-med1ated change 1n estrone disposition.
Shlverlck and Huther (1983) also measured estradiol metabolism In female
Holtzman rats given 2,3,7,8-TCDO at a dose of 1 yg/kg bw on days 4-19 of
gestation. At this fetal toxic dose, the catechol estrogen formation abil-
ity of Isolated liver microsomes from the dams was decreased 50% when mea-
sured on day 20 of gestation. These microsome preparations had a 4-fold
Increase 1n the 7a-hydroxy1at1on of testosterone, while there was no
change In the 16a- or 66-hydroxylase activity. Although steroid metabo-
lism was altered 1n microsomes Isolated from 2,3,7,8-TCDD-treated pregnant
rats, similar exposure of pregnant rats on days 4-15 of gestation did not
result 1n any change In circulating levels of serum 170-estradlol. The
authors suggested that other mechanisms besides liver metabolism of steroids
may be Involved In the fetotoxlc effect of 2,3,7,8-TCDO.
Gustafsson and Ingelman-Sundberg (1979) observed that 2,3,7,8-TCDO pro-
duced greater change In steroid metabolism 1n female Sprague-Dawley rats
than 1n male rats of the same strain, resulting In a liver enzyme pattern
displaying less sex differentiation than 1n unlnduced rats. Based on this
result, they propose that some of the effects of 2,3,7,8-TCDD result from an
Interaction with the hypothalamo-p1tultary axis, rather than from a direct
effect on steroid metabolism.
01350
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Since glucocorticoid hormones are known to have a catabolic effect on
lymphoid tissues, such as the thymus and spleen, and these tissues degener-
ate after exposure of rats to 2,3,7,8-TCDD, Neal et al. (1979) investigated
the ability of 2,3,7,8-TCDD to either stimulate the production or mimic the
effects of these hormones. In male Sprague-Dawley rats treated by gavage
with 2,3,7,8-TCDD at a dose of 50 pg/kg (the ~L0^), there was a slight,
depression in blood glucocorticoids during post-treatment days 1-4, followed
by an -2.5-fold increase on post-treatment days 7 and 14. While in competi-
tive binding assays between 2,3,7,8-TCDD and a synthetic hormone, dexametha-
sone, 2,3,7,8-TCDD had no affinity for the hormone receptor. Thus, 2,3,7,8-
TCDD may stimulate glucocorticoid production, but was not able to mimic the
action of these hormones by binding to the glucocorticoid receptor. It was
determined, however, that the Increase in glucocorticoids was likely not to
participate in the toxicity of 2,3,7,8-TCDD through adrenal hyperfunctlon,
since prior adrenalectomy did not provide any protection from the lethal
effects of 2,3,7,8-TCDD in rats.
Hyperthyroidism, observed In amlnals exposed to 2,3,7,8-TCDD, is
suggested to be Involved In the manifestation of the pathological conditions
associated with 2,3,7,8-TCDD exposure (Bastomsky, 1977). A partial protec-
tion from 2,3,7,8-TCDD Induced wasting syndrome and Immunotoxicity as a
result of thyroidectomy has been observed (Rozman et al., 1984; Pazdernik
and Rozman, 1905) although this process does not appear to involve any
change In the liver Ah-receptor regulated precesses (Henry and Gaslewicz,
1986). The data on the concentration of the Ah-receptor in various human
tissues are limited (Roberts et al.# 1985) and the toxic response in humans
has not yet been correlated with enzyme induction mediated by the
Ah-receptor.
01350
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Hakansson and Ahlborg (1985) pretreated male Sprague-Dawley rats with
2,3,7,8-TCDD at 10 yg/kg bw 4 days before the oral adminstration of 1200
IU/kg of retinyl acetate. One hundred ninety-two hours postadminstration of
retinyl acetate the 2,3,7,8-TCDD-pretreated rats excreted 41% if the retinyl
acetate compared to the control, excreting only 30%. After 2,3,7,8,-TCDD
treatment the decrease in vitamin A content was 39-53, 19-67 and 18-44% in
the liver, intestine and epididymis, respectively. 2,3,7,8-TCDD treatment
also Influenced vitamin A content in the thymus, initially increasing by 42%
in 6 hours and the decreasing by 40% in 192 hours as compared to the
controls. 2,3,7,8-TCDD pretreatment increased the vitamin A content in the
kidney 3-30 times that of the control. It is important to note that the
kidney becomes the primary vitamin A storage organ in vitamin A deficient
animals (Johnson and Baumann, 1947; Moore and Sharman, 1950). In a similar
study Thunberg and Hakansson (1983) has also found an increase of vitamin A
storage in the kidney after a single oral dose of 2,3,7,8-TCDD in male
Sprague-Dawley rats. Result from these observations suggest strongly that
pretreatment with a single oral dose of 2,3,7,8-TCDD can affect both storage
and excretion of retinyl acetate as well as the vitamin A storage in several
tissues.
Furthermore, since the underlying cause of the wasting syndrome is not
clearly understood, it remains to be determined if the severe body weight
loss associated with 2,3,7,8,-TCDD toxicity is the cause of the death of the
animal, or is an effect which caR be separated from lethality. Aust (1984)
suggested that 2,3,7,8-TCDD activated thyrotropin releasing hormone, which
has an anorectic action, and in conjunction with 2,3,7,8-TCDD-induced
vitamin A depletion results in loss of body weight. Regardless of the
01350
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04/12/88
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mechanism or length of exposure, the wasting sydrome appears to be an indi-
cation of impending death rather than an early sign of toxicity.
Summary
Carcinogenic PAHs induce cytochrome P-450-dependent monooxgenase AHH in
genetically responsive strains of mice, possibly by binding to a cytosolic
receptor protein that translocates to the nucleus to Initiate induction of
AHH (Nebert, 1979, 1982; Nebert and Gielen, 1972; Nebert and Jensen, 1979;
Nebert et al.t 1972, 1981, 1983). Since the carcinogenic and toxic effects
of PAHs require oxidation to reactive electrophi 1 s, it is likely that the Ah
receptor and AHH induction play an important role in mediating their toxi-
city and carcinogenicity.
Several studies with 2,3,7,9-TCDD in inbred mice support the Ah receptor
mediated hypothesis and have shown that effects associated with the Ah
receptor (porphyria, immunotoxicity, thymic atrophy, cleft palate) segregate
with the Ah locus (Beatty and Neal, 1976b; Poland and Glover, 1973a, 1980;
Jones and Sweeney, 1980). In responsive mice and Sprague-Dawley rats, the
Ah receptor can be induced with phenobarbital, which has been demonstrated
to increase the concentration of the receptor.
A number of reviews and comparative studies (Allen et al., 1979; Allen
and Norback, 1977; Kimbrough, 1974; Klmbrough et al., 1978; McConnell and
Moore, 1979; Taylor, 1979) indicate that the toxic halogenated compounds
(PCDDs, PCDFs, PCSs and PBBs) all elicit similar toxic responses (a wasting
syndrome, skin disorder, lymphoid atrophy and Immunodeficiency, porphyria,
endocrine and reproductive disorders, modification of chemical carcinogene-
sis and hepatic enzyme induction) In'Several different species. A common
01350
VII-15
04/08/88
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cytosolic receptor protein (Ah-receptor) appears to be present in species
that respond similarly to these compounds, indicating a common mechanism of
action (Safe, 1982; McConnell and Moore, 1979; Poland and Knutson, 1982).
