"Current Awareness" Program
So 1 .
Halogenated Phenoxy Acids,
Aromatic Ethers, Dibenzofurans
Dibenzo-p-Dioxins
-------
HALOGEMATED PHENOXY ACIDS, AROMATIC ETHERS,
DIBENZOFURANS AND DIBENZO-p-DIOXINS
CARCINOGENICITY.AND STRUCTURE-ACTIVITY
RELATIONSHIPS. OTHER BIOLOGICAL PROPERTIES.
METABOLISM. ENVIRONMENTAL SIGNIFICANCE.
Yin-tak Woo, Ph.D.
Joseph C. Arcos, D.Sc., and
Mary F. Argus, Ph.D.
Preparation for the Chemical Hazard
Identification Branch "Current
Awareness" Program
-------
5.2.2.3 Halogenated Phenoxy Acids, Aromatic Ethers, Dibenzofurans and
Dibenzo-p-dioxins.
5.2.2.3.1 Introduction.
Halogenated phenoxy acids, aromatic ethers, dibenzofurans and dibenzo-jv-
dioxins, the structures of which are depicted in Table XLVI, include a number
of controversial and notorious chemicals. Halogenated dibenzofurans and
/
dibenzo-jr-dioxins often occur as unwanted contaminants of polyhalogenated
biphenyls, halogenated aromatic ethers and phenoxy acids (see Section
5.2.2.3.5.2).
Chlorophenoxy acids were first developed in the early 1940's as plant
growth regulators shortly after the well known discovery, by Kogl and his
associates, of indole-3-acetic acid (also known as IAA or auxin) as a natural
plant growth hormone. They came into use as herbicides during World War II
when the optimization of food production with a reduced labor force was a
vital factor in the war effort. 2,4-Dichlorophenoxyacetic acid (2,4-D) and
2-methyl-4-chlorophenoxyacetic acid (MCPA) are two of the most widely used
herbicides for the control of broad-leaf weeds in many industrialized
countries (1-4). The production of 2,4-D in the United States increased from
450 metric tons in 1945 to a maximum of 36,000 metric tons in 1968; production
fell-to 20,000 metric tons in 1970 but gradually increased again to an esti-
mated 30,000 metric tons in 1977 (4). 2,4,5-Trichlorophenoxyacetic acid
(2,4,5-T), an effective herbicide against woody and herbaceous weeds, was
first produced commercially in the United States in 1944. The production of
2,4,5-T increased sharply between 1960 and 1968 (2, 5) when a 1:1 mixture of
jv-butyl esters of 2,4-D and 2,4,5-T was used as a defoliant in South Vietnam
under the names of "Agent Orange," "Herbicide Agent Orange," or "Herbicide
288
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TABLE XL VI
0-OVCOOH
2,4-D(X=CI;Y=CI;Z=H)
MCW(X=CHj;Y=CI;Z=H)
2,4,5-T(X=CI;Y=CI;Z=CI)
CHS
Dichtoroprop(X=CI;Y=CI;Z=H)
Mecoprop(X=CH3;Y=CI;Z=H)
Silvex(X=CljY=CI;ZECI)
CH,
-C-COOH
Clofibrate
[CHzJj-COOH
2,4-DB(X=CI;Y=CI)
MCPB(X=CH3jY=Cl)
Halogenoted Diphenyl
Ethers
Chlorinated
Dibenzofuran
CU
Chlorinated
Dibenzo-p-dioxin
-------
Orange." The amount of Agent Orange sprayed during the Vietnam war was esti-
mated to exceed 40 metric tons (Whiteside, cited in ref. 5). Concern over the
potential long-term health hazards of chlorophenoxy acids first arose becasue
v
of a study by the Bionetics Research Laboratory (6) indicating teratogenic
effects of technical-grade 2,4,5-T in rodents. Since 1969, the U.S.-
Government has restricted or suspended the use of 2,4,5-T in populated
areas. . Although the teratogenicity of technical-grade 2,4,5-T was later
attributed mainly to the action of an extremely potent teratogenic contami-
nant, 2,3,7,8-tetrachlorodibenzo-jv-dioxin (TCDD), there is some evidence that
some relatively uncontaminated chlorophenoxy acids may be teratogenic (albeit
weakly so) in certain animal species (see Section 5.2.2.3.2.2). Recent epi-
demiologic studies by Hardell and his associates (7, 8; see also Section
5.2.2.3.5.2) suggest that human exposure to chlorophenoxy acids (including
2,4-D and MCPA, which are usually not contaminated with TCDD) may represent an
increased carcinogenic risk for several types of tumors. These results,
coupled with the known carcinogenicity of clofibrate (see Table XLVI; see also
Section 5.2.1.7 in Volume IIIA) indicate that apart from possible contamina-
tion with TCDD, chlorophenoxy acids, as a class, may pose long-term health
hazard to humans and should be more thoroughly investigated.
Chlorinated dibenzo-j^-dioxins have long been recognized as possible
byproducts in the production of certain chlorinated phenols. 2,3,7,8-Tetra-
chlorodibenzo-jv-dioxin (TCDD), an extremely toxic compound, is known to be
formed in the manufacturing of products involving 2,4,5-trichlorophenol as an
intermediate. Since 1949, over 200 TCDD-related industrial accidents have
occurred around the world (9). The most characteristic and frequently
observed toxic effect in humans is chloracne, a severe form of dermatitis.
Suspicion of the possible long-term health hazards of TCDD arose when it was
289
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found in the Bi.oneti.es Research Laboratory study (6) that 2,4,5-T is terato-
genic in the rat and the mouse. In fact, later tests indicated that these
teratogenic effects may have been caused by trace amounts (27 i 8 ppm) of TCDD
present as a contaminant in the 2,4,5-T sample used in the Bionetics study.
The possibility that TCDD may be carcinogenic in humans was raised by Tung
(10) who reported an. increase in the incidence of liver cancers among -
Vietnamese during 1962-1968 when TCDD-contaminated defoliant, Agent Orange,
was sprayed in the countryside. Several well publicized incidents of public
exposure to TCDD the accidental release of a massive amount of TCDD into
the atmosphere in Seveso (Italy) in 1976; the finding of TCDH in Love Canal,
Niagara Falls; the spraying of TCDD-contaminated waste oil for dust control in
Missouri (see Section 5.2.2.3.5.2) further stimulated an explosive growth
in the investigations of the health hazards and environmental significance of
TCDD and related compounds. The deep concern over, and incense interest in,
TCDD and related compounds is reflected by the large number of review
articles, monographs and symposia (11-23K
5.2.2.3.2 Physicochemical Properties and Biological Effects.
5.2.2.3.2.1 PHYSICAL AND CHEMICAL PROPERTIES.
The physical and chemical properties of chlorophenoxy acids have been
described in detail by Melnikov (1). Some physicochemical properties of these
compounds are summarized in Table XLVII. 2,4-Dichlorophenoxyacetic acid
(2,4-D) is a relatively strong acrd with a pKa of 2.64. It reacts with
organic and inorganic bases to form stable salts (which are substantially more
soluble in water) and with alcohols to form esters. The lower alkyl esters of
2,4-D are relatively volatile (e.g., 2,4-D isopropyl ester has a vapor pres-
sure of 1.05 x 10"^ mm Hg at 25°C); the volatility decreases with an increase
290
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Table XLVII
Physicochemical Properties of Some Chlorinated Phenoxy Acids and Dibenzodioxinsa
Compound
M.W.
m.p.(°C) pKfl
Solubility
2,4-Dichlorophenoxy- 221.0
acetic acid (2,4-D)
2-Methyl-4-chlorophenoxy- 200.6
acetic acid (MCPA)
2,4,5-Trichlorophenoxy- 255.5
acetic acid (2,4,5-T)
2-( 2,4-Dichlorophenoxy)- 235.1
propionic acid
(Dichloroprop)
2-(2-Methyl-4-chlorophen- 214.6
oxy)-propionic acid
(Mecoprop)
2-( 2 ,4 , 5-Tr ichlorophenoxy)-
propionic acid (Silvex)
4-(2,4-Dichlorophenoxy)-
butyric acid (2,4-DB)
4-(2-Methyl-4-chlorophen-
oxy)-butyric acid (MCPB)
2,3,7,8-Tetrachlorodi- 322
benzo-jv-dioxin (TCDD)
Octachlorodibenzo-jr-dioxin 459.8
(OCDD)
141 2.64
120 3.27
158-159
117-118
94-95
Water, 0.54 g/1 (20°C); ethanol, 1.3 kg/kg; ether,
2.43 kg/kg; toluene, 0.67g/kg; soluble in most
organic solvents
Water, 0.63 g/1 (20°C); highly soluble in alcohol,
ether, carbon tetrachloride, benzene and organic
solvents
Water, 0.189 g/1 (20°C); highly soluble in alcohol,
ether, chloroform and benzene
Water, 0.35 g/1 (20°O; soluble in most organic
solvents
I
Water, 0.62 g/1 (20°C)
269.5 179-181 2.84 Water, 0.14 g/1 (25°C); acetone, 180 g/kg; methanol,
134 g/kg; ether, 98 g/kg; heptane, 0.86 g/kg
249.1 117-119 4.8 Water, 0.053 g/1; acetone, 100 g/kg
228.6 100-101
305
130
Water, 0.044 g/1; ethanol, 150 g/kg; acetone, 200
g/kg
Water, 0.2ug/l (0.2 ppb); j^-dichlorobenzene, 1.4 g/1;
benzene, 0.57 g/1; chloroform, 0.37 g/1; n-octanol,
0.048 g/1; acetone, 0.11 g/1
o-Dichlorobenzene, 1.83 g/1; xylene, 3.58 g/1;
chloroform, 0.56 g/1; dioxane, 0.38 g/1
aSummarized from data compiled by N.N. Melnikov [Residue Rev. 36, 157 (1971)]; C.R. Worthington (ed.): "The
Pesticide Manual," 6th ed., British Crop Protection Council, 1979; M.P. Esposito, H.M. Drake, J.A. Smith,
and T.W. Ownes: "Dioxins: Volume I. Sources, Exposure, Transport, and Control," EPA-600/2-80-156, U.S.
Environmental Protection Agency, Washington, D.C., 1980.
-------
molecular weight. Similar properties have been found with other chloro-
phenoxy acids. Chlorophenoxy acids are relatively susceptible to breakdown by
V
enzymes in plant tissues (e.g^, 24) or by microorganisms in the soil (25).
The physical and chemical properties of halogenated dibenzofurans and
dibenzo-£-dioxiris have not been thoroughly studied due to the extreme toxicity
of some members of these two classes of compounds. Theoretically, depending
on the number of chlorine atoms and the position(s) of substitution, there are
75 possible congeners or isomers of chlorinated dibenzo-j^-dioxins (2 mono-, 10
di-, 14 tri-, 22 tetra-, 14 penta-, 10 hexa-, 2 hepta-, and 1 octa-chloro-
isomers) and 135 chlorinated dibenzofurans (4 mono-, 16 di-, 28 tri-, 38
tetra-, 28 penta-, 16 hexa-, 4 hepta-, and 1 octa-chloro-isomers) (17). Some
40 of the 75 possible chlorinated dibenzo-jv-dioxins have been prepared and
identified (see ref. 22). 2,3,7,8-Tetrachlorodibenzo-jv-dioxin (TCDD) is the
prototype compound of the chlorinated dibenzo-jv-dioxins. It is remarkable for
its lack of reactive functional groups and its chemical stability (13). In
fact, it is thermally stable up to the high temperature of 700°C. Complete
decomposition occurs at 800°C (26). The half-life of TCDD in the soil has
recently been estimated to exceed 10 years (27). It is an extremely lipo-
philic compound, practically insoluble in water (0.2 ppb) and only sparingly
soluble in most organic solvents (see Table XLVII). Both chlorinated dibenzo-
_p_-dioxins (22, 26) and dibenzofurans (28) are, however, subject to photochemi-
cal degradation with dechlorination as the principal reaction. The photolytic
susceptibility of three chlorinated dibenzo-jp_-dioxins follows the order:
2,7-dichloro- > 2,3,7,8-tetrachloro- > octachloro- (26). A synoptic review of
photolytic studies on TCDD has been presented by Esposito jt_ atl_. (22).
291
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5.2.2.3.2.2 BIOLOGICAL EFFECTS OTHER THAN CARCINOGENICITY.
Toxic and Other Biochemical Effects. The toxicology of halogenated
»_
phenoxy acids has been reviewed in several publications (2, 29, 30). Table
XLVIII summarizes some representative acute toxicity data of 2,4-D, 214,5-T
and several related compounds in three mammalian species. In general, uncon-
taminated halogenated phenoxy 'acids are only moderately toxic to mammals
probably because of their rapid renal excretion (see Section 5.2.2.3.4).
