FINAL
United States CCAO-CIW-418
Environmental Protection March, V)88
Agency
«>EPA Research and
Development
DRINKING WATER CRITERIA DOCUMENT FOR
2,^-DICHLOROPHENOXYACETIC ACID (2,*-D>
FB89-1923i4a
Prepared for
OFFICE OF ORINKING WATER
Prepared by
Environmental Criteria and Assessment Office
Office of Health and Environmental Assessment
U.S. Environmental Protection Agency
Cincinnati, OH 45268
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50272-101
REPORT DOCUMENTATION »¦ REPORT NO 1
PAGE ! ;
2.
3. Recipient's Accession No.
PB89 1 9 2 3 4 R IK
4. Tj' jnd ,! entitle
''epori Da e
Drinking Water Health Criteria Document on 2,4-D
b.
7. Author(s)
8. Performing Organization Rept. No.
Environmental Protection Aqency
9. Performing Organization Name and Address
10. Project/Tesk/Work Unit No.
U. S. Environmental Protection Agency
Office of Drinking Water (WH-550D)
11. Contracts) or Grant(G) No.
4C1 M St. , S.W.
(C>
Washington, D.C. 20460
(G)
12!. Sponsoring Organization Name and Address
13. Type of feport & Period Covered
U. S. Environmental Protection Agecny
Office of Drinking Water (WH-55QD)
401 M St., S.W.
14.
Washington, D.C. 20460
15. Supplementary Notes
16. Abstract (Limit: 200 words)
The Office of Drinking Water (ODW), Environmental Protection Agency has
prepared a Drinking Water Criteria Document cn 2,4-D.
This Criteria
Dccument is an extensive review of the following topics:
Physical chemical properties of 2,4-D
Toxicokinetics and human exposure to
2,4-D
Health Effects of 2,4-D in humans and arimals
Mechanisms of toxicological effects of 2,4-D
Quantification of toxicological affects of 2,4-D
17. Dccument Analysis a. Descriptors ^ properties
exposure
toxicity
Drinking Water
Health Effects
b. Identifiers/Open-Ended Terms 2,4-D
c. COS ATI Field/Group
18. Availability Statement
19. Security Class (This Report)
21. No. of Pages
Non-Sensitive
1
20. Sesurity Class (This Page)
22. Price
Non-Sftnsiti vp
(See ANKI-Z39.18)
See Imtruction* on Reverse
OPTIONAL FORM 272 (4-77)
(Formerly NTIS-35)
Department of Commerce
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NOTICE
THIS DOCUMENT HAS BEEN REPRODUCED FROM
THE BEST COPY FURNISHED US BY THE SPONSORING
AGENCY. ALTHOUGH IT IS RECOGNIZED THAT CER-
TAIN PORTIONS ARE ILLEGIBLE, IT IS BEING RE-
LEASED IN THE INTEREST OF MAKING AVAILABLE
AS MUCH INFORMATION AS POSSIBLE.
I
ifc
-------
DISCLAIMER
This document has been reviewed 1n accordance with the l.S. Environ-
mental Protection Agency's peer 2nd administrative review policies and
approved for publication. Mention of trade names or commercial products
does not constitute endorsement or recommendation for use.
11
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FOREWORD
Section 1412 (b)(3)(A) of the Safe Drinking Water Act, as amended In
1986, requires the Administrator of the Environmental Protection Agency to
publish maximum contaminant level goals (MCLGs) and promulgate National
Primary Drinking Water Regulations for each contaminant, wrilch, 1n the
Judgment of the Administrator, may have an adverse effect on public health
and which 1s known or anticipated to occur 1n public water systems. The
MCLG Is nonenforceable and Is set at a level at which no known or antici-
pated adverse health effects 1n humans occur and which allows for an
adequate margin of safety. Factors considered In setting the MCLG Include
health effects data and sources of exposure other than drinking water.
This document provides the health effects basis to be considered In
establishing the MCLG. To achieve this objective, data on pharmacokinetics,
human exposure, acute and chronic toxicity to animals and humans, epidemi-
ology and mechanisms of toxicity are evaluated. Specific emphasis 1s placed
on literature data providing dose-response Information. Thus, while the
literature search and evaluation performed In support of this document has
been comprehensive, only the reports considered most pertinent In the deri-
vation of the MCLG are cited in the document. The comprehensive literature
data base 1n support of this document Includes information published up to
1984; however, more recent data may have been added during the review
process.