Structure activity studies have determined that the 2,3,7,8-(laterally
substituted) member is the most active of the PCDDs (McConnell et al.,
1978b). Of the PCDFs, 2,3,7,8-TCDF is the most active (Poland et al.t
1979). Activity of the toxic halogenated aryl hydrocarbons correlates well
with their ability to induce.AHH in chick embryos (McConnell et al., 1978b)
and rat hepatomas H-4-II-E cells (McConnell et al., 1978b; Yoshihara et al.,
1981). The activity of the various PCDD congeners has also been correlated
with their binding affinity with cytosolic receptor protein in mouse (Poland
et al., 1976, 1979; Bradlow et al., 1980; Bradlow and Casterline, 1979) or
rat (Bandiera et al., 1983) hepatic cytosolic receptor protein. Activity of
PCDD congeners is diminished by decreasing lateral substitution or increas-
ing CI substitution in the other positions (McConnell et al., 1978b).
Slight toxicity was observed in guinea pigs administered extracts of the
bile of dogs treated with 2,3,7,8-TCDD led Polger et al. (1982a) to conclude
that metabolism represents detoxification. The structural identification of
2,3,7,8-TCDD metabolites, however, has led investigators (Polger et al.,
1982b; Sawahata et al.( 1982) to hypothesize that reactive epoxide Inter-
mediates may form that account for a substantial amount of the toxicity
associated with 2,3,7,8-TCDD. Compared to other chemical carcinogens, how-
ever, the binding of 1,6-3H-2,3,7,8-TCDD derived radioactivity to hepatic
DNA was less by about four-orders of magnitude than the carcinogen DMN
(Poland and Glover, 1979). Much higher levels of radioactivity, however,
were found bound to cytosolic protein.
01350
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Since some of the effects of 2,3,7,8-TCDD toxicity resemble vitamin A
deficiency (epithelial keratosis, immunosuppression) Thunburg et al. (1979.
1980; Hakansson and Ahlborg, 1985) investigated the ability of single low
oral doses of 2,3,7,8-TCDD to reduce hepatic storage of retinol in rats. A
dose-related decrease in hepatic retinol was demonstrated. At the highest
dose of 2,3,7,8-TCDD, dietary retinol supplements could not fully compensate
for the 2,3,7,8-TCDD- induced hepatic losses of retinol.
Increased lipid peroxidation has been suggested as a possible mechanism
for 2,3,7,8-TCDD-induced toxicity (Sweeney and Jones, 1983). It was demon-
strated that iron deficiency, which reduces lipid peroxidation, reduced
2,3,7,8-TCDD-lnduced hepatotoxicity (Bus and Gibson, 1979; Sweeney et al.,
1979). Lipofuscin pigments, by-products of lipid peroxidation, accumulate
in the cardiac muscle of 2,3,7,8-TCDD treated rats (Albro et al.t 1978).
Butylated hydroxyanlsole, a known antioxidant, provided some¦protection to
mice treated with 2,3,7,8-TCDD (Sweeney and Jones, 1983). Dietary vitamin E
was not protective In this study. Recently, a dose-related increase in
liver microsomes was demonstrated in rats treated with oral doses of
2,3,7,8-TCDD (Stohs et al., 1983).
Some of the effects associated with 2,3,7,8-TCDD (hirsutism, diminished
libido) suggest that the compound may induce some of Us toxicity through
endocrine disturbances (Oliver, 1975). 2,3,7,8-TCDD has been shown to
retard catabolism of testosterpne (Nlenstedt et al., 1979) and estrone
(Shiverlck and Muther, 1982) and estradiol (Shiverlck and Muther, 1983).
Since glucocorticoids are known to have a lytic effect on lymphoid tis-
sues and lymphoid atrophy is part of the picture of 2,3,7,8-TCDD toxicity,
01350
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Neal et al. (1979) investigated the ability of the compound to stimulate
corticoid production or mimic its effect by competitively binding to recep-
tor sites. Rats treated with a single dose of 2,3,7,8-TCDD responded with
slightly decreased blood corticoids for ~4 days followed by a substantial
(-2.5-fold) increase on post-treatment days 7-14. 2,3,7,8-lCDD did not,
however, bind to receptor sites to which dexamethasone did bind. Gustafsson
and Ingelman-Sundberg (1979) proposed a hypothalamic-pituitary axis for the
action of 2,3,7,8-TCDD on steroid levels rather than a direct effect on
steroid metabolism.
01350
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VIII. QUANTIFICATION OF TOXICOLOGICAL EFFECTS
Introduction
The .quantifIcation of toxlcologlcal effects of a chemical consists of
separate assessments of noncarcinogenic and carcinogenic health effects.
Chemicals that do not produce carcinogenic effects are believed to have a
threshold dose below which no adverse, noncarcinogenic health effects occur,
while carcinogens are assumed to act without a threshold.
In the quantification of noncarclnogenic effects, a Reference Dose
(RfD), [formerly termed the Acceptable Daily Intake (AD I) ] Is calculated,
lhe RfD Is an estimate (with uncertainty spanning perhaps an order magni-
tude) of a dally exposure to the human population (Including sensitive
subgroups) that is likely to be without an appreciable risk of deleterious
health effects during a lifetime. The RfD Is derived from a no-observed-
adverse-effeet level (NOAEL), or lowest-observed-adverse-effeet level
(LOAEL), Identified from a subchronlc or chronic study, and divided by an
uncertainty factor(s) times a modifying factor. The RfD is calculated as
follows:
RfD = = mg/kg bw/day
[Uncertainty Factor(s) x Modifying Factor]
Selection of the uncertainty factor to be employed In the calculation of
the RfD is based upon professional Judgment, while considering the entire
data base of toxlcologlcal effects for the chemical. In order to ensure
that uncertainty factors are selected and applied in a consistent manner,
01360 VIII-1 * 07/22/87
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the U.S. EPA (198*9) employs a modification to the guidelines proposed by the
National Academy of Sciences (NAS, 1977, 1980) as follows:
Standard Uncertainty Factors (UFs)
Use a 10-fold factor when extrapolating from valid experimental
results from studies using prolonged exposure to average healthy
humans. This factor is intended to account for the variation
in sensitivity among the members of the human population. [ 1 OH]
Use an additional 10-fold factor when extrapolating from valid
results of long-term studies on experimental animals when
results of studies of human exposure are not available or are
inadequate. This factor 1s intended to account for the uncer-
tainty In extrapolating animal data to the case of humans.
[10A]
Use an additional 10-fold factor when extrapolating from less
than chronic results on experimental animals when there is no
useful long-term human data. This factor is Intended to
account for the uncertainty in extrapolating from less than
chronic NOAELs to chronic NOAELs. [1 OS]
Use an additional 10-fold factor when deriving an RfD from a
LQAEL instead of a NOAEL. This factor is intended to account
for the uncertainty In extrapolating from LOAELs to NOAELs.
[10L]
Modifying Factor (MF)
Use professional judgment to determine another uncertainty
factor (MF) that 1s greater than zero and less than or equal to
10. The magnitude of the MF depends upon the professional
assessment of scientific uncertainties of the study and data
base not explicitly treated above, e.g., the completeness of
the overall data base and the number of species tested. The
default value for the MF Is 1.