Acute toxic effects in mammals include various signs of muscular disorders
causing stiffness of extremities, ataxia, paralysis and eventually coma (31),
disruption of T-tubules in the myocardium, hypocholesterolemia and reduction
of serum triglyceride (30). Workers involved in the production of 2,4,5-T
were^reported to develop chloracne, liver disorders, neurologic changes,
metabolic disorders and porphyria (32); these effects appear to be due
primarily to the action of the extremely toxic contaminant, TCDD (see below).
The structure-activity relationships'of chlorophenoxy acids as herbicides
have been extensively studied (reviewed in refs. 1, 3). Chlorophenoxy acids
are structurally similar to the natural plant growth hormone, indole-3-acetic
acid (also known as auxin or IAA) and exert their herbicidal action by causing
lethally abnormal growth. Some of the important structural features of herbi-
cidal chlorophenoxy acids are: (a) the side-chain must possess a terminal
carboxyl group or a group that is easily converted to it within the plant
tissues; (b) the side-chain must contain an odd number of raethylene groups and
at least one hydrogen atom attached to the c(-carbon; chlorophenoxy acids with
3 or higher odd number of methylene groups owe their herbicidal activity to in
vivo |5 -oxidation to active chlorophenoxyacetic acid derivatives; (c) among
the chlorophenoxy acids with asymmetric carbon (e«g_. , dichloroprop, mecoprop,
292
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Table XLVIII
Acute Oral Toxicity of 2,4-D, 2,4,5-T and Related Compounds
In Mammalian Species8
(mg/kg)
Compound Mouse Bat Guinea pig
2,4-W.chlorophenoxyacetic acid (2,4-D)a
acid ' 368 (M) 375 (M) 469
sodium salt 375 805 (F) 551 (M)
isopropyl ester 541 (M) 700 550 (M)
mixed butyl esters 713 (F) 620 (F) 848 (F)
2-Methyl-4-chlorophenoxyacetic acid (MCPA)
acid 550 700
diethanolamine salt 550 800
2-(2,4-Dichlorophenoxy)propionic acid (Dichloroprop; 2,4-DP)
acid 400 800
2-(2-Methyl-4-chlorophenoxy)propionic acid (Mecoprop)
acid 650 700-1,500
diethanolamine salt 600 1,060
4-(2,4-Dichlorophenoxy)butyric acid (2,4-DB)b
acid 370-700
sodium salt 400 700-1,500
4-(2-Methyl-4-chlorophenoxy)butyric acid (MCPB)a
acid 680
sodium salt 700
2,4,5-Trichlorophenoxyacetic acid (2,4,5-T)a
acid 389 (M) 500 (M) 381 .
isopropyl ester 551 (F) 495 449 (F)
mixed butyl ester 940 (F) 481 (F) 750 (F)
mixed amyl ester 750 (F)
aSummarized from the data of V.K. Rowe and T.A. Hymas [Am. J. Vet. Res. 15,
622 (1954)].
^Summarized from the data compiled by J.P. Seller [Mutat. Res. 55, 197
(1978)].
s...
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silvex), only the (-O-forms of the stereoisomers are active; and (d) the
introduction of chlorine atoms into
The 2-, 4-; or 2,4-positions of the phenyl ring greatly enhances
activity whereas poor activity is found for compounds containing chlorine
atoms at the 2,6- or 3,5-positions. It has been hypothesized (Thimann's
theory) that the activity of IAA, chlorophenoxy acids, and other herbicides
t
such as 2,3,6-TBA (2,3,6-trichlorobenzoic acid) and picloram (2-carboxy-4-
amino-3,5,6-trichloropyridine) is dependent on the presence of a fractional
positive charge (e.g. , at the 1-position of IAA, 6-position of 2,4-D,
o
4-position of 2,3,6-TBA, 4-amino group of picloram) situated at a 5.5 A
distance from the negative charge of the carboxyl group. Presumably this
spacing of the polar centers is a requirement for the binding of these com-
pounds to receptor sites in plant cells so as to exert a phytohormonal action
(see ref. 3).
The toxicology of halogenated dibenzofurans and dibenzo-_p_-dioxins has
been a subject of extensive studies in recent years because of their environ-
mental significance and the extreme toxicity of some members of these two
X
classes of compounds. Numerous review articles, monographs and symposia (11,
12, 16-21, 30, 33) have covered this topic; only a brief account emphasizing
the comparative toxicity and structure-activity relationship is presented
below. 2,3,7,8-Tetrachlorodibenzo-jv-dioxin (TCDD), the most extensively
studied compound of the chlorinated dibenzodioxin series, has been widely
regarded as probably the most highly toxic compound synthesized so far. It
produces a gamut of toxic effects in a variety of animal species. Wide
species variations in lethality and pathologic responses have been observed.
The acute oral LDcg values (in ^jg/kg body weight) of TCDD in mammalian species
are: 0.6-2 for guinea pigs (34, 35) 22-60 for rats (34, 36); < 70 for monkeys
293
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(37), 115 for rabbits (34), 114-284 for mice (35, 38, 39), > 300 for dogs
(34), and > 3,000 for hamsters (40). The unusual resistance of hamsters to
TCDD toxicity may be partially, but not fully, explained by a much faster rate
^
of metabolism and elimination of the compound in this species (41). The acute
toxicity of TCDD in rats is decreased by pretreatment of the animals with
inducers of microsomal mixed-function oxidases (MFOs), but enhanced by MFO
inhibitors, suggesting that the metabolism of TCDD is mainly detoxifying in
nature (36). Most of the pathologic responses to TCDD involve epithelial.
tissues. The types of toxic responses include (a) hyperplasia and/or meta-
plasia of the skin (chloracne), gastro-intestinal raucosa, urinary tract, bile
duct and/or gall bladder, (b) hypoplasia, atrophy or necrosis of thymus, bone
marrow and testicles, (c) hepatomegaly or liver necrosis, (d) edema, and (e)
wasting (severe weight loss) accompanied by a depletion of adipose tissue;
many of these responses are highly species-specific (see refs. 18, 20). A
number of isosteric analogs of TCDD, such as 2,3,7,8-tetrachlorodibenzofuran,
2,3,6,7-tetrachloronaphthalene, and 3,3',4,4'-tetrachlorobiphenyl, appear to
have a similar spectrum of toxic effects as TCDD (20).
The structure-toxicity relationships of halogenated dibenzo-jv-dioxins,
dibenzofurans and related compounds have been extensively investigated by
McConnell, Moore and associates (18, 35, 37, 42). The acute, oral LD^Q values
of 13 chlorinated dibenzo-j^-dioxins and several related compounds in mice and
guinea pigs are summarized in Table XLIX. As the data in the Table indicate,
the degree of toxicity of chlorinated dibenzo-_p_-dioxin is dependent upon the
number and positions of chlorine substitutions. The most toxic compound
(TCDD) is at least 180,000 times more potent than the least toxic compound
(2,8-dichlorodibenzo-jr-dioxin). It is apparent that all the lateral positions
(2,3,7,8-) must be chlorinated to achieve the greatest degree of toxicity.
294
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Table XLIX
Estimated Single Oral 30-Day LD5Q Values of Chlorinated Dibenzo-_p_-dioxins
and Related Compounds3
Compound
Substituted Dibenzo-p-dioxin
2,8-Dichloro-
2,3,7-Trichloro-
2,3,7, 8-Te trachloro-
1,2,3,7, 8-Pen tachl oro-
1 , 2 , 4 , 7 , 8-Pentachloro-
1,2,3,4,7, 8-Hexachloro-
1,2,3,6,7, 8-Hexachloro-
1 , 2 , 3 , 7 , 8 , 9-Hexachloro-
1,2,3,4,6,7,8-Heptachloro-
1-Ni tro-3 , 7 , 8-trichloro-
l-Amino-3,7,8-trichloro-
l-Nitro-2, 3, 7 ,8-tetrachloro-
l-Amino-2,3,7,8-tetrachloro-
LD50
Mouse
> 10
0.88
0.94
> 14
2.11
3.19
> 3.67
> 5.4
> 14.2
(umole/kg)
Guinea pig
> 1,180
120.4
0.006
0.009
3.15
0.185
0.178-0.255
0.153-0.255
> 1.4
> 90
> 99
0.129
0.576
Relative
Po tency
in the
Guinea Pigb
0.01
0.09
1,883
1,256
3.6
61
44-63
44-74
8
0.13
0.11
88
20
Most Toxic Congener of Related Halogenated Aromatic Series
2,3,7, 8-Te trachlorodibenzof uran
3,3',4,4',5,5'-Hexachlorobiphenyl
2,3,6, 7-Te trachloronaph thalene
2,3,6, 7-Te trab romonaph thalene
0.023
1.39
> 11.3
0.547
507
8
1
21
aSummarlzed from E.E. McConnell, J.A. Moore, J.K. Haseman, and M.W. Harris
[Toxicol. Appl. Pharmacol. 44, 335 (1978)] and E.E. McConnell: In
"Halogenated Biphenyls, Terphenyls, Naphthalenes, Dibenzodioxins and Related
Products" (R.D. Kimbrough, ed.), Elsevier, Holland, 1980, p. 109.
"Relative to 2,3,6,7-tetrachloronaphthalene.
-------
Additional chlorine atoms (or amino or nitro groups) at ortho (or peri) posi-
tions reduce the toxicity but not to the extent caused by deletion or substi-
tution of a chlorine atom at one of the lateral positions. Interestingly, the
same structural requirements for toxicity are also observed in the halogenated
dibenzofuran, biphenyl and naphthalene series (see Table XLIX). The relative
toxicity of the most toxic chlorinated congener of each of the above series
follows the order: dibenzo-^-dioxin > dibenzofuran » biphenyl » naphtha-
*
lene. Considering the isosteric nature (a rectangular molecular shape of
o*
about 6 x 11 A with a halogen at each of the 4 lateral positions) of all these
compounds, the similarity of toxic effects, and other inferential evidence,
Poland and Knutson (20) proposed that they have a common mechanism responsible
for the toxicity. According to Poland and Knutson, these compounds bind to a
common cytosolic receptor and the receptor-ligand complex translocates to the
nucleus and mediates the ensuing gene expression causing two stages of pleio-
tropic responses a "limited" response involving a variety of biochemical
effects (see below) and a "restricted" response involving the expression of
normally restricted genes thus leading to a gamut of toxic effects. In this
connection the reader is cautioned that other isosteric compounds (such as
dibenzothiophene, thianthrene, thioxanthone, phenothiazine, anthraquinorie)
with lateral positions fully halogenated may be potentially toxic.
Other biochemical effects, produced by TCDD and its isosteric analogs,
arid the structure-activity relationships have been reviewed in detail by
Poland and associates (13, 20) and by Goldstein (43). Essentially, these
effects are: (a) endocrine effects (abnormal levels of estrogens, thyroxine
and corticosteroids); (b) vitamin A deficiency; (c) abnormal lipid metabolism
(causing fatty liver and possible increase in lipid peroxidation); (d) por-
phyria; (e) impaired biliary and renal excretory transport; and (f) induction
* These figures represent the approximate molecular size of biphenyl and
dibenzo-p-dioxin calculated with inclusion of the van der Waal radii.
295
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of microsomal mixed-function oxidases (MFOs). Among these, the induction of
MFOs by TCDD and congeners has been extensively studied (this topic will be
further discussed in Section 9.4 in a subsequent volume of this series). TCDD
* i
is generally considered to be probably the most potent inducer of MFOs; on a
molar basis, TCDD (median effective dose, EDj0 « 0.85 nmol/kg) is 30,000 times
more potent that 3-methylcholanthrene. The inducing effect of a single dose
of TCDD may persist for 35 days' in rats, reflecting the prolonged biological
half-life of the compound. The inducing effect of TCDD and congeners has been
shown to be mediated through a cytosolic receptor protein which binds the
ligand, translocates to the DNA and initiates enzyme induction. Structure-
activity relationship studies show a good correlation between their binding
affinities to the receptor protein and their potency to induce aryl hydro-
carbon hydroxylase (AHH) activity. The structural requirements for enzyme
induction are similar to those described for toxicity.
Mutagenicity. Owing to their environmental importance, halogenated
phenoxyacetic acids, dibenzodioxins and related compounds have been exten-
sively tested for mutagenicity. Table L summarizes the data available on Ames
Salmonella tests of a variety of halogenated phenoxyacetic acid, diphenyl
ether, dibenzofuran, and dibenzo-_p_-dioxin derivatives. As the data in the
Table indicate, some 20 derivatives of 2,4-D and 2,4,5-T have been tested;
interestingly, none of these compounds show any mutagenic activity.