When adequate health effects data exist, Health Advisory values for less
than lifetime exposures (1-day, 10-day and longer-term, ~10% of an
Individual's lifetime) are Included 1n this document. These values are not
used In setting the MCLG, but serve as Informal guidance to municipalities
and other organizations when emergency spills or contamination situations
occur.
Michael B. Cook
Director
Office of Drinking Water
in
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UULUficfti DtVELUHttENf
Michael L. Dourson, Oocument Manager
Environmental Criteria and Assessment Office, Cincinnati
U.S. Environmental Protection Agency
Helen H. Ball, Project Officer
Environmental Criteria and Assessment Office, Cincinnati
U.S. Environmental Protection Agency
Authors
Stephen J. Bosch
Syracuse Research Corporation
Syracuse, New York
(EPA Contract #68-03-3112)
Chapter IV was provided by the
Office of Drinking Water,
U.S. Environmental Protection Agency
Washington, DC
Scientific Reviewers
Michael W. Neal
Syracuse Research Corporation
Syracuse, New York
Steven D. Lutkenhoff
Environmental Criteria and Assessment
Office, Cincinnati
U.S. Environmental Protection Agency
David Bayllss
Charles H. RIs
Bernard Haberman
Carcinogen Assessment Group
U.S. Environmental Protection Agency
Washington, DC
Krlshan Khanna
Office of Drinking Water
U.S. Environmental Protection Agency
Washington, OC
James 0. Pierce
University of Southern California
Editorial Reviewers
Erma iDurden
Environmental Criteria and Assessment
Office, Cincinnati
U.S. Environmental Protection Agency
Judith Olsen
Environmental Criteria aud
Assessment Office, Cincinnati
U.S. Environmental Protection Agency
Document Preparation
Technical Support Services Staff: B, Zwayir, K. Davidson aid J. Moore
Environmental Criteria and Assessment Office, Cincinnati
iv
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TABLE OF CONTENTS
Page
I. SUMMARY 1-1
II. PHYSICAL AND CHEMICAL PROPERTIES II-l
DESCRIPTION II-l
PHYSICAI GERTIES II-l
SOLUBILi.r IN ORGANIC SOLVENTS II-l
pKa VALUES FOR 2,4-0 11-3
VAPOR PRESSURES OF 2,«-D ESTERS II-4
ULTRAVIOLET ABSORPTION OATA FOR 2,4-0 11-4
AQUEOUS DEGRADATION 11-4
DIOXINS IN 2,4-D 11 -7
SUMMARY 11-7
III. TOXICOKINETICS III-l
ABSORPTION III-l
DISTRIBUTION 111-3
METABOLISM 111-8
ELIMINATION 111-10
SUMMARY Ill-18
IV. HUMAN EXPOSURE 1V-1
To be provided by the Office of Drinking Hater
V. HEALTH EFFECTS IN ANIMALS V-l
ACUTE TOXICITY V-l
SUBCHRONIC TOXICITY V-4
CHRONIC TOXICITY V-l3
CARCINOGENICITY V-l 5
MUTAGENICITY V-21
TERATOGENICITY AND OTHER REPRODUCTIVE EFFECTS V-33
Teratogenicity V-33
Other Reproductive Effects V-50
SUMMARY V-52
VI. HEALTH EFFECTS IN HUMANS VI-1
ACUTE EFFECTS VI-1
SUBCHRONIC AND CHRONIC EFFECTS VI-4
Noncarc1nogen1c Effects VI-4
Carcinogenic Effects VI-8
SUMMARY VI-15
v
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TABLE OF CONTENTS (cont.)
