2,4,5-T: Position Document 1
2,4,5-T Working Group
U.S. Environmental Protection Agency
-------
2,4,5-T: Position Document 1
• • Page
I. Background 1
A* Chemical/Physical Characteristics.. 1
B. Manufacturing Process and Contaminants... 1
C. Formulation and Class ,.. 5
D. Registered Uses and Production 5
E. Metabolism in Experimental Systems , 7
F. Environmental Fate 10
(1) Persistence: Soils 10
(2) Persistence: Water 12
(3) Transport 15
(4) Bioaccumulation 15
G. Residues 19
(1) Soil 19
(2) Water 20
(3) Air 23
(4) Animals ,... 24
(5) Plants 27
(6) Humans 29
(.7) Animal Products 32
(8) Food 37
(9) Human Exposure Via Industrial
Accidents , 38
E*. Tolerances ,».. 42
I. Pesticide Episode Reports System (PERS).. 43
II. Regulatory History 45
III. Summary, of Scientific Evidence Relating to
Rebuttable Presumption. 50
A." . Oncogenic Effects.... 50
(1) 2,4,5-T 51
(a) Effects of Dietary 2,4,5-T
' . «0.05 ppm TCDD) on Rodents.... 51
(b) Effects of Subcutaneous Injec-
tion and Oral Administration of
2,4,5-T (30 ppm TCDD) on
—-— Rodents 54
(2) TCDD 56
(a) Oncogenic Effects of Low
Levels of TCDD on Rodents 56
(b) Effects Closely. Related to
Oncogenicity in Test Animals... 62
(3) Preliminary Epidemiological
Studies 64
B. Other Chronic or Delayed Toxic Effects... 66
(1) Pesticide-free TCDD 68
(a) Studies in which TCDD Produced
Teratogenic and/or Fetotoxic
Effects in Mice 68
-i-
-------
Page
(b) Studies in Which TCDD Produced
Teratogenic and/or Fetotoxic
Effects in Rats 7 1
(c) Summary 79
(2) 2>M,5-T (TCDD Contamination Ranging
From Undetectable to 30 ppm) 80
(a) Teratogenic and Fetotoxic Ef-
fects in Rodents 80
(b) Adverse Reproductive Effects
in Other Mammalian Test
Systems.. 98
(c) Adverse Effects in Avian
Species 99
(d) Studies in Avian Species in
Which Adverse Effects Were Hot
Observed...........*••••••••••• 101
(e) Summary.... 101
(3) Exposure Analysis 101
(a) Oral Exposure 102
(b) Dermal Exposure *35
(i) Spray Applicator: Back-
pack Sprayer 105
tii--) Spray Applicator: Trac-
tor-mounted, Low-boom
Spray Equipment 108
(iii) Aerial Application: Ex-
. posed Population Directly
>: .; Beneath Spray Plane 110
(c) Inhalation Exposure: Aerial
Application 113
(d) Cumulative Exposure.... 116
IV. Studies Relating to Possibe Adverse Effects.. 118
A. Mutagenicity .....* 118
,---. (D 2,1,5-T .' 118
(a) Positive Study 118
(b) Negative Studies 119.
(2) TCDD .. 121
(a) Positive Studies 121
"' (b) Negative Studies : 123
(3) Chromosomal Damage 124
B. Toxicity to Humans; TCDD • 125
CD Chloracne. 125
: (2) Porphyria cutanea tarda and
£-Amino-levulinic Acid Synthetase... 127
References 129
-ii-
-------
2,4,5-T: POSITION DOCUMENT 1
I. BACKGROUND
A* Chemical/Physical Characteristics
The herbicide commonly known as 2,H,5-T (chemical
name, 2,iJ,5-Trichlorophenoxyacetic Acid) has an empirical
formula -of CgH5Cl,0,» The pure acid form occurs as white
crystals and has a molecular weight of 255»^9» The melt-
ing point is 156.6°C. Its solubility in water is 278 parts
per million (ppm) at 25°C; it is also soluble in acetone,
ethanol, ether, and alkaline solutions (1). The esters of
2|4,5-T are formulated to be emulsifiable in water and
soluble in most oils, while its anine salts are soluble in
water but insoluble in petroleum oils (2, 3)»
-B-4 -Ma-nufacturing Process and Contaminants
2,4,5-T is produced commercially by a process using
1,2,4,5-tetrachlorobenzene as the starting material which is
reacted with methanol and sodium hydroxide under high temper-
ature and high"pressure to give the sodium salt of 2,^,5-tri-
chlorophenol (2,4,5-TCP).-'
2,1,5-TCP is the subject of a separate Rebuttable
Presumption Against Registration (RPAR) Position Document*
It is discussed in this document because both it and its
contaminant 2 ,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) may
be present in some commercial 2,4,5-T and in 2,4,5-T samples
used in animal experiments.
-1-
-------
This product is reacted with chloroacetie acid under
mildly alkaline conditions, Sulfuric acid (H SOJ Is then
added to the product of this step to produce 2,4,5-T. The
acid forn of 2,4,5-T can be readily reacted with a variety
of alcohols to produce a large selection of esters and with
amines to produce amine salts (3)*
During the first step in the manufacturing process
of 2,4,5-T, if temperature and pressure are not carefully
controlled, highly toxic contaminants, polychlorinated
dibenzo-p-dioxins, may be formed in large quantities* The
particular dioxin formed is dependent on the chlorophenols
present (4), The terra dioxin does not apply to any one
compound but to a group of related substances, which are
distinguished by the number and orientation of chlorine
atoms they contain. Dioxin toxicity also varies with the
position and numbers of chlorines attached to the phenol
rings.
In the 2,4,5-T manufacturing process an especially
toxic dioxin, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), is
formed when the reaction temperature is excessive (8, 9, 10,
11, 12), most commonly at temperatures above 160°C.
Halogens at the 2, 3, and 7 positions are known to produce
toxic dioxins (13)* In the case of TCDD, the chlorine atoms
-2-
-------
are attached at the 2, 3» 7» and 8 positions which are
\
considered the most toxic positions -possible (HO. The "
dioxin contaminant in 2,1,5-T is of particular concern
because of its extremely high toxicity, and because of the
apparent inability of manufacturers to produce 2,^,5-T
• »
2/
without the contaminant, TCDD (7).
TCDD occurs as a white crystalline solid. It is
99.5$ decomposed at 800°C. TCDD has the following solubility
in various solvents at 25°C (7).
• •
• .Solvent Solubility (wt. per cent)
Acetone 0.011 .
Benzene 0.057
Dimethylsulfoxide <0.01
Methanol 0.001
Water . " *' 0.00000002 (0.2 ppb)
It has been recognized for quite some time that
chlorinated dibenzo-p-dioxins occur as possible byproducts
(contaminants) in the manufacturing of chlorinated phenols
(15). The formation-of TCDD during production of 2,^,5-TCP
was demonstrated by Kimmig and Schulz (16). TCDD was
obtained from the pyrolyzing of 2,4,5-TCP by Higginbotham
2.1 Since manufacturers are unable to produce 2,4,5-T
without TCDD, all references to 2,4,5-T in this docu-
ment refer to 2,4,5-T contaminated with some level of
TCDD.
-3-
-------
et al» (11). They noted that the specific dioxin formed
depended on the chlorophenol pyrolyzed. Kearney et al.
(17), however, reported that TCDD is historically associated
with any pesticide derived from 2,1,5-TCP. A number of
researchers (12, 18, 19, 20, 21) have reported on the
formation of TCDD by thermal decomposition of the sodium
salt of 2,U,5-TCP under alkaline conditions during the
manufacturing process.
Since 1950, most of the chemical industry has known
that large quantities of TCDD may be formed as a byproduct
of the 2,1,5-TCP manufacturing process if the procedures are
not carefully controlled."" At one time 2,4,5-T was produced
which contained between 30 to 40 ppm of TCDD (7, 22, 55).
Between 1968 and 1969, one manufacturer had a 90$ decrease
in the amount of TCDD present in the 2,4,5-T it produced.
Different manufacturers produced 2,H,5-T with different TCDD
contents (17). - ;
After concern arose in 1969 about the extremely toxic
effects of TCDD, manufacturing methods were changed and
carefully controlled b.y manufacturers. By 1971 industry
had reduced TCDD content in commercial samples of 2,U,5-T to
less than 1 ppm (9, 23, 2*0. Current U.S» manufacturing
• . . -
specifications require 2,M,5-T presently being sold to
-n-
-------
contain less than 0.1 ppm TCDD (7). Several countries now
produce commercial 2,4,5-T containing less than.0.05 ppm
•
TCDD (25).
C. formulation and Class
2,4,5-T is classed and used as a selective herbicide,
<
especially for brush control (2). It is formulated in many
forms of salts and esters which are available as emulsifiable
concentrates containing 2, 4, or 6 pounds actual acid
equivalent per gallon and as oil soluble concentrates with 4
or 6 pounds active ingredient (AI) per gallon. The most
commonly used formulations are the low volatile esters (26),
i
2,4,5-T also occurs in registrations mixed with 2,4-D,
Dicamba, Picloram, Silvex, and 2-(2-methyl-4-chlorophenoxy)
propiOTiic acid (27)«
. . D* Registered Uses and Production
2.,4,5-T has been produced as a registered pesticide in
the United States since 1948, According to EPA records,
approximately 122 companies hold Federal registrations and .
formulate 424 registered products; eleven companies have
former state registrations-^ and formulate 21 products.
2i/ Pesticide products formerly registered under state
pesticide registration laws and shipped*or distributed
for sale solely within intrastate commerce are subject
to Federal pesticide regulations under 40 CFR Section
I62.1?(a). Application has been made to obtain Federal
registration for intrastate use of these products. For
a list of trade names under which 2,4,5-T is marketed,
see the registrant/product list.attached to this document.
-5-
-------
Section 7(c) of FIFRA requires manufacturers and
formulators to submit to EPA information on production,
sales, and distribution. Under FIFRA sections .7(c) and
10, this information may not be made available to the
public. A confidential memorandum containing this informa-
tion has been sent to the Deputy Assistant Administrator for
Pesticides (28). The Pesticide Review (29) reported that
11,626,000 pounds of 2,4,5-T acid, esters, and salts were
produced in the United States in 1969 and 12,335,000 pounds
in 1970. The Pesticide Review (29) also reported that tl.>
United States imported 738,907 pounds of 2,4,5-T during
1971 through 1974.-^ Of this total 155,342 pounds were
imported in 1974. This was down from nearly 392,000 pounds
in 1973 but up from the 5-year average of 148,000 pounds.
While The Pesticide Review (29) does not report export
figures for 2,4,5-T alone, it does report exports of 2,4-D
and 2,4,5-T together. Export of 2,4-D and 2,4,5-T was
reported at 6.8 million pounds in 1972; 21 million pounds in
1973; and almost 22 million pounds in 1974. ;
A great deal of variability exists in reports on
usage of 2,4,5-T. Agricultural end-use data obtained
from the National Study of Agricultural, Governmental,
4/ The level of TCDD in the imported 2,4,5-T was not
reported.
-6-
-------
and Industrial Uses of Pesticides, conducted by this
Agency (30), indicated the following uses of 2,4,5-T in
the United States in 1974.
Crop
Rangeland
Rice-
1
1
1
and Pastures 1
1
iKursery Crop I
[Turf and
1
Ornamentals 1
1
Blueberries- 1
1
[Estimated
I
total use 1
pounds AI
applied
968,000
..
16,000
12,000
200
.
8
996,000
in aqriculture for 1974 I
% total
aqriculture
97.24
1.54
1.20
0.02
-
100.00
a/ The Agency has looked at effects on aquatic organ-
isms representative of species likely to be exposed
from application of the triethylam.ine formulation of
2,4,5-1*to rice. The calculated concentration of this
formulation in e 6-inch layer of water at the highest
recommended use rate is 0.9 ppm. The LC-50 bioassay
values for bluegill and catfish are well above this
level (ranging from a 24-hcur LC-50 of 53 ppm for
bluegill to a 96-hour LC-50 of >72 ppm for bluegill
and channel catfish). Rainbow trout, which cannot be
considered "representative of the organisms likely to
be exposed" in the geographic areas where rice is grown,
have a 9-6-hour LC-50 ranging from 0.7 to 0.07 ppm.
b/ This is no longer a registered use.
In addition, this survey reported that 324,491
pounds of active 2,4,5-T were used by federal and state
agencies and 659,463 pounds by industry.
Other sources have reported usages for 1974 as
follow: rights-of-way, 4 million pounds; rangeland, 1.5 to
2.3 million pounds; rice, 220,000 pounds; and forestry,
50,000 pounds.
-7-
-------
E. Metabolism in Experimental Systems
Several studies have demonstrated that 2,4,5-TCP is
the primary degradation product or metabolite formed in the
breakdown of 2,4,5-T, by either physical or biological
mechanisms. Crosby and Wong (31) found that 2,4,5-TCP
was one of the major decomposition products in the photode-
composition of 2,4,5-T in water. Sharpee (32) found that
rcicrobial degradation of 2,4,5-T in culture, soil, and
aquatic ecosystems resulted in the formation of small
amounts of 2,4,5-TCP.
-7 a-
-------
Shafik et al. (33) dosed Sprague-Dawley rats by
gavage with 2,4,5-T at 50, 5, 0.05, .and 0.005 mg/kg for
three days. Two rats were dosed at each level. The authors
found that, at 0.005 mg/kg, excretion of 2,1,5-T in urine
was complete two days after the final dose. They also found
*
2,^,5-TCP excreted as a metabolite in the urine of rats
given 50 mg/kg, but no detectable 2,4,5-TCP was found at the
two lowest dose levels. A hydroxylated trichlorophenoxyacetic
acid and a hydroxylated trichlorophenol were identified, by
unconfirmed mass spectrometric analysis, as possibly being
• * *
two additional metabolites of 2,4,5-T.
Grunow et al. (3*0 studied seven male Wistar rats fed
a single 2,4,5-T dose at 50 mg/kg.body weight. They found
that the daily renal excretion of free 2,4,5-T was, in
general, at its maximum on the second day after feeding.
After seven days, free 2,1,5-T in the urine decreased to a
value below 2% for all animals. In addition to 2,4,5-T
excreted in the free form, the authors found it to be
•
excreted as derivatives which could be converted into
2,4,5-T by acid hydrolysis. They were able to identify one
of these as N(2,4,5-trichlorophenoxyacetyl) glycine.
Grunow and Bohme (35), in a study using Wistar .rats
and KMRI mice, fed doses of 2,4,5-T at 200 mg/kg body
weight. These authors isolated N(2,4,5-trichlorophenoxy
-------
acetyl) taurine as a metabolite of 2,4,5-T, in addition to
the metabolites named above.
•
Clark et al. (36) found residues of 2,4,5-TCP in the
muscle, liver, and kidney of sheep which were fed rations
containing 2,000 ppm of 2,4,5-T for 28 days. The 2,4,5-T
•
used in this study had a purity of 39% and contained no
detectable dioxin (detection limit! 0*5 ppm). -
Leng (37) conducted a feeding study during 1969 and
1970, in which dairy and beef cattle and sheep were given
2,4,5-T at levels from 10 to 2,000 ppm in the total diet for
intervals of two to four weeks at each level tested. The
author reported that no residues «0.05 ppm) occurred in
milk or cream of cows ingesting 10 to 30 ppm 2,4,5-T. At 100
ppm 2,^,5-T in the diet, traces of 2,4,5-TCP (0.06 ppm)
".*-..'• *
appeared in milk and cream. When given high 'levels of
2,4,5-T, equivalent to 300 and 1,000 ppm in the total diet,
residues of 2,4,5-T and 2,4,5-TCP ranged from 0.05 to 0.5
ppm in the milk of individual cows*
Fitzgerald et al. (38), studying the degradation
of 2,4,5-T in woody plants, reported that colorimetric
analysis suggested, and chromatographic analyses confirmed,
that the n-butyl ester of 2,4,5-T is degraded in sweet
gum CLlquidarobap stvraciflua) and southern red oak (Ouercus
falcata) to yield 2,4,5-TCP.
-------
F* Environmental Fate
(1) £ e r 3 i s t e n c e ; Soils
*
Soil surface and foliage are the major recipients of
phenoxy herbicides (39) whether applied by ground spray
systems or from aircraft. Once 2,4,5-T reaches the soil it
Bay be degraded chemically or biologically, volatilized and
moved to other areas, absorbed on soil colloids or in
organic matter, or leached to depths or locations where
it cannot be absorbed by plant roots (47),
Norris et al. (176) reported on the persistence
of 2,4,5-T in a Pacific Northwest forest. The authors
found that six months after application of 2,4,5-T at 2.24
kg/ha (2 pounds/acre), the level of herbicide in the forest
floor declined 90$; after one year, less than 0.02 kg/ha
remained in the forest floor* The authors found little
leaching of 2,4,5-T from the forest floor into soil, and
no residues were found deeper than 15 cm (maximum residue
found was 0.08 ppm) despite rainfall of 24 cm the first
month and 70 cm the first three months after application.
Norris et al, (176) stated that the rapid disappearance
of 2,4,5-T from the forest floor suggests abundant mi-
crobial activity. Norris (40) reported microbial ac-
tivity to be important in the disappearance of 2,4,5-T
from forest-floor material in the laboratory.
-10-
-------
Wiese and Davis (11) found that, in an agricultural
soil, 2,1,5-T remained in the upper .six inches even after
application of 1.5 inches of water over a short period of
time. ,
\
«
Helling et al. (39) found that 2,4,5-T is relatively
mobile in sandy soils but that movement decreases as organic
content increases. Thus 2,1,5-T is moderately mobile in
clay soils and only slightly mobile in muck (12).
Yoshido and Castro (13) studied the degradation
of 2,1-D, 2,1,5-T, and Picloram in two Philippine soils
•under upland and submerged conditions. . The authors found the
degradation of 2,1,5-T to be rapid in Maahas clay. Slightly
more 2,1,5-T residues were recovered in submerged than
in upland Maahas soil. In Luisiana soil under submerged
conditions, 2,1,5-T degraded rapidly in eight weeks after a
four-week lag period, while it degraded gradually under
upland conditions, with only about 10? of the 2,1,5-T
recovered after 12 weeks. .
Morton et al. (11), using technical grade 2,1,5-T
labeled in the carboxyl position with carbon-11, found that
its apparent half-life averaged 1.6 weeks in green tissues
of native grasses at College Station and Spur, Texas, and
1.7 weeks in litter tissue. The authors stated that the
-11-
-------
amount and frequency of rainfall were conducive both to
leaching and microbial decomposition of the herbicide, and
to growth of sideoats gramma plants, all of which were
factors contributing to rapid reduction of herbicide concen-
trations.
• * - . '
When considering the persistence of 2,4,5-T, the.
persistence of its manufacturing contaminant, TCDD, must
also be considered. Helling et al« (39) found that TCDD was
not photodecoaposed on soil. TCDD was found to be immobile
in Norfolk and Lakeland sandy loams, Hagerstown silty clay
•loam, Barnes clay loam, and Celeryville muck, and was not
.leached further into soil by rainfall or irrigation.
During surface erosion .of soil, however, lateral trans-
port of TCDD could occur.
The persistence of TCDD in Lakeland loamy sand and
Hagerstown silty clay loam at 1, 10, and 100 ppm was studied
by Kearney et al. (46) for 360 days. After one year these
researchers recovered 56 and 63$ of the originally applied
TCDD in Hagerstown and Lakeland soils, respectively.
Helling et al. (39) observed that TCDD's persistence was
predictable since it is insoluble in water.
(2) Persistence: Water
Current information indicates that, although some
2,4,5-T may enter streams flowing through or adjacent to
-12-
-------
areas being sprayed, residue levels in streams will be
very low. Norris (47) reported the results of an intensive
study of stream contamination from spray projects on range
and forest lands in Oregon which showed that peak concentra-
tions of phenoxy herbicides seldom exceeded 0,1 ppm and that
herbicide residues persisted for only a few hours in nearly
all streams. Norris (47) speculated, however, that applica-
tion of herbicides to marshy areas may result in high-level,
long persistence of chemical residues in nearby streams*
The Report of the Advisory Committee on 2,4,5-T
to the Administrator of the Environmental Protection
Agency (48) stated that all available data indicated that
the amount of 2,4,5-T entering water is small and does not
persist long. It is adsorbed on clay or absorbed by biota
within "a matter of days. ;
._. . .Phenoxy chemicals entering water may be lost by
volatilization, degradation', adsorption on sediment, adsorp-
tion by biota, and dilution as additional stream water
passes through the site. Almost all authorities agree that
there is adsorption on bottom sediment (48, 49, 50).
Kenaga (51) stated that esters of 2,4,5-T in most
•
kinds of water, except highly acidic waters, are usually
hydrolyzed within a matter of days. When the 2-ethylhexyl
-13-
-------
'ester of 2,4,5-T was applied to water in the laboratory at a
concentration of 1 ppm for an hydrolysis study, 58% remained
after 4 hours; 33? after 8 hours; and \2% after 16 hours.
Trichell et al. (52), studying the loss of herbicides
in runoff water, found 2 ug/ml of 2,4,5-T in runoff water 24
hours after it was applied at 2.24 kg/ha, after which 1.3 cm
of rainfall was simulated on sod-covered plots of 3% slope.
Four months after application, concentrations of 2,4,5-T in
runoff water had diminished to 0.04 ug/ml%
Edwards and Glass (53) monitored runoff and percola-
tion of 2,4,5-T at Coshocton, Ohio, for 14 months following
application of 11.2 kg/ha of 2,4,5-T and found that 5»5
g/ha, or over 0»05? of the herbicide, was lost from the
treated area. Host of the 2,4,5-T was removed in runoff
water during the first four months after application, and
more than half of the loss occurred the first month after
treatment.
Kearney et al. (46) concluded that contamination
of underground water supplies with TCDD seemed very unlikely,
since vertical movement of TCDD did not occur in a wide
range of soil types. The fact that no leaching occurred,
however, would not preclude runoff loss when soil erosion is
significant (39).
-14-
-------
(3) Jjraiisport
Isensee and Jones (54) measured uptake of TCDD from
soil by two crop species. Oats (Ayena sativa)- and soybeans
(Glvcine max) were grown in Lakeland sandy loam soil treated
with 0.06 ppm TCDD. The concentration of TCDD in soil
was approximately 4,000 times greater than the amount that
•
would be deposited in soil from an application of 2,4,5-T
(with 1 ppm TCDD) at a rate of 2 pounds/acre in the top
1/3 inch of the soil surface. The tops of these plants
were harvested at intervals to maturity. Mature oats and
soybean tops contained less than 1 part per billion (ppb)
•
TCDD. TCDD vars detected (with a detection limit of 1 ppb)
in mature oat grain, while no TCDD was found in the bean of
soybeans. The authors concluded that soil uptake of TCDD by
plants was highly unlikely, since little or no TCDD was
taken up by oats or soybeans under the conditions of this
experiment (54).
(4) Bioaccumulatjon
Woolson et al. (55) conducted a study to determine if
TCDD residues could be detected in bald eagle (ftaljaectas
leucooephalys) tissue extracts, as a representative of the
top of a food chain. Scientists at the Patuxent Wildlife
t
Center (U.S. Department of the Interior, Laurel, Maryland)
collected, and furnished to these researchers, 19 bald eagle
-15-
-------
r •
carcasses from Alaska, Maine, North Dakota, Wisconsin,
Michigan, Minnesota, Arkansas, Illinois, Missouri, Maryland,
Virginia, Iowa, New York, New Jersey, and Florida between
1966 and 1971. These states were selected as sampling
sites in order to provide a widely dispersed sample population.