Based on structural similarity to acridine compounds (see Section
5.1.2.2.2, Vol. IIB), TCDD is expected to be a frame-shift mutagen (15). The
evidence for mutagenicity of TCDD is, however, unconvincing with the Ames
test. Using a liquid preincubation procedure, Hussain j_t__al_. (59) reported
that TCDD was mutagenic without metabolic activation in Salmonella typhinrnrium
strain TA1532 (a frame-shift mutant) but inactive in strain TA1530 (a base-
296
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Table L
Mutagenlclty of 2,4-D, 2,4,5-T, TCDD and Related Compounds in the Ames Test
Compound
Mutagenicity References
2,4-D, 2,4,5-T and Related Compounds
4-Chlorophenoxyacetic acid
2,4-Dichlorophenoxyacetic acid (2,4-D)
acid or sodium salt
jn-butyl ester
isooctyl ester
3,4-Dichlorophenoxyacetic acid
2-Methyl-4-chlorphenoxyacetic acid (MCPA)
2-(4-Chlorophenoxy)propipnic acid
2-(2,4-Dichlorophenoxy)propionic acid (Dichloroprop)
2-(2-Methyl-4-chlorophenoxy)pripionic acid (Mecoprop)
4-Chlorophenoxyisobutyric acid (Clofibrate)
2-(2,4-Dichlorophenoxy)butyric acid (2,4-DB)
4-(3,4-Dichlorophenoxy)butyric acid
4-(2-Methyl-4-chlorophenoxy)butyric acid (MCPB)
2,4,5-Trichlorophenoxyacetic acid (2,4,5-T)
acid
jn-butyl ester
isobutyl ester
2-(2,4,5-Trichlorophenoxy)propionic acid (Silvex)
4-(2,4,5-Trichlorophenoxy)butyric acid
Halogenated Diphenyl Ether
Decabromodiphenyl ester
Chlorinated Dibenzofurans
Unsubstituted dibenzofuran
2,9-Dichlorodibenzofuran
3,6-W.chlorodibenzofuran
2,3,7,8-Te trachlorodibenzofuran
Octachlorodibenzofuran
Chlorinated dibenzo-p-dioxins
2,3,7,8-Tetrachlorodibenzo-£-dioxin (TCDD)
Octachlorodibenzo-p-dioxin
(44*)
(44*, 45-48)
(49)
(50)
(44*)
(51)
(44*)
(44*)
(44*, 52)
(53)
(44*, 45)
(44*)
(44*)
(44*. 54, 55)
(49, 54)
(49)
(44*)
(44*)
(56)
(57, 58)
(57)
(57)
(57)
(57)
(59, 60)
(56, 61, 62)
(60)
*Without metabolic activation
-------
pair substitution mutant). Seiler (60) confirmed the mutagenicity of TCDD in
TA1532 in a spot test but showed marginal or a lack of activity in two other
frame-shift mutants (TA1531, TA1534) and two base-pair substitution mutants
V
(G46, TA1530). Unpublished results of McCann (cited in ref. 15), however,
failed to show any mutagenic activity of TCDD in strains TA1532, TA1535,
TA1537 and TA1538 with or without metabolic activation in a plate incorpora-
tion test (which is more sensitive than the spot test). Negative findings
have also been reported by Nebert £t_jal_. (61), Geiger and Neal (62), and by
the U.S. National Toxicology Program (56) using strains TA98, TA100, TA1535,
TA1537 and/or TA1538. Strain TA1537 is a more sensitive direct descendant of
TA1532. Strain TA1538 and its plasmid-containing descendant, TA98, are also
highly sensitive to frame-shift mutagens. The assay by the U.S. National
Toxicology Program (56) used a liquid preincubation procedure, similar to that
used by Hussain jet_jil_. (59) for obtaining their positive finding. The
discrepancy betweem earlier and more recent studies may be due to differences
in solvent (dimethylsulfoxide in earlier studies; 1,4-dioxane in recent
studies), impurities or other factors. Nonetheless, the totality of the data
tends to support the view that, like most polyhalogenated aromatics (see
Section 5.2.2.2.2), TCDD has no appreciable rautagenic activity. It should be
noted that, because of its extremely high toxicity, TCDD can only be assayed
at raicrogram levels. It would be interesting to test congeners of TCDD with
lower toxicity. Thus far, the considerably less toxic octachlorodibenzo-jv-
dioxin has been tested and found to have questionable or no activity (60). In
the closely related dibenzofuran series, none of 4 chlorinated derivatives
exhibited any mutagenic activity (see Table L).
In addition to the Ames Salmonella test, 2,4-D (reviewed in ref. 4),
2,4,5-T (reviewed in ref. 5), TCDD (reviewed in refs. 15, 19, 21) and related
297
-------
compounds have been tested in a variety of test organisms including prophages
(59), other bacteria (e.g., ref. 45; see also refs. 4, 5), Drosophila (63; see
also refs. 4, 5), higher plants (64, 65; see also refs. 4, 5), cultured mam-
malian cells (45, 46, 66, 67) and experimental animals (54, 55, 68-71). With
a few exceptions, these compounds are generally inactive or marginally active
in most microbial and mammalian assays, but may display some activity in
certain plants. It 'should be noted that the mutagenicity of halogenated
phenoxyacetic acids in in vitro systems may be affected by the pH of the
incubation medium, because of the inability of anionic forms to penetrate the
cell membrane (48); also, studies with plants are often difficult to perform
because of the auxin-like action of these compounds (see ref. 4). Cytogenetic
studies have been conducted on the TCDD-exposed population in Seveso, Italy;
thus'far, the rate of chromosome aberrations appears to lie within the normal
range (23, 72).
Teratogenicity.
The teratogenicity of 2,4,5-T (technical grade) was first reported in a
1968' teratogen screening study conducted by the Bionetics Research Laboratory
(6) for U.S. National Cancer Institute. Subsequently, it was found that the
teratogenic effects observed were attributable, at least in part, if not
wholly, to the TCDD contaminant (30 ppm) present in the 2,4,5-T samples.
Further studies confirmed the teratogenicity of TCDD and established it as one
of the most potent animal teratogens known. Owing to their environmental
significance, the teratogenicity of 2,4-D, 2,4,5-T, TCDD and related compounds
has been extensively studied. A summary of the major findings of the terato-
genicity studies on these compounds is presented in Table LI.
2,4-Dichlorophenoxyacetic acid (2,4-D) and several and its alkyl esters
298
-------
p. i of 2
Table LI
Teratogenicity of 2,4-D, 2,4,5-T, TCDD and Related Compounds
Teratogenicity3
Compound Species (effects)
2,4-Dichlorophenoxyacetic acid (2,4-D)
2,4-D
2,4-D methyl ester
2,4-D ethyl ester
2,4-D butyl ester
2,4-D isooctyl ester
*
2,4-D dime thy lamine salt
2,4,5-Trichlorophenoxyacetic
2,4,5-T
2,4,5-T butyl ester
2,4,5-T isooctyl ester
t
Mouse
Rat
Hamster
Mouse
Mouse
Mouse
Rat
Mouse
Rat
-
Rat
acid (2,4
Mouse
Rat
Rabbit
Hamster
Sheep
Monkey
Mouse
Mouse
References
and Related Compounds
±
-
+ (SM)
± (SM)
-
-
-
+ (SM)
±
* (SM)
-
+ (SM)
,5-T) and Related
+c (CP, KA)
+ (CP, KA)
+ (CP)
+ (CP, SM)
± (CP)
+c (KA, VA)
-
-
+c (SM, EA)
-
-
-
+ (CP)
+ (CP)
(6)
(73)
(74)
(75)
(6)
(6)
(6)
(74)
(6)
(74)
(76)
(74)
Compounds
(6, 77, 78)
(78)
(79, 80)
(81, 82)
(83)
(6, 77)
(78, 84-87)
(87)
(75)
(88)
(89)
(90)
(85)
(85)
-------
Table LI (continued)
p. 2 of 2
Compound
2,4,5-T propylene glycol
butyl ether ester
2 ,4 , 6-Tr ichlorophenoxy-
acetic acid
Silvex
Phenoxyacetic acid
2,4, 5-Tr ich loropheno 1
Teratogenicity*
Species (effects) References
House
Mouse
Mouse
Rat
Mouse
Mouse
+ (CP) (85)
(6)
(85)
(85)
(82)
(82)
Chlorinated Dibenzo-p-dioxins
2-Chloro-
2,3-Dichloro-
2,7-Dichloro-
1,2,3,4-Tetrachloro-
2,3,7, 8-Tetrachloro-
Hexachloro-
(mixed isomers)
Octachloro-
Rat
Rat -
Rat
Rat
Mouse
Rat
Rabbit
Monkey
Rat
Rat
(68)
(68)
(34, 68)
(68)
+ (CP, KA) (78, 91, 92)
+ (CP) (79)
+ (KA, VA) (78)
± (VA) (68, 93)
+ (KA) (94)
+ (SPA) (95)
+ (CP, SM) (34)
(34)
aSymbols for teratogenicity: + = positive; - » negative; ± - incon-
clusive, marginal effect or may be considered as manifestation of
erabryotoxicity rather than teratogenicity.
Abbreviations used: CP = cleft palate; SM = skeletal malformations;
KA = kidney abnormalities; VA = visceral anomalies; EA = eye abnor-
malities; SPA = soft palate abnormalities.
cTeratogenicity attributable, at least in part, to the presence of
TCDD as contaminant.
-------
were tested for teratogenicity in several strains of mice in the screening
study of Bionetics Research Laboratory (6). Among these, 2,4-D and its iso-
octyl ester were found to have possibly weak teratogenic effects in some
strains of mice; however, the results were considered to have marginal signi-
ficance, requiring further studies for confirmation. The butyl ester of 2,4-D
was listed as fetotoxic, but probably not teratogenic, while the methyl and
ethyl esters of 2,4-D exhibited no fetotoxic or teratogenic effects under the
conditions of the study. Two other related compounds, 2,4-D isopropyl alcohol
and 2-(2,5-dichlorophenoxy)propionic acid, gave conflicting results from which
no conclusions could be drawn. In a three-generation study using Osborne-
Mendel rats, Hansen _e_t^ jrU (73) found no evidence in support of the terato-
genicity of 2,4-D. However, Khera and McKinley (74) reported that 2,4-D and
its butyl, isooctyl, butoxyethanol and dimethylamine derivatives (not believed
to contain TCDD contaminant) all induced fetopathy and increased incidence of
skeletal anomalies following daily oral administration of 100-150 rag/kg on
days 6-15 of gestation. In a more recent study by Unger et_^l_. (76), no
evidence of teratogenicity was noted in CD rats that received daily oral doses
of 2,4-D isooctyl ester equivalent to up to 87.5 rag/kg 2,4-D during days 615
of gestation. Low incidence of fetal anomalies was observed in the offspring
of Syrian golden hamsters receiving over 60 mg/kg 2,4-D during days 6-10 of
gestation; the effect was not clearly dose-dependent (75).
2,4,5-Trichlorophenoxyacetic acid (2,4,5-T) has been tested for terato-
genicity in six mammalian species. It is important to note that the purity of
2,4,5-T samples used in these studies plays a significant role in determining
the teratogenicity of the compound; technical grade 2,4,5-T may contain suffi-
cient amounts of TCDD contaminant (which is extremely teratogenic) to account
for a part of or all the teratogenic effects observed. Technical grade
299
-------
2,4,5-T was found to be teratogenic in several strains of mice inducing mainly
cleft palate and kidney abnormalities (6, 77, 78). Similar teratogenic
effects have been observed in a number of studies (78-82) using analytical
i
grade 2,4,5-T (containing less than 1 ppm TCDD) indicating that 2,4,5-T per se
is indeed teratogenic in mice. In the rat, however, only technical grade
2,4,5-T containing relatively high amounts of TCDD (30 ppm) was found to be
teratogenic (6, 77); a variety of'studies (78, 84-87) using more purified
2,4,5-T samples showed a complete lack of teratogenicity. Similarly, 2,4,5-T
appeared to be nonteratogenic in rabbits (87), sheep (89) and monkeys (90).