Page
VII. MECHANISMS OF TOXICITY VI1-1
SUMMARY VI1-6
VIII. QUANTIFICATION OF TOXICOLOGICAL EFFECTS VIII-1
INTRODUCTION VIII-1
NONCARCINOGENIC EFFECTS VI1I-6
QUANTIFICATION OF NONCARCINOGENIC EFFECTS VIIl-12
Derivation of 1-Day HA VIII-12
Derivation of 10-Day HA VII1-16
Derivation of Longer-Term HA .... . VIII-22
Assessment of Lifetime Exposure and Derivation of
a DMEL VI11-22
Conclusions VI11-27
CARCINOGENIC EFFECTS VIII-27
QUANTIFICATION OF CARCINOGENIC EFFECTS VIII-27
EXISTING GUIDELINES, RECOMMENDATIONS AND STANDARDS VIII-32
SPECIAL GROUPS AT RISK VIII-34
IX. REFERENCES IX-1
vl
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LIST OF TABLES
No. Title Page
II-") Physical Properties of Some 2,4-D Compounds ........ 11-2
II-2 Summary of Vapor Pressure Data Ccr Various 2,4-D Esters . . II-5
11-3 Conversion Factors for 2,4-D Esters 11-6
11-A D1ox1ns In 2,4-D Samples. . 11 -6
III-l Plasma Half-Life Values of 2,4-D After Administration of
a Single Oral Dose of 2,4-D Salt or Ester Equivalent to
100 mg 2,4-D/kg III-ll
V-l Acute Toxicity of 2,4-D Compounds V-2
V-2 Subchronlc Toxicity of Orally Administered
2,4-D Compounds V-7
V-3 Tumor Incidence in Rats Fed 2,4-D V-l9
V-4 Mutagenicity Testing of 2,4-D V-22
V-5 Mutagenicity Testing of 2,4-D 1n Plants V-25
V-6 Teratogenicity of Orally Administered 2,4-D and
Derivatives of 2,4-D V-34
VIII-1 Summary of Data Used to Derive HA and AADI VI11-28
v 11
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LIST OF ABBREVIATIONS
bv Body weight
CNS Central nervous system
CSF Cerebral spinal fluid
ONA Deoxyribonucleic acid
DWEL Drinking water equivalent level
EEG Electroencephalogram
FEL Frank-effeet level
GI Gastrointestinal
HA Health advisory
1.p. Intraperitoneal
1.v. Intravenous
LD50 Dose lethal to 5OK of recipients
LDH Lactic dehydrogenase
LOAEL Lowest-observed-adverse-eFI ect level
NOAEL No-observed-adverse-effeet level
NOEL No-observed-effect level
PCDO PolychlorInated d1benzo-p-cloxlns
ppb Parts per billion
ppm Part per million
ppt Parts per trillion
RfD Reference dose
RNA Ribonucleic acid
s.c. Subcutaneous
SGOT Serum glutamic oxalacetlc transaminase
SGPT Serum glutamic pyrjvlc transaminase
STEL Short-term exposure limit
v 111
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LIST OF ABBREVIATIONS (cont.)
Threshold limit value
Time-weighted average
Volume of distribution
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I. SUMMARY
2,4-D1chlorophenoxyacet1c acid (2,4-D) (molecular weight, 221; water
solubility, 540 mg/t at 20°C; pK., 2.87) Is a white to yellow crystal-
line powder that is used as a herbicide for both terrestrial and aquatic
plants and to preven r.charvest fruit dro|> 1n citrus trees, and also to
Increase the storage life of citrus fruit aid Increase the latex output of
old rubber trees. 2,4-0 has not been shown to contain 2,3,7,8-tetrachloro-
d1benzo-£-d1ox1n (2,3,7,8-TCDD), but does contain low concentrations (<60
ppb) of some other chlorinated dloxlns. 2,4-0 1s degraded rapidly 1n water
by chemical hydrolysis, photolysis; and biological processes (the major
removal mechanism).
Toxlcoklnetlc studies have shown that most of the 2,4-D that 1s orally
administered to animals (>90%) 1s excreted In the urine unchanged within
24-48 hours, suggesting fairly rapid and tomplete absorption and little
metabolism of the compound. 2,4-D Is widely distributed following absorp-
tion, but the highest concentrations are foun-I 1n the liver, klcney, spleen,
heart and lungs and the lowest level; 1n the nuscle, brain and fat. Elimin-
ation from animals at low levels of exposure ;<100 mg/kg) follows flrstorder
kinetics, but b1phasic patterns aru observed as concentrations Increase.