The eagle tissues were prepared and extracted as described
*
by Mulhearn et al« (56). Woolson et al. (55) detected no
dioxin residues at a level of 0,05 ppm TCDD, the lower limit
of detection for most pesticides in tissue samples .run by
the Patuxent Wildlife Research Center at that time. The
authors stated that the non-detection of dioxin residues
could imply that there was no dioxin build-up in the food
chain; that the build-up was less than the [then] current
detectable level of 0.05 ppm [50 ppb]; that the eagles
examined were not contaminated although other samples might
be; or that other species could feed on a different .food
chain to accumulate dioxins.
Isensee and Jones (57) exposed several organisms in
a model aquatic ecosystem to 1**C-labeled TCDD for up to 31
days to determine the distribution and bioacumulation
potential in the aquatic environment. Soil containing from
1 J|
0.0001 to 7.^5 ppm adsorbed C-TCDD was placed in aquaria,
containing eight snails fPhvsa sp,), a few strands of algae
(pedogonium c_ardi_a_c.um_), and 10 ml of old aquarium water
-16-
-------
containing various diatoms, protozoa, and rotifers. Fifteen
duckweed (L_emna minor) plants were also added to one aquarium,
Samples of daphnids were taken for analysis at 30 days, and
two aosquito fish (Gambusia affinis) were added to each
.tank. Three days later all of the organisms were removed
for analysis, and two fingerling channel catfish (Ictalurus
ounctatus) were'added to' each tank and exposed for six
days* .
The authors stated that all organisms in both treat-
ment and control tanks prospered during this exposure
. period, indicating that TCDD was not toxic at the concentra-
tions used* TCDD ac-c'Officiated in all organisms. At the
highest TCDD concentration (7*45 ppm) algae accumulated
'. 6»690 ± 960 ppb TCDD; snails, 1,820 ± 170 ppb; daphnids,
10,400 ± 480 ppbj and Gambusia, 1,380 i 220 ppb. Catfish
^ were not analyzed for TCDD residues* At the second highest
TCDD concentration (3.17 ppn), however, catfish accumulated
720 ± 130 ppb TCDD. The authors stated that accumulation in
all of the test organisms from soil containing 0.1 ppb TCDD
is important since this concentration approaches the concen-
tration which would occur under normal field use of 2,4,5-T.
The authors concluded that the data suggested that under
certain circumstances (discharge of storm runoff from
-17-
-------
recently treated rangeland into a small pond), water-eroded
surface soil or debris may contain enough TCDD for measurable
residues (parts per thousand [ppt] quantities) to accumulate
in fish or other aquatic organisms. However, the authors
speculated that TCDD, orginating from 2,4,5-T applications,
discharged into large lakes, streams, or estuaries would
probably become -sufficiently diluted so that no measurable
accumulation would occur.
• ^ ' '
As part of a broad study to determine whether 2,4,5-T
use leads to TCDD accumulation in the environment, Shadoff
et al. (58) collected samples of fish, mud, water, and human
nilk from areas in Texas and Arkansas. The Texas samples
of water, mud, catfish, and walleyed pike were collected
from the San Angelo Reservoir, an impoundment of the North
Concho River. The authors stated that this watershed has
" . - *
large acreages that have been sprayed with 2,H,5-T at 0.5
pounds/acre (2,4,5-T acid equivalent) for brush control.
These researchers also obtained six samples of human milk
from mothers residing in the general area of the San Angelo
Reservoir. In addition, bass from a 125-acre pond in the
heart of the Arkansas rice-growing area were collec.ted..-
Water from this pond is- used to flood rice fields treated
with the equivalent of 1.25 pounds/acre of 2,4,5-T acid,
four to eight weeks prior to flooding. The water is
-18-
-------
1 later drawn off the fields and pumped back into the pond for
re-use. In addition, the pond i3 supplemented by water from
wells and by water collected as .run-off fron surrounding
rice fields during the rainy season. The authors stated
that this cycle had been in use (including the proper use of
2,4,5-T) for 18 years up to the time of their study.
The authors stated that no TCDD was detected in the tissues
sampled, using a Gas Chromatography-Mass Spectrometry
procedure with a detection limit which averaged less than 10
ppt. No evidence was found that TCDD is accumulating in the
environment from the use of 2,4,5-T described in this
study*
G« Residues
(1) Soil
Koolson et al» (55) studied Lakeland sandy soil
to determine if TCDD residues could be detected in soil
receiving exceedingly large application of 2,4-D and 2,4,5-T,
The heaviest rate of 2,4,5-T application was 947 pounds/acre
applied aerially during 1962 through 1964, while the
lightest rate was_jj50 pounds/acre applied aerially during
1968 and 1969. .During this period, it was not uncommon
for commercial samples of 2,4,5-T to contain levels of
30 to 40 ppm TCDD.
-19-
-------
The authors were able to detect small amounts of
2,4,5-T in the soil samples. They observed that the residue
level decreased with time after application and stated
that leaching and microbial decomposition cpuld account for
this decrease. Using a detection limit of less than 1 ppb,
the authors did not detect any TCDD at any depth in 36-inch
core samples of the soil.
(2) Water
In October 1965, the U.S. Geological Survey initiated
*a limited program of pesticide monitoring on 11 waterways
in the western United States (59)* The streams, representing
agricultural areas where the probability of observing
pesticide residues would be greater, included the Missouri,
Brazos, Yellowstone, Sacramento, Colorado, Arkansas, Yakima,
Rio Grande, and Snake Rivers* Pesticides chosen for analysis
included the insecticides aldrin, DDD, DDE, DDT, dieldrin,
endrin, heptachlor, heptachlor epoxide, and lindane, and
the herbicides 2,4-D, 2,H,5-T, and silvex. The authors
reported that no herbicide was found at any time at any
station during the first year of the sampling program* The
lower limit of sensitivity (detection) was 5 ppt.
Manigold and Schulze (60), reporting on the results
of the U.S. Geological Survey stream monitoring program for
-20-
-------
the two-year period October 1966 to September 1968f observed
that beginning in August 196? 2,4-D, silvex, and 2,4,5-T had
been detected frequently* 2,4,5-T was found in 28 of the
320 samples and ranged from 0,01 to 0,07 ppb. The authors
stated that the established criteria permitted 100 ug/liter
(ppb) for herbicides. These authors reported that the
analytical procedures were changed from the preceding report
to use Law's sample clean-up procedure, which permits
routine detection of pesticides at 0.005 ug/liter in most
waters*
Norris (47) observed that peak concentrations of
phenoxy herbicides seldom exceeded 0*1 ppm in streams
contaminated from spray projects on range and forest lands
in Oregon,
Lawson (61) studied 2,4,5-T residues in storm runoff
from three small watersheds in Arkansas* Two watersheds,
one cleared and the other partially cut, were sprayed with
the isooctyl ester of 2,4,5-T, A third watershed, adjacent
to the two treated ones, was used as a control. Spraying
was done in September 1971, June 1972, and July 1973, either
to control woody sprouts and broadleaf vegetation or just to
provide herbicide application for-monitoring. The cleared
watershed was treated with 4 pounds acid equivalent per acre
and the partially cut site with 2 pounds/acre,
-21-
-------
In water samples taken after the first runoff-producing
storm in October 1971| Lawson (61) detected an average
of 2*1 ppm 2,4,5-T from the cleared watershed and 1*0 ppm
from the partially cut site. Maximum amounts detected were
2«2 and 1.3 pptn for the two areas. No 2,4,5-T was detected
from the control site* •
Only trace amounts (less than 0*2 ppm) were detected
from each of the two treated sites after the next runoff-
producing storms in November 1971» None was detected from
the control.
In approximately 90 samples taken after storms during
the period December 1971 through September 1973, no 2,4,5-T
was detected by Lawson (61) in the runoff from the treated
or control water sheds, . '.'•'" :r
Since TCDD is immobile in soil (39) and soluble in
water at only 0.2 ppb (7), the possibility of ground water
contamination is virtually nonexistent (.46)• TCDD could be
present in runoff when soil erosion is significant (39), and
thus TCDD contamination of water bodies could occur.
A recent National Academy of Sciences report on
drinking water stated that 2,4,5-T and TCDD have never b.een
detected in drinking water; the limit of detection was in
-22-
-------
the parts per trillion^ However, the report did project the
toxicity of 2,^,5-T and TCDD, their acceptable daily intake,
and suggested no-adverse-effect levels (62).
(3) lin
Prior to 1970, phenoxy herbicides were widely
used for early postemergence control of weeds in wheat*
Johnson (63) reported that air samples collected during
spring and summer in the state of Washington where these
crops are grown contained as much as 0.06 ug/m^ 2,4-D. and
2,4,5-1* Assuming that a man—inhales about 30 m' of air per
day, the authors estimated that exposure to 0*06 ug/m would
amount to inhalation of 1.8 ug phenoxy herbicide/day
or 0*025 ug/kg of body weight per day for a 70 kg man.
--Ambient air monitoring for pesticides in predominantly
agricultural areas of 28 states was conducted by the
National Air Monitoring Program in calendar years (CY) 1970
'through 1972 using ethylene glycol impinger type samples.
Table 1 records the arithmetic mean of residues of 2,4,5-T
detected in this program (6*0.
.23-
-------
Table 1. Air Monitoring Data for 2,M,5-T in 28 State
1
\ Name of
\ State or Citv
1 Louisana
{Montana
{New Mexico
{Idaho
{Illinois
{Oregon
{Tennessee
{Tennessee
i Qklahoma
2,H,5-T Ester I
Monitored For !
Isopropyl esterl
i
i
i
BOEE {
{
{
Isooctyl ester {
!
•a
ng/m
CY -1970
-
ND
- .
ND
ND
ND
1.1
ND
ND
ng/m3
CY iq?l
ND
ND
ND
• ND
3.6
0.5
ND
2.7
in. 6
ng/m3
CY 1Q72
1.9
0.8
1.0
1.7
ND
ND
ND
ND
ND
ND = Not Detected,
(H) Animals ; . .-
Phenoxy acetic acids are relatively strong acids,
and animals rapidly excrete them unchanged in their urine
(36). In their study of the fate of atrazine, kuron,
ailvex, and 2,U,5-T in the dairy cow, St. John et al. (65)
found that dairy cows given 2,1,5-T and silvex in their feed
at 5 ppm for four days, completely eliminated both 2,1,5-T
and silvex as soluble salts in the urine two days after
dosing stopped. .
Zielinski and Fishbein (66) treated female C57BL/6
mice with a single subcutaneous injection of 100 mg/kg body
weight of 2,M,5-T in dimethylsulfoxide solution. They
sacrificed the animals at various intervals after injection
and analyzed JLT^ toto for 2,U,5-T. The amounts recovered as
percentage of the amount injected indicated decreasing
levels at the following time intervals after dosing: at 0
hours, 77.1 ± 5.0J; at 16 hours, 56.9 ±. M.2%; and at 2M
hours 23.7 ±. 3.6J,
-------
In a preliminary report of a two-year chronic
toxicity feeding study, Dow Chemical USA (110)'reported
the following residue data for rats fed indicated TCDD
doses: 24,000 ppt in liver and 8,100 ppt in fat of females
ingesting 2,200 ppt/day; 5,100 ppt in liver and 1,700
ppt in fat of females ingesting 220 ppt/day; and 540 ppt in
liver and fat of females ingesting 22 ppt/day* The pre-
liminary report gives no residue data for treated males,
or for controls of either sex,
Piper et al. (67) studied the fate of 2,4,5-T follow-
ing oral administration to rats and dogs* Four groups of
three male and three female Sprague-Dawley rats (Spartan
strain) and two male and two female adult beagle dogs were
' 14 • ' " '
given single doses of C-labeled 2,4,5-T by intubation
at 5, 50, 100, and 200 mg/kg body weight in rats and
5 mg/kg body weight in dogs. The authors combined data
obtained for males and females since the pharmacokinetics of
2,4,5-T were essentially the same in each sex» In this
study, the clearance half-life for 5 mg/kg 2,4,5-T from dog
plasma was 77.0 hours; in rats the half-life was 4.7
hours at 5 mg/kg anff" ^.^'hours at 50 mg/kg. At doses of
100 and 200 mg/kg body weight, the clearance half-life for
rats increased to 19i4 and 25.2 hours, showing that the
-------
pharmacokinetics of 2,H,5-T varies with dose as well as with
species* The authors suggested that the half-life values
at 100 and 200 ng/kg body weight indicated that these doses
may have exceeded the excretory capacity of the rats»
Zitko (68) assayed chlorinated dibenzodioxin residues
in aquatic animals, but was unable to detect these compounds
(detection limit: 0»04 ug/g [ppm] for TCDD) in any of
several aquatic animals from Canadian locations. The author
had selected species from high trophic levels of the
aquatic food web to measure cumulative pesticide contamination*
More recently, using improved analytical methods for detection
of dioxin at ppt levels, Baughman and Meselson (69) found
mean TCDD levels ranging from 18 ppt to 810 ppt in fish and
crustaceans taken from Vietnamese rivers in August and
September 1970. TCDD levels tended to be higher in fish
*
from interior rivers than in those from seacoast locations.
In comparison, Baughman and Meselson found less than 3 ppt
TCDD in fish obtained in a market in Cape Cod, Massachusetts.
In another study, Matsumura and Benezet (70) placed TCDD-
coated sand directly in an aquarium containing brine shrimp,
mosquito larvae, and fish (silverside). TCDD pickup was low
in fish (2 ppb) and brine shrimp (157 ppb) under the experi-
mental conditions. But mosquito larvae, which are bottom
-26-
-------
feeders, showed a surprisingly high rate of pickup (4,150
ppb)« The authors concluded that TCDD was not likely to
accumulate in as many biological systems as DDT because of
TCDD1s low solubility in water and lipids, as well as its
low partition coefficient in lipids,
(5) Plants
Clark et al* (36) reported that, when herbicides
are applied to rangeland, the levels of phenoxy herbicides
available for ingestion by grazing livestock depend upon
the nature and degree of cover, the rate and mode of applica-
tion, time after application, and climate conditions.
Studies by Morton et al. (44) showed that residues on grass
immediately after application of 2,4,5-T are not likely
to exceed 100 to 150 ppm for each pound of actual herbicide
applied per acre. '.'••...'-
Leng (37) stated that herbicide residues in or on
plants declined rapidly, with a half-life of one to two
weeks, due to photodecomposition by sunlight, wash-off
by rain, metabolism by plants, and dilution from growth of
plants, 2,4,5-T was applied to grass in four states at an
application rate of 4 pounds/gallon, 3 gallons/acre;
initial residues immediately after treatment in California
averaged 684 ppm (or 57 ppm/pound applied per acre); 1,668
-27-
-------
ppm (or 139 ppn/pound) in Michigan; 1,H6H ppm (or 122
ppm/pound) in North Carolina; and 1,332 ppm or (111 ppn/pound)
in Texas. After two weeks, residues in the four locations
averaged 26 to 3H ppm/pound per acre* After 16 weeks, all
residues had declined to an average 3 ppm/pound applied per
* • '
acre*
• . *
Baur et al. (71) treated grass species indigenous
to Victoria County, Texas, with 2 pounds/acre 2,1-,5-T ester.
One month after application the concentration averaged
4,060 ng/g (ppb) for 2,A,5-T acid and 2,890 ng/g (ppb) for
2,4,5-T ester* Six months after application the concentra-
tion averaged 60 and 170 ng/g (ppb) for 2,M,5-T acid and
ester, respectively, •
•"'. Getzendaner and Hummel (72)"* described a 1969
study in which a 2,4,5-T propylene glycol butyl ether ester
formulation was sprayed on Texas-grass at an application
rate equivalent to 12 pounds of 2,M,5-T per acre; this rate
was 6 to 24 times the usual rate applied to grazing lands
for brush control. At this time, manufacturing specifications
Studies submitted by registrants as part of petitions
for residue tolerances are classified confidential, pending
outcome of litigation in U.S. District Court,
•28-
-------
for no detectable TCDD in 2,H,5-T used a method sensitive to
1 ppm. The authors found that residues of TCDD decreased
rapidly from about 500 ppt TCDD within one day of application,
to about 35 ppt TCDD after four weeks, and about 15 ppt TCDD
after 16 weeks. The TCDD decrease roughly paralleled the
loss of 2,1,5-T from the same grass,
'''•-.'••*"*.• • *"
(6) Humans- '
Matsumura (73) studied 2,4,5-T in the blood and urine
of human male volunteers who had ingested the chemical*
After ingesting 150 mg (2.2 mg/kg), the plasma concentration
of 2,1»,5-T in one subject reached a peak of 2U1 ug/ml after
four hours. A linear, semi-logarithmic concentration-time .
curve (a gradient of -0.065) four hours post-treatment
indicated first order elimination and absorption kinetics,
In a second part of this study, Matsumura gave
two male volunteers single oral doses of 100 mg 2,4,5-T»
Urine samples were collected over 72 hours. About H5% of
the original dose was found in urine collected during
the first 21 hours after treatment; 60$ had been recovered
36 hours after treatment; and after 72 hours, more than 80%
of the original dose-of 2,4,5-T had been recovered,
Gehring et al, (7^) also studied the fate of 2,4,5-T
following oral administration to man. Five male volunteers,
-29-
-------
ages 31 to 58 years, each ingested a single 5 mg/kg oral .
dose of analytical grade 2,4,5-T, with a purity greater than
99J and less than the detectable level (0.05 ppm) TCDD,
directly or as a slurry in milk. Blood, urine, and feces
were collected at intervals for up to 96 hours after
ingestion. Essentially all (88.5 ± 5.1$) of the 2,4,5-T .•
ingested by these subjects was excreted unchanged in the
urine after 96 hours* The plasma 2,4,5-T concentration
increased rapidly following ingestion and after 7 hours -
reached a peak of approximately 57 ug/ml, after which the
plasma contained 65J of the 2,4,5-T in the body, of which
99? was bound reversibly to protein,
* . . -
Kohli et al. (75) also studied absorption and
excretion of 2,4,5-T in man. Eight male volunteers, age 25
to 35 years, received a single oral dose (2, 3, or 5 •
.mg/kg) analytical grade 2,4,5-T with a purity greater than
99£* Urine was collected up to 96 hours, and blood samples
were collected up to 168 hours, 2,4,5-T was detected in
some two-hour urine samples, indicating rapid excretion of
the compound. More than half of the 2,4,5-T was excreted in
.the urine in the first 48 hours, although small quantities
were still being excreted at 96 hours.
-30-
-------
2,1,5-T appeared in all plasma samples one hour
after 2,4,5-T ingestion, indicating rapid absorption.
Maximum concentration (approximately 25 ug/ml for the 5
mg/kg dose) was reached between 7 and 24 hours after
/
ingestion and began to decline at a first-order rate
»
after 32 hours, .
* ... '"".."
These investigators concluded that 2,4,5-T was
readily absorbed from the gastrointestinal tract, that it
was eliminated unchanged in the urine, and that the half-life
for plasma clearance was 18.8 ± 3,1 hours. These authors
pointed out that, in general, higher recoveries were reported
by Gehring et al. (7*0 who used an electron capture detector,
.instead of the flame-ionization detector used in their
study. . •••;.' . -...••..
The National Hunan Monitoring Program for Pesticides,
through its cooperative arrangement with the Health and
Nutritional Examination Survey II (Hanes II project), is
currently analyzing human urine samples for silvex, 2,4,5-T,
and 2,4,5-TCP (64), The survey is scheduled for completion
in 1979, but some extremely tentative results are available.
No quantifiable 2,4,5-T residues have been detected in the
first 400 samples; however, trace amounts (<10 ppb) have
been found in a few samples, :
Dougherty and Piotrowska (177) reported on screening
of human urine for environmental contamination with toxic
-31-
-------
residues by negative chemical ionization mass -spectrometry.
The procedure is based on solvent extraction with minimal
clean-up followed by examination with negative chemical
ionization mass spectrometry for organochlorine residues and
related compounds with masses greater than 130 daltons*
Urine' for the screening procedure was obtained from students
at Florida State University (25 dorm residents; 21 football
team members; and 11 swimming team members). The authors
reported that the limited survey of human urines indicates *
contamination of the subjects with 2,4,5-T, pentachlorophenol,
•
other polychlorophenoxy acids, and numerous unknown compounds.
The authors indicated that 2,4,5-T was found in 36% (9/25) of
the dorm residents; 24? (5/21) of the football team; and 9%
(1/11) of the swimming team. The authors attempted to define
the source of the contamination by applying the same
screening procedure to environmental substrates and suggested
*
the food chain (beef fat in the case of 2,4,5-T) as one
significant source of the contamination. .
(7) Animal Products ' •.
Kocher et al. (76) surveyed beef fat from cattle
grazing on land where 2,4,5-T had been applied to determine
if TCDD was present in this tissue* None of the 2,4,5-T
samples used were available for analysis for TCDD content.
-32-
-------
The authors did not know whether the samples were produced
before 1972 (when maximum allowable TCDD content was 1 ppzn)
or after 1972 (when maximum allowable TCDD content was 0.1
ppm). None of the 16 samples from Sugarland, Texas, Missouri,
and Oklahoma showed TCDD residues when analyzed by a gas
chroaatography-mass spectrometry detection technique (detection
limits! 3 to 6 ppt). Three of the eight samples from
Mertzon, Texas, where animals had grazed for 30 days in a
fenced pasture sprayed in its entirety with 2,4,5-T, gave
positive responses at the detection limit of 3 to 4 ppt TCDD.
In another surveillance study, Mahle et al* (77)
analyzed milk from cows grazing on grass treated with
2,4,5-T in accord with normal agricultural practices.
Twenty-five samples were collected from different farms
in Oklahoma, Arkansas, and Missouri; these areas were
selected as representative of those where 2,4,5-T is used to
control broadleaf weeds and brush in pasture and rangeland.
Milk purchased in Midland, Michigan, an area where 2,5,5-T
is not used, provided control samples* Based on gas
chromatography-mass spectrometry data (detection limit:- 1
•
ppt), the authors stated that control samples were indistin-
guishable from the samples from treated areas and concluded
that TCDD was not present.
-33-
-------
The residue levels reported in animal products in
the studies cited below were obtained in laboratory
feeding studies and not from animals grazing on pastures
and rangelands treated at dosage rates recommended on
.registered product labels. Nevertheless, residues obtained
in these feeding studies could occur in the environment and
at these same levels since animals grazing on forage plants
immediately after treatment at recommended rates of applica-
tion could ingest 2,4,5-T in amounts similar to those fed in
the studies. _. —
• ' ~ .
Leng (37) found no residues greater than 0.05 ppm in
.milk or cream of cows ingesting 10 to 30 ppm 2,4,5-T. A.t
100 ppm 2,4,5-T in the diet, traces of 2,4,5-TCP (0.06 ppm) -
were found in milk and cream. When the diet contained high
-34-
-------
levels of 2,M,5-T, equivalent to 300 and 1,000 ppn in the
total diet, residues of 2,4,5-T and 2,4,5-TCP ranged from
0,15 to 0.5 ppm in milk of individual cows.
Leng T37)> reporting on residues in meat and meat
byproducts, stated that calves slaughtered after ingesting
t
300 ppm 2,4,5-T in the total diet contained average residues
of 0.12 to 0.28 ppm in muscle, fat, and liver, and 3.3 ppm
in kidney* Animals fed 900 to 2,000 ppm 2,4,5-T in the
total diet and slaughtered without withdrawal had propor-
tionally higher average residues in tissue. No residues rfer
detected (detection limit: 0*05 ppm) in most tissues when
animals were given untreated feed for one week after they
had been on the highest levels (1,800 and 2,000 ppm) of
2,4,5-T for four weeks. Residues of 2,^,5-T declined
rapidly in tissues as soon as animals started to eat
untreated feed.