Collins and Williams (75) reported that commercial samples of 2,4,5-T induced
eye abnormalities (absence of eyelid) and delayed head ossification in the
fetuses of Syrian golden hamsters; the effects were related to the amount of
TCDD contaminant present. In another hamster study, 2,4,5-T was not found
teratogenic (88). Besides 2,4,5-T, the butyl, isooctyl and propylene glycol
butyl ether esters of 2,4,5-T are all teratogenic in the mouse inducing mainly
cleft palate (85). In contrast, a number of closely related compounds, such
as 2,4,6-trichlorophenoxyacetic acid, silvex, phenoxyacetic acid and
2,4,5-trichlorophenol, are all inactive in the mouse suggesting that strict
structural requirements are needed for teratogenicity.
As may be expected from the fact that TCDD was discovered to be a terato-
gen because of its presence as an impurity in 2,4,5-T, TCDD has emerged as one
of the most potent animal teratogens known. The mouse appears to be the most
sensitive species. Studies by several groups of investigators (78, 79, 91,
92) concur that TCDD is teratogenic in mice at doses as low as 1 to 3
yug/kg/day. The principal teratogenic effects are cleft palate and kidney
anomalies. Kidney abnormalities have been observed even in mouse pups nursing
on TCDD-treated mothers (92). Rats are apparently more resistant to the
300
-------
teratogenic effects of TCDD than mice. In three rat teratology studies,
kidney abnormalities and intestinal hemorrhage were observed in one study (78)
while in the other two studies (68, 93) only intestinal hemorrhage (usually
considered as embryotoxicity rather than teratogenicity) was noted in off-
spring of rats given as much as 8 to 16 ^ig/kg/day. The kidney is also the
teratogenicity target organ of TCDD in the rabbit (94) whereas in the monkey,
TCDD induces abnormal development of soft palate (95). Besides TCDD, a number
of other chlorinated dibenzodioxins have been tested for teratogenicity (see
Table LI). 2-Chloro-, 2,3- and 2,7-dichloro-, 1,2,3,4-tetrachloro- and octa-
chloro-dibenzo-jv-dioxins are all nonteratogenic in the rat (34, 68). Only
hexachlorodibenzo-jr-dioxin (mixed isoraers) exhibits teratogenicity inducing
cleft palate and skeletal malformation in rats at a dose level of 100
/ig/kg/day (34). Thus, like other toxic effects, the teratogenicity of chlori-
nated dibenzo-_p_-dioxin is clearly dependent on the position and number of
chlorine substitutions on the dibenzo-jv-dioxin nucleus. Two isosteric analogs
of TCDD, 2,3,7,8-tetrachlorodibenzbfuran and 3,3*,4,4'-tetrachloroazoxybenzene
have recently been reported to be teratogenic in mice (96).
The potential fetal effect of paternal exposure to 2,4-D, 2,4,5-T and
TCDD mixture has recently been studied by Lamb et_ _al_. (97). Theoretically,
fetal effects might occur through the male if the chemical were transmitted to
the female via the seminal plasma resulting in direct exposure to the ova.
Groups of male C57BL/6N mice were exposed to mixtures of 2,4-D, 2',4,5-T and
TCDD shortly before mating. No teratogenic effects were observed in the
offspring sired by these males.
5.2.2.3.3 Carcinogenicity and Structure-Activity Relationships.
5.2.2.3.3.1 CARCINOGENICITY OF 2,4-D, 2,4,5-T AND RELATED COMPOUNDS.
301
-------
The potential carcinogenicity of 2,4-D, 2,4,5-T and six other related
herbicides (isopropyl, _n_-butyl and isooctyl esters of 2,4-D; dichloroprop and
its 2,5-dichloro isoraer; and silvex) was evaluated in a 1968 screening study
\.
sponsored by U.S. National Cancer Institute (98, 99). Two strains of mice, Fj
hybrids of C57BL/6 x C3H/Anf and C57BL/6 x AKR, were used in this study. The
compounds were given either by oral administration (gavage followed by diet)
at maximum tolerated doses daily for up to 18 months or by a single subcu-
taneous injection. The results of this study are summarized in Table LIT.
With one exception, none of these compounds displayed any significant carcino-
genic effects by either route. Only 2,4-D isooctyl ester (97Z pure;
impurities not specified) caused a significant increase in the incidence
(5/17) of reticulum cell sarcomas in female mice of the second strain after a
single subcutaneous injection of 21.5 rag/kg of the compound. The compound was
not carcinogenic in mice of the first strain by subcutaneous route and in mice
of either strain by oral route.
The carcinogenicity of 2,4-D (containing no detectable amount of TCDD)
was also tested in Osborne-Mendel rats (73). Groups of 50 rats (25 of each
sex) were fed for 2 years diets containing 0, 5, 25, 125, 625 or 1,250 ppra
2,4-D. The total number of rats with tumors at the end of the experiment in
the control and the five experimental groups was 15, 14, 18, 20, 23 and 22,
respectively. The tumors were randomly distributed among various tissues and
were of the types normally found in aging Osborne-Mendel rats. Although
statistically significant increase in the incidence of malignant tumors was
found in the male high-dose group (6/25 vs. 1/25 for control), the authors
(73) concluded that "a carcinogenic effect of 2,4-D has not been shown." In a
study by Arkhipov and Koslova (cited in ref. 2), random-bred rats were given
an amine salt of 2,4-D mixed in the feed at a concentration equivalent to 1/10
302
-------
Table LII
Carcinogenicity of 2,4-D, 2,4,5-T and Related Compounds in the Mouse and the Rat
Compound3
2,4-D acid
2,4-D isopropyl ester
2,4-D jn-butyl ester
2,4-D isooctyl ester
Dichloroprop
2-(2,5-Dichloro-phenoxy)-
propionic acid
2,4,5-T acid
Silvex
2 ,4,5-Trichlorophen-
oxyethanol
Clof ibrate
Species & Strain
Mouse, X or Y
Rat, Osborne-Mendel
Mouse, X or Y
Mouse, X or Y
Mouse, X or Y
Mouse, Y
Mouse, X or Y
Mouse, X or Y
Mouse, X or Y
Mouse, XVI I/G
Mouse, C3Hf
Mouse, X or Y
Mouse, Swiss
Rat, F344
Route
oral or s.c..
oral
oral or B.C.
oral or s.c.
oral
s.c. ,
oral or s.c.
oral or s.c.
oral or s.c.
oral
oral
oral or s.c.
oral
oral
Principal
Carcinogenicity Targets
None
Multiple sites (males)0
None
None
^
None
Hematopoietic system (females)
None
None
None
None
Multiple sites (females)
None
Liver (males)
Liver, pancreas, etc.
References
(98, 99)
(73)
(98, 99)
(98, 99)
(98, 99)
(98)
(98, 99)
(98, 99)
(98, 99)
(100)
(100)
(98, 99)
(101-103)
(104, 105)
aSee Table XLVI for structural formulas.
bStrain X - (C57BL/6 x CSH/AnfjFj; strain Y - (C57BL/6 x AKR)Fr
cNot considered carcinogenic.
-------
of the LDcQ. Two treated rats developed tumors (a mammary fibroadenoma and a
hemangioma of the mesenterium) after 27 months and one control rat had a
mammary fibroadenoma after 27 months. The significance of this study cannot
V
be assessed due to the incomplete reporting of the data.
Muranyi-Kovacs _et^jal.. (100) retested 2,4,5-T for possible carcihogenicity
in two strains (XVII/G and C3Hf) of mice. No carcinogenic effects were
observed in XVII/G mice. In C3Hf mice, however, 2,4,5-T (100 mg/1 in drinking
water for 2 months followed by 80 ppra in diet for life) caused a significant
increase in the incidence of various tumors in treated females (13/25 vs. 9/44
control). The tumors observed included 4 hepatomas, 3 leukemias, 3 cervical
tumors, 2 skin squamous cell carcinomas and 1 osteosarcoma. Since the 2.4,5-T
sample used in this study contained very low level « 0.05 ppm) of chlorinated
dibenzodioxins, the authors (100) attributed the carcinogenic effects to
2,4,5-T per se and recommended further testing of this compound in other
animal species.
Two compounds structurally related to 2,4-D and 2,4,5-T have been found
to be carcinogenic in rodents. 2,4,5-Trichlorophenoxyethanol (TCPE), a compo-
nent in the herbicide Buvinol, has recently been extensively studied by Sugar,
Toth and associates (101-103). Weekly oral administration of maximum
tolerated doses (67-70 mg/kg) of the compound to Swiss mice for one year led
to a significant increase in the incidence of liver tumors (48-58% for experi-
mental vs. 26% for control) in male animals. Although the TCPE sample used
was contaminated with trace amounts of 2,3,7,8-tetrachlorodibenzo-jv-dioxin
(TCDD), carcinogenicity studies using TCPE sample with different amounts of
TCDD contaminants strongly indicate that TCPE itself is hepatocarcinogenic.
Clofibrate (4-chlorophenoxyisobutyric acid ethyl ether) is another struc-
turally related compound which induces tumors in the liver, pancreas and in a
303
-------
number of other tissues in the rat. The details of these studies have been
described in Section 5.2.1.7.9 (p. 651 in Volume IIIA). It is important to
note that there is some epidemiological evidence that clofibrate (106) and a
number of chlorophenoxy acids (see Section 5.2.2.3.5.1) are potential human
carcinogens.
5.2.2.3.2.2 -<:A.RCINOGENICITY OF HALOGENATED DIPHENYL ETHERS AND
* '
DIBENZOFURANS.
There is virtually no information on the carcinogenicity of halogenated
diphenyl ethers and dibenzofurans. 4-Bromophenyl phenyl ether was tested in
the lung adenoma assay by Theiss _£t_ Jjj;/ (107), using male strain A/St mice as
the test species. Multiple injections of the compound up to total doses of
920-3,600 mg/kg body weight had no significant effect on the incidence or
multiplicity of lung adenomas. Whereas the study may suggest a lack of car-
cinogenic activity of the compound toward the mouse lung, no conclusion can be
made regarding its carcinogenicity toward other organs. No chlorinated
dibenzofurans have thus far been tested for possible carcinogenicity at the
time of this writing despite the fact that they are structurally closely
related to chlorinated dibenzo-jr-dioxins. The unsubstituted parent compound,
dibenzofuran, was tested by skin painting in a small number of stock mice; no
carcinogenic activity was detected (108).
5'.2.2.3.3.3 CARCINOGENICITY OF TCDD AND RELATED COMPOUNDS.
Carcinogenicity Studies on TCDD.
Investigation of the possible carcinogenicity of TCDD (2,3,7,8-tetra-
chlorodibenzo-jv-dioxin) was prompted by the report of Tung (10), indicating
increased liver cancer incidence among Vietnamese exposed to TCDD-contarainated
defoliant, "Agent Orange." To date, six carcinogenicity studies (not includ-
304
-------
ing promoter or modifier studies) on TCDD have been reported; the major
findings of these are summarized in Table LIII. Van Miller _et_^l_- (116) were
among the first to report an increased incidence of tumors in rats exposed to
V
low levels of TCDD. In this study, groups of 10 male Sprague-Dawley rats were
fed diets containing 0.001, 0.005, 0.05, 0.5, 1 and 5 ppb TCDD for up to 78
weeks. The number of tumor-bearing rats at the termination of the experiment
was 0, 5, 3, 4, 4 and 7, respectively; none of the 10 control rats bore
tumors. With the exception of the 5 ppb group, the tumors appeared to be
randomly distributed among various tissues suggesting that TCDD may be a
promoter of neoplastic changes rather than a complete carcinogen. In the 5
ppb group, six liver tumors (2 cholangiosarcomas and 4 neoplastic nodules) and
four pulmonary squamous cell tumors were found among the seven tumor-bearing
rats. Thus, the results indicate that TCDD may bring about an increase of
tumor incidence in rats at dietary levels as low as 0.005 ppb (equivalent to
weekly exposure of 0.001 yug/kg body weight), but the induction of tumors in
specific organs (i.e., liver and lung) requires relatively high (5 ppb) dose
levels.
i
The carcinogenicity of TCDD in Sprague-Dawley rats has been confirmed by
Kociba _et__al^. (114, 115) in a 2-year ingestion study. However, statistically
significant increases in tumor incidences were observed only in rats exposed
to 0.1 yug/kg/day. The significant neoplasms include: hepatocellular car-
cinoma (11/49 experimental vs. 1/86 control) and squamous cell carcinoma of
the lung (7/49 experimental vs. 0/86 control) in females; stratified squamous
cell carcinoma of the tongue (3/50 experimental vs. 0/85 control) in males;
and stratified squamous cell carcinoma of the hard palate/nasal turbinate
(8/99 experimental vs. 0/171 control) in rats of either sex. In rats given
0.01 ^g/kg/day TCDD, the only significant effect was an increase in the inci-
305
-------
Table LIII
Carcinogenicity of TCDD and Related Compounds in the Mouse and the Rat
Dibenzo-jv-dioxin
Derivative Species & Strain Route Principal Organs Affected References
Unsubstituted
compound
2,7-Dichloro-
2,3,7,8-Tetra-
chloro-
Mouse, Swiss Webster Topical None? (preliminary)*
Mouse, B6C3Fi Oral None
Rat, Osborne-Mendel
Oral
None
Mouse, Swiss Webster Topical None? (preliminary)9
Mouse, B6C3Fj Oral
Rat, Osborne-Mendel Oral
None
Mouse, Swiss Webster Topical) Skin (females)
Mouse, Swiss
Mouse, B6C3FJ
Oral
Oral
Rat, Sprague-Dawley Oral
Rat, Sprague-Dawley Oral
Rat, Osborne-Mendel Oral
Liver (males)
Liver (males and females);
thyroid gland (females)
Liver, lung (females); hard
palate/nasal turbinates,
tongue (males and females)
Liver, (high dose); multiple
sites (low dose)
Thyroid gland (males);
liver (females)
(109)
(110)
(110)
(109)
Liver, hematopoietic system (111)
(males)
Hexachloro- Mouse, Swiss Webster Topical None
(1:2 mixture of Mouse, B6C3Fj Oral Liver (males and females)
1,2,3,6,7,8- and Rat, Osborne-Mendel Oral Liver (females)
1,2,3,7,8,9-
isomers)
Octachloro-
Mouse, Swiss Webster Topical None (preliminary)8
(111)
(112)
(103)
(113)
(114, 115)
(116)
(113)
(117)
(118)
(118)
(109)
aThe data of these studies are considered inconclusive by U.S. National Cancer
Institute/National Toxicology Program.