The uncertainty factor used for a specific risk assessment is based
principally upon scientific judgment rather than scientific fact and
accounts for possible intra- and interspecies differences. Additional
}
considerations not incorporated in the NAS/ODW guidelines for selection of
an uncertainty factor Include the use of a less than lifetime study .for
deriving an RfD, the significance of the adverse health effects and the
counterbalancing of beneficial effects.
01360
VI1I-2
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From the RfD, a Drinking Water Equivalent Level (DWEL) can be calcu-
lated. The DWEL represents a medium specific (I.e., drinking water)
lifetime exposure at which adverse, noncarclnogenlc health effects are not
anticipated to occur. The DWEL assumes 100% exposure from drinking water.
The DWEL provides the noncarclnogenlc health effects basis for establishing
a drinking water standard. For Ingestion data, the DWEL Is derived as
fol1ows:
DWEL = lRfD) x (BodY we1qht in kfl) = mq/l
Drinking Water Volume in l/day
where:
Body weight = assumed to be 70 kg for an adult
Drinking water volume = assumed to be 2 fc/day for an adult
In addition to the RfD and the DWEL, Health Advisories (HAs) for expo-
sures of shorter duration (1-day, 10-day and longer-term) are determined.
The HA values are used as Informal guidance to municipalities and other
organizations when emergency spills or contamination situations occur. Ihe
HAs are calculated using an equation similar to the RfD and DWEL; however,
the NOAELs or LOAELs are Identified from acute or subchronic studies. The
HAs are derived as follows:
HA _ (NOAEL or LOAEL) x (bw) _ ,
(UF) x ( . a/day)
Using the above equation, the following drinking water HAs are developed
for noncarclnogenlc effects:
1. 1-day HA for a 10 kg child ingesting 1 i water per day.
2. 10-day HA for a 10 kg child ingesting 1 l water per day.
3. Longer-term HA for a 10 kg child ingesting 1 i water per day.
4. Longer-term HA for a 70 kg adult Ingesting 2 si water per day.
01360
VIII-3
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The 1-day HA calculated for a 10 kg child assumes a single acute
exposure to the chemical and is generally derived from a study oF <7 days
duration. The 10-day HA assumes a limited exposure period oF 1-2 weeks and
is generally derived Prom a study oP <30 days duration. The longer-term HA
is derived Por both the 10 kg child and a 70 kg adult and assumes an
exposure period of ~7 years (or 10% of an individual's lifetime). 1.he
longer-term HA is generally derived from a study of subchronic duration
(exposure Por 10% oP animal's lifetime).
The U.S. EPA categorizes the carcinogenic potential of a chemical, based
on the overall welght-of-evldence, according to the following scheme:
Group A: Human Carcinogen. Sufficient evidence exists from
epidemiology studies to support a causal association between
exposure to the chemical and human cancer.
Group B: Probable Human Carcinogen. Sufficient evidence of
carcinogenicity In animals with limited (Group B1 ) or Inade-
quate (Group B2) evidence in humans.
Group C: Poss ible Human CareInogen. Limited evidence of
carcinogenicity In animals in the absence of human data.
Group D: Not Classified as to Human Carcinogenicity. Inade-
quate human and animal evidence of carcinogenicity or for which
no data are available,
Group E: Evidence of Noncarc inogen1c1ty for Humans. No
evidence of carcinogenicity in at least two adequate animal
tests 1n different species or in both adequate epidemiologic
and animal studies.
If toxlcologlcal evidence leads to the classification of the contaminant
as a known, probable or possible human carcinogen, mathematical models are
used to calculate the estimated excess cancer risk associated with the
Ingestion of the contaminant In drinking water. The data used in these
01360
VI11-4
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estimates usually come from lifetime exposure studies using animals. In
order to predict the risk for humans From animal data, animal doses must be
converted to equivalent human doses. This conversion includes correction
for noncontinuous exposure, less than lifetime studies and for differences
in size. The factor that compensates for the size difference is the cube
root of the ratio of the animal and human body weights. It is assumed that
the average adult human body weight is 70 kg and that the average water
consumption of an adult human is 2 1 of water per day.
For contaminants with a carcinogenic potential, chemical levels are
correlated with a carcinogenic risk estimate by employing a cancer potency
(unit risk) value together with the assumption for lifetime exposure from
ingestion of water. The cancer unit risk is usually derived from a linear-
ized multistage model with a 95% upper confidence limit providing a low dose
estimate; that is, the true risk to humans, while not identifiable, is not
likely to exceed the upper limit estimate and, in fact, may be lower.
Excess cancer risk estimates may also be calculated using other models such
as the one-hit, Welbull, logit and probit. There is little basis in the
current understanding of the biological mechanisms Involved in cancer to
suggest that any one of these models is able to predict risk more accurately
than any other. Because each model 1s based upon differing assumptions, the
estimates derived for each model can differ by several orders of magnitude.
The scientific data base used to calculate and support the setting of
cancer risk rate levels has an inherent uncertainty that Is due to the
systematic and random errors In scientific measurement. In most cases, only
studies using experimental animals have been performed. Thus, there is
01360
VI11-5
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uncertainty when the data are extrapolated to humans. When developing
cancer risk rate levels, several other areas of uncertainty exist, such as
the Incomplete knowledge concerning the health effects of contaminants in
drinking water, the impact of the experimental animal's age, sex and
species, the nature of the target organ system(s) examined and the actual
rate of exposure of the internal targets in experimental animals or humans.
Dose-response data usually are available only for high levels of exposure
and not for the lower levels of exposure closer to where a standard may be
set. When there is exposure to more than one contaminant, additional
uncertainty results from a lack of information about possible synergistic or
antagonistic effects.
Noncarclnoqenlc Effects
The characteristic effects of exposure to 2,3,7,8-TCDD Include thymic
atrophy and weight loss (see the General Toxicity Section 1n Chapter V). In
rats and rabbits, and to a lesser extent In guinea pigs and monkeys, liver
damage is a major pathological symptom. Death 1s commonly preceded by a
prolonged period of weight loss, during which time severe deterioration of
the animals Is observed; however, no specific lesion has been identified as
the cause of death. Death generally occurs 1-7 weeks following an acute
exposure. This unusual characteristic of 2,3,7,8-TCDD toxicity has resulted
In many short-term studies that report only minor effects at doses near, or
sometimes many-fold greater that the LD^. 2,3,7,8-TCDD is also an
immunosuppressant in mice, rats and guinea pigs.
The acute toxicity of 2,3,7,8-TCDD varies among species tested. Acute
oral LD5Q5 ranging from 0.6 ^g/kg bw for male guinea pigs to 5051
vg/kg bw for hamsters have been reported (see Table V-l). The relative
01360 VI11-6 * 09/23/87
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sensitivity of man to 2,3,7,8-TCDD toxicity, compared with other species,
cannot be determined from the existing data.
Short-Term Exposure. The data used for the determination of a 1-day
HA' are summarized in Table V1II-1. LD^ data, while not useful in the
derivation of an HA, are a useful way to compare species susceptibility and
are, therefore, included in the table for comparison purposes. Four studies
were found that identified NOAELs or LOAELs, which could be useful in the
derivation of an HA (Harris et al., 1973; Madge, 1977; Smith et al.f 1981;
Turner and Collins, 1983).