Clark and Palmer (78) found 0.08 ppm 2,4,5-T in
omental fat of each of two sheep given four oral doses of
either 0.15 or 0,75 mg/kg of the propylene glycol butyl
ester of 2,4,5-T. They also found 368 ppm 2,4,5-T in
kidneys of animals killed by four daily 250 mg/kg doses of a
2,4,5-T ester.
Clark et al. (36) found 2,4,5-T no higher than 0.05
ppm in muscle or fat of sheep held one week on untreated
1
•35-
-------
feed. Residues of the metabolite 2,4,5-TCP wer-e not detected
in the fat of any of the aniraals4 They also found that the
2|4,5-T level in liver and kidneys was less than 0.05 ppm
after the animals were on untreated feed for seven days*
Leng (79) found low levels of 2,4,5-T in muscle and
fat of calves .receiving 300 to 900 ppm 2,4,5-T in the diet
and much higher residues in tissues of animals fed 1,800
ppm 2,4,5-T for 28 days* Calves fed 300 ppm 2,4,5-T showed
0.12 and 0.28 ppm 2,4,5-T in muscle and fat, respectively.
Calves fed 900 ppm showed 0.24 and 0*38 ppm in muscle and
fat, respectively. And at 1,800 ppm in the diet, calves
showed 1*2 and 2*0 ppm in muscle and fat, respectively. In
this same study, Leng found relatively low residues of
2|JJ,5-T in liver at feeding levels of 300 and 900 ppm (0»2
and 1.0 ppm, respectively) and sharply increased residues
(7*9 ppm) at 1,800 ppm, indicating that the threshold level.
may have been exceeded at this higher dosage level* Residues
of 2,4,5-T in kidney appeared to be proportional to the
level in the diet.
Eighty-five samples of beef fat were analyzed
for TCDD content under the auspices of the EPA Dioxin
Implementation Plan (see Section II). These beef fat
-36-
-------
samples included 18 samples from control areas and 67
samples from areas previously treated with 2,4,5-T.
Hone of the 18 control samples had detectable amounts
of TCDD at a detection limit of 10 ppt. Of the 67 samples
from areas previously exposed to 2,4,5-T, one showed a
positive TCDD level of 60 ppt; two appeared to have TCDD at
20 ppt; and fiv.e may have had TCDD levels which ranged from
5 to 10 ppt» The values for these five samples were at or
below, the limits of detection of 10 ppt. Forty-three beef
liver samples were analyzed and showed no TCDD residues at a
detection limit of 10 ppt»
(8) Food
Evidence that very little 2,4,5-T gets into food is
seen in results of Market Basket Surveys conducted by
'the Food ajxd Drug Administration (FDA), Of the 134 total
"diet samples involving 1,600 food composites (Market Basket
<
Survey) analyzed from 1964 through April 1969, only three
contained 2,4,5-T* Two were dairy products containing 8 to
13$ fat with 0.008 and 0»19 ppm in the fat. A single
meat, fish, and poultry composite from Boston consisting of
17 to 23? fat was found to contain 0.003 ppm 2,4,5-T on
a fat basis (81, 82, 83, 84).. •
FDA Market Basket Survey samples from 1969 through
July 1974 showed no 2,4,5-T residues (detection limit: 0.02
-37-
-------
ppn) in 155 total diet samples involving 1,869 food composites
(85, 86, 87, 88, 89).
(9) Human Exposure via Industrial Accidents
There have been a number of industrial accidents
during manufacture of chlorinated phenols that have resulted
in human exposure to TCDD.
Whiteside (90) reported on a 19^9 explosion at a
chemical'plant producing 2,^,5-T in Nitro, West Virginia*
The release -of intermediate chemicals led to 228 cases of
\
chloracne among exposed workers, Whiteside stated that
symptoms of effected workers-included skin eruptions,
shortness of breath, intolerance to cold, palpable and
tender liver, loss of sensation in extremities, damage to
peripheral nerves, fatigue, nervousness, irritability,
insomnia, Toss of libido, and vertigo*
Goldmann (91) reported on a 1953 accident at a
2,^,5-TCP production plant in Germany. Temperature and
pressure rose explosively in the autoclave, forming previously
unknown, very toxic chlorinated hydrocarbons; H2 persons
contracted serious cases of dermatitis, in which Hi persons
suffered consequent damage to internal organs, and seven
persons experienced disturbances of the nervous system.
-38-
-------
A similar accident occurred in Amsterdam in 1963 when an
explosion in a 2,4,5-T factory resulted in 50 workers
contracting chloracne (90).
In 1954, 31 workers in a Hamburg, Germany, chemical
plant producing 2,4,5-T from technical 2,4,5-TCP contracted
chloracne (10, 16, 92) and suffered the physical and psycho- .
logical symptoms associated with it (93)• Kimmig and Schulz
(10) extensively investigated the workers' conditions
and conducted experiments treating the skin of a rabbit's
ear with chemicals to which workers had been exposed.
These researchers tentatively identified the causative agent
of the chloracne as TCDD.Bauer et al. .(15) conclusively
identified TCDD as the cause of chloracne,
In 1964 workers in a 2,4-D and 2,4,5-T plant in
the United States developed chloracne (93> 9*0. Eleiberg
et al. (94) found evidence of porphyria cutanea tarda (PCT)
of varying'degrees of severity in 11 out of 29 workers. PCT
had never before been described as related to chloracne, nor
had it been ascribed to industrial exposure in the United
States. The authors stated- that either the finished chemicals
or some intermediate were responsible for both diseases.
The Fine Chemicals Unit of Coalite and Chemical
Products Limited located at Bolsover, Derbyshire, in England
had been producing 2,4,5-TCP for nearly three years without
' -39-
-------
.Incident when an explosion occurred at midnight on April 23>
1968* As a result of this exothermal reaction, TCDD had
accidentally been produced* Workers at this'plant were
accidentally exposed to TCDD, and 79 cases of chloracne were
recorded, many of them severe (9» 95K
Beginning in May 1971 an accidental poisoning episode
occurred in the United States that affected humans, horses,
and other animals* The exposure was related to the spraying
of waste oil, contaminated with TCDD, on riding arenas to
control dust% Three days after spraying, sparrows and
other birds were found dead on the arena floor* Of 85
horses exercised within the arena, 62 became ill, and 48
died* The first horse died on June 20, 1971% Horses
continued to die as late as January 1974, Human illnesses
were less severe, but did include one case of hemorrhagic
cystitis in a 6-year-old girl who frequently played in the
-ar«na. Analysis showed the arena contained 31.8 to 33,0
ug/g TCDD (96, 97, 162), .
Beale et al, (98) presented follow-up information on
the 6-year-old girl involved in this accidental poisoning.
These authors stated that the girl's symptoms resolved in
three to four days and did not recur. Results of a repeat
voiding cystogram three months later appeared normal,
Cystoscopy at this time did, however, demonstrate numerous
punctate haemorrhagic.areas in the bladder, especially in
: -HO-
-------
the region of the trigone* Five years later, an investiga-
tion showed that this girl had grown normally;- results of a
physical examination, including a detailed neurological
examination, were normal, Cystogram and liver-function
tests were also normal, as was the urinary excretion of
uroporphyrins, coproporphyrins, and thyroid function,
On July 10} 1976, an accident at the ICMESA chemical
plant in the Seveso Region of Italy released 2 to 10 pounds
of TCDD over a wide area (90, 99» 100)» Hundreds of animals
died, many area residents reported skin disorders, and an
area of 110 hectares was evacuated (101), Reports of the
immediate symptom^ _and^indications of many long-term effects
are just becoming available.
Seveso inhabitants initially experienced numerous,
burnlike skin lesions which gradually receded; Whiteside
(90) believed this type of lesion was probably due to direct
contact with the sodium hydroxide and phenolic components of
the fallout. Two and a half months after the explosion,
however, children and young people in the zone most affected
by the fallout developed symptoms of true chl.oracne, a sign
of dioxin poisoning, on their faces,-arms, and bodies* By
November 1976, 28 people had developed confirmed cases of
chloracne, and the number rose to 38 by December 1976; one
year later, the number of confirmed cases of chloracne was
130.
-------
A number of Seveso women were pregnant at the
time of the accident, Whiteside (90) reported that the
number of legal and illegal abortions performed after the
accident probably totalled 90» Results of a survey by an
epidemological commission showed that 183 babies were
delivered in the two months following the accident, and that
there were 51 spontaneous abortions as distinct from induced
abortions (approximately double the rate of spontaneous
abortions previously reported for the area)^ Whiteside
(SO) reported that eight cases of birth abnormalities have
been noted to. date among babies born to women in the Seveso
area who were pregnant at the time of the explosion.
Physicians in the Seveso~~area have had difficulty relating
this directly to the explosion, however, since this incidence
of birth abnormalities was not disproportionate to the usual
incidence of abnormal births,
H, Tolerances
There are no tolerances established for 2,4,5-T in or
on food crops. Likewise, no tolerances have been set
specifically far—TCDD--i-a—or--on food crops. However, 40 CFR
Section 180,302 does establish a tolerance of 0.05 ppm for
hexachlorophene on cotton seed (a nonhuman dietary food
• *
item), with a stated limitation that the technical grade
-42-
-------
hexachlorophene used in the formulation shall not contain
more than 0»1 ppn TCDD« The limitation does hot constitute
a tolerance (102).
1. •" pesticide Episode Reports System (PERS)
EPA's Pesticide Episode Response Branch of 'the Office
of Pesticide Programs maintains a Pesticide Episode Reports
System (PERS) which collects reports of pestibide exposure
affecting humans, domestic animals, livestock, and wildlife
(103)% According to their records, there were 96 episodes
from 1966 to April 1977 involving 2,4,5-T*
Many of these 96 episodes recorded effects in more
than one area of the environment* Plant damage was reported
60 times, effects on humans 16 times, water contamination
.14 times, effects on domestic animals and soil contamination
7 times each, general environmental contamination 3 times,
V"-V. *•
-an-d fish kills and complaints against use of 2,4,5-T twice
each.
There was substantial evidence in 13 of the 96
episodes link-ing 2,4,5-T to the episode's effects; there was
circumstantial evidence in 20 of the episodes for involvement
of 2,4,5-T; there was insufficient evidence in 62 of the
episodes to prove or disprove involvement of 2,^,5-T; and
one episode had no verification status listed.
-H3-
-------
Of the 13 episodes for which there was substantial
evidence linking 2,4,5-T to the episode's effects, two
. involved humans (including one suicide); 2,4-D was also
involved in both episodes. Three episodes involved plant
damage from drift of herbicides; 2,4,5-T residues were
found in plant samples in two episodes; 2,4-D was also
involved in one of these episodes. Two episodes involved
fish kills resulting from accidental spills into streams,
with 2,4-D involved in both incidents; in one of these
episodes, 6,000 fish (90$ juvenile salmon) were killed;
residues of both 2,4-D and 2,4,5-T were found in these fish,
Two incidents involved soil contamination when two warehouses
were destroyed by a tornado and fire; many other pesticides
were Involved In both instances. Two episodes involved
domestic animals; in one, 24 cows died after herbicide
application. Arsenic residues were found in two cows,
and arsenic contamination of the herbicide mioc was suspected,
In the other instance, 8 cows drank water contaminated with
2,4,5-T; residue levels of 0.03 and 0.02 ppm were found in
the milk five and eight days, respectively, after the
incident. Two hundred and forty gallons of milk were
dumped. One incident involved water contamination as a
result of a warehouse fire; many other pesticides were also
involved*
-------
II. KECULATORY HISTORY
2,4,5-T was developed during World War-II and was
first registered as a pesticide on March 2, 19^8 (3)» Since
then, it has been the subject of several Federal regulatory
actions.
On April 13, 1966, the United States Department of
Agriculture (USDA) and the Food and Drug Administration
(FDA) published an announcement in the Federal Register
abolishing the "No Residue and Zero Tolerance" concepts as
scientifically untenable* Future registrations would be
granted on the basis of either "Negligible Residue" or
"Permissible Residue," Industry was given until December
31, 1967, to comply by obtaining tolerances for residues of
2,^,5-T in all treated food, feed products, and byproducts
*.(in addition no registrations would be continued beyond
December 31, 1970).
Following this action, a series of Pesticide Registra-
tion (PR) Notices were issued over several years, extending
certain "no residue" and "zero tolerance" registrations
beyond the December 31, 1967, deadline for obtaining
residue tolerance, (These and all following PR Notices
are cited in Reference 104,) Among uses of 2,4,5-T extended
beyond the deadline were uses on pasture grasses and
rangeland; on apples (Mclntosh), blueberries (low bush),
cereal grains (undesignated), rice, and sugarcane; and in
lakes and ponds*
-------
PR Notice 70-8 issued by the USDA on March 10, 1970,
identified data needs for certain compounds* .'2,4,5-T
was identified as one of the compounds requiring further
teratogenic studies*
PR Notice 70-11 published on April 20, 1970, suspended
2,4,5-T products bearing certain directions for use.
The suspended uses were all uses in lakes, ponds,' or on
ditch banks; and liquid formulations for use around the
home, recreation areas, and similar sites*
PR Notice 70-13 issued by the USDA on Hay 1, 1970,
cancelled 2,4,5-T products bearing certain directions
for use. The cancelled uses were all granular 2,4,5-T
formulations for use around the home, recreational areas,
and similar sites; and all 2,4,5-T uses on food crops
intended for human consumption. -
All registrants wera advised of these actions, and
two of the 2,4,5-T registrants, Dow Chemical and Hercules
Incorporated,- excercised their right under Section 4(e) of
the Federal Insecticide, Fungicide, and Rodenticide Act
(FIFRA) [7 U.S.C. 135 et seq.] to petition for referral of
the cancellation (rice use only) to an Advisory Committee.
As provided by Section 4(c) of FIFRA (1964 amendment),
& nine-member Advisory Committee of scientists was appointed
-46-
-------
to consider all relevant facts, submit a report and recommen-
dations regarding registration for certain uses of 2,4,5-T,
and state the reasons or bases for these recommendations.
Their report was submitted to the Administrator of the
Environmental Protection Agency on May 7, 1971 (48)* The
Committee recommended that use of 2,4,5-T be permitted in
forestry, range land, and rights-of-way providing that the
limit of 0.1 ppm of contamination with TCDD be set for all
future production of 2,4,5-T; that 2,4,5-T be applied no
more than once a year at any one site; and that 2,4,5-T be
applied with proper caution so that it will not contaminate
other areas where it may come into contact with humans,
The Committee also recommended that this action be
reviewed again when existing deficiencies in information
about possible magnification of TCDD in the food chain
have been'rectified by specific research.
In the meantime, PR Notice 70-22, published by the
USDA on September 28, 1970, addressed the presence of
chlorodioxin contaminants in economic poisons. This notice
stated that the BJS.DA had determined that certain toxic
chlorodioxins (such as TCDD) may be present as contaminants
in the basic materials used in formulating 2,4,5-T and
silvex. The notice also stated that the presence of such
f
-47-
-------
chlorodioxins constituted a possible hazard to nan since they
had been found to be extremely toxic to laboratory animals,
and that appropriate regulatory action would be taken under
provisions of FIFRA since products containing chlorodioxins
are considered to be in violation of FIFRA%
Dow Chemical obtained an injunction against EPA in
July 1972, enjoining further administrative action against
2,4,5-T* The United States Court of Appeals for the Eighth
Circuit overturned the injunction in 1973, and administrative
proceedings were allowed to go forward.
On July 20, 1973, a notice of intent to hold public
hearings on all uses "o'f ~2,4,5-T was filed with the EPA
Hearing Clerk under Section 6(b)(2) of FIFRA, as amended
1972* All federally approved uses of 2,4,5-T were to be
explored in a public hearing scheduled for April 1974,
following completion of an intensive monitoring program for
detecting dioxin in the ppt range (38 FR 19869, July 29,
1973)*
On May 10, 1974, the information hearing was expanded
to include all ins-ecticides and herbicides having 2,4,5-TCP
In their manufacturing process. These included silvex,
erbon, and ronnel, as well as 2,4,5-T and 2,4,5-TCP, all
of which may contain TCDD.
-48-
-------
On June 24, 1974, EPA withdrew cancellation and
information-gathering proceedings initiated against 2,4,5-T
and related compounds because of its inability to monitor
food for TCDD residues with the necessary analytical
precision. Although the 2,4,5-T notice of hearing was
withdrawn, the Agency stated that it "will continue its TCDD
residue monitoring program and will take such further action
as it deems appropriate once the results of the monitoring
project are available" (39 FR 24050 June 28, 1974).
On July 25-26, 1974, the Agency held a Dioxin Planning
Conference in Washington, D.C., primarily for those parties
having an interest in the withdrawn 2,4,5-T/dioxin hearings,
to address data analysis and retrieval (in the areas of
analytical methodolgy, toxicology, and monitoring) with
emphasis on analytical methodology for TCDD at the ppt
level. As a result, the Agency established a Dioxin Implemen-
tation Pla-n (DIP) intended to identify a preferable analytical
methodology to monitor human and environmental samples for
JCDD.
On-going TCDD studies under the DIP include: an
analytical method validation study to produce statistically
defensible data; monitoring for resioues in human milk in
the Pacific northwest; additional beef fat residue studies;
-49-
-------
additional technical pesticide residue studies; and an
environmental monitoring program for TCDD residues in soil,
water, and biota*
III. SUMMARY OF SCIENTIFIC EVIDENCE RELATING TO
E PRESUMPTION '
The following adverse effects of 2,4,5-T and/or
TCDD have been found to exceed the criteria for issuance of
a rebuttable presumption as stated in Section 162.11 of the
Code of Federal Regulations (CFR 40)« Because of industry's
apparent inability to produce 2,4,5-T without TCDD contamina-
tion, none of the studies cited are for pure 2,4,5-T. The
effects of TCDD must also be considered when assessing
2,4,5-T by the Agency's risk criteria*
'•' .. A» pncogenic Effects .' .. '_ ' .
40 CFR Section I62.11(a) (3) (ii) (A) provides that a
rebuttable presumption shall arise "if a pesticide's
ingredient(s)... (i)nduces oncogenic effects in experi-
mental mammalian species or in man as a result of oral,
inhalation or dermal exposure...." Section 162.3(bb)
defines the term oncogenic as "the property of a substance
or a mixture of substances to produce or induce benign
or malignant tumor formation in living animals."
-50-
-------
The studies summarized below indicate that 2,4,5-T
containing less than 0.05 ppm TCDD and/or TCDD alone have
oncogenic effects in two mouse strains and one rat strain.
Since 2)4,5-1, as currently formulated, contains TCDD (at a
maximum amount of 0.099 ppm), a rebuttable presumption
against the registration of 2,4,5-T products has arisen
because of the oncogenic effect of 2, 4,5-1 and its contami-
nant, TCDD,
(1)
(a) Effects of Dietary _2,ltf5-T «0.05 ppm TCDD)
on Rodents
In their bioassy of 2,4,5-T for carcinogenicity in
.«z
mice, Muranyi-Kovacs et al* (105) administered 2,4,5-T
(containing <0.05 ppm TCDD^'to inbred C3Hf and XVII/G mice.
The mice were given 100 mg/liter of 2,4,5-T in the drinking
water for two months beginning at six weeks of age. During
the succeeding 15 to 20 months, the mice were given 2,4, 5-T
mixed in the diet at a concentration of 80 ppm ad libitum.
' In C3Hf mice, 48* of the treated females (12/25) and
55$ of the treated males (12/22) developed tumors, compared
with control values of 21 J (9/44) and 49* (21/43), respec-
tively (Table 2)% The differences between the number of
tumors observed and the number expected were significant for
female mice at all sites (p < 0.03) and for the combined sexes
This TCDD level is less than the 0.1 ppm TCDD currently
found in most commercial formulations (see Section I.B).
-51-
-------
7/
(p < 0.01). For non-incidental tumors, the differences
were significant for each sex and the combination; no
8/
significant differences were found in incidental tumors.
No other strain-sex combination yielded statistically
significant values (106). Rare types of tumors, not seen in
the control animals, were observed in the treated C3Hf
females.
Table 2. "Oncogenic Effects of 2.4.5-T on Mix
•t^ain
C3HF
•
VII/G
^^
*
| ! Dietary!
I j Level !
i ; i
i M i 0 ,'
i i 80 !
i F i 0 i
! ! so i
IM! o j
! i so i
IF! c !
II 1
1 i On '
i i OU i
Mean
Survi-
val Timej
fdavs)
630
51lJc/
680
620
521 -
583
569
. •
641^
Mice with
and Lung
Leukemia
and Liver
Tuaors !
No/Total
21/43
12/22
9/44
-^272-.
25/32
15/20
21/40
16/1Q
No^i *
149
!55
121
(48
J78
i
}75
,'53
1
!8a
Incidence of
Total
• 22
13
9
n
27
16
24
16
i i
! Lunc 1
i 2 j
i - i
! 5 !
! - i
j 22 j
1 1
! 20 !
I |
f 15 f
i
Liver !
19 I
10 i
3 i
4 !
4 i
-- |
— i
1
__ i
Tunors
i
Leuker">.a !
t
— — i
2 1
1 !
I
T t
- 1
1 i
2 j
i
"1 f
i
i
:
Other1
id/ ;
• i
1-' I
— !
6£/ |
\&/ i
• i
217 i
i
_— '
Data from Kuranyi-Kovacs (105).
Estimated daily oral dose = 12 mg/kg body weight.
Effective number of mice are mice surviving longer than 300 days or developing a tumor
fore 300 days of age.
J>leomorphic salivary gland tumor. ' •
ribrosarcoma; one hyperplastic urinary bladder and one hyperplastic forestomach not
eluded.
One osteogenic sarcoma; two sarcomas; two cutaneous tumors; one cervical tumor.
Forestomach tumor.
Urinary bladder papilloma; two hyperplastic lesions of'urinary bladder not included.
^"wo hemangiomas. ^ t_. _^__ __ .' •
p < 0.01 compared with controls. ~~ ' ""-.
p < 0.001 compared with controls.
The investigators found no significant sex-related
Incidental tumors are tumors discovered at necropsy
an animal which died from some other cancer; non-
eidental tumors are tumors diagnosed during life or
Ich caused the death of the animal.
-52-
-------
A decrease in survival time for mice with tumors was
noted in both male and female treated C3Hf mice when compared
with controls. C3Hf treated male mice survived an average
of 511 days compared with 630 days for control male mice.
According to the evaluation by EPA's Carcinogen Assessment
Group [CAG] (106), this difference was significant (p <0.001)
Treated female C3Hf mice survived 620 days compared with 680
days for control females. Chemically induced oncogenic
effects typically show long latency periods. The finding
of reduced longevity among treated animals as compared
with controls complicates the assessment of the potential
oncogenic effects of 2,4,5-T.
In XVII/G mice, 84? of the treated females (16/19)
and 75? of the treated males (15/20) developed tumors,
compared with control values of 53? (21/40) and 78? (25/32),
respectively. • ~. . . . '. - . . •_"
An increase in survival time for mice with tumors
over controls was noted among the XVII/G treated animals.
There was an average survival time of 583 days for treated
male mice compared with 521 days for control male mice.
Treated females survived 641 days compared with 569 days for
control females. According to CAG (106), the difference
was significant (p < 0.01) in females.