Suggestive evidence of a carcinogenic effect (see text).
-------
dence of hepatocellular hyperplastic nodules (18/50 experimental vs. 8/86
control). At the dose level of 0.001 ug/kg/day, no adverse effects were
noted.
In a recent carcinogenesis bioassay of TCDD conducted for U.S. National
Toxicology Program (113), Osborne-Mendel rats were given TCDD by gavage at
weekly doses of .0.01, 0.05 or 0.5 jig/kg body weight for 104 weeks. Signi-
f
ficaht neoplastic changes occurred only in the high dose group, showing
increased incidences of follicular cell adenomas of the thyroid gland (10/50
experimental vs. 1/69 control) and of hepatocellular neoplastic nodules (12/49
experimental vs. 5/75 control) observed in male and female rats, respec-
tively. Thus, the liver and the thyroid gland appear to be the target organs
of TCDD in Osborne-Mendel rats.
The carcinogenicity of TCDD has also been demonstrated in mice. Toth et
al. (103) administered TCDD (0.007, 0.7 and 7.0 yug/kg body weight) to Swiss
mice by gavage once a week for one year and observed the animals for the rest
of their lifespan. A significant increase (21/44 experimental vs. 7/38
control) in the. incidence of liver tumors was noted in the medium dose
^
group. Mice in the high dose group had considerably shortened lifespan
because of severely ulcerating skin lesions and lethal amyloidosis. In both
the gavage and dermal studies of the U.S. National Toxicology Program (112,
113), TCDD was found to be carcinogenic in mice at or close to the maximum
tolerated doses. In the gavage study (113), B6C3Fj mice were given weekly
doses of 0.01, 0.05 or 0.5 yug/kg (males) and 0.04, 0.2 or 2 ^ug/kg (females)
TCDD for 104 weeks. Significantly higher incidences of hepatocellular car-
cinomas (males: 17/50 experimental vs. 8/73 control; females: 5/46 experi-
mental vs. 0/69 control) were observed in the high dose groups. In female
mice,' follicular cell adenomas of the thyroid gland also occurred at signifi-
306
-------
cantly higher incidence in the high dose group (5/46 experimental vs. 0/69
control). In the dermal study (112), Swiss-Webster mice received topical
applications of 0.001 ug (males) or 0.005 ug (females) TCDD, 3 times per week
v
for 99 or 104 weeks. The only significant finding was the induction of skin
tumors located on the back at or near the site of application. Most, of the
skin tumors were fibrosarcomas with an occasional fibroma or kerato-
acanthoma. The incidence of fibrosarcomas of the integumentary system was
significantly higher in female mice (8/27 experimental vs. 2/41 control). An
increase in this type of tumor was also noted in male mice (6/28 experimental
vs. 3/42 control) but the increase was not statistically significant.
/
Thus, the totality of the studies indicates that TCDD is carcinogenic in
at least two animal species; however, doses close to the maximum tolerated
dose (MTD) are often required for the manifestation of its carcinogenic
activity. Depending on the species or strain of the animal, the principal
target organs are the liver, the lung, the thyroid gland, and nasal/oral
cavities by oral administration; the skin appears to be the only affected
tissue by dermal route.
Carcinogenicity Studies on TCDD-Related Compounds.
Besides TCDD, the unsubstituted parent compound (dibenzo-jv-dioxin) and
several other chlorinated derivatives have been tested for possible carcino-
genicity by the U.S. National Cancer Institute/National Toxicology Program
(see Table LIU). In 1973, King _£t__al_- (109) reported the preliminary data of
a skin carcinogenesis study on the unsubstituted parent compound and the
2,7-dichloro- and octachloro- derivatives. Swiss-Webster mice were topically
treated with 0.2 ml of acetone solutions containing 0.2, 3.0 and 80 rag/ml of
the test compound, three times per week. None of these compounds exhibited
307
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any evidence of "complete" carcinogenicity toward the skin by the 59-64th week
of the 78 week study. Among the mice that were histopathologically examined,
a malignant lymphoma of the lymphocytic type was found in a mouse treated with
V
unsubstituted dibenzo-jv-dioxin; two mice treated with octachlorodibenzo-j£-
dioxin bore subcutaneous tumors. The final results of these studies-are not
available and are listed as "data inconclusive" in the June 15, 1982 issue of
the "Carcinogenesis Testing Program Chemicals on Standard Protocol" released
by the U.S. National Toxicology Program.
Dibenzo-j^-dioxin and its 2,7-dichloro derivative have also been tested in
feeding studies (110, 111). Osborne-Mendel rats and B6C3F^ mice were given
diets containing 5,000 or 10,000 ppm of the respective compound for 110-117
^
weeks (rats) or 91-101 weeks (mice). No compound-related carcinogenic effect
was observed with dibenzo-p_-dioxin. The 2,7-dichloro derivative was also
concluded to be noncarcinogenic in rats of either sex and in female mice.
However, in male mice, the combined incidence of leukemia and lymphoma was
significantly higher in the low dose group (7/50 experimental vs. 0/50
control) and the combined incidences of hepatocellular adenomas and carcinomas
were also significantly increased (control, 8/49; low dose, 20/50; high dose,
17/40). Thus, the data suggest that 2,7-dichlorodibenzo-jv-dioxin may be
i
carcinogenic for male B6C3Fj mice.
A 1:2 mixture of 1,2,3,6,7,8- and 1,2,3,7,8,9-isomers of hexachlorodi-
benzo-jr-dioxin has been tested for possible carcinogenicity in Osborne-Mendel
rats and B6C3F, mice by gavage twice weekly for 104 weeks (118). The doses
administered were 1.25, 2.5 or 5/ig/kg/week for rats and male mice and 2.5, 5
or 10 /ig/kg/week for female mice. No compound-related tumors were observed in
male rats. In female rats, however, the hexachlorodibenzo-jv-dioxins induced a
significantly increased incidence of hepatocellular neoplastic nodules or
308
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carcinoma (combined incidences: control, 5/75; low dose, 10/50; mid dose,
12/50; high dose, 30/50). In mice of either sex, dose-related increases in
the incidences of hepatocellular carcinomas or adenomas (males: control,
15/73; low dose, 15/50; mid dose, 14/49; high dose 24/48; females: control,
3/73; low dose 4/48; mid dose, 6/47; high dose, 10/47) were observed. In
direct comparison to the control groups, the increased incidences in the high
t
dose groups were statistically significant. Thus, the hexachlorodibenzo-jr-
dioxin isomer mixture is carcinogenic in female rats and in mice of either
sex, the liver being the only carcinogenicity target organ. In contrast to
the gavage study, no carcinogenic effects were noted in a dermal study in
which Swiss Webster mice were skin-painted with 0.01 yug of the hexachloro-
t
dibenzo-jv-dioxin mixture 3 times per week for 104 weeks (117).
The data reviewed above allow a ranking of the carcinogenic potencies of
the above compounds and of TCDD. Considering the doses required to elicit
significant carcinogenic effects and the incidences or types of tumors, the
carcinogenic potency of the dibenzo-jv-dioxins appears to follow the order:
2,3,7,8-tetrachloro- > hexachloro- mixture > 2,7-dichloro- > unsubstituted
compound. Interestingly, this relative order of carcinogenic potency corre-
lates with the relative order of toxicity or biochemical effects such as
enzyme induction suggesting a possible commonality in the mechanisms of
action.
5.2.2.3.3.4 MODIFICATION OF CARCINOGENESIS BY TCDD.
2,3,7,8-Tetrachlorodibenzo-jv-dioxin (TCDD) has been extensively tested
for its ability to act as a turaorigenesis promoter, tumor-initiator or cocar-
cinogen. Somewhat conflicting results have been reported. It appears that
the modifying effect of TCDD is dependent on the type of carcinogen admini-
309
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stered and the target organ involved. In some cases, TCDD appears to inhibit
rather than to enhance the carcinogenic effects of other chemicals.
t.
In two-stage skin carcinogenesis studies using CD-I mice, DiGiovanni et
al. (119)'showed that TCDD (2 ^ig per mouse) was at most a weak tumor initi-
ator, inducing papillomas in only 14Z of the mice with an average of 0.1
papilloma/mouse after promotion,for 32 weeks with 12-0-tetradecanoylphorbol-
12-acetate (TPA). When applied concurrently with the known tumor-initiator,
7,12-dimethylbenz[a]anthracene (DMBA), TCDD only slightly enhanced the initi-
ating activity of DMBA in an additive manner. Berry jst_ _a_l/ (120) found TCDD
to be completely Lnactive as a tumorigenesis promoter in DMBA-initiated mice
when applied one we>k after DMBA at a dose of 0.1 yug per mouse twice weekly
for 30 weeks. Interestingly, when applied shortly before DMBA initiation and
subsequent promotion by TPA, TCDD (0.1-2/ug per mouse) exhibited a potent
anticarcinogenic effect (121-123). The anticarcinogenic effect was both time-
and dose-dependent. Maximum inhibition (89-97%) was observed with TCDD
administered 3-5 days prior to DMBA initiation. When applied 5 minutes before
DMBA, no inhibition was noted. A similar anticarcinogenic effect of TCDD has
been found in studies using benzofalpyrene or 3-methylcholanthrene as the
tumor initiator (123, 124). It has been suggested (121, 124) that TCDD exerts
its anticarcinogenic effect by inducing epidermal monooxygenase enzymes that
detoxify hydrocarbons or alter the type of hydrocarbon-DNA adduct.
At variance with the above data, the study of Kouri J-t__al_. (125) indi-
cates that TCDD (1 or 100 /ug/kg), administered intraperitoneally two days
prior to a single subcutaneous injection of 3-raethylcholanthrene, appears to
have no appreciable modifying effect on the induction of subcutaneous tumors
by the hydrocarbon in B6C3Fi and D2 mice. When administered simultaneously,
however, TCDD significantly potentiated the carcinogenic effect of 3-methyl-
310
-------
cholanthrene in D2 mice. The authors (125) suggested that TCDD may act as a
cocarcinogen possibly by inducing epidermal aryl hydrocarbon hydroxylase. No
consistent cocarcinogenic effect was noted in B6C3F, mice.
In a 'two-stage hepatocarcinogenesis study, Pitot jet__a_K (126) found that
TCDD is a potent promoting agent for diethylnitrosaraine-induced hepatocarcino-
genesis. In this study, female .Charles River rats were partially hepatecto-
mized and exposed to a single initiating dose (10 mg/kg) of diethylnitrosamine
followed by biweekly subcutaneous injections of 0.14 or 1.4yug/kg TCDD for
seven months. No hepatocellular carcinoma and few preneoplastic foci occurred
in rats receiving diethylnitrosamine or TCDD alone. In the groups that
received both the nitrosamine and TCDD, increased incidences of hepatocellular
foci, neoplastic nodules (low dose group: 3/5; high dose group: 1/7), and
carcinomas (high dose group: 5/7) were observed. The potent promoting activ-
ity of TCDD led the investigators (126) to suggest that the hepatocarcinogenic
effect observed in chronic studies of TCDD (see Section 5.2.2.3.3.3) may arise
from its promoting activity rather than its activity as a complete carcinogen.