Harris et al. (1973) administered a single oral dose of 2,3,7,8-TCDD in
acetone:corn oil to groups of CD rats of mixed sex. Weights were determined
at least once each week. Rats given 50 or 100 yg/kg bw demonstrated a
decreased weight gain and Increased mortality. In the high-dose group,
mortality approached 50% with a mean time interval until death of 18.3
days. A dose of 25 yg/kg bw, the LOAEL in this study, resulted in a
decreased body weight in females at 1 week postdosing and a decreased rate
of weight gain in males for 2 weeks postdosing. After 2 weeks, both male
and female rats gained weight at the same rate as the controls. Doses of 1
or 5 yg/kg bw had no effect on body weight.
Madge (1977) investigated the effect of 2,3,7,8-TCDD on intestinal
absorption In CD-I mice. Mice were given single oral doses of 10, 25, 75,
150, 200 or 300 yg 2,3,7,8-TCDD/kg bw. , Absorption of D-glucose, D-galac-
tose, L-argenine and L-histldlne was measured 7 days later, using the
everted intestinal sac technique. Absorption of D-glucose was decreased at
all dose levels; however, absorption of the other compounds was not affected
01360
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TABLE VIII-1
j Acute Toxicity of 2,3,7,8-TCDD
OJ
cr
o
Route Dose Duration Duration Effect
Species of Exposure ^g/kg/day of Exposure of Experiment Level Endpolnts Reference
Rat
oral
25
1 day
B-9 weeks
L0AEL
Decreased body weight
Harris et al., 1973
Rat (male)
oral
22.0
1 day
2-8 weeks
LD50
Schwetz et al., 1973
Rat (female)
oral
45.0
1 day
2-8 weeks
ld50
Schwetz et al., 1973
Rat
l.p.
2.5
1 day
8 weeks
L0EL
Increased serum
triglycerides
Poll et al.. 1980
House
l.p.
1
1 day
NA
L0AEL
Decreased macrophage
and natural killer
cell number
Mantovanl et al., 1980
house
oral
10
1 day
7 days
L0AEL
Decreased Intestinal
absorption of d-glucose
Hadge, 1977
House
oral
50
1 day
12 weeks
L0AEL
Porphyria
Smith et al.. 1981
House
oral
15
1 day
12 weeks
N0AEL
Porphyria
Smith et al.. 1981
House
oral
284
1 day
30 days
lo50
HcConnell et al., 1978a
House
oral
114
1 day
2 months
ld50
Vos et al.. 1974
Guinea pig
(female)
oral
0.1
1 day
42 days
L0AEL
Hi Id histopathologic
effects on liver
Turner and Collins,
1983
Guinea pig
(male)
oral
0.6
1 day
2-8 weeks
ld50
Schwetz et al.. 1973
Guinea pig
(female)
oral
2.1
1 day
2-8 weeks
ld50
Schwetz et al., 1973
Guinea pig
(male)
oral
2.0
1 day
30 days
ld50
HcConnell et al., 1978a
Rabbit
oral
115
1 day
2-8 weeks
ld50
Schwetz et al.. 1973
Rabbit
dermal
275
1 day
3 weeks
ld50
Schwetz et al., 1973
Hamster
oral
5051
1 day
55-71 days
ld50
Henck et al., 1981
Hamster
oral
1157
1 day
50 days
ld50
Olson et al., 1980b
Hamster
l.p.
>3000
1 day
50 days
LD50
Olson et al., 1980b
Honkey
oral
<70
1 day
>35 days
ld50
HcConnell et al., 1978b
l.p. - Intraperitoneal
-------
by any of the treatment. The decrease in D-glucose absorption was dose-
related over the range of 0-75 yg/kg bw. . In this study, 10 yg/kg bw
consti tuted a LOAEL.
Smith et al. (1981) investigated the effect of 2,3,7,8-TCDD on hepatic
porphyrin levels in C57B1/1Q and DBA/2 mice. A single oral dose was admin-
istered in arachis oil (0, 5. 15, 50, 75, 150, 300, 600 or 1200 yg/kg bw)
and hepatic porphyrin levels were determined at intervals for <12 weeks.
Ihere were large strain differences in susceptibility to porphyria
induction, with the C57B1/10 strain being -20 times as sensitive as the
DBA/2 strain. In this study, the lowest dose that induced porphyria was 50
yg/kg bw. Thus, 50 yg/kg bw was a LOAEL and 15 yg/kg bw represented a
N0AEL.
Turner and Collins (1983) administered single oral doses of 0.1, 0.5,
2.5, 12.5 or 20 yg/kg bw of 2,3,7,8-TCDD in aqueous methyl cellulose to
groups of 4-6 female guinea pigs. Survivors were killed 42 days after dos-
ing and examined for histopathologic changes in the liver. Four of the 6
animals in the highest dose group and 1/5 In the 12.5 yg/kg group died
before the end of the observation period. Mild histopathologic changes
Including steatosis (fatty change), focal necrosis and cytoplasmic degener-
ation were noted in animals from all treated groups, but not in controls.
The authors Indicated that qualitative differences among the dosage groups
were not detectable by light microscopy.
All of the LOAELs and NOAELs determined for rats and mice are above the
LDp-g for guinea pigs (0.6-2.1 yg/kg). Although no NOEL or N0AEL 1s
01360
VI11-9
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available for guinea pigs, a LOAEL of 0.1 yg/kg can be derived from the
study of Turner and Collins (1983).
Studies of appropriate duration for determining a 10-day HA are
identified in Table V111 -2 .
Longer-Term Exposure. The health effects of long-term exposure to
213,7,8-TCDD. are summarized in Table V111-3. Most of the long-term studies
have been performed using rats. There Is, therefore, only limited informa-
tion on the chronic toxicity of 2,3,7,8-TCDD in other species. Of the
studies included in Table VIII-3, only those by Kociba et al. (1978a,b,
1979 ) and Murray et al. (1979) were done with administration of 2,3,7,8-TCDD
in the diet on a daily basis.
The U.S. EPA developed an ADI based on noncarcinogenic effects as
Indicated in U.S. EPA (1984). For consistency, the rationale used by U.S.