-53-
-------
(b) Effects of. Subcutaneous Injection and Oral
Administration of 2.4.B-T (30 Dom TCDD)
*
on Rodents
Innes et al, (107) studied the tumorigenicity of
2,i7.5-Tf containing about 30 ppm TCDD, in two hybrid
strains of mice, designated as "Xn and "Y", after Oral or
subcutaneous administration of the maximum tolerated dose
(Table 3)» The testing was performed at Bionetics Research
Laboratories, under contract from the National Institutes
of Health* Results of the studies were calculated comparing
treated groups with matched and pooled controls.%/
In the subcutaneous study, mice were given a single
injection of 21,5 mg/kg of 2,4,5-T in a dimethyl sulfoxide
(DMSO) solution at approximately 18 months of ageo Seventeen
percent (3/18) of the treated "Y" males developed pulmonary
adenomas, compared with 1J (1/71) of the matched controls and
3% (1/122) of the pooled controls* This increased incidence
of pulmonary adenomas was significant relative to -both con-
trol groups [p = 0.024 matched and p = 0,04 pooled] (106)»
In'the oral study, 21.5 mg/kg of 2,4,5-T in gelatin
/
was administered daily by stomach tube, beginning at seven
Si/ Because this was a large scale screening study,
several control groups were used. No significant dif-
ferences were found among these groups*
-54-
-------
days of age. After weaning, 60 ppm of 2,4,5-T was mixed
In the diet and provided ad Xi_bi_t_um until the end of the
study at approximately 18 months. Gross and histological
examinations were made of all major organs and visible'
lesions; thyroid glands were not examined. According to
CAG's evaluation (106), there were no significant differences
between 2,1,5-T treated and control groups of mice with
respect to tumors at specific sites or total number of
tumor-bearing animals*
Table
Tumors in Mice Ineestine_
StrairL
X
•
Y
1 ! ! Mice with Specific Tusors
jDose
S3x! (orn)
M ! 0
S (matched)
1 o
! (pooled)
! 60
F ! 0
! (matched)
; o
{(pooled)
! 60
M i 0
! (matched)
! 0
!( pooled)
! 60
F j 0
\ (matched)
i 0
! (pooled)
Mice with Tumors iReticulum Cell! Tumor Tvpe Pumonaryj
No/Total Mo! 5 ! Sarcoca ! Adenoma & Carcinoma iHeoatoma
5/15 ! 33 i 0
! !
22/79 ! 28 ! 5
! i .
6/18 ! 33 ! 1
2/18 ! 11 ! 1
i i ;;
8/87 ! 9 i 4
! !
1/81 ! 6 ! —
3/18 ! 17 i —
! !
16/90 i 18 ! 1
i !
3/18 ! 17 i 2
1/15 I 7 i 1
i !
7/82 ! 9 i 3
\ \
! 60 ! 2/18 ! 11 ! 1
2 i 3
5
' .-- ' •- .':."•
1
1
3
1
3
10
' ~_
— —
"
3
—
8
4
• _
•
—
5
1
—
1
— !
-55-
-------
. (2) TCPP
(a) Qncqgenlc Effects of Low Levels of
on Rodents .
Van Miller et al» (109) recently reported the results
of a two-year feeding study with male Sprague-Dawley rats.
t
Ten groups of ten animals per group were fed ground chow
containing 0, 1, 5, 50, or 500 ppt (= 10"12 gram TCDD/gram
food), and 1, 5, 50, 500, or 1,000 ppb ( = 10 gram TCDD/gram
food) TCDD.
Food intake (10 i 4 g/day) was significantly lower in
rats ingesting the three highest dose levels (50, 500, or
1,000 ppb TCDD) than in controls (21 ± 2 g/day), and none of
the rats in these' three groups gained weight after the start
of the experimental diet. All rats receiving these three
dose levels died between the second and fourth week of
treatment* . .
1
On the other hand, food intake for rats on other dose
levels was similar to controls "(20 ± 2 g/day)» Weight
gain was significantly less only for rats given 5 ppb TCDD
(391 ± 54 g) as compared to controls (531 ± 44 g)» In these
-56-
-------
seven groups only one animal died before the 30th week, and
that death occurred in the 500-ppt group at the l?th week.
In the 5- and 1-ppb groups, all animals died by the 90th
week of the experiment. Table U shows the mortality figures
for all groups.
)>./
Si/
£/
£./
Jl/
j./
Table 4. Mortality in Rats Ingesting
Levels of TCDD
!
Dose I
J2/ i
1 ppt*7 1
5 ppt" !
50 ppf2-7 !
5 0 0 p p u !
1 ppb*7 i
ii/ J
5 ppb i
4 / 1
50. ppb i
500 ppb-17 !
1 , 000 t>ob~" !
^Veek of ..
First Death
68
86
33
69
it".
31
31
• • '-'3 ..."
2 - •"• '-
2
iNo, Rats Dead
! at QSth Week-7
I 6/10 (60*)
! 2/10 (20*)
! 4/10 (40*)
! 4/10 (40*)
5/10 (50*)
! 10/10 (100*)
! 10/10 (100*)
i
j. 10/10 (100*)
i 10/10 (loo*)
1 10/1.0 (100?)
I
1
t
1
i
1
1
1
j
1
1
1
1
1
1
Surviving animals sacrificed at 95 weeks.
Control group. Diet contained no TCDD.
Approximate weekly dose was 0.0003 ug/kg body wt,
001 ug/kg body wt.
01 ug/kg body wt.
1 ug/kg body wt.
4 ug/kg body wt.
0 ug/kg body wt.
Approximate weekly dose was
Approximate weekly dose was
Approximate weekly dose was
Approximate weekly dose was
Approximate weekly dose
Approximate weekly dose
0
0
0
0
was 2
was 24 ug/kg body wt.
Approximate weekly dose was 240 ug/kg body wt.
Approximate weekly dose was 500 ug/kg body wt.
-57-
-------
' Laparotomies were performed on all rats surviving
through the 65th week, and all tumors observed were biopsied.
Rats were maintained on these diets until the 78th week and
were then placed on the control diet. Surviving animals
were killed at 95 weeks. Complete necropsies were done at
death or sacrifice, and tissue samples were microscop-
ically examined. Special staining methods were used to "aid
in the diagnosis of neoplasms*"
Tumorigenic and toxic effects were observed in rats
in the six lowest dose groups. The overall incidence
of neoplasms in the six experimental groups was 38? (23/60),
compared with 0% Co /TO ) In—the control group. The difference
is statistically significant (106). Neoplastic nodules and
cholangiocarcinomas of the liver were observed in 40? (4/10)
6f the rats ingesting 5 ppb TCDD; two animals had both
neoplastic nodules of the liver and cholangiocarcinomas.
. " i
One rat (10J) in the 1 ppb group had hepatic carcinoma
compared to none of the controls. Hepatic tumors were not
found in other dose groups (Table 5).
Table 5. Liver'Togror's 'In Rats Ingestin
1
JDose (orb)
i 0
I 1
i ,
{ Rats With
I Neoplastic
! Nodules
! No. ! ?
! 0/10 i o
I 0/10 ! o
| | h/
fiats With
Cholangio-
carcinoma's
No . ! I
0/10 ! 0
1/10 j 10
2/10 I 20-k'
Nodules plus
Carcinomas
No. i ?
0/10 ! o !
1/10 ! 10 j
a/io ! 4o-x 1 '
Data from Van Miller (109).
Jb_/ Two animals had both neoplastic nodules of the liver
and cholangiocarcinomas,
-58-
-------
Tumors developed in M6J (23/50) of the rats ingesting
5, 50, or 500 ppt and 1 or 5 ppb TCDD, compared to none
(0/10) in the control rats* Van Miller et al* noted that
"nineteen (57?) Csic - Agency calculation is 5W (19/35)3
of the animals that died in the six groups fed subacute
levels of TCDD had neoplastic alterations." Carcinomas were
observed in the ear duct, kidney, and liver. Three retriperi-
toneal histiocytomas were described as metastasizing to the
"lungs, kidney, liver, and skeletal musculature*" According
to CAG's evaluation (106), statistically significant increases
in tumors at all sites were found in rats fed 5, 500, 1,000,
and 5,000 ppt as compared with control'animals (p=0.05)
[Table 6], Three .of the ten deaths which occurred in the
5-ppb dose group were attributed to apiastic anemia. One
animal in the 500-ppt group had a severe liver infarction,
; Dow Chemical USA (110) has provided EPA with a
preliminary report of a study of TCDD's chronic toxic effects
in Sprague-Dawley rats* Groups of 50 rats of each sex were
fed 0.1, 0«01, or 0.001 ug TCDD/kg body weight daily for two
years* To provide these dose levels, the concentrations of
TCDD in the diet were approximately 2,200, 210, and 22 ppt*
Eighty-six animals of each sex were used as controls.
Dow (110) reported "discernible increases" in the
incidence of hepato.cellular carcinomas of the liver
and of squamous cell carcinomas of the lung, hard palate/nasal
-59-
-------
Table
1 I
1 &/ !
!Dose !
i o . !
! 1 PPt !
!
I 5 ppt !
150 ppt 1
1500 ppt !
I 1 ppb i
i 5 DDb i
6.
Total
Tumors in Rats .Ii
Destine: T
C1 T\ TY^^
I Rats With
Beni
0
0
1
2
2
b
8
en
i
1
,1
j
Tumors
Halicnant Total
o i o
0 ! 0
! A /
5 i 6s"'
1 1 f
2 ! #>•
5 j 5A/
2 ! 10
' Tumors
! No.
! o/io
1 o/io
i
! 5/10
i 3/10
1 4/10
! 4/10
! 7/10
{
o^x
0?
e/
50$-'
30J
40?^
40J
70?
i
i
i
i
i
•
!
!
1
i
I
Data from Van Miller (109).
Ji/ Rats administered 50, 500, and 1,000 ppb were all
dead within four weeks.
£/ Forty male rats used as controls for another study '.
were received at the same time and kept under identical
conditions did not have neoplasms when killed at 18 months.
jj/ One rat had ear duct carcinoma and lymphocytic leukemia,
The following tumor "types were each observed in one rat:
adenocarcinomas (kidney), malignant histiocytoiaa (retroperi-
toneal), angiosarcoma (skin), and Leydig cell adenoma (testis)
£/ Three rats died with aplastic anemia.
£/ The following tumor types were each observed in one rat:
fibrosarcona (muscle), squamous cell tumor (skin), and
astrocytoma (brain).
£./ The following tumor types were each observed in one rat:
fibroma (striated muscle), carcinoma (skin), sclerosing
seminoma (testis), and adenocarcinoma (kidney)*
JL/ One rat had a severe liver infarction,
i/ One rat .had cholangiocarcinoma and malignant histio-
cytomas (retriperitoneal)» The following tumor types were
each observed in one rat: angiosarcoma (skin), glioblastoma
(brain), and malignant histiocytoma (retroperitioneal).
J./ One rat had squamous cell tumor (lung) and neoplastic
nodule (liver). Two rats had cholangiocarcinoma and neo-
plas-tic nodule diver)* Three rats had squamous cell tumors
(lung)* One rat had neoplastic nodule.
-60-
-------
turbinates, and tongue in rats at 0*1 ug/kg. They also
reported decreased incidences of pituitary, uterine,
mammary gland, pancreatic, and adrenal gland tumors at this
dose level, Dow also reported that this dose level produced
increased mortality, decreased body weight gain, and changes
in blood chemistry values which suggested severe toxicity,
Hepatocellular nodules and alveolar hyperplasia were
observed.in the 0.01 ug/kg group* A squamous cell carcinoma
of the hard palate was observed in one female receiving this
dose; Dow considered this unrelated to TCDD treatment
because a similar tumor occurred in "other concurrent
studies," At 0,001 ug/kg there were no "discernible effects
in male rats and an increased incidence of [reversible]
swollen hepatocytes in female rats,"
•';.'. Dow's preliminary report does not include control
data, quantitative data on tumor incidence, or statistical
analyses. CAG has not evaluated this study. Table 7
describes the available tumor information. Dow has submitted
the final report for this study, which CAG is currently
reviewing.
-61-
-------
Table 7« Tumors in Sprague-Dawley Rats
a/
Ingesting TCDD
J_ pose L 1
v i pot I Tumors L
10 ; I 0 i I
lo.ooi - ! 22 ! 1
10.01 1 210 ! Hepatocellular Nodules !
I I I Squamous Cell Carcinoma-^-' I
j I I Alveolar Hyperplasia \
JO.I I 2,220 ! Hepatocellular Carcinoma"" !
Sauanous Cell Carcinoma
-^-
Data from Dow Chemical USA (110), a preliminary report,
Hardpalate squamous cell carcinoma observed in only
one female rat»
Observed only in females.
Squamous cell carcinoma observed in lungs, hardpalate/-
nasal turbinate, or tongue.
(b) Effects Closely Related__to Oncogenicitv in Test
.'"^'>>. Animals _ .
Many chemically non-reactive carcinogens are
enzymatically converted to biologically active carcinogens,
The eazyme aryl hydrocarbon hydroxylase (AHH) is strongly
implicated in this process (112). For example, the incidence
of bronchiogenic carcinomas in humans (113) and mouse
sarcomas induced by 3-methyl-cholanthrene (II1*) have been
related to the level of inducibility of AHH (99).
Kouri et al» (11M) studied AHH induction in human
lymphocyte cultures by TCDD. The authors stated, "TCDD
itself is not a potent carcinogen in mice; however, the
synergistic action of TCDD with 3-methylcholanthrene (MC)
produces cancer in different strains of mice in direct
-62-
-------
proportion to the degree of elevation of the induced hydroxy-
lase activity and associated cytochrome pj-450 content,"
Their study showed a positive correlation between basal
enzyme activity and enzyme levels maximally inducible by
either TCDD or MC» They also found that TCDD is about 40 to
i
60 times more potent than MC as an inducer of hydroxylase
activity in cultured human lymphocytes* These authors
further suggested that, because of the relatively high
levels of TCDD in certain parts of the world, TCDD
•
may also present considerable long-term risk because of
possible synergism in chemically initiated oncogenesis, in
addition to short-term risks posed by its toxic and terato-
genic properties. .
The implication of TCDD in AHH inducibility has
also been reported by Poland and Glover (115, 116) and
Poland et al. (117)« In their studies on chick embryo
livers, Poland and Glover (115) found that all dioxins
which are potent inducers have halogens at three of the
four lateral ring positions and at least one nonhalogenated
carbon atom, Poland and Glover (116) compared the po-
tency of TCDD as an inducer of hepatic AHH with that of
MC, the most commonly employed inducing agent. They
stated that analysis of the data by a computer program
for bioassay showed that TCDD was 28,640 times as potent
-63-
-------
as MC on a molar basis, (The 95? confidence interval
ji
of the potency ratio,is 2,07 .to 3,95 X 10 ,) The index of
precision, A , was 0,18. Poland et al. (117) suggested that
a hepatic cytosol species which binds TCDD is the receptor
for the induction of hepatic aryl hydrocarbon hydroxylase.
Allen et al. (118) conducted a study in which female
rhesus monkeys were fed diets containing 500 ppt TCDD for
nine months. Anemia, thrombocytopenia, and leukopenia were
the most debilitating changes* The altered lymphopoiesis
could be associated .with immune suppression. The authors
reported widespread hypertrophy, hyperplasia, and metaplasia
in the epithelium of monkeys exposed to TCDD, and related
this to data showing increased tumor frequency in TCDD fed
rats* . ,/...--. • •.. '.'-..:"••-. . • ..-.-'.• /••.-..
(-3) preliminary Epideniolo.gical Studies
Two epidemiolgical studies lend -support to a finding
of~in<;reased tumorigenicity due to 2,4,5-T exposure. The
English summary of a Swedish paper by Hardell (108) stated
that "there were seven cases of malignant mesenchymal tumors
in [87] persons [who had been] exposed to 2,^,5-T over a
-64-
-------
period of 10-20 years." In five of the cases, exposure had
been direct and comparatively massive* The latent period of
10 to 20 years is in agreement with that assumed for chemical
carcinogenesis. The statistical distribution of 7 of the
87 patients deviated from the national average with a
dominance of humors in males,
Tung (120) reported an elevated incidence of primary
liver cancers among Vietnamese following the Hide application
s
of "Agent Orange" as a defoliant during the years 1961 to
1962, "Agent Orange" is composed of equal parts 2,4,5-T and
2,4-D (2,4-dichlorophenoxyacetic acid) and is contaminated
With TCDD. During 1962 to 1968, 10J (791/7911) of all
cancers were liver cancers, compared with 3$ (159/5*142)
during 1955 to 1961, The latent period involved is shorter
than that normally assumed for chemical carcinogenesis; the
possibility of a shorter latent period for some chemicals,
however, cannot be eliminated. Neither of these studies is
sufficient to be the basis of any firm conclusions concerning
a causal connection between 2,4,5-T and cancer. But in view
of the results obtained in experimental animals, they
•
warrant noting.
-65-
-------
The Working Group concludes that there is sufficient
evidence to indicate that 2,4,5-T, containing TCDD at
levels as low as 0»05 ppm, and TCDD alone can produce
oncogenic effects in mammalian species. Since 2,4,5-T, as
currently formulated, contains TCDD (at a maximum amount of
0*099 ppm), a rebuttable presumption against registration of
2,4,5-T products has arisen because of the oncogenic effects
of 2,4,5-T~and TCDD.
•v-.
B« Other Chronic or Delayed Toxio Effects
40 CFH Section 162.11(a)(3)(ii)(B) provides that "a
rebuttable presumption shall arise if a pesticide's
ingredient(s)..,(p)roduces any other chronic or delayed
toxic effect in test animals at any dosage up to a level, as
determined by the Administrator, which is substantially
higher than that to which humans can reasonably be anticipated
to be exposed, taking into account ample margins of safety."
This section reflects concern that chronic exposure to
chemicals may result in injury to the reproductive system
and/or the fetus and provides that a rebut'table presumption
shall arise if chronic chemical exposure in test animals
produces such results.
The studies summarized below show that 2,4,5-T
containing 0.5 ppm or less TCCD produces teratogenic and/or
fetotoxic effects in mice at 30 mg/kg, in rats at lOO.mg/kg,
in hamsters at 40 mg/kg, and in birds at 1 mg/kg. Other
-------
studies show that pesticide-free TCDD is fetotoxic and/or
teratogenic at doses as low as 0»125 ug TCDD/kg in rats and
0»1 ug TCDD/kg in nice. Specifically, these studies
show that exposure to TCDD and/or 2,4,5-T containing TCDD
during pregnancy is associated with statistically significant
increases in the incidence of cleft palate, kidney anomalies,
skeletal and intestinal tract anomalies, and embryonic
resorption. (Maternal toxicity has also been observed in
many of these studies, primarily in the form of reduced
weight gain and increased liver-to-body weight ratio.
Whenever it has appeared particularly relevant, details have
been cited in the individual studies*)
The Working Group has concluded from these studies
that 2,4,5-T containing TCDD, 2,4,5-T without detectable
dioxin, and TCDD alone produce fetotoxic and teratogenic
effects in mammals. The Working Group has also concluded
that an ample margin of safety does not exist for the
population at risk (women of child-bearing age) for dermal
and inhalation exposure and for cumulative oral, dermal, and
inhalation exposure to both 2,^,5-T and/or TCDD. For these
reasons, the Working Group irecommencLa issuance of a rebuttable
presumption based on the fetotoxic and teratogenic effects
of 2,U,5-T and/or TCDD.
-67-
-------
(1) Pesticide-free TCDD
A Bionetics Research Institute study on 2,^,5-T
provided the first indication that TCDD adversely affected
mammalian development (123)• In this study, detailed with
later confirming studies in Section III,B.(2) below, 2,4,5-T
significantly increased the frequency of cleft palate,
kidney anomalies, and fetal mortality in the litters of
treated dams. The 2,4,5-T used in this study contained
approximately 30 ppm TCDD* Subsequent studies, detailed in
this section, using pesticide-free TCDD have established
that TCDD alone produces these effects, and that the TCDD
contaminant may be the principal chemical determinant
of the fetotoxic and teratogenic effects in mammals exposed
to the pesticide 2,4,5-T.
(a) Jgtudles _ln which TCDD Produced Tera'togenlc
'.-•'••" and/or Fetotoxic Effects in Mice
Courtney and Moore (128) studied TCDD's embryotoxic
and teratogenic effects in three mouse strains (Table 8).
Test animals were administered 1 or 3 ug TCDD/kg body
•weight subcutaneous!^ in_.s£lutions of 100? dimethylsulfoxide
(DMSO) on days 6 to 15 of gestation. DMSO was administered as
the control. TCDD produced cleft palates in all three
strains. At 3 ug/kg, 30J (3/10) of the CD-I litters had
fetuses with cleft palates compared to OJ (0/9) of the
controls; 71% (5/7) of the C57BL/6 litters had cleft palates
-68-
-------
, ...»•- ,
.:,....-"•"•*:.....'-'•'" .„...'•-•"
-- — "
of
.69-
-------
TCDD was administered orally at 25 to 400 ug/kg body weight
r
and subcutaneously at 25 to 200 ug/kg.
Mortality per litter increased .with the dose and
reached 97? (oral) and 76? (subcutaneous) in the litters
administered TCDD, as compared with a mortality of 6 and
14? in the oral and subcutaneous control groups, respec-
tively. The most common anomalies observed were cleft
palates and malformed kidneys. All of the fetuses in
the 200 and 400 ug/kg (oral) and 200 ug/kg (subcutaneous)
groups exhibited cleft palates as compared to 0? of the
controls. Of the fetuses in the 200 ug/kg (oral) group,
100? had kidney malformations as compared to 1? of the
controls. Other anomalies observed were hydrocephalus,
open eye, and club foot. Edema and petechiae were also
.observed in fetuses administered the high doses.
Table Q. Fetotoxic and Teratogenio Effects of TCDD in CD-I Mice"
i : i
i Dose !
Uug/kg i Route of Ad-
Joer dav) J ministration
! 25 i Oral
! 50 i Oral
MOO i Oral
! 200 i Oral
1^400 ! Oral
25 I Subcutaneous
50 ! Subcutaneous
100 iSuhcutaenous
200 iSubcutaenous
5f i Oral
corn oil,1
(0.1 ml)!
_DKSO~ J Subcutaneous
-
% Average Fetal
Mortal itv/Litterj
. 6
13
14
87
o? ...
36
56
72
76
6
14
Average # j
Abnormal
Fetuses
ner Litter
. 4.6
8.1
8.3
1.5
.0.4
6.7
5.0
3.5
0.8
0.2
Abnormal i
Cleft
Palate
a
K
O
19
66
100
100
82
79
85
100
0
i o
Anomalies/Total
Kidney
Anomalies
34
72
71
100
•50
53
58
95
1
1
' 0
Fetuses
Club
Foot
*
3
7
13
14
50
11
17
0
18
4
i i
! 1 !
Data from Courtney (133)
DMSO r dimethylsulfoxide
-70-
-------
Moore et al. (174) also fpund that TCDD caused
fetotoxic and teratogenic responses in C57BL/6 mice at
1 ug/kg administered on days 10 through 13 of gestation.
Compared with 0? incidence (0/27) in the control litters,
t
94? (15/16) of the treated litters exhibited kidney
anomalies, and 19? (3/16) -had cleft palates. At 3 ug/kg,
the incidence of these anomalies was 100? (14/14) and 86$
(12/14), respectively.
Neubert and Dillman (127) tested the embryotoxic
and teratogenic effects of TCDD in NMRI mice (Table 10).
In one test, pregnant mice were given varying doses of TCDD
(0»3 to 9 ug/kg) by intubation on days 6 to 15 of gestation*
At 9 ug/kg, 100? (3/3) of the viable litters had resorptions;
•
67? (6/9) of all litters had total resorptions* Oil control
values were 32 and 0? for litters with resorptions and
litters vith total resorptions, respectively. Cleft palate
was observed in all of the litters and 82? of the fetuses
at 9 ug/kg; comparable oil control values were 6 and 0.7?,
respectively* Statistically significant (p < 0.01)
proportions of the fetuses evidenced cleft palate at 3, 4.5,
and 9 ug/kg (3, 13, and 82?, respectively) when compared
with the oil control.