5.2.2.3.4 Metabolism and Mechanism of Action.
\
Halogenated phenoxy acid derivatives are absorbed readily from the
gastrointestinal tract of humans or animals after oral dosing (e.g., 127,
128), but slowly upon dermal contact (129). The pharmacokinetics of the salt
or ester forms of phenoxy acids is similar to those of the free acids (e.g.,
4, 128-130) because of rapid hydrolysis (131). Owing to their relatively high
hydrophilicity, these compounds are rapidly excreted, mostly unchanged, in the
urine (127-130). There is some evidence that 2,4-D and 2,4,5-T are rapidly
excreted through an active organic anion transport system in the renal
proximal tubules (132, 133); this renal transport system may, however, be
311
-------
saturated or impaired upon exposure to high doses (4, 134). Biotransformation
of halogenated phenoxy acids does not seem to occur to any significant extent
in mammalian species. The usual metabolic routes of most aralkoxy compounds,
ring hydroxylation and cleavage at the ether linkage, do not appear to occur
to any significant extent for phenoxy acids (reviewed in ref. 4). There is
some suggestive but unconfirmed evidence of the possible formation of
4-chloro-£-cresol from MCPA in t'he rat (135). Depending on the dose applied,
a portion of halogenated phenoxy acids may be excreted as conjugates, mainly
with glycine, taurine or glucuronic acid (reviewed in ref. 4).
The mode of herbicidal action of halogenated phtnoxy acids suggests that
these compounds may also be potentially genotoxic to animal cells if suffi-
cient amount reaches the genome. In vitro studies using cultured chicken
muscle cells revealed that non-cytotoxic concentrations of 2,4-D inhibit cell
differentiation and cell mitosis, probably by affecting the template activity
of the deoxyribonucleoprotein matrix (136). Bednar et al. (137) showed that
2,4-D may be activated by a H702~Peroxidase system to electrophilic reactive
intermediate(s) (possibly an epoxide) that bind covalently to soluble RNA in
an in vitro system. Differential 'labeling studies suggest that only the
benzene ring moiety is incorporated whereas the acetic acid moiety is not.
This activation is considered to be the mechanism whereby the ultimate phyto-
hormonal effects in plants are produced. Conceivably, the same mechanism can
be potentially genotoxic in mammalian tissues. It remains to be investigated
whether mammalian tissues are capable of activating chlorophenoxy acids in
this manner. A number of chlorophenols, the potential metabolites of chloro-
phenoxy acids, have been shown to be carcinogenic in animal bioassays. The
details of these studies are discussed in Section 5.2.2.5. Vainio et al.
(138) have recently demonstrated that, like clofibrate (see Section .
312
-------
5.2.1.7.9), 2,4-D and MCPA are capable of inducing peroxisome proliferation in
Chinese hamsters. Clofibrate has been suggested to exert its carcinogenic
action through excessive production of intracellular hydrogen peroxide causing
DNA damage. It is probable that a similar mechanism may account for the
carcinogenic action'of other chlorophenoxy acids.
Relatively little is known about the metabolism of TCDD (reveiwed in ref.
139). Earlier pharmacokinetic studies in rats (140-143), mice (144), guinea
pigs (145) and monkeys (142) indicate that TCDD is excreted very slowly,
suggesting relatively high metabolic stability of the compound. Olson et al.
(41) showed that the Syrian golden hamster, which is unusually resistant to
TCDD toxicity, excretes TCDD in the urine and feces at a much faster rate than
other species. High-pressure liquid chromatographic analysis of urine and
feces indicated the presence of water-soluble metabolites. No metabolites
were found in extracts of liver or adipose tissue, suggesting that the
metabolites are readily excreted in the urine and bile and that metabolism may
be the rate-limiting step for excretion. A recent study by Ramsey et al.
(146) indicates that TCDD is also metabolized in rats to more polar metabo-
lites which are excreted as such or as conjugates of glucuronide. Using an in
vitro system with isolated rat hepatocytes, Sawahata et al. (147) isolated two
polar metabolites and identified them as l-hydroxy-2,3,7,8-tetrachlorodibenzo-
jp_-dioxin and 8-hydroxy-2,3, 7-trichlorodibenzo-jr-dioxin.
The mechanism of carcinogenic action of TCDD and related compounds is
poorly understood. An in vivo covalent binding study by Poland and Glover
(148) indicated that 3H-labeled TCDD does not bind to rat liver DNA or ribo-
somal RNA to any significant extent. The maximum estimate (assuming that all
unextractable radioactivity represents covalent binding) of covalent binding
of TCDD to DNA is less than 1 molecule of TCDD per 1011 nucleotides, or 4 to 6
313
-------
orders of magnitude lower than that observed for most chemical carcinogens.
As discussed in Section 5.2.2.3.2.2, despite the favorable molecular size and
shape of TCDD as "an intercalator, there is no convincing evidence that TCDD is
mutagenic. At least three alternative mechanisms have been hypothesized
(148): (a) TCDD is a potent inducer of microsomal mixed function oxidases,
and this induction might enhance the rate of formation of reactive inter-
mediates from potentially carcinogenic endogenous, dietary and environmental
compounds, (b) TCDD may act as a promotor and stimulate previously initiated
cells to divide, and (c) TCDD may act in a hormone-like manner, trophically
stimulating the target tissues. As discussed in Section 5.2.2.3.3.A,, the
finding of Pitot _et^jal_. (126) that TCDD is a potent promotor of diethyl-
nitrosamine-induced hepatocarcinogenesis supports hypothesis (b). However,
TCDD is devoid of tumorigenesis promoting activity in skin carcinogenesis
studies using 7,12-diraethylbenz[a]anthracene as the tumor-initiator (120); in
fact, under some circumstances, TCDD appears to inhibit rather than to enhance
the carcinogenic effects of a number of skin carcinogens (see above in this
Section). In addition to the hypothesized mechanisms discussed above, it
should be pointed out that TCDD is a potent immuno-suppressant in several
species of animals; an impairment of immune competence is often associated
with an enhancement of tumor development.
5.2.2.3.5 Environmental Significance.
5.2.2.3.5.1 EPIDEMIC-LOGIC EVIDENCE.
The potential carcinogenic risk of human exposure to halogenated phenoxy
acids, dibenzofurans and dibenzodioxins has been a subject of great interest
and concern in recent years. Studies by a number of Swedish investigators
indicate that human exposure to phenoxy acids may be associated wih an
314
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increase in the risk for several types of tumors, although firm epidemiologic
evidence of a causal relationship to specific compounds is still lacking.
In 1977, Hardell (149) noted that several Swedish patients with soft-
tissue sarcomas were reported to have had. previous heavy exposure to chlori-
nated phenoxy acids. Phenoxy acid herbicides (mainly 2,4-D, 2,4,5-T and MCPA)
have been used to control unwanted hardwoods or weeds in Swedish forestry and
t
farming since the beginning'of the 1950's. A subsequent matched case-control
study by Hardell and Sandstrom.(150) of 52 patients (from northern .Sweden)- -
with a known history of exposure to chlorinated phenoxy acids or phenols
revealed an approximately 6-fold increase in the risk for this type of
tumor. It was not known, however, whether impurities (such as chlorinated
dibenzodioxins and dibenzofurans) present in these herbicide preparations
played any contributory role. A more recent case-control study, by Eriksson
_et__al_. (151), on patients in southern Sweden indicated about the same extent
of increase in risk for soft-tissue sarcomas. Moreover, persons exposed to
2,4-D, MCPA, dichloroprop and mecoprop (these phenoxy acids are generally not
considered to be contaminated by chlorinated dibenzodioxins or dibenzofurans)
appeared to have approximately the same degree of increased carcinogenic risk
as those exposed to 2,4,5-T, suggesting that the phenoxy acids per se may be
the suspected carcinogen.
Besides soft-tissue sarcomas, human exposure to phenoxy acids or chloro-
phenols may also be associated with an increased risk to non-Hodgkin's malig-
nant lyraphoraa of the histiocytic type. Hardell (152) first reported that
among 17 Swedish patients admitted for treatment of histiocytic lymphoma, 11
reported such exposure. The median latent period was 15 years. A matched
case-control study by Hardell et_ al_. (7) gave a calculated relative risk of
4.8 for phenoxyacetic acids. Olsson and Brandt (153) reviewed the case
315
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histories of 123 patients with non-Hodgkin's lymphoraa and found 5 patients
with cutaneous lesions as the sole clinically detectable manifestation of. the
\.
malignancy. Four of these 5 patients have one commonality repeated skin
exposure to 2,4-D, 2,4,5-T or MCPA for a period of 18-20 years. Axelson et
al. (8) followed a cohort of 348 Swedish railroad workers who were exposed to
herbicides, phenoxyacetic acids and/or aroitrol, during the period 1957-1972.
An excess of stomach cancer (3 observed cases versus 0.41 expected) was noted
among those exposed to phenoxyacetic acids. An increased mortality from lung
cancer was also reported among a number of German pesticide workers who were
exposed to 2,4-D and MCPA, as well as other pesticides (154). Another chlori-
nated phenoxy acid derivative, which has been implicated as being potentially
carcinogenic in humans (106, 155) is clofibrate, a drug that has been used for
the treatment of hyperlipoproteinemia in the United States and Europe (see
Section 5.2.1.7.9 and "Notes added after completion of Section 5.2.1.7" in
Vol. IIIA). Increased incidence of cancer of the liver, gall bladder and
intestines were reported in patients treated with clofibrate (106). The
totality of these data suggest that further epidemiologic studies are needed
and that chlorinated phenoxy acid derivatives must be handled with caution
irrespective of whether they are contaminated with chlorinated dibenzodioxins
or not.
Epidemiologic studies on potential carcinogenic risk of human exposure to
chlorinated dibenzodioxins are mostly incomplete at the time of this
writing. Tung (10) reported an increase in the incidence of primary carcinoma
of the liver among cancer patients admitted to hospitals in Vietnam during the
period 1962-1968. He attributed this increase to TCDD exposure as a result of
the spraying, for military purposes, of defoliant (later found to be contami-
nated with TCDD) in Vietnam during the 1960's. No epideraiologic data were,
-------
however, provided in this study. Jirasek .et^^U (cited in ref. 2) followed up
55 subjects of a cohort of 78 TCDD-exposed Czechoslovakian workers for 5-6
years and observed two cancer deaths, both from bronchogenic carcinoma. Based
on 1965 World Health Organization lung cancer mortality rate for
Czechoslovakia, the expected number of lung cancer deaths for a cohort of this
size would be 0.12 (2). Thiess and Goldmann (cited in ref. 2) noted 4 cancer
f ' BM^^M^K^^HB^V
deaths (1 lung, 2 gastric and 1 colonic carcinoma) among 53 TCDD-exposed
German workers; the study is still in progress. Zack and Suskind (156)
followed, over a period of nearly 30 years, 121 workers who developed chlor-
acne resulting from TCDD exposure in a 2,4,5-trichlorophenol process accident
at a chemical plant in Nitro (West Virginia, USA) in 1949. Analysis of the
mortality data indicated no apparent excess in deaths from malignant neoplasms
(9 observed versus 9.04 expected). There were 5 lung cancer deaths versus
3.02 expected and 1 skin cancer death versus 0.15 expected. The investigators
cautioned that the results of this study cannot be considered conclusive
because of the small size of the cohort and the relative small number of
deaths observed. Bishop and Jones (157) reported two cases (compared to 0.28
cases expected) of non-Hodgkin's lymphoma of the skin among 158 workers in a
plant which manufactured pentachlorophenol and its sodium salt. Chlorinated
dibenzo-_p_-dioxins, particularly the octachloro and the hexachloro congeners,
occurred as contaminants up to about 200 ppm at the intermediate stages of
manufacture and at 5 ppm in the final products. The tragic accident of mas-
sive release of TCDD and related compounds into the atmosphere of Seveso
(Italy) in 1976 attracted a flurry of epidemiologic studies (e.g., 72, 158;
see also ref. 159). Thus far, data have not revealed any evidence for car-
cinogenicity (72). However, in view of the fact that the long latent periods
are usually needed for tumor development, it is premature to draw any conclu-
317
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sion at this stage. The U.S. National Institute for Occupational Safety and
Health (NIOSH) is currently conducting an extensive epidemiologic study
involving about 3,000 chemical workers who had been or were suspected to have
been exposed to TCDD; the results of this study are not expected to be
released until the mid-1980's.