Limited human toxlcoklnetlc data ar«> consistent with the animal data, but
considerable Interlndlvldual variations are ncted 1n rates of absorption and
elimination and the amount of the 2,4-0 excreted as conjugates.
01400
1-1
04/07/88
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Acute exposure to 2,4-0 by oral administration or Injection by various
routes results In progressive symptoms of muscular Incoordination, hind-
quarter paralysis, stupor, coma and death In animals. Myotonia Is a domi-
nant effect of exposure, and lethal levels of 2,4-0 have been shown to cause
kidney and skeletal muscle damage 1n rodents. Oral LD^Q values are gener-
ally In the range of 350-500 mcbw for rodents; significant differences
In toxicity are not apparent between 2,4-0 and Its salts and esters.
Subchronlc oral administration of 2,4-0 at dally doses of -15.0 mg/kg/
day caused alterations In hematology, 1n kidney and brain weights, and
alterations In pituitary and adrenal weights, as well as In the liver
enzymes LDH, SG0T, S6PT and alkaline phosphatase, which were biologically
and stat- Istlcally significant 1n mice and rats. Effects at 45 mg/kg/day
or higher Included GI disturbances as well as acute toxicity to hepatic
tissues. Effects of higher doses included GI Irritation and mild hepatic
effects as well as symptoms and signs characteristic of acute exposures.
Dogs appear to be more sensitive and guinea pigs less sensitive to
subchronlc oral administration of 2,4-0. Repeated s.c. or 1.p. injection of
100-200 mg/kg bw 2,4-0 cause pathological and functional effects 1n the
liver, kidneys, lungs, thyroid, and nervous system of rats and mice, but
systemic toxicity Is not produced by dally dermal application of 2,4-D
dlmethylamlne salt or esters to rabbits.
Chronic oral administration of 2,4-0 at levels of up to -78 mg/kg bw/day
has no effect on hematological Indices, clinical chemistry Indices or non-
tumor pathology In rats. Administration of 2,4-0 In the diet of dogs for 2
years at levels up to -14.5 mg/kg bw/day did not produce adverse gross or
hlstopathologlcal effects.
01400 1-2 04/07/88
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— »- u.s. -bi.i i i k 11 iiy aitu ui»eso ived reports of Indue .1on of lympho-
sarcoma 1n rats that were administered 2,1-0 1n the diet a: levels In the
range of 0.25-62.5 mg/kg bw/day for 2 years, but administration of 2,4-0 or
the Isopropyl, butyl or Isooctyl esters by intubation before weaning (*6-100
ing/kg bw/day) and subsequently 1n the diet (-14-42 mg/kg bw/day) for 73-90
weeks was not tumorlgenlc. Rats or mice led 2,4-0 amine salt at 0.10% of
the LDgg level for life reportedly did not develop a significant increase1
In tumors. Single s.c. Injections of 2,4-) isooctyl ester here associated
with reticulum cell carcinomas 1n mice after 78 weeks of latency, but
similar Injections of 2,4-0 acid or the 1)opropyl or Isobutyl esters were
not tumorlgenlc. Repeated dermal applications of 2,4-0 to mice produced
skin papillomas only when treatment was preceded by application of the
Initiator 3-methylcholanthrene. A 1985 1n jus try sponsored rat and mouse
bloassay Is available but on an Interim basis has not been critically
evaluated In this document. Note Is made that EPA's Office of Pesticide
Programs, 1n a March 23, 1988 Federal Register Notice propeses that the
available animal evidence be viewed as inadequate to assess the carcinogenic
potential 1n animals.
Five epidemiologic studies provide evidence of cancer induction from
exposure to a class of compounds-chlorophenuxy herbicides. Hoth EPA and
IARC have Judged this evidence to be limited (iccordlng to welghl-of-ev1dence
guidelines. The evidence for 2,4-0 alone prior to a 1986 study by Hoar et
al. was clearly Inadequate, thi Hoar et al. study raises questions as to
whether the epidemiologic evidence for 2,4-0 is now more substantial.