-71-
r
?-,
-------
Table 10. Ecabryotoxic and Teratogenic
Effects of TCDD on KMRI Mice3-7
!
iLitters Af fectqd/ Viable Litters!
l Resorptions I Cleft Palate I
!(u*/k>?)
; o
oil
0.3
3.0
4.5
9.0
! Q.O
# !
23/95!
21/65!
7/13!
16/24!
5/12!
3/3 !
V6 !
%
24
32
' 54
67
42
100
50
! f
! 6/95
! 4/65
! 0/13
i 7/24
! 6/12
! 3/3
! <5/6
I
6
6
0
29
50
100
8?
i
i
\
\
i
!
!
i
Data fron Neubert and Dillnan (127).
All doses administered on days 6 to 15,
except second 9.0 ug/kg dose which was
administered on days 9 to 13.
In this study, a single oral dose of 45 ug/kg TCDD on
day 6 produced resorption in 100$ of the viable litters; 23
ug/kg on day 10 led to 50$ resorptions. Seventy-one per
cent of the. viable litters had embryos with cleft palate
when 45 ug/kg was given as a single dose on day 11. Control
values were 24$ for litters with resorption and 6$ for
litters with cleft palates. -
;.. Smit-h et al. (135) administered 0.001, 0.01, 0.1,
i.O, and 3.0 ug TCDD/kg body weight per day to CF-1 mice by
garage from days 6 through 15 of gestation (Table 11). The
percentage of resorptions per implantation was significantly
higher in treated mice than in the controls only in the 1.0
ug/kg group. Cleft palate occurred in 71$ of the litters
treated at 3.0 ug/kg and in 21$ of the litters treated at
.1.0 ug/kg; bilateral dilated renal pelvises occurred in 28$
-72-
-------
of the litters treated at 3,0 ug/kg, and in 5% of the
'litters treated at 1,0 ug/kg. No significant increase in
either cleft palate or dilated renal pelvis was observed at
0*1| 0.01, or 0.001 ug/kg. None (0/3*0 of the control
litters had cleft palate or abnormal kidneys.
Table 11. • Fetotoxic- and Teratog°nic_Effects of TCDD in CF-1 Mice
{Incidence of Cleft!
{Palate in Litters i
Dose 'per Live Litters !
( utr/ke) ! *
0 i 0/34
0.001 i 2/41
0.01 ! 0/19
0*1 i 1/17
I 1.0 ! 4/19
^o ho/u
• * 1
ff 1
0 i
5 i
o !
6 i
2& \
7& i
Litters With
Resorbed Fetuses
tier Live Litters
* i *
25/34 i 74
30/41 ! 73
17/19 i 89
16/17 ! 94
18/19 I 95
11/14 ! 78
Litters With Dilated
Renal Pelvis. per
Live Litters
# . ! i
0/34 0
0/41 0 I
0/19 0 !
0/17 0
1/19 \ 5
1 4/14 ! 2&k/ i
%J Data fron Smith et al.
.by Statistically different
probability test (p < 0,05)
(135).
from controls by the Fishers exact
Neubert et al. (175) estimated the ED-50 for cleft
palate in fetuses to be 40 ug TCDD/kg per day (Table 12).
The no-effect-level during days 6 to 15 of gestation was
estimated to be 2 ug/kg per day for NMRI mice. No pronounced
fetal mortality was observed when 3 ug TCDD/kg body weight
was administered on days 6 to 15 of pregnancy.
Table 12. Occurrence of Cleft Palate in Offspring of Hloe Fed
!
{Strain!
CD-1 j
1
DBA !
1
NMRI \
1
IC57B1 j
1 !
Dose
(ue/ke)
0
3
0
?
0
?
0
1
% Cleft Palates per {Affected Litters/Total Litters
Total Fetuses Exanined !
<0.3 1 '
? !
<1 !
U !
0.7 !
3 !
<1 !
1 22 !
*
0/29
V10
0/23
2/9
10/160
7/2>4
0/23
5/7
! *
! o
! 30
i - 0
! 22
i 6
! 2Q
i 0'!
! 71 !
JEL/ Data from Neubert et al. (175)»
-73-
-------
Khera and Ruddick (6) studied the perinatal ef-
fects of TCDD in Wistar rats. In one test, rats were orally
administered 0.125, 0»25, 0.5, and KO ug TCDD/kg per day on
days 6 through 15 of gestation (Table 14), Visceral
lesions were observed at 0.25 ug/kg and above; slight
decreases in fetal weight were also seen. Postnatal effects
of prenatal exposure to TCDD were studied by allowing
offspring of treated dams to be reared by untreated dams .
until weaning. Reduced survival, body weight gain, and
reproductive ability in the progeny were observed after
maternal treatment with 0.5 and 1*0 ug/kg« No fetotoxic
effects were observed at 0*125 ug/kg.
In a second experiment, rats were treated orally
with 1, 2, 4, 8, and 16 ug TCDD/kg body weight per day
on days 6 through 15 of gestation. TCDD treatment reduced
Tetal weight^ and the number of live fetuses per litter, and
• • " "
produced visceral lesions in 50? (3/6) of the 1,0 ug/kg
fe~tus-es and 1*3$ (3/7) of the 2.0 mg/kg fetuses, as compared
to none (0/10) in the controls* The incidence of skeletal
anomalies was comparable to that in the controls at all dose
levels* Doses of 1 ug/kg or more produced maternal toxicity;
4 ug/kg or more produced 100J embryomortality. The authors
concluded that oral treatment of pregnant Wistar rats with
0«25 ug (or more)/kg per day on days 6 to 15 of gestation
adversely effected rat development,
-75-
-------
Teratogenic Effects of TCDD in Wistar Rats2-
1
i
ise I
VkR) !
S-l I
Un- I
eated i
.ntrol i
•eated!
jutrol i
17125 i
3.25 I
0.5 I
est 2 !
41n- I
-reated I
Control !
s'reated !
control!
1.0 i
6*0 S
4.0 i
8.0 I
16.0 T
Avg." tf Live
Fetuses/Litter
10.7
11.0
10.6
10,9
10.5
P. "3
11.5
9.8
6.5
6.0
0
0
0
Avg. Fetal
Weight
( eratns)
4.82
4.51
4.64
4.79
4.46
u:io
4.68
4.77
4.17
3.31
. .
Fetuses with
Skeletal Anomalies/-
Total tf Examined
#
5/107
21/116
3/121
6/109
10/105
6/81
8/116
9/89
7/80
7/57
*„
5
18
2
6
10
7
7"
10
9
12
h
1
i
[Fetuses with Micro-
iscopic Visceral
i Lesions/Total tf
! * !
i >
I 0/13 !
I
i i
i 0/11 I
i i
! 0/38 i
! 1/33 !
3/31 !
! V10 i
i i
! 0/10 l
Ii
I
I i
! 0/10 !
! i
} 3/6 I
3/7
i
i
I i
Examined
%
0
0
0
3
10
?0
0
0
50
43
I
]
J Data from Khsra and Ruddick (6); treated controls given anisole-corn oil.
Courtney and Moore (128) administered TCDD to CD rats
subcirfrane-ously in solutions of 100? DMSO on days 6 through
15 of gestation (Table 8). DMSO was administered as the
control. 'Kidney anomalies were found in four of the six
litters (67?) whose dams were administered 0,5 ug/kg as
compared to OJ (_0/9) in the controls. TCDD did not affect
fetal mortality, fetal weight, or cleft palates in the
fetuses,
Dow Chemical USA (110) conducted a three-generation
reproductive study on Sprague-Dawley rats continuously fed
the equivalent of 0,001, 0.01, or 0,1 ug TCDD/kg per day.
-76-
-------
A preliminary report cites reduced fertility and litter
survival in f rats as the reasons for discontinuing the
0.1 ug/kg dose level; significantly reduced fertility was
also observed at 0.01 ug/kg. "Clearly evident" indications
of toxicity at 0.01 ug/kg among f and f litters included
smaller litter size at birth, plus decreased survival and
growth of neonates. Dilated renal pelvis was observed in
each of the three f rats at 0,1 ug/kg which survived to
adulthood* Increased frequency of this anomaly was also
seen among weanlings at lower doses; however a dose-
related or generational correlation could not be made.
In summary, Dow conclu-ded thart~nthe reproductive capacity
~of rats ingesting TCDD was clearly affected at dose levels
of 0.01 and 0.1 ug/kg per day, but not at 0,001 ug/kg per
day , through three successive generations," The pre-
liminary report did not include the numerical data neces-
~— ™. - . . ^
sary for Agency evaluation. Analysis will continue as
these become available.
-77 & 78-
-------
Adverse reproductive effects due to TCDD.have also
been observed in hamsters and chickens* Gastrointestinal
hemorrhage was noted in hamster fetuses after administra-
tion of TCDD at 0.5 ug/kg per day on days 6 to 10 of gesta-
tion (48; 62). Buu Hoi et al, (111) established that
0.02 ug/kg TCDD caused teratogenic effects in chick embryos,
Bowes et al. (137) and Verrett (136) confirmed these
results. They found abnormalities in the beaks, eyes,
and feet of chick embryos after TCDD exposure. !
(c) Summary •
Studies have established that TCDD is fetotoxic
•and teratogenic at doses as low as 0.125 ug/kg in rats
(129) and at 0,3 ug/kg in mice (127); preliminary data from
Dow (110) indicates that TCDD may have effects at 0.01 ug/kg
in rats. Cleft palate and kidney anomalies have been
observed in rats, mice, and hamsters. No fetotoxic or
teratogenic effects have been observed at doses of 0,03
ug/kg in rats (129) and 0,1 ug/kg in mice (135), Table 15
lists the no-effect-levels in rats and mice for teratogenicity
from TCDD, -
-79-
-------
Table 15. Ho-Effeet-Levels for Teratogenesis from TCDD
(Route of Ad-
Soe c i e s 1 ci i n i s t_p at ion
Wo-Effect-Level!
per dav
Reference
i Subcutaneous
I Oral
! Oral
House {Subcutaneous
• i.Y-WJv. Or.ai"V
I Oral
<0.5 .'Courtney and Moore (128)
0.125 {Khera and Ruddick (6)
0,03 iSparschu et al« (129)
<1»0 {Courtney and Moore (128)
<0.3 iNeubert and Dillman (127)
0. 1 .'Smith et al. (US)
(2) £, 4, 5-T f TCDD _Contaplnatlo_n_ Ranging Fron"T?ndeteotable
to ^0 ppm) .' . .
(a) Teratogenic and, Fetotoxlc .Effects In RodgnJbs
Courtney et al* (123) developed the first evidence
that a 2,4,5-T pesticide product was teratogenic and fetotoxic
(Table 16). - The 2,4,5-T used in this study contained
approximately 30 ppn TCDD. The pesticide was administered
daily either orally or subcutaneously on days 6 to 14 of
gestation in C57BL/6 mice, days 6 to 15 in AKR mice, and
d-aya 10 to 15 in Sprague-Dawley rats. Subcutaneous adminis-
tration of, 113 mg/kg body weight resulted in significant
increases in the incidence of cleft palate and cystic
kidneys — ^ in the embryos of both strains of mice, and
fetal mortality in the C57BL/6 mice. Oral administration of
_Lfi/ Results of this study were published by the Department of
Health, Education, and Welfare (121) and by Clegg (122)*
JJ/ In a recent report on studies measuring renal alkaline
phosphatase in fetal mice, Highman et al, (45) attributed
the increased incidence of "cystic kidneys" in the offspring
of 2,4,5-T treated animals to retarded development, rather
than true teratogenesis. Reduction in fetal weight and
increased incidence of cleft palate were also observed
among the fetuses of treated dams.
-80-
-------
Table 16. Teratocenic Evaluation of
In
1
! Route of
£se {Adtninis-
."sin Itration
j7BL/6£/ i
•ntreated!
>ntrol {Subcutaneous
>*trol i Stomach tube
i
'eated {Subcutaneous
^eated {Subcutaneous
i
^eated ! Stomach tube
i
fated j Stomach tube
^7BL/6 !
>ntreated{
introl {Subcutaneous
i
'•ated {Subcutaneous
i
i
:RJ/ j
^treated i
>ntrol 'Subcutaneous
i^rol {Stomach tube
•eated {Subcutaneous
-eated (Stomach tube
i
!
i
Dose If Lit"
\
— ! 72
£/ ! 106
&./ ! 32
21.5^: 6
ns.o^7! 18
46.4i/| 6
113.CT1 1 12
i
i
— ! 8
L/ \ 10
ns.o^7! 10
i
i
i
{ 58
£/ ! 72
&/ ! 12
113.0117! 14
m.o^! 7
Per Litter
Avg. # Live! Abnormal
Fetuses {Fetuses
i *
i
j
5.8 ! 11
5.5 ! 12
7.1 i 14
7.7 ! 12
4.4 ! 5^
8.5 j yf
4.8 ! 7017
i
i
i
5.1 ! 31
6.1 ! 8
7.7 ! 7717
i
i
j
7.1 ! 5
6.9 ! 4
8.8 ! 0
6.9 i 2917
^_? ! 551/-
% Fetuses
with
Cleft {Cystic
Palate iKidnev
1
<1 } 1
<1 { 2
0 ! 1
0 ! 0
4 / J i /
221/| ^J/
2 ! 3317
23l/j ^
i
I
i
0 i 7
0 { 0
29^7{ 60^-7
1
I
j
<1 { <1
0 { 0
28^7| 1
5^\ 0
Fetal
Mortality
26
29
15
3
42
8
47i/
'
36
23
11
16
15
9
23
4-JJ/
Abnormal
Litters
38
42
41
50
86^7
10C;
100"^
71
30
100i/
19
24
0
71 i
10017
Contained approximately 30 ppa TCDD.
Data from Courtney et al» (123).
Jreated from day 6 through 14 of pregnancy. Killed on day 18 of gestation.
Treated from day 9 through 17 of pregnancy. Killed on day 18 of gestation.
Treated from day 6 through 15 of pregnancy. Killed on day 19 of gestation.
Dose, 100 ul DMSO per mouse,
Dose, 100 ul honey solution (honey to water, 1:1) per mouse.
Administered as a solution of 2,4,5-T in 100? DMSO in a volume of 100 ul per mouse,
0?,4,5-T was suspended in a honey solution (honey to water, 1:1) in a volume of 100
j mouse.
P = 0.01. , .
P = 0.05.
ul
-81-
-------
the same dose caused increased incidence of cleft palate and
fetal mortality in both strains and cystic kidneys in
C57BL/6 mice* Courtney et al. also reported increases in
liver-to-body weight ratios in fetal mice,
«
These investigators also found that 4,6, 10, or 46.4
ing/kg 2,4,5-T given orally to Sprague-Dawley rats produced
kidney anomalies and other embryotoxic effects at all levels
(Table !?)« The occurrence of hemorragic gastrointestinal
tracts in rat fetuses was also reported* •
Roll (125) found embryotoxic and teratogenic effects
in NMRI mice after prenatal exposure to 2,4,5-T containing
0.05 jt 0.02 ppm dioxin (Table 18), 2,4,5-T at 20 to 130
ing/kg body weight was administered orally to the dams on
each of days 6 to 15 of gestation. At 90 or 130 mg/kg,
the percentage of resorptions and/or dead fetuses was
markedly increased relative to the controls; however,
maternal toxic effects were also observed at these dose
levels.-^ Statistically significant, dose-related
reductions in fetal weight were observed at 20 mg/kg
and above. .
JL2/ Although the LD-50 for female NMRI mice had been
previously determined to be 778 ing/kg, an increased maternal
mortality rate was seen at 130 mg/kg and weight gain was
depressed at doses above 60 mg/kg (125),
.82-
-------
Table 17. Terato.cenic Evaluation of g.U.S-T^in Rats^7
• !
1
!
{Route of
st ' lAdminis-
irnal Itration
tV 1
ntreated!
ntrol {Stomach tube
eated j Stomach tube
1
0ted {Stomach tube
Dose
(icc/k^)
...
-------
three of the litters treated with 90 mg/kg of sample (B);
none was seen in the controls. Fetal weight was significantly
depressed in all treated groups compared with the oil
control. •
The percentage of fetuses with cleft palate was
significantly higher than the .control group in all 2,^,5-T
groups treated with ^5 tig/kg or more. In the group treated
vith 120 mg/kg 2,H,5-T containing <0.02 ppm dioxin, 5M$
(7/13) of the litters and 11* (U/145) of the fetuses
exhibited cleft palate compared with oil control values of
s
6$ (A/65) and 0»7*_(5/669V,__respectively.
These investigators also tested the butyl ester
of.2,4,5-T and found similar effects. In experiments
combining 2,4,5-T and TCDD, potentiation of teratogenic
effects was observed. Sixty mg/kg of 2,4,5-T (sample
A) combined with 0«3 ug/kg TCDD increased cleft palate
frequency among fetuses from 5 to 1MJ, In this study no
cleft palates were observed among fetuses treated only with
0.3 ug/kg TCDD,
-85-
-------
Tabl» TQ. Esbrvntoylo Effects of 2.'
1
1
i
iPioxin
{Content
stsent Ifonn)
JNone I —
ion
|2 4
I'
1
1
1
1
1
J2 i
I
1
i?,4
control! —
,5-T U)! <0.02
i
1
1
1
i
j
,5-T (B)| 0.05
i -
1
! 1 Resoortion (PESJ
'Dose
i
! 0.4 nl
[ 8.0
Il5.0
Iso.o
1 45.0
[60.0
Iso.o
1 120.0
J30.0
I
I60.0
i
190.0
i i
,5-T fC)!unknowr.!QO.O
\% Litters!? EES/Inplan-
iwith RES Station Sites
1 1
i ' i
1 24
i 32
! 35
! 38
! 56
1 55
! 63
1 53
! 54
! 44
1
! 71
I 71
i
1
j
j
j
i
1
i
i
i
i
I
t
4
4
3
5
7
6
11
8
10
6
7
8-
n
! RES/Single
! Litter w/RES
! fM)
! 0.6
I 0.5
1 0.4
1. 0.5
! 0.8
i 0.6
i 1.2"
! 1.1"
! 1.3^
! 0.6
! 0.4
i ,.0^
! i.iX
i i
i Fetal !
! Weight i
if crass)!
! 1.26^1
! 1.30 !
! i^l
I ..«"!
I 1.09^1
! 0.98*/!
1 1.01^1
1 1.02^1
1 0.95^1
i 1.11^!
i I.IT^I
I 0.99^1
! ^.o^• 1
Cleft Palat
J Litters!?
with C? Iwi
6 i
6 !
<7 1
8 |
• 11 I
16 |
20 i
35 i
54 |
22 !
71 |
86 {
i
72 f
e fCP)
Fetuses
th C?
0.6
0.7
<1
1
,
3^
5^
8&/
1tli/
2
9^
23^
•><£•'
Pata from Neubert and Pi11nan (127); 2,4,5-T sample (b) received from Roll (125).
p^ 0.01. . .
'•'--• Bage et al. (132) injected NMRI mice subcutaneously
with 50 and 110 ng/kg 2,4,5-T (<1.0 ppm dioxin) on each of
days 6 through 14 of gestation* At 110 mg/kg, 2,4,5-T was
teratogenic, causing fetal death, cleft palate, and other
anomalies. .
Courtney and Moore (128) studied the effects of
2,4,5-T Urn LTD-T 'fiindom-b'red mice, two strains of inbred
Bice, DBA/2J and C57BL/6J, and CO rats (Table 20).
-86-
-------
J
9
1
1
(Soecies
I CD-1
| House
i£xpt. 1
1
!
1
1
i
I
Table 20. Eabrvotoxlc
I
1
}
! Coanound
!
1
I DMSO
12.4,5-T
KTech.)
12,4,5-T
KTech.)
|2,4,5-T
If Tech.)
lErpt. 2! DMSO
i
I
jExpt. 3
i
1
I
1
I
J
|
1 DBA/2
I Mouse
j
IC57B1/6
I House
i
!2,4,5-T
j(Analy.)
! DMSO
12,4,5-T
KTech.)
i2,4,5-T
KAnaly.)
}
!2,4,5-T
!(Analv.)
i DMSO
12,4,5-T
!(T°ch.)
! DMSO
!2,4,5-T
i
iCTech.)
! CD fiat [Sucrose
I
t
I
1
|
I
I
1
12,4,5-T
KTech.)
!2,4,5-T
KTech.)
12,4,5-T
KTech.)
|2,4,5-T
KTech.)
Dose
(nc/lre)
50
100
150^
100
100—
100
125
...
100
—
100
—
10
21.5
46.4
80.0s/
% Fetal
Mortality
Effects of Analytical and Techr
[Fetal
[Weight
!?er Litter Ksraas)
6.6
6.6
7.5
51.7
8.8
9.6
8.4
.
11.6
12.9
25.1
27.0
10.8
15. P
3.4
1.8
1.4
3.8
52.1
i
\
\ 1.35
! 1.26
|
i 1.00
I
! 0.91
t
! U02
i 0.73"
t
t
i 1.09
J 0.85"
i
I 0.86"
I
! JS/
! 0.71
1
t
i o.as
! 0.6T"
i
i 0.99
i
| o 7«^7
i 2.48
i 2.40
t
! 2.54
t
i
! 2.20
-W
I 2.30
i
£\eft Palate (C?)
% Litters!
Affected !
!
{
i
0 i
0 !
[
33 !
{
100 !
|
0 i
89 I
!
0 !
40 !
- !
40 |
I
!
78 !
!
o :
27 !
i
0 i
J
HO \
0 i
0 !
j
o :
o i
i
o -!
!
1
1
if C? per!J
ij«a 2
. l|,5-T
Kidnev A
a.'
no~alies
Littersi* Affected F
Affected [Affected
Litter
0
0
3.0
5.3
0
4.4
0
2.0
2.0
5.4
0
1.0
0
1.3
0
0
0
0
0
1
.1-..
i
i
!
I
1
1
i
1
!
i
i
i
[
i
i
i
|
1
1
1
!
I
i
t
I
I
i
!
I
i
i
i
i
i
j
i
i
1
j
I
0
0
0
0
33
78
63
80
100
67
13
9
9
0
0
20
38
14
50
[per
!Litt
i
i
1
1
!
i
S
]
1
!
1
j
l
i
i
1
|
1
1
s
j
i
1
!
i
I
i
}
j
!
I
•
i
i
i
{
Affected
AT*
0
0
0
0
1.0
1.7
2.0
2.4
4.2
4.3
1.0
1.0
1.0
o
0
1.0
1.3
2.0
4.0
i
i
etusesj
!
i
i
i
!
!
s
i
I
}
1
\
I
i
i
1
{
I
!
j
i
!
j
{
!
J
!
i
I
i
j
{
i
i
|
3j Data fron Courtney and Moore (128).
J>/ Investigators thought this data to be close to a maternal toxic dose.
SJ Maternal LD-40.
Jj/ p < 0.05.
-87-
-------
2,4,5-T containing 0.5 ppm (technical) or 0.05 ppm (analytical)
TCDD was administered subcutaneously to mice at 50 to 150
eg/kg in DMSO and orally to rats at 10 to 80 mg/kg in
sucrose on each of days 6 to 15 of gestation. At 100 mg/kg
or more, both 2,4,5-T samples produced significant reductions,
which appeared to be dose related, in fetal weight in all
strains of mice; rats were not affected. 2,1,5-T was
fetocidal at two doses, but the investigators considered
this effect to be due to maternal toxicity.
Both 2,U,5-T samples produced cleft palate in mice.