5.2.2.3.5.2 ENVIRONMENTAL .SOURCES, OCCURRENCE AND EXPOSURE.
Human exposure to chlorophenoxy acids is mainly associated with the use
of these compounds as herbicides. A number of chlorophenoxy acids, parti-
cularly 2,4-D and MCPA, are still widely used to control broad-leaf weeds (2-
4). Apart from occupational exposure, the population-at-large may.be
seasonally exposed during lawn spraying. Chlorophenoxy acids are not known to
persist in the environment; human exposure from the general environment
appears to be limited. The possible occurrence of chlorophenoxy acids in the
finished drinking water of U.S. cities has been monitored by the U.S.
Environmental Protection Agency (160). In a 10-city survey, trace amounts of
2,4-D (40 nanogram/liter) and silvex (20 nanogram/liter)'were detected in the
drinking water of one city. An expanded survey of 53 cities in the mid-west
region indicated the presence of 2,4-D in the raw water of only one city; none
was detected in the finished drinking water. The U.S. Food and Drug
Administration (161-163) has monitored the level of 2,4-D, 2,4-DB and 2,4,5-T
in the average American diet in its "Market Basket" study. With one excep-
tion, no significant amounts of any of these herbicides were detected in 12
different categories of foodstuffs destined to adults, infants and "toddlers"
(young children). In one case, trace amounts of 2,4-D (0.025 ppra) were
detected in the "sugar and adjuvant" category of toddler diet. Based on this
information, the possible daily intake of 2,4-D by toddlers was estimated to
be 0.0058 xig/kg body weight (163). Another minor source of exposure is from
318
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the use of clofibrate for the treatment of hyperlipoproteinemia (see Section
5.2.1.7.9).
The intensity of concern over the environmental exposure to halogenated
dibenzo-jr-dioxins and dibenzofurans is generated by the extreme toxicity of
some members of these two series of compounds, their frequent occurrence in
certain industrial chemicals and associated wastes and their persistence in
i
the environment. Detailed reviews on environmental sources and occurrence and
on exposure to halogenated dib.enzo-_p_-dioxins and dibenzofurans (17, 19, 22,
-23, 164, 165) have been published. A brief perspective on these topics is
given below.
Chlorinated dibenzo-jv-dioxins have long been known to occur as unwanted
byproducts in chemical manufacturing processes involving chlorinated
phenols. Figure 21 depicts the pathways for the synthesis of 2,4,5-trichloro-
phenol, the herbicide 2,4,5-T and the bactericide hexachlorophene. The" con-
version of 1,2,4,5-tetrachlorobenzene to 2,4,5-trichlorophenate is known to be
an accident-prone reaction; at temperatures above 230°C, dangerous exothermic
decomposition reaction may occur leading to further increase in temperature
and gas formation. At temperatures above 180°C, 2 molecules of 2,4,5-tri-
chlorophenate may combine to form the highly stable TCDD. Similarly, a
variety of other chlorinated dibenzo-_p_-dioxins may be pyrolytically formed
from other chlorophenols, with at least one chlorine and one phenolic group
substituted ortho to each other (e.g., 2,7-dichlorodibenzo-_p_-dioxin from
2,4-dichlorophenate; octachlorodibenzo-jv-dioxin from pentachlorophenate) (see
22, 164, 165). Chlorinated diphenyl ethers are also precursors of chlorinated
dibenzo-£-dioxins (165, 166); chlorinated diphenyl ethers with a chlorine atom
and a phenolic group at positions ortho to the intercyclic bond are often
called "pre-dioxins" and are believed to be an intermediate in the thermal
319
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OCH2COOH
.Cl
HOOfaCHgOH
SoOH
Cl
Hexgchlorgehene
TCDD -
Fig. 21. Formation of TCDD during synthesis of 2,4,5-TCP, 2,4,5-T
or hexachlorophene. The abbreviations used are: 1,2,4,5-TCB = 1,2,4,5-
tetrachlorobenzene; Na 2,4,5-TCP = 2,4,5-trichlorophenol sodium salt
(sodium 2,4,5-trichlorophenate); 2,4,5-TCP = 2,4,5-trichlorophenol;
2,4,5-T » 2,4,5-trichlorophenoxyacetic acid; TCDD = 2,3,7,8-tetrachloro-
dibenzo-p_-dioxin.
-------
synthesis of chlorinated dibenzo-_p_-dioxins from chlorophenols. A number of
commercially used chemical processes with a reasonable potential for dioxin
byproduct contamination have been surveyed and assessed (167).
The mechanism of formation of halogenated dibenzofurans has been reviewed
in details by Choudhry and Hutzinger (165). Morita _et__a_l/ (168) showed that
heating a polychlorinated biphenyls (PCBs) mixture for one week at tempera-
t
tures between 270°C and 300°C gives rise to a variety of chlorinated dibenzo-
furans, including up to 80 ppm of the highly toxic 2,3,7,8-tetrachlorodibenzo-
furan. Pyrolysis of individual PCB congeners leads to the formation of
specific chlorinated dibenzofurans via intramolecular cyclization; for
example, 2,4,5,3*,4'-pentachlorobiphenyl has been shown to be a precursor of
2,3,6,7- and 2,3,7,8-tetrachlorodibenzofuran (169). Chlorinated diphenyl
ethers are also excellent precursors for chlorinated dibenzofurans; yields as
high as 4.5% were obtained in Che pyrolysis of these precursors at 600°C
(166). Dibenzofurans may be formed during thermal cracking of phenols or
cresols (170); Buser (171) postulated that pyrolysis of chlorobenzenes may
generate chlorinated dibenzofurans via chlorophenols.
There are numerous reports of occurrence on the chlorinated dibenzo-jv-
dioxins and dibenzofurans in various industrial and commercial products. An
average of 1.86 ppm TCDD (maximum 47 ppm) was found in surplus Agent Orange
(1:1 mixture of 2,4,5-T and 2,4-D) preparations stockpiled after the Vietnam
War (14). A survey by Woo Is on £t__al_. (172) of 42 samples of 2,4,5-T manufac-
tured from 1966 to 1970 showed that 13 of these samples contained 10-100 ppra
TCDD, 7 had less than 10 ppm and 22 had less than 0.5 ppm. Commercial chloro-
phenols also contain various chlorinated dibenzo-_p_-dioxin congeners (22).
Some pentachlorophenol products manufactured before 1970 contained as much as
3,600 ppm octachlorodibenzo-jv-dioxin (see ref. 22). An exhaustive list of
320
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commercial products that may be contaminated with chlorinated dibenzo-j^-
dioxins has been compiled by Esposito ^t__al_. (22). Since the time that the
highly hazardous nature of TCDD was widely publicized, manufacturers have
V
significantly reduced the TCDD content in various products. The 2,4,5-tri-
chlorophenol produced around 1977 in the United States reportedly contained an
average of 0.026 ppm TCDD (173). Chlorinated dibenzofurans have been detected
in various commercial samples o'f polychlorinated biphenyls (PCBs) as well as
in the tissues of individuals ("Yusho" disease) who had been exposed to PCBs
(174-176; see also refs. 17, 169).
Human exposure to chlorinated dibenzofurans and dibenzo-jv-dioxins may
occur in the workplace as well as in nonoccupational settings. Since 1949,
over 200 TCDD-related industrial accidents have occurred around the world
(9). A partial list of reported incidents of occupational exposure to chlori-
nated dibenzo-jv-dioxin during routine chemical manufacturing has been compiled
by Young .et^jil,. (177). Industries or occupations using chemicals which may be
contaminated with chlorinated dibenzo-jv-dioxins cr dibenzofurans include
textiles, leather tanning, wood preserving, pulp and paper, pesticide formu-
lators and applicators, automotive, construction, drug and cosmetics, paint,
farming, railroad maintenance, transformers and capacitors manufacturing or
repairing, chemical laboratories and waste management (17, 22). In addition, a
group of U.S. military personnel may have been exposed to TCDD during handling
and spraying of Agent Orange in the Vietnam War. As mentioned in Section
5.2.2.3.5.1, the U.S. National Institute for Occupational Safety and Health is
currently conducting an extensive epidemiologic study of potential long-term
health hazards of occupational exposure to TCDD.
The general public may be exposed to chlorinated dibenzo-jv-dioxins or
dibenzofurans in localized areas in the vicinity of industrial or transporta-
321
-------
tion accidents, in areas where contaminated industrial wastes were improperly
disposed or in areas where contaminated herbicides were sprayed. There are a
number of well-publicized episodes of such exposures (reviewed in ref. 22).
In 1976, a massive amount (estimted to be somewhere between 300 g and 130 kg)
of TCDD was released into the atmosphere over an area of about 700 acres in
Seveso, Italy during an industrial accident. Numerous investigations have
f - *
been devoted to studying the toxicological effects caused by TCDD contamina-
tion (23, 72, 158, 159, 178-180). Thus far, the most evident acute pathology
is the induction of chloracne, especially in children. Although there are
some reports (72, 158) of the apparent lack of genotoxic effects of TCDD in
the exposed population, it is probably still too early to draw any conclusion
about the chronic effects of this incident at this time. In 1979, a railroad
accident at Sturgeon, Missouri, caused the spillage of a tank car of jsr-chloro-
phenol; subsequent analysis of the contents showed the presence of 37 ppb
TCDD. The presence of TCDD in ^-chlorophenol is somewhat unexpected. Details
of the incident have not been released because of pending legal action (22).
Leakage of PCBs into rice oil during processing caused an epidemic of toxic
effects ("Yusho" disease) in Japan in 1968 (181; see also Section 5.2.2.2);
chlorinated dibenzofurans were detected in the contaminated rice oil (181,
182) and in tissues of "Yusho" patients (176). The most notable incident of
public exposure to TCDD-contarainated industrial wastes involved the spraying
of TCDD-contarainated waste oil to control dust in horse arenas and private
roads (183). Up to 100 sites in the state of Missouri may have been contami-
nated with TCDD since 1971 (184, 185). Chlorinated dibenzo-j^-dioxins
(including TCDD) have also been found in two chemical landfills in Niagara
Falls, New York. One of these, Love Canal, was until recently the site of a
residential community. About 30 tons of 2,4,5-trichlorophenol wastes were
322
-------
reportedly buried in the Love Canal (22). The incineration of TCDD-
contaminated chemical wastes has been suspected to contribute to the presence
of TCDD in the atmosphere in the vicinity of a chemical plant in Michigan
(22). The use of TCDD-contaminated defoliant in Vietnam is another well-
publicized incident of public exposure and is the subject of a U.S. Air Force
technical report by Young _£t__aJL (177). The spraying of 2,4,5-T and silvex in
/
forest areas in Oregon led to a heated public debate and culminated in an
emergency ban in 1979 on the continued use of these herbicides in these areas
(22). Many other incidents' of possible public exposure to TCDD through the
use of herbicides have been reported throughout the world (22).
Besides exposure in localized areas, public exposure may also occur from
the general environment; relatively litte information is available to assess
the extent of such exposure. Miller (186) suggested that a worldwide back-
ground of atmospheric TCDD contamination may exist as a result of the
incineration of TCDD-contarainated Agent Orange by the U.S. Air Force.
Pyrolysis of chlorinated benzenes, phenols, phenoxy acids, biphenyls and
aromatic ethers may also generate chlorinated dibenzofurans and/or dibenzo-jv-
dioxins (166, 169, 187-189; reviewed in refs. 17, 164). There is some sugges-
tion that chlorinated dibenzo^pj-dioxins may be formed de novo in the burning
of fossil fuel (190); however, the actuality of such a route of formation
remains questionable (191-193). Chlorinated dibenzo-jv-dioxins have been
detected in the water effluents from some chemical plants; however, there
appears to be no indication of their possible occurrence in drinking water
supplies (22).
There are several reports on the possible contamination of human food
sources with trace levels of TCDD. Beef fat taken from cattle grazed on
pasture treated with 2,4,5-T occasionally contained TCDD at 3-70 parts per
323
-------
trillion levels (22, 194, 195). Food samples collected from south Vietnam in
1970 (196) and from Seveso, Italy in 1976 (197) contained higher levels of
TCDD. As discussed in Section 5.2.2.2.5.2, PCBs may be present in a variety
V
of foodstuffs. In view of the contamination of PCBs with chlorinated dibenzo-
furans, it is possible that trace amounts of chlorinated dibenzofurans may
also be found in human food sources.