>1400
1-3
04/13/88
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On an interim basis this document defers an evaluation of the human weight
of evidence by the CAG In recognition of additional data forthcoming from
NCI epidemiologic investigators. Note Is made that the Office of Pesticide
Programs (March 23, 1988) has proposed that the human data base Including
the Hoar et al., 19B6 findings be considered Inadequate for OPP regulatory
use. The Pesticide Programs also propose that new animal studies be
conducted and that the cancer evidence be reconsidered depending on the
findings of such studies or the availability of newer epidemiologic
Information.
2,4-D has been tested for mutagenicity In a variety of assays (e.g.,
plant, bacteria, yeast, fruit flies. In vitro and In vivo mammalian sys-
tems), but there Is a preponderance of negative and Inconsistent results In
the animal assays. It appears that these varied results may be attributed
to differences In pH. At physiological pH, 2,4-D exists In the Ionized
state, where It less readily crosses cell membranes than when In the non-
Ionized state; the Inconsistent results may Indicate that sufficient levels
of 2,4-D did not reach the target sites.
Teratogenicity testing with several species of rodents Indicates that
2,4-D and several of Us esters and other derivatives are embryotoxlc, but
only weakly teratogenic or non-teratogenlc. Malformations generally con-
sisted of cleft palate and other skeletal effects, but the threshold for
adverse fetal effects Is not clearly defined; sporadic evidence of mild
fetotoxlclty was reported In orally treated rats at doses as low as 12.5-25
mg 2,4-D/kg bw/day for both 2,4-D and 2,4-D esters. Mult1generat1on studies
Indicated that 2,4-D caused Increased mortality 1n preweanllng rats, but
produced no adverse effects on Utter size or fertility.
01400 " 1-4 04/13/88
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reports oi numans wno acute iy mgesieu iuiunons o* were exposed
to 2,4-D formulations via Industrial or agiIcultural exposure Indicate that
symptoms of gastritis, vomiting, loss of tonsclousness, neurological signs
(e.g., reflex disorders) and muscular paralysis precede death. Autopsies of
fatal poisoning cases have shown *1desprea1 pathologic effects (e.g., con-
gestion and hyperemia of most organs, hepiUc necrosis). An Inadequately
reported epidemiology study conduced that chronic exposure to 2,4-0 did not
produce adverse clinical effects.
A non-lethal single oral dose 1n mice tfat represents a lwest-observed-
adverse-effect level (LOAEL) was lined to de*1ve a 1-day HA for Ingestion of
2,4-0 In drinking water of 1.1 nig/i for children. A 10-day HA of 0.30
mg/i for children was derived froin subchror»1c NOAELs in rats. A lifetime
Drinking Water Equivalent Level (MEL) of C.35 mg/t Is reconmended at this
time based on a subchronlc rat N0AEL.
01400
1-5
04/13/86
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II. PHYSICAL AND CHEMICAL PROPERTIES
Description
2,4-D1chlorophenoxyacet1c acid (2,4-0) 1s a white to yellow crystalline
powder that 1s used almost exclusively as an herbicide and to'Increase the
latex output of old rubber trees, prevent preharvest drop 1n citrus trees
and Increase storage life of citrus fruit (Hawley, 1977; Ayers et al.. 1976;
Bovey and Young, 1980). It Is also used as an herbicide for aquatic plants.
The chemical structure of 2,4-0, the Chemical Abstracts Service (CAS)
Registry Number and the Registry of Toxic Effects of Chemical Substances
(RTECS) number are given below.
CI
CI
CAS Number: 94-75-7
RTECS Number: AG6825000
Physical Properties
The physical properties of 2,4-0 and four derivatives are presented 1n
Table II-l.
Solubility 1n Organic Solvents (Herbicide Handbook. 1979)
acetone - 850,000 mg/l at 25*C
ethanol (95X) - 1,300,000 «g/l at 25'C
isopropanol - 316,000 lug/1 at 31 *C
benzene - 10,700 mg/l at 28*C
01410
II-l
02/14/87
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TABLE
Physical Properties of
II-l
Som 2,4-0 Compounds*
Melting lolling Vapor
flolecular Molecular Point Point Pressure Physical Mater
CoMpfuntfs Structure formila Weight (*C) (*C) Density (Torr) State Solubility
2,4-0
Ar0CH?C00H
2,4-0 Sodltfi ArOCH^COOKa
Salt
2,4-0 Dlaethyl- ArOCHjCOONHitCHjlz
aslno Salt
CflMfcCljOj 221.04 140-141
m-itt
(tech.)