For CD-I dams treated with 100 mg/kg of either 2,4,5-T
sample, 40$ of the1 I'l-t-^er-s—and two fetuses per affected
litter evidenced cleft palate compared with 0? in the
control (Expt, 3)« No cleft palates were observed among the
rat fetuses. To verify this observation, a second group
of rats was given two 150 mg/kg doses of technical 2,^,5-T
subcutaneously at the time of palate closure (days 13
to 1*0. Again, no cleft palates were observed; however,
there was a significant increase in fetal mortality among
treated animals (1*1$) when compared with the controls
(OJ). "" *""""^"^ _
Fetuses of CD-1 mice treated with analytical 2,4,"5-T
also showed increased incidences of kidney anomalies;
the'response to technical 2,4,5-T was not as great. At 100
ttg/kg, 100$ of the litters and *U2 fetuses per affected
litter of dams treated with analytical 2,4,5-T displayed
-88-
-------
kidney anomalies, compared with 80$ and 2.4 for technical
2,4,5-T and 63? and 2.0 for controls (Expt. 3)* The effect
in inbred strains of nice was comparable with control
values. In rats, technical 2,4,5-T at all dose levels
produced higher incidences of litters affected and numbers
of fetuses per litter affected than seen in the control
animals. The maximum effects on kidney anomalies in rats '
were 50$ of. the litters and 4.0 fetuses per litter at 80
mg/kg, compared with 0$ in the control litters.
In another study using CD-1 mice, Courtney (134)
administered 0.45 to 1.0 mM/kg body weight per day of
•
2,4,5-T (0.05 ppm dioxin) either orally or subcutaneously
during various segments o"f the gestation period (Table 21).
Cleft palate was seen in all groups treated with 2,4,5-T;
there were no instances of this anomaly within the control
groups. At 0.8 mM/kg, 48? of total fetuses and 37$ of the
litters . evidenced this malformation. Statistically signifi-
cant (p S. 0.05) increases in the percentage of fetuses dead
and/or resorbed were observed at the highest doses. All
dose levels had adverse effects on fetal weight. The author
noted that by slightly altering experimental conditions, the
cleft palate effect and the effects on- fetal mor-tality and
fetal weight could be produced independently.
J_3./ Maternal toxicity was also observed, evidenced by
reductions in maternal weight gain and increased liver-
to-body weight ratios (134). • "
-89-
-------
DOS*
;/
'Days
'#/tot
al #•
73)
?"-'«/ 21/59
/'«•«' Sr
I 4 ^
/
a p.
siu«
89
;SFe^ ;;.*
.Monf-,-,., . ~
/Fetal
72
!igftt
IPs
tusea
6
t7
13
17
U
29£/
/ n ^^~~
/ °»95 /
0.94
1.07
0.89
1 /N A '
• 0.8? i
i •°' i
/0.87 /
— — ^.
16
lin
1
— i
1 —
/
/
/
1
/
1
«**t
-_
6
U
:/
•03
:ers;
37
«•
-
.„,
- ,
-
.-
«.
-90-
• •-
-------
per litterT"* The larger proportion of malformed fetuses
in the treated groups resulted from either an increased
incidence of skeletal anomalies also seen in the controls or
a low incidence of abnormalities not observed in the controls.
The former category included wavy ribs, retarded ossification,
extra ribs, and a variety of sternal defects; the latter
included fused ribs, small-sized distorted scapula, malformed
humerus shaft, and bent radius or ulna. Abnormal kidneys
. were observed in 7 to 45$ of the examined fetuses treated
with sample T-1, compared with a control value of 20 to 3-5J.
Table 22. Effects of 2.4.S-T on Wistar Hat Fetuses2-7
iCompundjDose
! |(mg/kg)
! !
! T-1 {Treated
1- } Control
! 50
! 100
T-2 ! Treated
{Control
! 25
.J 50 __
! 100
•Treated
{Control
! 25
--"- \ 50
{ 100
! ISO
T-3 {.Treated
{ Control-
! 25
! 50
! 100
! 1SO
! T-4^7 ! SO
tf of
Litters
14
1
Q
10
13
12
Q
10
11
14
10
s
10
14
2
12
1
8
Avg. # p
Viable
Fetuses
11.1
12.9
9.2
10.5
11.7
8.6
12.6
12.7
11.5
11.0
•11.6
11.8
12.2
11.0
12.6
11.0
11.3
>er Litter
Dead
Fetuses
0.6
1.3
1.P '
0.6
0.8
0.5
' 2.4
0.7
0.5
1.4
0.6
2.2
0.7
•
0.9
0.5
0.9
1.0
1.1
Fetal
Weight
4.65
4.84
5.34
5.06
5.15
4.S7
4.67
5.15
4.91
4.35
5.31
5.00
4.75
5.00
3.00
4 04
Avg. % Mal-
formed Fetuses
oer Litter^-
15
24
10
15
9
26
10
28
36
17
11
56
37
P1
in i
Data from Khera.and HcKinley (130).
One or more skeletal malformation (viable fetuses).
No treated control given. .
14/ Statistical significance was determined using the
average value per dose level. Data from T-4 were not
used in this analysis.
-91-
-------
, In the postnatal portion of the study, after
normal delivery, survival rate, sex ratio, and pup weight
/
on days 1 and 21 were compared. Although treated pups
surviving from day 2 to 21 were slightly smaller at some
dose levels, there were no significant differences from
controls for any variable. In some experiments, litters
were standardized at 8 pups on day 2, and the remaining
littermates examined for defects. The increased incidences
of malformations among treated groups were comparable
to those found in the prenatal study. Assuming the same
incidence for pups not examined, the investigators con-
cluded that there we.r§_np_re.al differences in survival
rates among control and treated groups* The butyl ester of
2,4,5-T produced similar toxic effects. :
Sokolik (131) orally administered 100 and 400 mg/kg
and 50 and 200 mg/kg of 2,4,5-T and its butyl ester to rats
of the Rappolovo line on each of days 1 to 14 or 1 to 16 of
pregnancy. At TOO mg/kg, 2,4,5-T produced embryos with a
combination of deformities including absence of lower jaw,
abnormal hind limbs, and exophthalmos. At 400 mg/kg, the
embryos of treated rats evidenced cleft palate, hydrocephalus,
hydronephrosis, and abnormalities of the upper limbs which
included tridactyly, webbed toes, and abnormal shortness*
-92-
-------
The butyl ester of 2,4,5-T was more toxic than
the parent compound, causing more than 30$ embryonic mortality
at 200 mg/kg. The lower dose, 50 ng/kg, also caused
high mortality among the embryos. Cleft palate, hydronephro-
sis, hydrocephalus, and extensive gastrointestinal hemorrhages
were also observed within the treated groups. From these
results, the author concluded that 2,4,5-T and its derivatives
have a high potential 'for teratogenic activity,
Collins and Williams (124) tested seven samples
of 2,4,5-T from different sources for embryotoxic effects in
golden Syrian hamsters (Mesocric.e.tu.s auratus) [Table 23].
The dioxin contents ranged from not detectable (detection
limit < 0.1 ppm) to 45 ppm. Daily oral doses of 20 to 100
og/kg body weight were administered in acetone:corn oil:car-
boxymethyl cellulose (1:5*8:10) on days 6 to 10 of gestation.
2,4,5-T with no detectable dioxin significantly (p < 0.05)
reduced fetal weight and fetal viability per litter at all
levels tested*
Total fetal mortality was greatly increased at all
levels when compared with controls and was dose-dependent,
as was the effect on fetal viability. The increased
incidence o-f gastrointestinal hemorrhage also appeared to be
dose related. At 100 mg/kg, "pure" 2,4,5-T caused increased
incidences of malformations and reductions in the number
of live fetuses per litter. One "pure" sample, F, at
100 mg/kg significantly reduced fetal weight from 1.8
-93-
-------
fable 23.
i
i
•i
}
fCoEDOund
{Control
/ A
f ••
^
i
! B
-
C
•
D
m
F
4*
Ezbrvotoxic Effects of
i
i
i
i
|
i
i
i
i
i
I
1
j
j
f
f
i
j
j
i
!
i
i
|
i
j
i
i
j
Dioxin
Content
(DOS)
—
45
2.9
0.5
0.1
NI^7
•
ND
ND
>
i
i
i
J
i
i
i
1
i
i
i
j
i
i1
i
i
j
j
I
i
f
i
i
i
f
i
i
i
I
i
J
i
f
i
i
Dose
(n£/k£)
20
40
80
100
40
80
100
20
40
80
100
40
80
100
40
80
100
100
100
1
% Total,'
Mortal-,'
itv
3 U '
32.3 I
74.3 /
94.4 /
100.0 !
7.2 }
9.8 }
1
11.4 !
8.5 i
4.0 !
43.6 j
j
57.2 !
2.4 {
33.3 i
j
47. 1 /
•10.7 •'
i
29.9 i
i
56.3 1
31.3 /
30,0' I
2,4,5-T in
Fetuses
i
Avg # Live}
per Litter}
i
11.0 !
7.3 /
3.7 i
0.8 j
0 !
9.1 !
10.4 j
i
12,8 /
12.6 j
13.4 /
6.6 i
I
5.1 !
11.4 }
7.8 j
,
6.0 /
11.2 i
j
8.7 i
1
6.3 !
7.3 /
8.4 ,'
Kansters"^
u
Avg Weigh t-^
(grams )
1.8
1.7
1.7
1.6
••»•.
1.7
1.7
1.7
I.T^
1.647
u^
$J
1.5
1.8
1.7
' ^J /'
i » O
1.5^
j /
1.5~
&/
1.5
1.6-7
1.6—
,
_L? Fetal Via-
/ 1
.'bility per
{Litter
i
96.7
/ 68. 117
I 25.8^
I 5.3"
( Ci*
• 93. 1
/ 90.1^
i » t
\ DO cS-'
, 00, ~
/ on p£/
i yo.<>
•' 95.9
/ 58,3^
{ jj/
' 40,2
97.8
/ 68.3d7
| J M
\ 57.2s-
/ 88. 217
| . *
1 69.1
I ji/
% Abnor-
malities
per Live
Litter
3.5
25.0"
33.3SL/
100.0"
0
12.5
. /
50,-Cr-
0
11.1
40.0^
A/
40.0
0
0
0
0
0
d/
' Hemorrhages!
per Total
Live Fetuses
O.3.?
28.4
75.7
42.9
0
2.4
13.0
8.6
2.5
12.5
7.6
0
2.1
5.6
1.5
4.2
I 53. 1 ' 36. 4 I 0
i 71. $•' 1 40. O^7 J 6.8
I 68.3d7 I 0 / 16.7
i
•
i
i
I
i
i
i
i
i
i
i
i
i
i
i
i
r
i
1
i
i
i
/
|
|
/
|
/
i
/
i
i
i
')ata from Collins and Williams (124).
ipparently normal weights for samples A and B attributed to edema.
'ot detected.
< 0.05.
-94-
-------
to 1.6 grams, reduced fetal viability from 96.7 to 71.4?,
and increased abnormalities from 3.5 to 40J. The anomalies
•
associated with 2,4,5-T containing no dioxin were exencephaly,
eye abnormalities, delayed head ossification, and hind
limb deformities.
Increasing the level of dioxin contamination increased
fetal mortality and the incidence of abnormalities per
litter; fetal viability was reduced, A clear correlation
was found between the level of dioxin and abnormalities per
litter. Although the incidence of hemorrhages also increased,
no relationship between it and dioxin level could be found.
Bulging eyes (ab-s-eno-e .-of .eyelid) and delayed ossification
were'the most common anomalies seen among fetuses exposed
to dioxin-contaminated 2,4,5-T; exencephaly, edema, cleft
palate, ectopic heart, and fused ribs were also observed.
Emerson et al. (141) found no adverse effects of
commercial 2,4,5-T, containing 0.5 ppm TCDD, on fetal
\
development in Sprague-Dawley derived rats and New Zealand
white rabbits. Daily oral doses of 2,4,5-T in gelatin were
administered to the rats at 1 to 24 mg/kg on days 6 to 15 of
gestation; to t"R=e~r"aT5i1t:S~~a'€":::3b to 40jng/kg on days 6 to 18
of gestation. The investigators found no maternal or
embryonic toxic effects in either species, nor was 2,4,5-T
-95-
-------
considered teratogenic under the conditions of these experi-
ments. The most frequently observed abnormalities were
accessory ribs, hydronephrosis, and retardation in the
development of the sternebrae. With the exception of partial-
ly ossified sternebrae in both species and bilateral accessory
ribs in the rabbit, the incidence of these anomalies was
greater in the control animals than in the examined treated
groups.
Sparschu et al. (140) orally administered 2,4,5-T,
containing 0.5 ppm TCDD, to rats in daily doses of 50
%
and 100 mg/kg on days 6 to 15 and 6 to 10 of gestation,
respectively. Results-are given in Table 24. At 50 mg/kg,
there were no significant maternal or embryonic toxic
effects attributable to 2,4,5-T except for an increased
incidence of delayed skull ossification, and a single fetus
with intestinal hemorrhage. At 100 mg/kg, 2,4,5-T was toxic
to both dams and fetuses.^""*
JL5./ The high rate of maternal mortality caused dosing
to be stopped on day 10, instead of day 15. Significant
reductions in weight gain were also observed.
-96-
-------
Table 24. Kffgefcs of 2.4.5-T on Fetal Devlopnent of Rats
I
1
u
i
j
\%
I
JL
Parameter
Viable fetuses
Total
Mean oer litter
Resorptions
Litters
Total fetuses
j
t
i
I
i
I
i
jFetal weight (grams) j
! Male i
1 Female 1
jSex Ratio (M:F)
i
i
i Abnormalities !
{(} fetuses examined) !
j Poorly ossified sternebrae!
! Fifth 1
!
I
!
-1
i
1
I
a/
JL/
£/
Second and fifth
Multiple
Malaligned sternebrae
Delayed ossification
Interparietal
Parietals :
Frontals
Data from Sparschu et al
All viab-le fetuses from
• p < 0.05
1
i
T
j
i
i
i
. (1
one
Dose (
0
252
11
68
6.7
4.41
4. 17
53:47
15.2
3.0
8.3
0.8
3.8
3.0
0.8
40).
litter.
mg/kK oer dav)
! 50
i
! 203
! 11
I
i 61
i 12.1
I 4.38
I 4.15
! 44:56
1
! 22.1
! 4.2
j 12.6
i 2.1
{
J16.85./
il6.8^
1 7.4-a/
100
13* '
100
3.57^
2^:77
57.1^
14.3
14.3
28.6^X
2B.6^\
K'J l£./ !
D I . I i
I I *l * T
Resorptions were observed in all litters; 75? were
totally resorbed. Fetal weight was significantly (p <
0.001) reduced in both sexes and the sex ratio was shifted
in favor of females. Abnormalities observed which had
significantly (p < 0.05) higher incidences than in the
-97-
-------
controls were poorly ossified and malaligned sternebrae and
delayed skull ossification. The investigators concluded that
the delayed ossification observed in this study was a
reversible manifestation, rather than a true teratogenic
effect,
(b) .Adverse Reproductive Effects in. Other Mam-
mal Jan., Test Systems
Adverse reproductive effects of 2,4,5~T exposure
have been observed in other mammalian test systems.
Lloyd et al» (173) reported on In vivo enzymatic studies
showing reduced uptake and metabolism of testosterone
by the prostate gland in male nice treated orally with
doses of 2,4,5-T (6.25, 12.5,. pi* 25 mg/kg, ten times
-daily)*
lefiaenko (151) reported on the effects of acute
and chronic exposure to the butyl ester of 2,4,5-T on
gonadal and somatic tissue in an jn vivo cytogenetic
study in male albino rats. Chronic effects on the go-
nads were observed after exposure to 0,1 ug/kg for two
and one half months. Adverse effects (seen at seven months,
when the experiment was terminated), which were considered
persistent effects, included testicular atrophy, decreased
sperm count, desquamated tubules, and aberrant cells
in the germinal epithelium. Chromosomal aberrations
were also observed during the chronic phase of the ex-
periment. EPA .evaluation of this study found inadequacies
in the methodology which would prevent the drawing of
firm conclusions from this data (106).
-98-
-------
Recent studies in rats by Sjoden and Soderberg
[cited in (25)3 appear to show that prenatal exposure.
to 2,4,5-T leads to behavioral abnormalities and changes
in thyroid activity and brain seritonin levels in the
progeny. Single oral doses of TOO mg/kg were administered
to the dams on days 7» 8, or 9 of pregnancy.
(c) Adverse Effects in, Avian Species
Embryotoxic effects in avian species due to 2,4,5-T
exposure have been reported. Verrett (136) studied the
effects of 2,4,5-T, containing either 27 or 0.5 ppm TCDD,
on chicken eggs. The 2,4,5-T was injected through the
air cell of the eggs, either preincubation or on the
fourth day of incubation. The sample containing 27 ppm
TCDD was found to be more lethal (LD-50 = 25 ug/egg) than
tOie less contaminated sample (LD-50 a 100 ug/egg). Both
samples produced teratogenic effects, including chick
edema, eye defects, beak defects (primarily cleft palate),
and short, twisted feet resulting from tendon slippage.
Teratogenic effects were observed at doses as low as
.1 ppn (50 ug/egg) with the sample containing 0.5 ppm
TCDD and as low as 0.125 ppm (6.25 ug/egg) with the sample
containing 27 ppm TCDD.
Lutz and Lutz-Ostertag (138) studied the action
of 2,4,5-T, in aqueous solution afc a concentration of
2 to 10 g/liter, on the embryonic development of quail
(Coturnix coturnix japonica), chicken (Sallus gall.u.s),
-99-
-------
pheasant (Phasianus .colchicus), and two partridge species
(Alectoris ru.fa and Perdrix; perdrix). The 2,4,5-T was
adoinistered by dipping, spraying, and organo-typic cultures.
Abnormal genital tracts were observed in all species,
indicating abnormal sexual differentiation. Further,
morphological changes in the tastes often gave the appearance
of true testicular atrophy. In another study, 2,4,5-T
affected fertility in birds of both sexes (139).
-100-
-------
(d) Studies in Avian Species in Which.Adverse
Effects Were Not Observed
Using 2,U,5-T contaminated with less than 0»1 ppm
dioxin, Strange and Kerr (112) found no abnormal development
in chicken embryos. Doses of 12,5, 25, 50, 75, 100, and 125
mg/kg were injected into eggs on days 0 and 5 of incubation;
observations were made HB hours later* At this, developmental
stage, kidneys were not sufficiently developed to detect the
tubule lesions reported by Bjorklund and Erne (143)*
(e) Summary
Studies have established that 2,4,5-T is fetotoxic
and teratogenic at doses as low as 35 mg/kg (0.05 ±. 0.02
ppm TCDD) in mice (125); *1.6 mg/kg (approximately 30 ppm
TCDD) in rats (123); and 20 mg/kg (0,5 ppm TCDD) in hamsters
(12*0 » Cleft palate and kidney anomalies have been .observed
in mice, rats, and hamsters. No fetotoxic or teratogenic
effects (no-effect levels) have been observed at doses of
20 mg/kg (0.05 ± 0.02 ppm TCDD) in mice (125) and 25 to 150
mg/kg (0.05 ± 0.02 ppm TCDD) in rats (125).
(3) Exposure Analysis
In order to determine whether a rebuttable presumption
should be issued based on reproductive and fetotoxic effects,
pursuant to Section 162.11 (a)(3)(ii)(B), the Working Group
must determine whether or not an ample margin of safety
-101-
-------
exists between the levels of 2,4,5-T and/or TCDD which
produce reproductive and fetotoxic effects, and the level(s)
to which humans can reasonably be anticipated to be exposed.
The cancellation of uses of 2,^1,5-T on food crops
intended for human consumption and for use around the
home, recreation sites, aquatic areas, and ditch banks in
1970 was thought to have eliminated the potential exposure
to that portion of the population at risk (women of child-
bearing age).
-lOla-
-------
Social changes over the last few years, however,
have given wonen the opportunity for employment in areas
that once were considered open only to men. Since women
of child-bearing age are now employed in occupations such
as pesticide applicators, operators of highway construc-
tion and maintenance equipment, foresters, and chemical
formulators, they have become part of the population at
risk with potential exposure to 2,4,5-T and/or TCDD.
In order to determine whether an ample margin of
safety exists, the Working Group must first determine
how much 2,4,5-T a woman could be exposed to through
oral, dermal, or inhalation erposure. For each of these
analyses, the Working Group assumes a woman to weigh
60 kg. The following calculations are based on an exposure
analysis for 2,4,5-T and TCDD performed by EPA's Criteria
and Evaluation Division [CED] (164).
(a) •Oral Exposure .
For purposes of this analysis, the Working Group
considered currently registered uses where the possibility
of oral exposure to 2,4,5-T and/or TCDD existed. Treat-
ment of range and pasture land could result in oral ex-
posure through ingestion of meat and milk from animals
grazing on the treated area. Since actual data on residues
of 2,4,5-T in animals grazing on treated rangeland is
-102-
-------
unavailable, for purposes of the 2,4,5-T oral exposure
analysis, the Working Group used residue information
obtained in a feeding study (37) in which cattle were
fed. considerably higher amounts of 2,4,5~T than they
would normally be exposed to in grazing on treated land.
«
The following calculations are based on the average quanti-"
ties of food eaten per day (1.5 kg), as reported by Lehman
(144, 165).
Table 25. 2.4r5-T Oral Exposure Analysis
{ Whole Milk Meat (Beef).'
{No-adverse-effeet 20 mg/kg 20 mg/kg j
{level for terato-
jgenicity in mice
{Average level of 0.103 ppm"2" 0.2
{2,4,5-T identified
\% of food item in 19.6% 4,6?
{total human diet : '
{Average amount of 1.5 kg 1.5 kg
{food eaten per day
I
{Exposure to 2,4,5-T 0.0005 0.0002 !
'per dav rag/kg mg/kg {.
£./ Animals were fed at 300 ppm 2,4,5-T in the diet for 2 to
3 weeks. This is a worst case assumption for cows grazing
on freshly-treated pasture without a withdrawal period; all
milk and meat was obtained from such cows. Meat (beef)
includes muscle, fat, and liver tissues which constitute the
major portion of edible meat*
To find the average daily intake of a single food
item, multiply the average daily food intake by the percent
of that item in the total diet: for milk, 1.5 kg X 19.6?
=0.294 kg; and for meat (beef), 1.5 kg X 4.6? s 0.069 kg.
-103-
-------
The quantity of 2,4,5-T in .the average daily diet
equals the average daily intake of each food item multi-
plied by the level of 2,4,5-T in the food item: for
milk, 0.294 kg X 0,103 ppm = 0.03 og; and for meat (beef),
0.069 kg X 0.2 ppm =0,014 mg.
* .
The theoretical exposure of an average woman equals
the amount of 2,4,5-T in the daily diet divided by the
weight of the average woman: for milk, 0.03 og / 60 kg
= 0.0005 mg/kg; and for meat (beef), 0.014 mg / 60 kg
s 0.0002 mg/kg; total exposure from milk and beef products
could be 0,0007 mg/kg per day,
x Existing data on TCDD residues in animals grazing
on treated rangeland are too meager to use for an analysis
of TCDD exposure to humans through ingestion of meat or
milk from animals so exposed.
The W.orking Group considers that the difference
between the no-adverse-effect level of 2,4,5-T for terato-
genic effects (20 mg/kg) and the calculated oral exposure
level for 2,4,5-T (0,0007 mg/kg per day) does constitute an
ample margin of safety. Since this risk criterion for other
chronic adverse effects has not been met or exceeded, a .
rebuttable presumption does not arise,
-104-
-------
(b) D'errsal Exposure
In order to conduct these analyses, the Working
Group nust determine the amount of 2,4,5-T and/or TCDD
which would come in contact with the skin and the amount
that would be absorbed.