EPA for the ADI calculation in U.S. EPA (1984) Is used for the RfD
calculation herein. The rationale as presented In U.S. EPA (1984) is as
follows:
2,3,7,8-TCDD displays an unusually high degree of reproductive
toxicity. It is teratogenic, fetotoxic and reduces fertility. In
a 3-generation reproductive study, Murray et al. (1979) reported a
reduction in fertility after dally dosing at 0.1 or 0.01 yg
2,3,7,8-TCDD/kg in the F] and generations of Sprague-Dawley
rats. Although Murray et al. (1979) considered 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 yg/kg dose. The re-evaluated data would suggest
that equivocal adverse effects were seen at the lowest dose (0.001
yg/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
01360 VI11-10 04/14/88
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TABLE VI11-2
Effects of 4-13 Weeks Exposure to 2,3,7,8-TCDD
w
cr>
o
No. of
Species Route of Oose Treatments/ Duration Effect Endpolnts Reference
Exposure wg/kg Ueek of Exposure Level
Rat
oral
0.01 5
13 weeks
N0AEL
Decreased body weight
Koclba et al.. 1976
Rat
oral
0.1 5
13 weeks
L0AEL
Decreased body weight
Koclba et al.. 1976
Rat
oral
0.5 2
13 weeks
N0AEL
Toxic hepatitis
NTP, 1980a
Rat
oral
1.0 2
13 weeks
L0AEL
Toxic hepatitis
NTP, 1980a
Rat
oral
0.1 7
30 days
L0AEL
Decreased thymus weight
Harris et al., 1973
Rat
oral
1.0 1
6 weeks
N0AEL
Decreased body weight
Harris et al., 1973
Rat
oral
0.1 ~ 7
30 days
L0AEL
Thrombocytopenia
Zlnkl et al., 1973
Rat
oral
5.0 1
6 weeks
L0AEL
Decreased body weight and thymus weight
Vos et al.. 1973
Rat
oral
1.0 1
6 weeks
N0AEL
Decreased body weight and thymus weight
Vos et al.. 1973
Rat
oral
0.001 7
3 generation
L0AEL
Decreased body weight, decreased fertility,
decreased fetal survival
Hurray et al., 1979
House
oral
1.0 2
13 weeks
L0AEL
Toxic hepatitis
NTP, 1980a
House
oral
5.0 1
4 weeks
N0AEL
Porphyria
Goldstein et al.. 1978
House
oral
25.0 1
4 weeks
L0AEL
Porphyria
Goldstein et al.. 1978
House
oral
5.0 1
4 weeks
L0AEL
Decreased thymus weight and graft-versus-host
response
Vos et al., 1973
F
House
oral
1.0 1
4 weeks
N0AEL
Decreased thymus weight and graft-versus-host
response
Vos et al.. 1973
House
oral
1.0 1
4 weeks
L0AEL
Decreased resistance to Salmonella
Thlgpen et al.. 1975
House
oral
1.5 1
4 weeks
L0AEL
Increased endotoxlc (E. coll) suscepttb11Hv
Vos et al., 1978a
House
oral
10 ppb 7
5 weeks
L0AEL
Decreased tetanus response, antigenic RBC response,
Hlnsdl11 et al., 1980
sensitization to DNFB, resistance to Salmonella
Infection, resistance to Listerla Infection
-------
TABLE VI11-2 (cont.)
o
—J
CJ
q No. of
Species Route of Dose Treatments/ Duration Effect Endpolnts Reference
Exposure yg/kg Week of Exposure Level
House l.p. 0.4 1 4 weeks LOAEL
Guinea oral 0.008 1 8 weeks NOAEL
pig
Guinea oral 0.04 1 8 weeks LOAEL
pig
l.p. = Intraperitoneal
Decreased cytotoxic T-cell response Clark et al.. 1981
Thymus weight and tuberculin hypersensitivity Vos et al., 1973
Thymus weight and tuberculin hypersensitivity Vos et al., 1973
i
o
<£>
N
i\i
CO
V
co
-------
TABLE VI11-3
Effects of Long-Term Oral Exposure to 2.3.7.B-TCDD
No. of
Species Dose Treatments/ Duration Effect Endpolnts Reference
(iig/kg) Week of Exposure Level
Rat
Rat
Rat
Rat
Rat
Rat
Rat
Rat
Rat
Rat
House
house
0.001
0.01
0.1
0.1
0.001
0.01
0.01
0.0S
1.0
0.01
0.01
0.007
3 generation LOAEL
16 weeks NOAEL
16 weeks LOAEL
28 weeks LOAEL
104 weeks NOAEL
104 weeks LOAEL
104 weeks NOAEL
104 weeks LOAEI
45 weeks LOAEL
45 weeks LOEL
104 weeks LOAEL
52 weeks exposure, LOAEL
104 weeks study
duration
Decreased body weight, decreased fertility,
decreased fetal survival
Elevated porphyrin levels
Elevated porphyrin levels
Fatty changes In liver, decreased body weight
Degenerative and necrotic changes In the liver
Degenerative and necrotic changes In the liver
Toxic hepatitis
loxlc hepatitis
Porphyrla
Hepatic enzyme Induction, liver weight
Toxic hepatitis
Dermatitis and amyloidosis
Hurray et al.. 1979;
Goldstein et al., 1982b
Goldstein et al.. 1982b
King and Roesler, 1974
Koc Iba et al., 1978a,b,
19/9
Koclba et al., 1978a,b,
1979
NTP, 1982a
NTP, 1982a
Cantonl et al.. 1981
Cantonl et al., 1981
NTP. 1982a
loth el al.. 1978. 1979
-------
effects in rhesus monkeys Fed a 50 ppt 2,3,7,8-TCOD diet for 20
months. This corresponds to a calculated daily dose of 0.0015
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. Since the data from the
limited study by Schantz et al. (1979) are supportive of the
findings by Murray et al. (1979), it seems reasonable to determine
an ADI based on the LOAEL.
Quantification of Noncarcinoqenic Effects
Derivation of 1-Day HA. The data on very short-term exposures, 1-H
days, are sufficient for the derivation of a 1 -da y HA. As previously
discussed the available studies establish LOAELs for rats and mice that are
greater than the LD^q for guinea pigs. A N0AEL is not available for
guinea pigs, but a L0AEL of 0.1 yg/kg can be derived from the study of
Turner and Collins (1983). This L0AEL can be used to calculate a 1-day HA,
using an uncertainty factor (UF) of 1000 for an animal LOAEL.
For a 10 kg child:
where:
0.1 yg/kg bw/day = L0AEL from the study of Turner and Collins
(1983)
1-day HA =
(0.1 uq/kq bw/dav x 10 kg)
1000 x 1 si/day
= 0.0010 yg/8.
= 1x10 3 yg/i
10 kg
= Assumed body weight of a child
1 SL/day
= assumed water consumption by a 10 kg child
1000
= uncertainty factor, represents two 10-fold
factors to account for both intra and
interspecies variability to the toxicity
of chemicals, and a 10-fold factor to
account for a L0AEL being used instead of
a N0AEL
01360
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This HA is equivalent to 0.0010 yg/day or 0.00010 yg/kg bw/day
Derivation of 10-Day HA. A 10-day HA is calculated by dividing the
1-day HA by 10 to convert the 1-day HA to a 10-day HA. Therefore, along
with using an uncertainty factor of 1000 for an animal L0AEL (I.e., 10-fold
for intra- and 10-fold for interspecies variability to the toxicity of a
chemical in lieu of specific data, and an additional 10-fold because the
estimate is based on a L0AEL rather than a N0AEL), a 10-day HA can be calcu-
lated from the L0AEL of 0.1 yg/kg/day reported by Turner and Collins
(1983).
For a 10 kg child:
10 day HA =
0.001 yg/kg bw/day x 10 kg
1000 x 1 i/day x 10
= 0.0001 yg/SL
1x10 4 yg/i
where:
0.001 yg/kg bw/day = 1-day HA for child
10 kg
assumed body weight of a child
1 i/day
assumed water consumption by a 10 kg
child
1000
uncertainty factor, represents two
10-fold factors to account for both
Intra and Interspecies variability to
the toxicity of chemicals, and a 10-fold
factor to account for a L0AEL being used
Instead of a N0AEL
10
conversion of 1-day HA to 10-day HA.
This HA is equivalent to 0.0001 yg/day or 0.00001, yg/kg bw/day.