CioHnC)2M°) 2M.12 85-07
HO
(at 0.4 torr)
1.57
(at 30*C)
HA
CjN;C1jOjMa 243.03 211-218 HA
HA
RA
MA
white S40 ¦g/t at 20-C
crystals, (flelnlkev, 1971);
odorless 72S «g/t at 2S'C
when pure (ftalley and Uhlti
1165J; 900 ag/l
at 2S*C (Herblclt
Handbook, 1979)
white
27.S g/100 «
crystals H»0 at 0*C:
33.5 «/100 g
H?0 at 20*C:
SO.t a/100 a
H?0 at 30*C"
10~» at 28*C& white 300 «/100 g
odorless HjO at 20*C
crystal-
i«.. ..iij
2,4-0 Butoxy- ArOC«2COOCH2CH2OCH2CH?CH2CH3 CuHijCljOa 321.20 MA
ethyl Ester
2.4-0 n-Butyl ArOCHjCOOCHjCHjCHpCNj
Ester
Ci2NuCl203 277.15 9
158-182
(at 1-1.5 torr)
185-190
(at 5.5-7 torr)
146-147
(at 1 torr)
HA see Table 11-2 viscous, 12 «g/t at 25*C
colorless,
odorless
liquid
triien pure
1.235-1.245 see Table II-2 clear, 4* ag/ft at 25*C
at 25*C
colorless
irfien pur#
iwi; ner»ici«e Handbook, 1979; MelnHov, 1971; Zepp et al., 1975
No reference or experlNntal wthod was provided for this value
tech. « technical
Ar •
rAr
V
CI
CI
-------
toluene - 6,700 mg/i at Z5*C
xylene - 5,800 mg/i at 25*C
diethyl ether - 270,000 mg/t at 25'C
dloxane - 785,000 mg/l at 31"C
n-heptane - 1,100 mg/t at 25*C (Helnlkov, 1971)
carbon te*r-:hlor1de - 1,000 mg/l at 25*C
carbon dl uiflde - 5,000 mg/l at 29'C
P*a Values for 2.4-0
The pK values for 2,4-0 1n water at 25'C are listed below with the
O
method of determination (2,4-0 1s a weakly acidic herbicide, pK Is the
negative log of the Ionization constant):
pKa Method Reference
2.87+0.6
spectrophotometry
Cessna and Grover, 1978
2.73
potentlometrlc
Nelson and Faust, 1969
2.96
potentlometrlc
Wedding et al.t 1954
2.90
conductlmetrlc
Hatell and Undenfors, 1957
2.92
conductlmetrlc
Wershaw et al., 1967
3.28
unspecified
Andus, 1949
According to Cessna and Grover (1978), their spectrophotometry method
yields the most accurate pK value claiming that potentlometrlc tratlon
d
1s not Ideal for many herbicides because of their low solubilities; iat the
conductlmetrlc method, although suitable at low concentrations, has to be
performed at a number of dilutions, with each conductlmetrlc value requiring
different activity corrections; and that activity corrections are quite
tedious. This results In the wide range of pK values (Cessna and Grover,
a
1978).