* '
(i) Sprav Applicator; Back-pack, Sprayer
For purposes of this analysis, the Working Group
assumes the applicator to be a 60-kg woman of child-
bearing age, and the site of application either a right-
of-way or spot treatment of pasture or rangeland. The
equipment is a back-pack sprayer (166K The following
calculations of exposure are based on dilution for spray-
ing of three pints of formulated product- per 32 pints of
water. Typical 2,4,5-T formulations, based on inspection of
a large number of registered labels (164), range from 4 to 6
pounds active ingredient (acid equivalent) per gallon. The
product used in this exposure analysis has an assumed
concentration of 4 pounds 2,4,5-T per gallon. Label recommen-
dations vary from a recommended dilution of 0.094 to 4
pounds acid equivalent per 32 pints of water. A dilution
rate of 1.6 pounds per 32 pints has been selected as represen-
tative of a typically-used spray mixture. .
Wolfe et al. (166) studied dermal exposure to
fenthion during hand back-pack spraying for mosquitoes
-105-
-------
for ten situationsi Exposure ranged from 0.1 to 6.3 mg/hr,
with a mean value of 3»6 mg/hr (6 ml/hr). Method of applica-
tion was a hand pressure sprayer, using a 0,06$ spray.
Workers wore short-sleeved, open-necked shirts with no
gloves or hat. Based on Wolfe's data, CED (164) calculated
a dermal exposure of approximately 0.177 pints per day. CED
(16M also determined that approximately 10? of the 2,4,5-T
and TCDD coming in contact with the skin of the applicators
would be absorbed even after washing, based on absorption
studies with other pesticides (1H5, 1^6, 163).
Table 26. Back-pack Spraver Dermal Exposure,Data
TCDD
jUse Dilution rate — — — 5 pints 3 pints
I ' (U6 pounds (0,00000016
j 2,4,5-T) per pounds TCDD)
I •-••-. 32 pints per 32 pints
{ water water
I '<"-• ' '• - ~ . •
{Amount of diluted 0,18 pint 0*18 pint
I material gotten ..... r •;
|-on skin daily . :
I . • v .-. -. :
\% Diluted material 10$ 10J
labsorbed
! • '• ••• . , .
lExposure level 409 ng O.OM09 ug
!
iDose level 6,8 mg/kg 0.0007 ug/kg
I
.lNo-Adverse-Ef fec.t, ____ ..^^^.Q.JDLg/kg 0,03 ug/kg
I level for terato- :^- —
Jeenic effects
The following calculations (see Table 27 for mathe-
matics) will give the daily dermal exposure for both 2,4,5-T
•106-
-------
and TCDD: 1) convert the dilution rate to grams; 2) multi-
ply this figure by 1,000 (for 2,4,5-T) to convert to milli-
grams and by 1,000,000 (for TCDD) to convert to micrograms;
3) multiply this figure by the daily dermal dose of diluted
material; 4) multiply this figure by the percent absorbed;
and 5) divide this figure by the weight of the applicator
for the daily exposure to 2,4,5-T or TCDD per 8-hour working
day,
Table 27
I 2,4,5-T
11) 1.6 pounds/32 pt X 454 g/-
{ pound s 22*70 g/pt;
I
|2) 22.70 g/pt X 1,-9.04X-ttg/g =
I 22,700 mg/pt;
!
!3) 22,700 mg/pt X 0.18 pt =
I 4,086 mg;
!4) 4,086 mg X 10$ = 408.6 mg
TCDD
1) 0.00000016 pounds/-
32 pt X 454 g/pound -•
0*00000227 g/pt;
2) 0.00000227 g/pt X
1,000,000 ug/g =
2.27 ug/pt;
3) 2.27 ug/pt X 0.18 pt
0.41 ug;
4) 0.41 ug X 10? s
! ' j 0,041 ug;
!5) 408.6 mg / 60 kg = !5) 0.041 ug / 60 kg =
I 6.8 mg/kg per day I 0.0007 ug/kg per dav
The Working Group considers" that the difference
between the no-adverse-effect level of 2,4,5-T for tera-
togenic effects (20 mg/kg) and this calculated dermal
exposure level for 2,4,5-T (6.8 mg/kg), as well as the
difference between the no-adverse-effect level of TCDD for
teratogenic effects (0.03 ug/kg) and this calculated expo-
sure level for TCDD (0.0007 ug/kg), do not constitute an
ample margin of safety. The Working Group therefore recom-
mends issuance of a rebuttable presumption against pesticide
-10 7-.
-------
products contaanj.u& &,-.,., . ....
Section 162.11(a)(3)(ii)(B).
(ii) Sprav Applicator: Tractor-mountedf Low-boon
Spray Equipment
For the purpose of this analysis, the Working Group
assumes the applicator to be a 60-kg female of child-
bearing age clearing brush on either rangeland or rights-
of-way. The same product cited above (2,4,5-T at 4 pounds/gal)
is being used, and the dilution rate is 1.6 pounds of
formulation to 32 pints of water1 (equal to 4 pounds of
2,4,5-T per 10 gallons of water). Based on exposure studies
using similar equipment but a different herbicide (14?)» the
Working Group determined that, during an eight-hour working
day, the applicator would get 0*048 pints of diluted
material on her skin. The Working Group determined that 10J
of the pesticide on the skin would be absorbed (145, 1*16, 163).
Table 28* Dermal Exposure Data (Tractor Mounted Equipment)
I
SUse Dilution rate
|
lAmount of diluted
imaterial gotten
|on skin daily
i
1$ Diluted material
{absorbed
I
lExposure level
1 :
{Dose level
i
i
1 No-Ad verse-Effect
llevel for terato-
Ipenic effects
2T4r<5-T TCDD
3 pints 3 pints
(1.6 pounds (0.00000016
2,4,5-T) per pounds TCDD)
32 pints per 32 pints
viater „ water
0.048 pint 0.048 pint
'
10$ 10$
• • .
109 mg 0.0109 ug
1.8 ing/kg 0.00018 ug/kg
20 mg/kg 0.03 ug/kg
j
-108-
-------
The following calculations (see Table 29.for mathe-
.»
matics) will give the daily dermal exposure for both <2,4,5-T
and TCDD: 1) convert the dilution rate to grams; 2) multi-
ply this figure by 1,000 (for 2,4,5-T) to convert to milli-
grams and by 1,000,000 (for TCDD) to convert to nicrograms-;
3) multiply this figure by the daily dermal dose of diluted
\
material; 4) -multiply this figure by the percent absorbed;
and 5) divide this figure by the weight of the applicator
for the daily exposure to 2,4,5-T or TCDD per 8-hour working
day.
. Table 2Q 1
2f 4.5-T _!.
1) 1.6 pounds/32 pt X 454 g/-
pound = 22.70 g/pt;
2) 22.70 g/pt X 1,000 mg/g =
22,700 mg/pt;
3) 22,700 mg/pt X 0.048 pt r
1,089.6 mg;
4) 1,089.6 mg X 10J =
108.96 mg;
5) 108.96 mg / 60 kg =
1.8 ffig/kg per.day
TCDD
1) 0.00000016 pounds/-
32 pt X 454 g/pound =
0,00000227 g/pt;
2) 0.00000227 g/pt X
1,000,000 ug/g =
2.27 ug/pt;
3) 2.27 ug/pt X 0.048 pt
0.109 ug;
4) 0.109 ug X 10$ =
0.011 ug;
' 15) 0.011 ug / 60 kg s
...'.. 0.00018 ug/kg per, day
The Working Group considers that the difference
between the no-adverse-effect level of 2,4,5-T for tera-
togenic effects (20 mg/kg) and this calculated dermal
exposure level for 2,4,5-T (1.8 mg/kg), as well as the
difference between the no-adverse-effect level of TCDD for
teratogenic effects (0.03 ug/kg) and this calculated expo-
sure level for TCDD (0,00018 ug/kg), do not constitute an
-109-
-------
ample margin of safety. The Working Group therefore recom-
mends issuance of a rebuttable presumption against pesticide
products containing 2,4,5-T and/or TCDD pursuant to 40 CFR
Section 162.11(a)(3)(ii)(B).
(iii) Aerial Applicat ion; EXPOs_e d.,P_opu 1 ation
Directly Beneath Spray Plane
Caplan et al. (167), working with aerially applied
malathion in oil sprays applied at 0,46 pounds per 0.76
gallons water/acre, determined a dermal exposure to persons
directly beneath the spray plane for bare skin (head, neck,
shoulders, forearms, hands, and thighs) of 3*556 mg/day.
With these data, an equivalent dermal exposure for 2,4,5-T
and TCDD, aerially applied at 4 pounds acid equivalent
2,U,5-T per TO gallons water/acre, can be determined,
Table ?0. Dermal Exposure Pat?. (Agrj.g.1 fit? plication^
jDermal exposure to
{aerially applied
{malathion
\
i
lUse Dilution rate
!
1
! •
\
I
\% Diluted materiaT*"
jabsorbed
I
{Exposure level
1 ' . ."
I Dose level
1
1
! No-Ad verse-Effect
{level for terato-
jKenic effects
3.556 mg/0.46
per acre
2f4T5-T
4 pounds
2,4,5-T per
10 gallons of
water/acre
---TO? - •
.
• • ' - . - .- . ..
3«1 mg
0.051 mg/kg
" ' . •
20 mg/kg
pounds malathion
TCDD
0.0000004
pounds TCDD
per 10 gal-
lons of water
per acre
10$
.-•
0,0003 ug
5 X 10"6
ug/kg
0.03 ug/kg
!
I
-110-
-------
The following calculations (see Table 31. for .mathe-
matics) will give the daily dermal exposure for both 2,4, 5-T
and TCDD: 1) divide the dermal exposure to malathion
by the malathion application rate and multiply by the
application rate of 2, 4, 5-T and TCDD to obtain the dermal
exposure; for TCDD, multiply this figure by 1,000 to convert
to microgramsj 2) multiply this figure by the percent
absorbed; and 3) divide this figure by the weight of the
applicator for the daily exposure to 2,4, 5-T or TCDD per
8-hour working day.
Table 31
2f 4r5-T - j • TCDD |
1) 3.556 mg/0.46 pounds X SD.3.556 mg/0.46 pounds X f
4 pounds = 31 ng; j 0.0000004 pounds =
.1 0.000003 mg X 1,000 =
I 0.003 ug;
2) 31 mg X 102 = 3.1 mg; !2) O.OC3 ug X 10$ =
j 0.0003 ug;
3) 3.1 mg/ 60 kg = J3) 0.0003 ug / 60 kg r
0.051 mg/kg per day { _ v ,--6 .. , ,
! 5 X 10 ug/kg per day !
• The Working Group considers that the difference
between the no-adverse-effect level of TCDD for teratogenic
effects (0.03 ug/kg) and this calculated dermal exposure
level for TCDD (5 X 10 ug/kg) does constitute an ample
nargin of safety. The Working Group also considers,
however, that the difference between the no-adverse-effect
level of 2, 4, 5-T for teratogenic effects (20 mg/kg) and this
calculated dermal exposure level for 2,4, 5-T (0.051 mg/kg)
-111 & 112-
-------
does not constitute an ample margin of safety. The Working
Group therefore recommends issuance of a rebuttable presumption
against pesticide products containing 2,4,5-T pursuant to
40 CFR Section 162.11(a)(3)(ii)(B).
(c) Inhalation.. Exposure; Aerial Application
There are no studies available on inhalation exposure
of 2,4,5-T. There are, however, several studies on inhala-
tion exposure to malathion (167, 168) which CED used as a
model for this 2,4,5-T exposure analysis (164)* Caplan et
al. (167) determined an air concentration, for unprotected
persons directly beneath the spray plane during application
and for two hours afterward, of 0*067 mg malathion/m^ from
aerial application of 0.46 pounds Al/gallon per acre. The
collection period spanned the course of the actual application
time plus two hours thereafter. The authors considered the-sam-
p.ling technique to be equivalent to average inspiration through
the nostrils. This inhalation exposure (amount available for
inhalation) was 12$ of the applied malathion, Caplan et al.
further reported that the average median diameter ( = volume
median diameter, or vmd-^1 was 109 microns. Based on work
by Akesson and Yates (168), CED (164) estimated that the
size of the malathion droplets which could be inhaled was
16/ The vmd is that droplet size-which divides the total
volume of drops in half, i.e», 50? of the volume is in
drops above the vmd size and 50? below it»
-113-
-------
under 60 micron?. Since 2,4,5-1 is typically applied as a
medium or coarse spray, while malathion is applied as a fine
spray, the percent of 2,4,5-T droplets small enough to be
inhaled (under 60 microns) would be less than the percent of
malathion droplets small enough to be inhaled. According to
Akesson and Yates (168), 2% of Z,4,5-T spray droplets would
be available for. inhalation (or 1/6 the amount of malathion
droplets available for inhalation), on a "worst case" basis.
T a b 1 e 32» Inhalation Exposure Data (Aerial Application)
JAir concentration of
{aerially applied
Imalathion
{Use Dilution rate
I
{Lung Absorption
{Rate
Breathing Rate
\
{Exposure level
! ..._,_
Dose level
I
{No-Adverse-Effect
{level for terato-
'genic effects
0.067 mg/m with application
rate of 0.46 pounds malathion
per -gallon per acre
2,4T5-T
4 pounds
2,4,5-T per
10 gallons of
viater/acre
100?
1.8 nr/hr
0.34 mg
per 2 hr
0.023 mg/kg
per 8 hr
20 mg/kg
?CDD
0.0000004
pounds TCDD
per 10 gal-
lons of water
per acre
10055
1.8 m°/hr
0.000032
ug per 2 hr
2 X 10 ug/kg!
per 8 hr
5
0.03 ug/kg
.114-
-------
1 The following calculations (see Table 33 for mathe-
matics) will give the daily inhalation exposure for both
2,4,5-T and TCDD: 1) multiply the air concentration of
malathion by the amount of 2,4,5-T and TCDD applied, then
multiply this figure by 1/6 for the inhalation exposure to
2,4,5-f and'TCDD; for TCDD, multiply this figure by 1,000 to
convert to micrograms; 2) multiply this figure by the
breathing rate; 3) multiply this figure by eight [8] to
get the 8-hour exposure total;'and 4) divide this figure
by the weight of the applicator for the inhalation exposure
to 2,4,5-T or TCDD per 8-hours exposure.
• Table 33. '
2. /I.5-T
1) 0.067 mg/cu m per 0.46
pounds X 4 pounds = 0.58
mg/cu m X 1/6 s 0.097
- mg/cu m; ;
2) 0.097 mg/cu m X 1.8 cu m/-
h'r = 0.17 mg/hr;
3) 0.17 mg/hr X 8 = 1.36 mg;
TCDD
1) 0.067 mg/cu m per 0.46
pounds X 0,0000004
pounds = 0.000000058
ng/cu n X 1/6 s
0.000000009 ng/cu m X
1,000 = 0.000009 ug/cu m;
2) 0.000009 ug/cu m X
1.8 cu ra/hr =
• 0.000016 ug/hr;
3) 0.000016 ug/hr X
8 = 0,000128 ug;
!4) 1.36 mg / 60 kg = !4) 0.000128 / 60 kg =
I 0.026 mg/kg exposure } -6
J _ per day . I 2 X 10 ug/kg -per
The Working Group considers that the difference
between the no-adverse-effect level of TCDD for teratogenic
effects (0«03 ug/kg) and this calculated dermal exposure
level for TCDD (2 X 10 ug/kg) does constitute an ample
margin of safety. The Working Group also considers,
-115-
-------
however, that the difference between the no-adverse-effect
level of 2,4,5-T for teratogenic effects (20 ag/kg) and this
calculated dermal exposure level for 2,4,5-T (0.026 mg/kg-^-M
does not constitute an ample margin of safety. The Working
Group therefore recommends issuance of a rebuttable presumption
against pesticide products containing 2,4,5-T pursuant to i|0
CFR Section 162.11(a)(3)(ii)(B). .
(d) Cumulative Exposure '
The Working Group has also considered the possibility
of a single individual being exposed through two or more of
the above routes. The results (derived from Tables 27> 29»
and 31) are shown in Table 3^. The Working Group also notes
that possible cumulative exposure to several dioxin-containing
pesticides could increase the total body burden and increase
total risk from dioxin exposure. . :
V . • - . ' ''-'.- • • :
The Working Group considers that the differences
between the no-adverse-effect level of TCDD for terato-
genic ef.fects (0.03 ug/kg) and the calculated cumulative
exposure levels for TCDD in Situations 2 and 3 (see Table
34) do constitute an ample margin of safety. The Working
Johnson (63) [see Section I.G.(3)], in a review article,
calculated a daily inhalation exposure to phenoxy herbicides
of 0.025 ug/kg for a 70-kg adult. The calculations were
based on actual air monitoring data of air samples collected
.in two wheat-growing areas in the state of Washington during
spring and summer and analyzed for phenoxy herbicides. The
author did not specify how soon after application the
samples were taken.
-116-
-------
Table .34. ._.Cumulative Exposure to 2,4f5-T and TCDD
I Situation #1 ; 2.4.5-T i Situation # i : TCDD
(Oral- 0.0007 mg/kg }0ral-
JDermal- 6.8 mg/kg IDermal- 0.0007 ug/kg
* 4 | •
llnhal.- 0.2 mg/kg5- llnhal.- negligible3-
ICum. = 7.0 mg/kg iCum. = 0.0007 ug/kg
i i
I Situation *2; 2.4.5-T i Situation #2: TCDD
SOral- 0.0007 mg/kg !Oral-
iDermal- 1,8 lag/kg • iDermal- 0.00018 ug/kg
llnhal.- Oi.05a/ llnhal.- negligible-7
{Cum. = 1.85 mg/kg |Cum. = 0.00018 ug/kg
! Situation #?: 2.4.5-T i Situation ?n; TCDD
JOral- 0.0007 mg/kg iOral- .
!i g
Dermal- 0.051 mg/kg IDermal- 5 X 10 ug/kg
llnhal.- 0.026 mg/kg llnhal.- 2 X 10~6 ug/kg
I I £
{Gun. = 0.0777 nz/ke !• Cua. r 7 Y 10 ug/kg
&/ Calculations were made on a worst-case basis as 3$
of dermal exposure based on Wolfe (179) who states, "over
97$ of the pesticide to which the body is subjected during
most exposure situations, and especially to applicators of
liquid sprays, is deposited on the skin." TCDD inhalation
exposure values were negligible: Situation #1, 21 X 10~
ug/kg; Situation #2, 54 X 10~7 ug/kg.
Croup also considers, however, that the differences between
the no-adverse-effect levels of 2,4,5-T and TCDD for terato-
genie effects (20 mg/kg and 0.03 ug/kg, respectively) and
the calculated cumulative exposure levels for 2,4,5-T in
Situations 1, 2, and 3 and TCDD in Situation 1 (see Table
34) do not constitute an ample margin of safety. The
Working Group th~er~efore- recommends issuance of a rebuttable
presumption against pesticide products containing 2,4,5-T
pursuant to 40 CFR Section 162.1 1 (a)(3)(ii)(B).
-117-
-------
IV. STUDIES RELATING TO POSSIBLE ADVERSE EFFECTS
This section addresses other types of adverse
effects of 2,-^,5-T for which the Working Group has deter-
mined that insufficient evidence exists to initiate a
rebuttable presumption. The Agency solicits comments
from registrants and other interested parties on the
evidence listed below, and requests submission of any
additional studies or relevant information on 2,4,5-T
and/or TCDD relative to these potential adverse effects.
A. Mutaggnicitv _ . ....
Section 162.11(a)(3)(ii)(A) provides that a rebuttable
•presumption shall arise if a pesticide's ingredient(s),
aetabolitefs), or degradation product(s) induce mutagenic
effects, as determined by multitest evidence. .
(1) 2f 4.5-T
(a) .' positive Study
Majumdar and Golia (178) fed male Pro soph 11 a me1ano-
jgaster either 250 or 1,000 ppm dioxin free 2,4,5-T (obtained
from Eastman Kodak) for 15 days. They were then mated to
sets of virgin females to generate three 4-day broods of
offspring. F flies were allowed to mate, and F flies were
scored for X-linked recessive lethals. No differences among
-118-
-------
broods were noted, and data from all broods were pooled.
The percent lethals in controls, 250 and 1,000 ppm groups
were dose-related and were 0.05> 0.26, and 0.66$, respectively.
The control vs, 1,000 ppm lethal rates were significantly
different from one another (p < 0,01), Ethyl methane
sulfonate (250 ppm) was included as a positive control;
it yielded 13.70? lethals. The total number of flies
in each experimental group was no larger than 2,000.
*
(b) Negative Studies,
The mutagenicity of 2,4,5-T was evaluated by Ercegovich
et al» (148), employing the procedure of Ames, using five
strains of galmonella typhimurium without activation. They
concluded that 2,4,5-T is not.mutagenic.
•- ••'*- • " . ' "
Fujita et al. (149) reported chromosomal abnormalities
in Jji vitro cytogenetic studies of human lymphocytes
• -7 -4
exposed to 10 to 10 M of 2,4,5-T, which contained 0.09
ppm TCDD. Breaks, deletions, and rings were observed.
,.£hromatid breaks increased with increasing concentrations
of 2,4,5-T% It was not possible to distinguish whether this
was a toxic effect or a potential genetic effect (150).
.119-
-------
Majundar and Hall (169) reported on .the cyto-
genetic effects of 2,4,5-T—^ on in vivo bone-marrow
cells of Mongolian gerbils. The animals were injected
with total amounts of 2,4,5-T at the rate of 50, 150,
250, 350, or 500 mg/kg body weight over the 5-day period
of the study. Increasing numbers of chromatic gaps,
breaks, and fragments were observed at 250, 350, and
500 mg/kg doses. No exchange figures or isochromosozne
gaps or breaks were observed. This is not a definitive
experiment for indicating the potential of 2,4,5-T for
causing heritable chromosome damage (170)» Toxicity
effects of the chemical could give similar results (170).
Davring and Hultgren (171) reported on an JLn
ylvo study on the cytogenetic effects on bone-marrow
cells of tyu_4 musculus (male mice) induced by a Swedish
TO/
commercial 2,4,5-T ester formulation-^ and its compo-
nents. The study showed that 2,4,5-T commercial pro-
ducts can affect chromosomal and reproductive mechanisms,
Two different strains of mice were used with similar
results for both. These results correlated with effects
seen in Drosophila. The authors stated that chromatid
The 2,4,5-T used in this study was purchased from
Eastman Kodak Company, Rochester, N.Y,, and contained
no measurable amount of TCDD. The authors do not indi-
cate the limit of sensitivity.
JL2/ The concentration of TCDD was guaranteed to be .
less than 0.1 ppm in the product,
-120-
-------
protocol were given, and statistical methods were apparently
not employed in assessing the data.
The second test by Hussain et al. (24) studied
reverse mutation from histidine dependence to histidine
independence in Salmonella tvphimurlum (Strains TA 1532 and
TA 1530). TCDD was positive in TA 1532 but negative in TA
1530. This indicated that TCDD acts as a frameshift mutagen.
ICR-170 was used as a positive control in the test with TA
1532. No positive or negative controls were tested in IA
1530*
In the third test Hussain et al, (24) observed slight
prophage induction in £» o o1i K-39. However, data from this
test were difficult to evaluate because the solvent used,
dimethyl sulfoxide, causes cellular effects,
A preliminary report on the chromosomal analysis of
hospital.patients exposed to TCDD in the accident at the
Seveso, Italy, factory was presented at the Department of
Health, Education, and Welfare meeting on October 12, 1976
(152). An increased number of chromosomal lesions (gaps,
chromatidic and chromosomal breaks, and rearrangements) were
observed in somatic cells of the 2- to 28-year-old males
and females tested. Cytogenetic studies of tissues from
therapeutic abortions performed on women who were exposed to
TCDD during the accident indicated that there was chromosomal
-122-
-------
damage to cells in maternal peripheral blood, and placental
and fetal tissues. These preliminary results were based on
a small number of samples, and no specific data are available
at this time (150).