01360
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Derivation of Longer-Term HA. The studies Included in lable V111 - 3
are considered for calculation of a longer-term HA. This is based on a
LOAEL of 0.00T yg/kg for reproductive effects in the 3-generation repro-
ductive study in rats by Murray et al . ( 1 979 ) along the rationale discussed
in the Longer-Term Exposure Section. Although DeCaprio et al. (1986) found
NOELs of 0.61 and 0.68 mg/kg/day, respectively, for male and female guinea
pigs in a 90-day ingestion study, this dose is slightly below the LOAEL of 1
mg/kg/day (0.001 yg/kg/day) observed in the Murray et al. (1979) study.
Using the LOAEL of 0.001 yg/kg bw/day, because the Murray et al.
(1979 ) study suggested that a dose of 0.001 yg/kg bw/day may be a LOAEL
for reproductive effects, and dividing this by an uncertainty factor of 1000
for an animal LOAEL, a longer-term HA can be calculated.
For a 10 kg child consuming 1 l of drinking water daily, the longer-term
HA is calculated as follows:
Accordingly, for a 70 kg adult consuming 2 a of drinking water daily, the
longer-term HA for an adult is calculated as follows:
Longer-term HA =
(0.001 uq/kg/day) (10 kg)
(1000) (1 i/day)
= 0.00001 yg/2.
= 1x10 5 yg/fi.
Longer-term HA =
(0.001 yg/kg bw/day x 70 kg)
1000 x 2 l/day
= 0.000035 yg/ft,
= 3.5x10 5 yg/9.
01360
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This longer-term HA is identical to the 10-day HA for adults and Is equiva-
lent to an ADI of 70 pg/day or 1.0 pg/kg bw/day. This ADI is the same as
that estimated in the AWQC document for TCDD for comparison purposes to the
criteria derived from carcinogenicity data (U.S. EPA, 1984).
Assessment of Lifetime Exposure and Derivation of a DUEL. 2,3,7,8-
TCOO may be classified in Group B: Probable Human Carcinogen, according to
U.S. EPA's proposed weight-of-evidence scheme for the classification of
carcinogenic potential (U.S. EPA, 1986). Because of this, caution must be
exercised in making a decision on how to deal with possible lifetime exposure
to this substance. The risk manager must balance this assessment of
carcinogenic potential against the likelihood of occurrence of health effects
related to noncarclnogenic endpol'nts of toxicity. In order to assist the
risk manager in this process, drinking water concentrations associated with
estimated excess lifetime cancer risks over the range of 1 in 10,000 to 1 in
1,000,000 for the 70 kg adult, drinking 2 a of water per day, are provided
in the following section. In addition, in this section, a DWEL is derived.
A DWEL is defined as the medium-specific (in this case, drinking water)
exposure that is Interpreted to be protective for noncarclnogenic endpoints
of toxicity over a lifetime of exposure. The DWEL is determined for the 70
kg adult ingesting 2 % of water per day. Also provided is an estimate of
the excess cancer risk that would result if exposure were to occur at the
DWEL over a lifetime.
Neither the risk estimates nor the DWEL take relative source contribu-
tion into account. The risk manager should do this on a case-by-case basis,
considering the circumstances of the specific contamination incident that has
occurred.
01360
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The U.S. EPA has developed, for comparison with cancer-based criteria, a
presumed safe daily intake level based on noncarc1nogenic effects as
indicated in U.S. EPA (1 984). For consistency, the rationale used by the
U.S. EPA for the calculation of this value in U.S. EPA (1984) is used here
for the DWEL calculation. The rationale as presented in U.S. EPA ( 1984) is
as follows:
2,3,7,8-TCDD displays an unusually high degree of reproductive
toxicity. It is teratogenic, fetotoxic and reduces fertility. In
a 3-generation reproductive study, Murray et al. (1979) reported a
reduction in fertility after dally dosing at 0.1 or 0.01 yg
2,3,7,8-TCDD/kg in the F-j and generations of Sprague-Dawley
rats. Although Murray et.al. (1979) considered the lowest dose
tested, 0.001 yg/kg, to be a no-observed-effect level (NOEL), a
re-evaluation of these data by Nisbet 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 yg/kg dose. The re-evaluated data would suggest
that equivocal adverse effects were seen at the lowest dose (0.001
yg/kg/day) and that this dose should, therefore, represent a
lowest-observed-adverse-effect level (L0AEI). 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 daily dose of 0.0015 yg 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. Since the data from the limited study by
Schantz et al. (1979) are supportive of the findings by Murray et
al. (1979), it seems reasonable to determine an RfD based on the
L0AEL.
From these results, a L0AEL of 0.001 yg/kg was identified. Using this
L0AEL, the DWEL is derived as follows.
Step 1 - RfD Derivation
RfD - (Q-QQ1 yq/kg/day) = 1x10*6 pg/kg/day
(1000)
01360
VI11-18
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-------
wher e :
0.001 vg/kg/day = LOAEL from Murray et al. (1979)
1000 = uncertainty factor appropriate for use with a
LOAEL from an animal study
Step 2 - DWEL Derivation
DWEL = (1*10 6 yQ^g/day) (70 kg) = 0.000035 »q/i
(2 t/day)
= 3.5xl0~5 vq/l
where:
lxl0~6 yg/kg/day = RfD
70 kg = weight of protected individual
2 a/day = assumed volume of water ingested by an adult
The estimated excess cancer risk associated with lifetime exposure to
drinking water containing 2,3,7,8-TCDD at 3.5x"l0~5 pg/2. is ~2xl0~4.
This estimate represents the upper 95% confidence limit from extrapolations
prepared by the U.S. EPA's Carcinogen Assessment Group using the linearized,
multistage model. The actual risk is unlikely to exceed this value, but
there is considerable uncertainty as to the accuracy of risks calculated by
this methodology.
Carcinogenic Effects
A number of epidemiological studies have attempted to relate 2,3,7,8-
TC0D exposure to human health effects (see the Epidemiological Studies
Section). These studies are limited by small sample sizes, short follow-up
period and exposure to multiple compounds. These epidemiological studies do
not unequivocally establish a relationship between 2,3,7,8-TCDD and the
01360
VI11-19
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-------
development of tumors in humans, though an association has been' suggested
with soft-tissue sarcomas (Hardell and Sandstrom, 1 979; Eriksson et al . ,
1979, 1981), lymphomas (Hardell et al., 1980, 1981) and stomach cancer
(Axelson et al.f 1980; Theiss and Frentzel-Beyme, 1977).
In comparison, a number of cancer bioassays have clearly demonstrated
the carcinogenic potential of 2,3,7,8-TCDD in animals (Kociba et al.,
1978a,b; Van Miller et al. , 1977a,b; Toth et al., 1979; NIP, 1980a,b).
These studies are summarized in Table VIII-4. Oral administration of
2,3,7,8-TCDD, either in the diet or by gavage, results in the production of
hepatocel 1 ular carcinomas in female rats and both sexes of mice (Kociba et
al., 1978a,b; NTP, 1980a; Toth et al., 1979). Follicular-cell adenomas of
the thyroid have been observed in both male rats and female mice (NTP,
1980a). Various squamous cell carcinomas have also been reported in both
sexes of rats (Kociba et al., 1978a,b).
Toth et al. (1979) administered weekly gavage doses of 0.0, 0.007, 0.7
and 7.0 yg/kg bw to groups of 45 male Swiss mice for 1 year. The rela-
tively short duration of exposure limits the sensitivity of this assay for
determining the carcinogenic potency of 2,3,7,8-TCDD.