01410
11-3
07/30/84
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Vapor Pressures of 2.4-D Esters
Vapor pressures of several 2,4-C esters
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TABLE 11-2
Summary of Vapor Pressure Oata for Various 2,4~D Esters*
Vapor Pressure
Temperature
Ester
*
(mm
Hg)
26.6
Boiling point determinations
Mulllson and Hummer, 1949
12.70
X
10~»
25
Transpiration
Warren and Gillies, 1952
2.30
X
10~>
25
Gas liquid chromatography
Jensen and Schall, 1966
Ethyl
0.86
X
10~»
26.6
Boiling point determinations
Mulllson and Hummer, 1949
11.50
X
10"»
25
Transpiration
Warren and Gillies, 1952
1.10
X
10"*
25
Gas liquid chromatography
Jensen and Schall, 1966
n Propyl
7.20
X
10~"
25
Transpiration
Warren and Gillies, 1952
Isopropyl
1.20
X
10"9
26.6
Boiling point determinations
Mulllson and Hummer, 1949
10.50
X
10"»
25
Transpiration
Warren and Gillies, 1952
1.05
X
10~2
25
Transpiration
Vernettl and Freed, 1963
1.40
X
10"'
25
Gas liquid chromatography
Jensen am chall, 1966
4.60
X
10~5
25
Gas liquid chromatography
Flint et a , 1968
2,32
X
10"«
25
Knudson cell
Kybett et a.., 1976
n-Butyl
4.50
X
10~»
25
Radioactive tracer
Warren and Gillies, 1952
3.92
X
10~»
25
Transpiration
Vernettl and Freed, 1963
3.97
X
10"«
25
Gas liquid chromatography
Jensen and Schall, 1966
6.16
X
10"'
25
Knudson cell
Kybett et al., 1976
n-Pentyl
3.00
X
10~»
25
Transpiration
Warren and Gillies, 1952
n Hexyl
2.10
X
10"»
25
Iransplratlon
Warren and Gil lies, 1952
fi ¦' y 1
1.35
X
10"»
25
Radioactive tracer
Warren and Gillies, 1952
0.02
X
10 s
2S
Gas liquid chromatography
Jensen and Schall, 1966
h tuecyl
0.40
X
10"«
C J
Transpiration
Warren and Gillies, 1952
2-Ethyl hexyl
2.00
X
10~*
25
Gas liquid chromatography
Flint et al., 1968
Isooctyl
2.00
X
10"*
25
Gas liquid chromatography
Flint et al., 1968
7.06
X
10*
25
Knudson cell
Kybett et al., 1976
Butoxy ethyl
1.70
X
10"»
25
Radioactive tracer
Warren and Gillies, 1952
4.50
X
10~«
25
Gas liquid chromatography
F lint et al., 1968
Propylene glycol
3.00
X
10"*
25
Gas liquid chromatography
Flint et al., 1968
butyl ether
'Source: NRCC, 1978
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TABLE II-3
Conversion factors for 2f4-D Esters
Ester
Conversion factor
Methyl
i ppm =9.66 mg/m3
1 mg/m3 - 0.104 ppm
Ethyl
1 ppm = 10.2 mg/m*
1 mg/m' * 0.0978 ppm
n-Propyl
Isopropyl
1 ppm = 10.8 mg/m3
1 mg/m3 = 0.0926 pam
n-Butyl
1 ppm « 11.4 mg/m3
1 mg/m3 = 0.0879 pwn
n-Pentyl
1 ppm = 11.95 mg/m1
1 mg/m* - 0.0837 pj>m
n-Hexyl
1 ppm = 12.5 mg/m3
1 mg/m3 * 0.0798 pi>m
n-Heptyl
1 ppm =13.1 mg/m3
1 mg/m3 * 0.0763 ppm
TrIdecyl
1 ppm = 16.5 mg/m3
1 mg/m3 = 0.0605 ppm
2-Ethyl-hexyl
Isooctyl
1 ppm = 13.7 mg/m3
1 mg/m3 - 0.0731 ppm
Butoxy ethyl
1 ppm ¦ 13.2 mg/m3
1 mg/m3 * 0.0413 ppm
Propylene glycol butyl ether
1 ppm * 14.4 mg/m#
1 mg/m3 = 0.0694 ppm
01410
] 1-6
07/30/84
-------
pH 9.0 and 6.0 were 1.1 hours and 44 days, 17.0 hours and 710 days, 5.2
hours and 220 days, 5.2 hours and 220 days and 37 hours and 1500 days,
respectively. 2,4-0 esters are photolytlcally degraded In water by >290 nm
light, but limited data suggest that this rate Is significantly slower than
hydrolysis (Blnkley and Oakes, 1974a t b; Zepp et al.. 1975). Although
abiotic degradation of 2,4-D may be rapid and Is clearly a significant
removal mechanism, 2,4-0 appears to be degraded predominantly by biological
processes {half-life of 1-2 weeks once blodegradatlon Is detected) (Aly and
Faust, 1964; Demarco et al., 1967; Boethllng and Alexander, 1979).