(b) Negative Studies
Khera and Ruddick (6) conducted dominant lethal tests
in which male Wistar rats received TCDD at dosages of 4 and
8 ug/kg per day. The studies indicated that no dominant
lethal mutations arose during the 35 days after treatment.
The period examined corresponded to postmeiotic stages of
spermatogenesis,
A cytogenetic screening study of the effects of TCDD
on bone marrow cells of male Osborne-Mendel rats was per-
formed by the Food and Drug Administration (119). Two
separate experiments were performed. The first was a multiple
dose test in which 10 ug TCDD/kg per day was administered by
intubation for 5 consecutive days* In the second test,
single doses of 5, 10, and 15 ug TCDD/kg were administered
intraperitoneally and 20 ug/kg (the highest dose) was
administered orally. There was no evidence "-•from these
studies to indicate that TCDD produced cytogenetic damage in
the bone marrow of these male rats. Toxicity, which was
indicated by a slight weight loss, was noted in rats that
received a single dose of 15 or 20 ug/kg (the highest
dose levels).
-123-
-------
Green (119) conducted a short-term investigation of
several dioxins, using male Osborne-Mendel rats, to determine
what potential these substances had to produce cytogenetic
damage in rat bone-marrow. In one study all of the dioxins
were tested by being intubated in the rats for five consecu-
tive days at 10 ug/kg per day. A second study involved TCDD
alone administered orally at 20 ug/kg and intraperitoneally
at 5, 10, and 15 ug/kg. The author found no evidence that
any of the substances tested produced cytogenetic damage in
the bone marrow of male rats,
In conclusion, although Hussain et al» (2*0 have
demonstrated that TCDD does appear to act as a point .(gene)
mutagen, the evidence is weak for heritable genetic effects
since the level of mutagenic testing is meager and there
were some major deficiences in some tests. However, the
study by Hussain et al. does not fulfill the criterion of
multitest•evidence as prescribed in HO CFR 162.11. Although
TCDD does appear to have the potential to act as a chromosomal
mutagen from the In vivo cytogenetic studies (152), specific
data are not yet available from the Seveso accident.
(3) Chromosomal Damage
The Working Group also wishes to call attention
to three studies [previously discussed in Sections III.B.(2)(b),
IV. (1), and IV.(2Mb)], which indicate that 2,H,5-T and/or
-------
TCDD may cause chrooosomal damage. Fujita et al. (
reported chromosomal abnormalities in jLn, vitro- tests on
human lymphocytes exposed to 2,4,5-T; abnormalities included
breaks, deletions, and rings. Yefimenko (16?) reported
damage.to bone-marrow cell chromosomes (including breaks,
true aberrations, or rearrangements) in jjl vivo tests on rat
gonadal and somatic tissue exposed to butyl ester 2,4,5-T.
The preliminary HEW report (152) on the Seveso incident
indicated an increased number of chromosomal lesions (gaps,
chromatic breaks, and rearrangements) in somatic cells of 2-
to 28-year-old humans exposed to TODD.
The Working Group concludes that there is a data gap
on mutagenic -effects and that further evidence and testing
is needed on the mutagenicity of 2,4,5-T and TCDD. The
Working Group would like to evaluate more detailed and
specific information as it becomes available from the Seveso
.accident. Relevant information or studies on the mutagenetic
effects' of 2,4,5-T and/or TCDD should be submitted to the
Agency, and the option for re-evaluating their mutagenic
properties must be left open should more conclusive evidence
become available. • .
B. Toxicitv to Humans: TCDD
(t) Chloracne
A number of researchers have reported illness ascribed
to TCDD (90, 93, 95, 153). Most of these toxic effects have
-125-
-------
occurred in chemical plant workers after accidental exposure
to the dioxin. While a number of ill effects have been
reported, the most widely known is chloracne,
Chloracne is a severe skin disease resulting from
exposure to highly chlorinated dibenzo dioxins. It is a
disease of the follicular and sebaceous glands. Its
symptoms and signs include skin lesions, follicular hyper-
•
keratosis, and the formation of large sebaceous cysts,
inflammed tubercles, and pustules. In addition to these
symptoms, chloracne is often accompanied by a brownish
keratinization of the skin, cystitis, pyelonephritis,
depression, hirsutism, fatigue, neurological disturbances,
raised cholesterol levels, liver damage, and psychological
manifestations (10, 15, 16, 154, 155, 156, 157). Several
researchers have observed that chloracne is not only irritat-
ing and persistent but also very difficult t'o cure. It is
one of the most frequently contracted forms of occupational
dermatitis, occurring primarily in chemical plant employees
«
engaged in the production of 2,4,5-T and 2,4,5-TCP (16, 95,
155, 156, 158).
• • • ' • ' ' . *
The first report on a toxic material being the
causative agent for an occupational skin disease appears to
have been by Dr. Karl Herxheimer in 1899. Dr. Herxheimer
diagnosed the cause of dermatological problems in a German
-126-
-------
factory worker as exposure to chlorine ions in the produc-
tion of caustic potash (159). It is from, this early
diagnosis that we get the name chloracne. During the early
1950's there were a series of industrial accidents in
Germany resulting in an outbreak of chloracne in the
employees of chemical plants manufacturing 2,4,5-T and
2,4,5-TCP. The. symptoms of the employees of one of these
factories in Hamburg, Germany, were extensively ' investigated
by Kimming and Schulz (16)* These researchers, using
the rabbit ear test, proved that the cause of the chloracne
was a contaminant found in crude 2,4,5-TCP and not the
formulated 2,4,5-TCP. Later on, Bauer et al, (15) conclusively
identified TCDD as the causative agent of chloracne,
'•."- (2) Porr>hvrla cutanea tarda and P -Aminolevulinic
••'.-.-."•:'•' " Acid Svnthetase .. ' -. • '. .'''•'.
_ '.... . Porphyria cutanea tarda (PCT) , a form of hepatic
porphyria, is another disease caused by exposure to TCDD
and often accompanies chloracne. PCT occurs primarily in
industrial workers associated with the manufacture of
'2,4,5-T (93,~9T
The symptoms of porphyria cutanea tarda, a defect in
hepatic metabolism of porphyrins, are fragility of the
skin, photosensitivity of the skin, hyperpigmentation,
over-production of porphyrins, hirsutism, and neurological
-127-
-------
and intestinal disorders (9^i 160). It is also characterized
biochemically by an increase in the activity of the mitochon-
drial enzyme 6-aminolevulinic acid (ALA) synthetase, which
is the first and rate-limiting enzyme in heme biosynthesis
(160)» TCDD was thought to be a potent inducer of ALA
activity in chick embryo liver (115)* Goldstein et al. (161)
r—
reported that TCDD was found to induce ALA synthetase and
hepatic prophyria in mice. These researchers stated that at
that time [1973] TCDD was the most potent porphyrogenic
chemical known* Poland and Kende 1976 (*J) found that the
duration of ALA induction from TCDD exposure is prolonged,
most likely due to the long biological half-life of TCDD,
These researchers also found that ALA synthetase inducers
have halogen atoms occupying at least three of the four
lateral ring positions (positions 2, 3> 7, and 8), and that
there is at least one free, nonhalogenated ring position.
TCDD fulfills all of these requirements.
»128-
-------
2,4,5-T: Position Document 1
References
•1, Raw, G.R., ed. 1970. CIPAC handbook, Vol. I,
analysis of technical and formulated pesticides*
Collab. Intntl. Pest. Anal. Council Ltd,,
Hertfordshire, England".
*2. Meister, R.T., ed, 1977. Farm chemicals handbook,
Meister Publishing Co., Willoughby, Ohio.
3» Report on 2,4,5-T: a report of the Panel on
Herbicides of the President's Science Advisory
Committee. 1971. Executive Office of the President,
Office of Science and Technology, Washington, D,C,
*4. Poland, A,, and A, Kende, 1976. 2,3,7,8-tetra-
chlorodibenzo-£.-dioxin: environmental contaminant
and molecular probe. Fed, Proc, 35:2404-2411.
65« Crossland, J., and K,P, Shea. 1973. The hazards of
impurities. Environment 15(5):35-38.
C6, Khera, K»S., and J.A, Ruddick, 1973. Polychlorodi-
benzo-£.-dioxins: perinatal effects and the dominant
lethal test in Wistar rats. Pages 70-84 in E.A.-
-,. . Blair, ed. , Chlorodioxins - origin and fate. Advances
•--:'•-. in Chemistry Series, No. 120. Am. Cheni. Soc, ,
;••',': . . Washington, D.C. - -. ' .- . .
'7, Harvey, R.G. 1973, Dioxin, a contaminant in 2,4,5-T.
Presented at Wisconsin Pest Control Conference with
Industry: January 17, 1973, (unpublished.)
*8. Fishbein,. L. 1973» Mutagens and potential mutagens in
the biosphere: I. DDT and its metabolites, polychlori-
nated biphenyls, Chlorodioxins, polycyclic aromatic
• . hydrocarbons, haloethers. Sci, Tot. Environ. 4:305-340.
*9» Milnes, M.H.. ..1971,. Formation of 2 , 4,7 , 8-tetrachlorodi-
• benzodioxin by thermal decomposition of sodium 2,4,5-
trichlorophenate. Nature 232:395-396.
°References marked with an asterisk are protected from
unauthorized duplication by copyright, A.copy of the Position
Document and all references are avilable for public inspec-
tion in the Office of Special Pesticide Reviews (WH-566),
Office of Pesticide Programs, Environmental Protection Agency,
East Tower, Room 447, 401 M Street S,W., Washington, D,C.
20460.
-129-
-------
•10. Schulz, K.H. 1968. On the clinical aspects and
etiology of chloracne. (Transl, form German.)
Arbeitsnedizin Sozialmedizin Arbeitshygiene 3(2):
25-29.
Bll» Higginbotham, G.R., A. Huang, D, Firestone, J. Verrett,
J, Ress, and A,D, Campbell. 1968. Chemical and
toxicologicial evaluations of isolated and synthetic
chloro derivatives of dibenzo-J2.-dioxin. Nature 220:
702-703.
*12. Muelder, W,Wv, and L,A. Shadoff. 1973* The prepara-
tion of. uniformly labeled 1'*C-2,7-dichlofodibenzo-.a-
dioxin and ^C-2,3,7,8-tetrachlorodibenzo-£-dioxin.
Pages 1-6 in, E.A. Blair, ed., Chlorodioxins - origin
and fate. Advances in Chemistry Series, No. 120.
Am, Chem. Soc», Washington, D,C»
e!3» Burger, E.J., Jr. 1973. Summary: conference on
dibenzodioxins and dibenzofurans, National Institute •
of Environmental Health Services, April 2-3, 1973.
Environ. Health Perspec. 5:279-282.
cll|. Schwetz, B.AT ,""7r7H7~Norris, G.L» Sparschu, V.K. Rowe,
P»J, Gehring, J.L. Emerson, and C.G, Gerbig. 1973.
Toxicology of chlorinated dibenzo-£.-dioxins. Environ,
Health Perspec. 5:87-99.
e!5, Bauer, H» , K.H. Schulz, and D, Spiegelberg* 1961.
Occupational intoxication in the production of
chlorinated phenol compounds. (Transl. from German*)
Arch. Indust. Path. Indust. Hyg. 18:538-555.
Kiramig, J., and K»H. Schulz, 1957. Occupational
acne (chloracne) caused by chlorinated aromatic
cyclic ether. (Transl, from German.) Dermatologica
115:5^0-546.
Kearney, P.C., E.A. Voolson, A.R. Isensee, and C.S,
Helling, 1973. Tetrachlorodibenzodioxin in the
environment-^, sources, .fate, and decontamination,
Environ. Health Perspec, 5:273-277.
°18« Buser, H, 1975. Polychlorinated dibenzo-£.-dioxins:
separation and identification of isomers by gas
chromatography-mass spectrometry. J. Chromatog.
114:95-108. '
-130-
-------
•19. Kimbrough, G.D. 1972. Toxicity of chlorinated
hydrocarbons and related compounds: a review
including chlorinated dibenzodioxins and chlorinated
dibenzofurans. Arch. Environ. Health 25(1) :125-131 •
»20. Firestone, D,, J. Ress, N.L. Brown, R.P. Barren,
and J.N. Damico. 1972. Industrial chemicals:
determination of polychlorodibenzo-£-dioxins and
related compounds in commercial chlorophenols.
J. Ass. Offic. Anal. Chem. 55(1):85-92»
B21. Jones, E.L., and H. Krizek. 1962. A technic
for testing acnegenic potency in rabbits, applied
to the potent acnegen, 2,3>7,8-tetrachlorodibenzo-
jj.-dioxin» J. Invest. Dsrmatol. 39:511-517*
C22» Anonymous. 1970. Another herbicide on the black-
list. Nature 226:309-311*
C23. Grieg, J.B., G. Jones, W»H, Butler, and J.M. Barnes,
1973. Toxic effects of 2,3,7,8-tetrachlorodibenzo-
i-dioxin. Fd. Cosmet, Toxicol. 11:585-595.
e24. Hussain, S,, L« Ehrenberg, G. Lofroth, and T.
Gejvall, 1972. Mutagenic effects of TCDD on
bacterial systems* Anibio 1(1):32-33»
*25. World Health Organization, International Agency for
Research on Cancer. 1977* IARC mongraphs on the
evaluation of the carcinogenic risk of chemicals
to man: some fumigants, the herbicides 2,4-D and
2,'i»,5"-T, chlorinated dibenzodioxins and miscel-
_._. laneous industrial chemicals, Vol. 15. International
Agency .for Research on Cancer, Lyon, [France],
c-26, Thomson, W.T, 1975. Agricultural chemicals: book
II herbicides. Thomson Publications, Indianapolis,
Indiana.
27. Memo: Representative 2,Jt,5-T labels, dated June 30,
1977* From Harvey L. Warnick, Project Manager,
Office of Special Pesticide Reviews, to Files*
28. Memo: 2,H,5-T production, dated September 30, 1977.
From Harvey L, Warnick, Project Manager, Office of
Special "Pesticide Reviews, to Edwin L. Johnson,
Deputy Assistant Administrator, Office of Pesticide
"•Programs, (CONFIDENTIAL.)
.131-
-------
29* U.S. Department of Agriculture, Agricultural
Stabilization and Conservation Service. -1976. The-
pesticide review 1975. U.S. Department of Agriculture,
Washington, D.C.
30. Memo: Request for usage data - 2,4,5-T, 2,4,5-TP,
erbon and ronnel, dated September 16, 1977. From
A. Hale Vandermer, Program Planning and Analysis
Officer, Human Effects Monitoring Branch, to Harvey
L« Warnick, Project Manager, Office of Special
Pesticide Reviews, . .
*31» Crosby, -D.G,, and A.S. Wong, 1973. Photodecom-
position of 2,4,5-trichlorophenoxyacetic acid
(2,4,5-T) in water. J. Agr. Food Chem, 21(6):
1052-1054.
*32. Sharpee, K. 1973. Microbial degradation of phenoxy
herbicides in culture, soil, and aquatic ecosystecs,
Ph.D. Thesis, 94 pp. University Microfilms, Ann
Arbor, Mich*
*33. Shafik, M.T., H^C. Sullivan, and H.F, Enos. 1971*
A method for determination of low levels of exposure
to 2,4-D and 2,4,5-T» Intntl. J. Environ. Anal.
Chem. 1:23-33.
634. Grunow, W., Chr. Borne, and B. Budczies. 1971.
Renal excretion of 2,4,5-T by rats. (Transl.
• -.from German.) Fd. Cosmet. Toxicol, 9:667-670.
*35. Grunow, W., and Chr. Bohme. 1974. Metabolism of
,. - .2,4,5-T and 2,4-D in rats and mice, (Transl.
from German.) Arch. Toxicol. 32:217-225.
*-36» Clark, D,E., J.S. Palmer, R.D. Radeleff, H.R.
Crookshank, and P.M. Farr. 1975. Residues of
.' chlorophenoxy acid herbicides and their phenolic
metabolites in tissues of sheep and cattle. J.
Agr. Food Chem. 23(3):571-578.
C37. Leng, M.L, 1972. Residues in milk and meat and
safety to livestock from the use of phenoxy
herbicides in pasture and rangeland. Down to
- Earth 28(1):12-20.
C38. Fitzgerald, C.H., C,L. Brown, and E.G, Beck.
1967. Degradation of 2,4,5-trichlorophenoxyacetic
acid in woody plants. Plant Physiol, 42:459-460.
-132-
-------
B39. Helling, C.S,, A,R. Isensee, E»A. Woolsori, P.D.J.
Ensor, G.E. Jones, J.R. Plimmer, and P.C. Kearney.
1973. Chlorodioxins in pesticides, soils, and plants.
J. Environ, Quality 2(2) :171-178.
•40. Morris, L.A. 1966. Degradation of 2,4-D and
2,4,5-T in forest litter. J. Forest, 64(7):475-476.
•41. Wiese, A.F., and R.G. Davis. 1964. Herbicide
•movement in soil with various amounts of water*
Weeds 12(2) :101-103.
«42. Anonymous* 1.971. A close look at TCDD, Agr,
Res. 20(4):8-10.
*43. Yoshida, T., and T.F. Castro. 1975. Degradation
of 2,4-D, 2,4,5-T, and picloram in two Philippine
soils. Soil Sci. Plant Nutr. 21(4):397-404,
*44. Morton, 'H.L., E.D. Robison, and R.E, Meyer.
1967. Persistence of 2,4-D, 2,4,5-T, and
dicamba in range forage grasses. Weeds 15(3) •
268-271.
*45, Highman, B., T.B, Gaines, and H.J. Schumacher.
1977» Retarded development of fetal renal alkaline
phosphatase in -mice given 2,4,5-trichlorophenoxyacetic
;: . ' acid, J. Toxicol. Environ. Health 2:1007-1018.
*46. Kearney, P.C,, E*A» Woolson, and C.P, Ellington,
- Jr. 1972. Persistence and metabolism of Chloro-
dioxins in soils. Environ, Scii, Technol. 6(12):
1017-1019.
*47, Norris, L.A. 1971. Chemical brush control: asses-
sing the hazard. J. Forest. 69(10):715-720.
.48. Report of the Advisory Committee on 2,4,5-T to
The Administrator of the Environmental Protection
Agency. May 7, 1971. (unpublished.)
*49. Bailey, G.W., A.D. Thrustori, Jr., J.D. Pope, Jr,,
and D.R. Cochrane. -1970. The degradation kinetics
. of an ester of silvex and the persistence of silvex
in water and sediment. Weed Sci. 18(3):413-418,
C50. Frank, P.A., and R.D. Comes. 1967. Herbicidal
residues in pond water and hydrosoil. Weeds 15(3):
210-213.
-133-
-------
«»51. Kenaga, E.E. 1974, 2,4,5-T and derivatives:
toxicity and stability in the aquatic environment.
Down to Earth 30(3):19-25. .
*52. Trichell, D.W., K.L. Morton, and M.G, Merkle, 1968.
Loss of herbicides in runoff water. Weed Sc-i,
16(4): 447-449.
*53, Edwards, W.M., and B.L. Glass. 1971. Methoxychlor
and 2,4,5-T in lysimeter percolation and runoff
water. Bull. Environ. Contain. Toxicol. 6(1):
*54, Isensee, " A.R, , and G.E. Jones. 1971. Absorption
and translocation of root and foliage applied 2,4-
dichlorophenol , 2 ,7-dichlorodibenzo-j>.-dioxin and
2,3 ,7 ,8-tetrachlorodibenzo-.2.-dixoin, J. Agr. Food
Chem. 19(6):1210-1214.
C55. Woolson, E.A., P.D.J. Ensor, W.L. Reichel, and A.L.
Young. 1973* Dioxin residues in lakeland sand and
bald eagle samples. Pages 112-118 in E.A, Blair,
ed» , Chlorodioxins - origin and fat.e. Advances in
Chemistry Series, No. 120. Am. Chem. Soci , Washington,
*56. Mulhern, B.M,, W.L. Reichel, L.N. Locke, T.G,
,' . Lament, .A, Bel.isle, E» Cromartie, G»E« Bagley,
and R.M. Prouty. 1970* Organochlorine
..''. residues and autopsy data from bald eagles, .
1966-68. Pest, Mon. J. 4( 3) :141-14H.
C57« Isensee, A.R., and G.E. Jones, 1975» Distri-
bution of 2 ,3 >7 , 8-tetrachlorodibenzo-jE.-dioxin
-.'.. (TCDD) in aquatic model ecosystem. Environ,
. • Sci. Technol. 9 (7) : 668-672.
C58. Shadoff, L.A., R.A, Hummel, L. Lamparski, and J,
H, Davidson. Undated. A search for 2,3,7,8-
tetrachlorodibenzo-p-dioxin (TCDD) in an environ-
ment exposed annually to 2 , M ,5-trichlorophenoxy-
acetic acid ester (2,4,5-T) herbicides, (In Press,
Bull, Environ. Contam. Toxicol,)
C59. Brown, E., and Y.A. Nishioka, 1967. Pesticides
in selected western streams - a contribution to
the National Program, Pest, Mon. J. l(2):38-46,
C60. Manigold, D.B., and J.A Schulze. 1969, Pesticides
in selected western steams - a progress report,
Pest. Mon. J. 3(2) :12M-135.
-134-
-------
jl. Lawson, E.R. 1S76. 2,4,5-T residues in storm
runoff from small watersheds, J» Soil Wa.ter
Conserv. 31(5) :217-219»
*62» National Research Council, Safe Drinking Water
Committee. 1977» Drinking water and health:
Part II. National Academy of Sciences, Washington,
D.C,
*63. Johnson, J.E. 1971. The public health impli-
cations of widespread use of the phenoxy herbicides
and picloram, .BioSci. 21(1):899-905.
64, Memo: Environmental monitoring data on 2,4,5-T,
2,4,5-TP (silvex), erbon, and ronnel, dated July 6,
1977. From Frederick W, Kutz, Acting Chief,
Ecological Monitoring Branch, to Harvey L« Warnick,
Project Manager, Office of Special Pesticide Reviews.
*65. St» John,.L,E., Jr., D.G» Wagner, and D*G. Lisk.
1964. Fate of atrazine, kuron, silvex, and 2,4,5-T
in the dairy cow, J. Dairy Sci. 47(11):1267-1270,
*66. Zielinski, W,L», Jr., and L. Fishbein. 1967* Gas
chromatographic measurement of disappearance rates
of 2,4-D and 2,4,5-T acids and 2,4-D esters in mice.
J. Agr, Food Chem. 15(5):841-844,
*67» Piper, W*N., J.Q. Rose, M.L. Leng, and P,J, Gehring.
: 1973. The fate of 2,4,5-trichlorophenoxyacetic
acid (2,4,5-T) following oral administration to
rats and dogs, Toxicol. Appl. Pharmacol. 26:339-351.
*68. Zitko, V. 1972. Absence of chlorinated dibenzo-
dioxins and dibenzofurans from aquatic animals,
Bull, Environ. Contain. Toxicol. 7:105-110.
C69» Baughman, R., and M, Meselson, 1973. An analytical
method for detecting TCDD (dioxin): levels of TCDD
in samples from Vietman. Environ, Health Perspec.
5:27-34.
C70. Matsumura, F., and H.J. Benezet, 1973. Studies on the
b'ioaccumulation and microbial degradation of 2,3,7,8-
tetrachlorodibenzo-£.-dioxin. Environ. Health Perspec.
5:253-258.
C71. Baur, J.R., R.W. Bovey, and J.D. Smith, 1969. Herbicide
concentrations in live oak treated with mixtures of
picloram and 2,4,5-T. Weed Sci. 17(4):567-570.
. -135-
------- |