324
-------
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339
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Noted Added After Completion of Section 5.2.2.3
Several recent monographs on the toxicological and environmental aspects
of 2,3,7,8-tetrachlorodibenzo-_p_-dioxin (TCDD) and related compounds have been
published (1-4). The U.S. National Institute for Occupational Safety and
Health issued a "Current Intelligence Bulletin" on TCDD (5) and a special
issue of Chemical and Engineering News was devoted to discussion on TCDD
(6). Reuber (7) has recently written a comprehensive review on the carcino-
genicity and toxicity of 2,4-dichlorophenoxyacetic acid (2,4-D).
In contrast to the lack of conclusive evidence for mutagenicity of TCDD
in the Ames test, Rogers _et_ al_. (8, 9) reported that TCDD is mutagenic in
^^ \
L5178 mouse lymphoma cells. The mutagenic potency and the dose-response curve
of TCDD appears to be comparable to those of classical intercalating agents
such as proflavin. Tenchini et al. (10) completed a comparative cytogenetic
study on maternal and fetal tissues derived from TCDD-exposed and nonexposed
pregnant women (during the Seveso incident in Italy), who underwent induced
abortions. In agreement with previous reports, no significant clastogenic
effects of TCDD on maternal peripheral blood and placental tissues were
observed. However, a significant increase was noted in the frequencies of
aberrant cells and in the average number of aberrations per damaged cell in
the aborted fetal tissues. Although a causal relationship cannot be estab-
lished, the results suggest a possible transplacental clastogenic action of
TCDD. Two nitrated derivatives of dibenzo-jv-dioxin (2-nitro- and 2,3-di-
chloro-7-nitro-) were found to be potent frameshift mutagens in the Ames test
(11). Although this type of mutagenic activity was attributed to the aromatic
nitrb" group, the presence of the chlorine substituents made the compound also
slightly active as a base-pair substitution type mutagen. An isosteric struc-
tural analog of TCDD, 3,4,3',4'-tetrachloroazobenzene, is a weak frameshift
-------
mutagen in the Ames test (12). The less chlorinated homolog, 4,4'-dichloro-
azobenzene, and its azoxy derivative, 4,4'-dichloroazoxybenzene, are consider-
ably more active, inducing both frameshift and base-pair substitution type
mutations.
Additional teratogenicity testing of 2,4-D has been carried out by
Rodwell et_al. (13) using F344 rats. The animals were given 8, 25 or 75
mg/kg/day 2,4-D orally from day 6 through day 15 of gestation. There was no
evidence of teratogenicity. 2,4-Dichlorophenol, a metabolite of 2,4-D was
also nonteratogenic (14). In both studies, the highest dose levels were
sufficiently high to cause a slight degree of maternal toxicity. Further evi-
dence for the potent teratogenicity and of TCDD and its structural analogs has
been obtained in several recent studies. Giavini j2t_ jil_. (15) found that a
2-week exposure of female rats to TCDD shortly before mating (not during
gestation) interfers with normal embryofetal development. At a daily dose of
2 ug/kg, a variety of maternal and fetal toxic effects as well as malforma-
tions occur, indicating that TCDD accumulated before mating may exert terato-
genic/embryotoxic effects during the organogenesis period. D'Argy et al. (16)
showed that the teratogenic effect of TCDD in mice is strain-specific. A
single dose of 30 ug/kg TCDD on day 12 of gestation induced cleft palate in
75-100% of NMRI mice but had no significant effects in DBA mice (which lack
the cytosolic receptor for TCDD). Two isosteric structural analogs of TCDD
2,3,7,8-tetrachlorodibenzofuran (TCDF) and 3,3'4,4'-tetrachloroazoxybenzene
(TCAOB) have been shown to be teratogenic in strain C57B1/6N (17) and NMRI
(16) mice, respectively. The spectrum of teratogenicity targets of these two
compounds is similar to that of TCDD suggesting a common mechanism of
action. The teratogenic potency of TCDF and TCAOB is about 10-30 and 260
times lower than that of TCDD, respectively.
-------
No new animal carcinogenicity studies on 2,4-D, 2,4,5-T and TCDD have
been found in the literature since the completion of Section 5.2.2.3. Deca-
bromodiphenyl ether (crude grade; 77.4% pure, contains 21.8% nonabromo- and
0.8% octabromo- homologs), a fully halogenated structural analog of TCDD was
reported to be noncarcinogenic in the Sprague-Dawley rats after feeding for 2
years daily doses of 0.01, 0.1 or 1 mg/kg of the compound (18, 19). The only
adverse effect noted was an accumulation of bromine in the liver and adipose
tissues of rats of the high dose group. The amine salt of 2,4-D was reported
to promote 3-methylcholanthrene-initiated skin carcinogenesis in CBA x C57/BL
hybrid mice (Archipov and Kozlova, cited in ref. 7). The promoting activity
of 2,4-D is relatively weak. Skin papillomas occurred in 17.7% of mice
treated with initiating doses of 3-raethylchplanthrene (0.5% solution in ben-
zene) followed by 2,4-D (10% solution in acetone); none were found in mice
treated with either agent alone. Poland et al. (20) studied the capacity of
TCDD to promote skin carcinogenesis in HRS/J mice and found evidence of a
genetic control of the susceptibility to TCDD-induced tumor promotion linked
to the control of hair growth. Following initiation with 7,12-dimethyl-
benz [ajanthracene (DBMA), repeated topical application of TCDD failed to
produce any skin tumor in HRS/J haired mice. In contrast, the same regimen of
DMBA and TCDD treatment produced skin tumors in 15 of 19 HRS/J hairless
mice. A similar, dose-dependent promoting effect of TCDD in HRS/J hairless
mice was observed using N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) as the
initiator. Compared to the classical promoter, 12-0-tetradecanoylphorbol-13-
acetate (TPA), TCDD was about 100 times less active. The capacity of several
congeners or analogs of TCDD to promote skin tumorigenesis was also tested;
the isosteric analogs, TCDF and 3,4,5,3',4',5'-hexabromobiphenyl, were both
active promoters whereas 2,7-dichlorodibenzo-jv-dioxin and 2,4,5,2 ' ,4',5'-hexa-
-------
bromobiphenyl had no activity. The results indicate that TCDD and related
halogenated aromatic hydrocarbons comprise a class of potent tumor promoters
whose expression is genetically controlled. It is interesting to note that
the HRS/J hairless mouse is considered to be a good model for dermatoxicologi-
cal study of human skin.
A variety of new epidemiological studies have been conducted to assess
further the carcinogenic risk of human exposure to 2,4-D, 2,4,5-T, TCDD and
related compounds. In contrast to an elevated risk to soft-tissue sarcomas
and malignant lymphomas (see Section 5.2.2.3.5.1), Hardell £t__aj^. (21, 22) did
not find any significant increase in the incidence of colon, nasal and naso-
pharyngeal cancers in Swedish workers exposed to chlorophenols and chlorophen-
oxy acids. Combining the mortality data from three separate studies (each
reporting a lack of carcinogenic risk) of four small cohorts of U.S. workers
involved in the production of 2,4,5-trichlorophenol and 2,4,5-T, Honchar and
Halperin (23) noted an unusually high incidence of soft-tissue sarcoma (three
cases out of 105 deaths; approximately 43 times higher than expected). Sub-
sequently, four additional cases of soft-tissue sarcomas were reported among
U.S. workers who were possibly exposed to chlorophenols, 2,4,5-T or TCDD (see
ref. 24). However, a more recent detailed review of work exposure record and
re-examination of pathological specimens led to the conclusion that only two
of the seven cases had both confirmed exposure to 2,4,5-trichlorophenol or
2,4,5-T and diagnosis of soft-tissue sarcoma (J.D. Millar, cited in ref. 5).
Moses et al. (25) reported the health status of 226 U.S. workers in a chemical
plant (in Nitro, West Virginia) where 2,4,5-T (believed to be heavily contami-
nated with TCDD) had been manufactured from 1948 to 1969 and where an explo-
sion of reactor vessel containing 2,4,5-T occurred in 1949. Over 52% of the
workers developed chloracne which persisted for an average of 26 years. Based
-------
on worker recall of job assignment, the development of chloracne was posi-
tively correlated to the extent of exposure. Twenty-five subjects reported a
positive cancer history. Among these, eleven had bladder cancer; however, all
had had also an occupational history of exposure to 4-aminobiphenyl, a known
bladder carcinogen. Skin cancer was reported by 12 subjects; however, there
appeared to be no association between chloracne and skin cancer. Other types
of cancers reported included two cases of laryngeal cancer, one case each of
kidney, lymphoma, bowel, leukemia, and prostate cancer. No attempt was made
in this study to assess whether the occurrence of cancer was related to expo-
sure. An extensive epidemiological investigation ("Project Ranch Hand II") of
health effects in U.S. Air Force personnel following exposure to herbicides
(most notably Agent Orange) during the Vietnam War has recently been completed
(26). A group of 1,241 men who handled herbicides daily for up to 4 years
(probably the most heavily exposed group in the Air Force) was selected and
matched by age, race and occupational category to a comparison group of non-
exposed personnel. The men were subjected to a complete physical examination
including an evaluation of several major organ systems and their functions,
neurological and psychological assessment. Members of the Ranch Hand
(exposed) group did not develop chloracne or porphyria cutanea tarda, two of
the most characteristic toxic effects of TCDD. Analyses of cancer data showed
significantly more nonmelanotic skin cancer in the Ranch Hand group; however,
these analyses have not been adjusted for the possible impact of sunlight
exposure, the prime etiology of skin cancers. There were no statistically
significant differences in the occurrence of malignant or benign systemic
1
tumors between the two groups. The report concluded that there is insuffi-
cient evidence to support a cause and effect relationship between herbicide
exposure and adverse health effects in the Ranch Hand group.
-------
The metabolism and disposition of TCDD and related compounds has been
further studied. Poiger et_j_l_. (27) identified the chemical nature of five
polar metabolites of TCDD found in the bile of dogs as 2-hydroxy-3,7,8-tri-
chlorodibenzodioxin, 2-hydroxy-l,3,7,8-tetrachlorodibenzodioxin, and
dihydroxytrichlorodibenzodioxin (probably arising from ring epoxidation), and
4,5-dichlorocatechol and dihydroxytetrachlorodiphenyl ether (which may be
formed by cleavage of the ether bridge(s)). Once formed, the metabolites
appear to be readily excreted with no evidence of bioaccumulation (28) sug-
gesting that metabolism of TCDD may be a rate-limiting step in its elimina-
tion. Significant differences in the disposition of TCDD in C57B1/6J and
(C57B1/6J x DBA/2j)Fi mice as compared to DBA/2J mice were noted by Gasiewicz
et al. (29); the half-life of TCDD in these three strains was estimated to be
11.0, 12.6 and 24.4 days, respectively. The difference may be due, in part,
to the sequestration of TCDD in the adipose tissue which is more abundant in
the DBA/2J strain. Similar strain differences were observed in the disposi-
tion of 2,3,7,8-tetrachlorodibenzofuran (TCDF) although TCDF was excreted at a
much faster rate than TCDD (30). The half-life of TCDF was estimated to be
around 2 days in C57B1/6J mice and 4 days in DBA/2J mice.
The ability of chlorophenoxyacetic acids to induce peroxisomal enzymes in
the rat liver has been studied by Kawashima et al. (31) prompted by the find-
ings of positive correlation between peroxisome proliferation and carcino-
genesis (see Section 5.2.1.7.9 and Notes Added After Completion of Section
5.2.1.7; see also ref. 32). Both 2,4,5-T and, to a lesser extent, 2,4-D were
shown to induce peroxisomal ft-oxidation. 2,4-D differed from 2,4,5-T in not
causing hepatomegaly or catalase induction. The less chlorinated 2-chloro-
and 4-chlorophenoxyacetic acid and the unsubstituted phenoxyacetic acid were
inactive. Thunberg (33) observed that TCDD treatment leads to substantial
-------
reduction in the level of vitamin A in the liver and serum of a variety of
animal species and proposed that the TCDD-induced vitamin A deficiency may
play a role in the carcinogenesis or tumor promotion by the compound. Vitamin
A has been shown to suppress or prevent chemical carcinogenesis in a number of
animal studies; conversely, vitamin A deficiency has been associated with
increased susceptibility to carcinogenesis. The mechanism of promotion of
carcinogenesis by TCDD has been studied by Boreiko and Dorraan (34). Unlike
12-0-tetradecanoylphorbol-13-acetate (TPA), TCDD fails to inhibit intercel-
lular communication in cultured C3H10T1/2 cells. The interference of inter-
cellu.lar communication has been proposed as a mechanism of tumor promotion by
a variety of promoters (35).
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-------
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10
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