Van Miller et al. (1977a,b) maintained groups of 10 male Sprague-Dawley
rats on diets containing 0.0, 0.001, 0.005, 0.05, 0.5, 1.0, 5.0, 50, 500 or
1000 ppb 2,3,7,8-TCDD for 78 weeks. Based on the food consumption of two
rats from each group, these dietary levels resulted in doses of 0.0, 0.003,
0.001, 0.01, 0.1 , 0.4, 2.0, 24, 240 and 500 yg/kg bw/week, respectively.
«
01360
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-------
TABI F VIII 4
Carcinogenicity Bloassays of 2.3.7.8-tCDD by Oral and Dermal Exposure
Specles
Route
Dose Range
DuratIon
of Treatment
DuratIon
of Study
AnImals/
Group
Tumor Types Reported
Reference
Rat (male)
gavage 0-0.5 |ig/kg bw/week 104 weeks 1 OS-107 weeks
Rat (female) gavage 0-0.5 pg/kg bw/week 104 weeks 105-107 weeks
Rat (male)
Rat (male)
diet 0-1000 ppb
78 weeks 95 weeks
diet 0-0.1 iig/kg bw/day 105 weeks 105 weeks
Rat (female) diet 0-0.1 pg/kg bw/day 105 weeks 105 weeks
House (male) gavage 0-0.5 pg/kg bw/week 104 weeks 105-107 weeks
House (female) gavage 0-0.2 pg/kg bw/week 104 weeks 105-107 weeks
House (male) gavage 0-7.0 pg/kg bw/week 365 days 424-649 days
House (male) dermal 0-0.03 pg/week 104 weeks 104 weeks
House (female) dermal 00.015 pg/week 104 weeks 104 weeks
50
50
10
50
50
50
50
45
30
30
Folllcular-cell adenoma
or carcinoma of the
thyroid
Neoplastic nodule or
hepatocellular carcinoma
of the liver
All tumors
Squamous cell carcinoma
of the hard palate and
the tongue, adenoma
of the adrenal cortex
Mepatocellular carcinoma,
squamous cell carcinoma
of the tongue and lung
Hepatocellular carcinoma
Hepatocellular carcinoma,
folllcular-cell adenomas
of the thyroid
Liver tumors
Fibrosarcoma of the
Integumentary system
Fibrosarcoma of the
Integumentary system
NTP. 1982a
NTP, 1982a
Van HI 1ler et al.
1977a,b
Koc Iba et al.,
1979
Koclba et al.
1979
NTP. 1982a
NTP. 1982a
Toth et al.. 1979
NTP, 1982b
NTP, 1982b
-------
The small group sizes (10 rats/group) and relatively short exposure times
[78 weeks) limit the usefulness of this study in quantitative risk assess-
ment .
Both the NTP (1982a) study and the Kociba et al. (1978a,b) study contain
sufficient animals/group and involved sufficiently long dosing schedules to
be used for quantitative risk assessment. Kociba et al. (197 8a, b) main-
tained groups of 50 Sprague-Dawley rats on diets providing doses of 0.0,
0.001r 0.01 or 0.1 yg 2,3,7,8-TCDD/kg bw/day for 2 years. The high dose
resulted in reduced lifespans for the female rats, reduced body weight gain
in both sexes and signs of tissue toxicity. Statistically significant
increases in hepatocellular neoplastic nodules were observed in females at
doses of 0.1 and 0.01 yg/kg bw/day. At the high-dose, increases were
observed In stratified squamous cell carcinomas of the hard palate and/or
nasal turbinates in both sexes, in keratinizing squamous cell carcinomas of
the lungs in females, In squamous cell carcinomas of the tongue in males and
in hepatocellular carcinomas in females.
In the NTP bloassay (NTP, 1982a), groups of 50 Osborne-Mendel rats or
Swiss mice were dose twice weekly by gavage with 2,3,7,8-TCDD in a 9:1 corn
olliacetone solution. The rats and male mice received TWA doses of 0.0,
0.0014, 0.0071 and 0.071 yg/kg bw/day. The corresponding doses for female
mice were 0.0, 0.0057, 0.029 and 0.29 yg/kg bw/day. Dose-related depres-
sions In mean body weight were. reported for both sexes of rats. In male
rats, a dose-dependent Increase In the Incidence of folllcular-cell adenomas
or carcinomas of the thyroid was observed. The incidence of subcutaneous
tissue Mbromas was significantly increased in the high-dose group. In
female rats, observed Increases In'the incidence of subcutaneous tissue
01360
VI11-22
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-------
fibrosarcomas, adrenal cortical adenomas and hepatocellular carcinomas and
neoplastic nodules were statistically significant in the high-dose group.
In mice, statistically significant increases in tumor incidences were ob-
served only in the high-dose group. An Increase in hepatocellular carci-
nomas and neoplastic nodules was noted in the males. In females, increases
were observed in hepatocellular carcinomas and adenomas, fibrosarcoma,
histiocytic lymphoma, thyroid foilicular-cell adenoma and cortical adenoma
or carcinoma.
Quantification of Carcinogenic Effects
A summary of 95% upper-limit human carcinogenic potency estimates for
2,3,7,8-TCDD derived from the Kociba et al. (1978a,b) and NCI (NTP, 1980a)
studies in rats and mice, with two pathologists' findings for the Kociba
study, are given in Table VIII-5. These potency estimates have been calcu-
lated using the linearized multistage model by a previously described
methodology (Federal Register, 1980). The largest of these potency factors
(q^*) comes from data in an Independent pathologist's (Dr. R. Squire)
review of the Kociba 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 pathologists' estimates after
mortality adjustment is as follows:
qi* = [(1.51.x 10s) x (1.61 x IG5)]"^ r 1.56 x 10s (mg/kg/day)_1
These potency estimates were derived from the Kociba feeding study. The
responses and parameters of the Kociba feeding study in female rats are
given in Table VIII-6. The number with tumors refers to the number of
animals with at least one of liver, lung, hard palate or nasal turbinate
tumors. Adjustment for early mortality refers to eliminating those animals
01360
VIII-23
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TABLE VI11-5
Summary of Human Potency Estimates for 2,3,7,8-TCDQa
Species Sex
Pathologi st
Human Potency Estimate
q-j* in (mg/kg/day)_1
Reference
Rat M
Kociba
1.47
X
10"
Kociba
et al.,
1978a,b
Squire
1.73
X
10*
Rat F
Kociba
Unadj usted
Adjusted for
early mortality
2.52
1.51
X
X
105
10sb
Koc i ba
et al.,
1978a,b
Squire
Unadj usted
Adjusted for
early mortality
4.25
1 .61
X
X
105
10sb
Rat F
NTP-reviewed
3.28
X
104
NIP, 1982a
Mouse M
NTP-rev1ewed
7.52
X
104
NTP, 1982a
Mouse F
NTP-reviewed
4.56
X
104
NTP, 1982a
aSource: U.S. EPA, 1984
^Values used to
determine the geometric mean of
1.
,56x10s (mg/kg/day) 1
01360
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TABLE VIII-6
Responses and Parameters of the Kociba Feeding Study*
Dose No. with Tumors/No. Examined
(mg/kg/day) Adjusted for Early Mortality
Squi re Koclba
0 16/85 9/85
0.001 x 10"3 8/48 3/48
0.01 x 10"3 27/48 18/48
0.1 x 10"a 34/40 34/40
le = 720 days Wh = 70 kg
Le = 720 days W0 = 0.450 kg
L = 720 days R = 5000 a/kg
*Source: Kociba et al.t 1978a#b
01360
VIII-25
09/23/87
-------
that died during the first year of study. The first tumor appeared in the
high-dose group during the thirteenth month.
With these parameters, the mean 95% upper-limit carcinogenic potency
factor for humans, q^*, is 1.56x105 (mg/kg/day p1. For a 70 kg human
drinking 2 a. water/day, the water concentration should be <2.2xlQ~6
pg/l in order to keep the upper-limit individual lifetime cancer risk
<10~5. Water concentration corresponding to excess cancer risk of 10~4
and 1CT6 are, therefore, <2.2xl0~5 and <2.2xl0~7, respectively.
Existing Guidelines, Recommendations and Standards
The U.S. EPA has established the limits of 1.3xl0~7, 1.3xlO~B or
1.3x10"® ug 2,3,7 ,8-TCDD/S. in ambient waters, based on an assumed
dally consumption of 6.5 g of contaminated fish and shellfish and ? I of
drinking water (U.S-. EPA, 1984). Under these conditions, 94.2% of the total
exposure would result from the consumption of aquatic organisms. The
recommended levels correspond to estimated human lifetime excess cancer
risks of 1Q~S, 10~6 or 10~7, respectively. These values are
considerably lower than the HAs for drinking water that are recommended in
this document (see the Summary Section of this chapter), reflecting the high
bioaccumulatlon of this compound in aquatic species.
An AD I of 1Q~4 yg 2,3,7,8-TCDD/kg bw/day has previously been pro-
posed by the National Academy pf Sciences Committee on Drinking Water and
Health (NAS, 1977 ). This ADI was based on a 13-week rat feeding study by
Kociba et al. (1976) and was proposed before convincing evidence for the
carcinogenicity of 2,3,7,8-TCDD had accumulated.
01360
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Agency
FDA
CDC
EPA
I ARC
N10SH
Advi sory
The FDA advises that fish containing >50 ppt of 2,3,7,8-lCDD
should not be consumed and those containing >25 ppt, but <50
ppt, should not be consumed more than twice a month (FDA,
1983). This is reflected in a Canadian limit of 20 ppt in
the Lake Ontario commercial fish imported into the United
States (NRCC, 1981).
Levels >1 pbb in residential soil are levels of concern
(Kimbrough et al., 1984)
Group B2 - Sufficient animal data to indicate
carcinogenicity, plus Inadequate human evidence which
suggests that 2,3,7,8-TCDD is probably a human carcinogen.
(U.S. EPA, 1986)
IARC Group 2B - Sufficient animal data to indicate
carcinogenicity, plus inadequate human evidence which
suggests that 2,3,7,8-TCDD is probably a human carcinogen.
(U.S. EPAt 1986)
It is recommended that 2,3,7,8-TCDD be considered a
potential occupational carcinogen and exposure should be
limited to the fullest extent feasible (NIOSH, 1984).
Special Cons 1 derations
Synergistic Effects.
Enzyme Induction -- 2,3,7,8-TCDD has been demonstrated to signifi-
cantly alter the toxicity of other toxicants, primarily as a result of
enzyme induction (see the Interactive Effects Sect.ion in Chapter VI). These
changes may either increase toxicity, if metabolism is predominantly an
activation pathway, or decrease toxicity if metabolism is predominantly a
detoxification mechanism. Thus, Grieg (1972) has demonstrated a 54%
decrease in the duration of zoxazolamine-induced paralysis and a 2-fold
Increase in hexabarbltone sleepigg time.
Cocarclnoqenesls and Promotion -- In addition to being a complete
carcinogen, 2,3,7,8-TCDD has been demonstrated to function as a promoter of
DEN-lnitiated hepatocarclnogenesis (Pi tot et al., 1980). Positive results
01360
VII1-27
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For tumor promoting activity have also been obtained when 2,3,7,8-TCDD was
tested on the skin of mice homozygous for the "hairless" trait (Poland et
al.p 1982). Another attempt to demonstrate the tumor promoting ability of
2,3,7,8-TCDD on mouse skin (Swiss-Webster mice) have produced negative
results (NTP, 1980b; Berry et al., 1978, 1979 ). 2,3,7,8-TCDD has also been
demonstrated to be cocarcinogenic with 3-methylcholanthrene (Kouri et al.,
1978).
High Risk Subpopulatlons. The data from human studies are Insuffi-
cient to establish the existence of sensitive subpopulatlons, though there
1s suggestive evidence that children may be more sensitive than are adults
(see the High Risk Subpopulatlons Section in Chapter VI).
Summary
The recommended HAs developed 1n this document are summarized in Table
VIII-7. The 1-day HA 1s based on a single-dose LOAEL in the most sensitive
species, the guinea pig (Turner and Collins, 1983). The 10-day HA is calcu-
lated by dividing the 1-day HA by 10. A DWEL for noncarc inogenlc effects
from lifetime exposure is derived from the LOAEL in the 3-generation repro-
ductive study by Murray et al (1979) along the rationale developed by the
U.S. EPA; however, the carcinogenicity risk assessment based on the
linearized multistage model and the carcinogenicity data in the Kociba et
al. (1978a,bt 1979) study Indicates lower HAs for lifetime exposure.
01360
VI11-28
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TABLE VI11-7
Summary of Calculated Health Advisories for 2.3,7,8-TCDD
Health Advisory
NOEL/NOAEL
LOEL/LOAEL
Species/
Route
Effect
Calculated Level (pg/l
for Safe Exposure
Child
Adult
Reference
I-day HA
10-day HA
Longer-term HA
Lifetime DUEL
0.1 pg/kg/day
No adequate data3
0.001 pg/kg/dayb
guinea
ply/oral
reproductive effect
reproductive effect
10 4 excess cancer risk
10 1 excess cancer risk
10 • excess cancer risk
1.0x10 »
1.0*10""
1.0x10*
3.5x10*
3.5x10*
2.2x10"*
?.2x10'*
2.2xl0"7
Turner and
Collin. 1903
Hurray
et al.. 1979
Hurray
et al.. 1979
KocIba
et al..
1978a ,b
KocIba
et al.,
1978a,b
KocIba
et al. .
1978a,b
dThe 10-day HA Is derived by dividing the 1-day HA by 10.
busing 0.001 pg/kg/day as the L0AEL, the RfD Is determined as follows:
RfD = 0,001 pg/k9/day = lxl0«. ^/kg/day
1000
where 1000 Is an uncertainty factor appropriate for use with a L0AEL from an animal study.
-------
IX. REFERENCES
Abernathy, D.J., W.F. Greenlee, J.C. Hubard 'and C.J. Boreiko. 1985.
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) promotes the transformation of
C3H/10T1/2 cells. Carcinogenesis. 6: 651-653.
Aitio, A. and M.G. Parkki. 1978. Organ specific induction of drug metabo-
lizing enzymes by 2,3,7,8-tetrachlorodibenzo-p-dioxin in the rat. Toxicol.
Appl. Pharmacol. 44(1): 107-114.
Albro, P.W. 1979. Problems in analytical methodology: Sample handling,
extraction, and clean up. Ann. NY Acad. Scl. 320: 19-27.
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