D1ox1ns In 2.4-D
A number of studies have reported the presence of chlorinated dlbenzodl-
oxlns In phenoxy herbicides Including 2,4-D (Table II-4). In addition to
these samples with positive contamination, Cochrane et al. (1980) analyzed 1
mixed butyl ester, 3 dlmethylamlne salts and 10 acid samples that contained
no detectable dloxlns. Thomas (1980) detected dlchlorodlbenzodloxlns In 3
of 30 samples. Norstrom et al. (1979) analyzed five older 2,4-D samples and
found no dloxlns. There are few toxicology data available on these dloxlns.
Summary
2,4-0 Is a crystalline powder used as an herbicide for both terrestrial
and aquatic plants. It does not volatilize from water (although some of the
esters are volatile) but 1s not persistent In water. Blodegradatlon 1s the
major removal mechanism from water, although chemical hydrolysis of the
esters 1s rapid at basic pH and photolysis may be significant. 2,4-D has
not been shown to contain 2,3,7,8-TCDD but does contain low concentrations
(<60 ppb) of some other chlorinated dloxlns.
01410
11-7
02/14/87
-------
TABLE 11-4
Dloxtns In 2,4-D Samples
Concentration of PCDDs
2,4~D 1,3,6,9- or 2,3,7,8- Country of
f,4-D Form Acid X 01- Tr1- 1,3,6,8-Tetra- Tetra- He*a- Manufacture Reference
(ppb) (ppb) (ppb) (ppb) (ppb) (ppm)
Actd
44.6
NO
NO
NO
NO
NA
Canada
Cochrane
et a
.. 1980
NR
NO
ND
ND
NO
NA
Canada
Cochrane
et a
.. 1980
NR
NO
NO
ND
ND
NA
Canada
Cochrane
et a
.» 1980
41.3
NO
NO
ND
ND
NA
Canada
Cochrane
et a
.. 1980
NR
NO
NO
ND
ND
NA
Canada
Cochrane
et a
1980
41,7
NO
NO
NO
ND
NA
Canada
Cochrane
et a
.. 1980
37.9
NO
NO
NO
ND
NA
Canada
Cochrane
et a
., 1980
NR
ND
NO
NO
ND
NA
Canada
Cochrane
et a
19B0
42.44
316
490
132
NO
NA
Canada
Cochrane
et a
.. 1980
43.10
275
587
136
ND
NA
Canada
Cochrane
et a
., 1980
NR
ND
NO
ND
ND
NA
Canada
Cochrane
et a
1980
NR
NO
ND
NO
ND
NA
Canada
Cochrane
et a
., 1980
42.9
409
551
210
ND
NA
r»ni
»tt
200
104
238
109
NAb
474
346
226
ND
ND
Canada
Ccchrcr.r -» -»
I70C
*9
I11
ND
ND
Canada
Cochrane et al.
1902
632
1752
ND
NO
Canada
< jthrane et al.
1902
639
315
NO
ND
Canada
hrane et al.
1982
825
852
ND
ND
Canada
wchrane et al.
1982
929
486
ND
ND
Canada .
Cochrane et al.
1902
NAb
384
ND
ND
Canada
Cochrane et al.
1981
450
8730
ND
ND
Canada
Cochrane et al.
1981
-------
TABU II-4 (cont.)
Concentration of PCODs
2,4-0 for*
2.4-0
1,3,6,9- or
2,3,7,8-
Country of
Acid X
01-
Trl-
1,3,6,8-Tetra-
Tetra-
Heua-
Manufacture
Reference
(ppb)
|ppt>>
(ppb)
Not analyzed due to coelutlng Interferences
HA - Not analyzed; NO - Not detected at <1 ppb; N8
Not reported
-------
III. TOXICOKINETICS
Absorption
Available data for nonrumlnant mammal? Irdlcate that absorption of 2,4-0
from the GI tract 1s rapid and virtually complete.
Erne (1966a) administered various forms of 2,4-0 by gavage to rats and
pigs that were fasted overnight. When a single oral dose oE 2,4-D tr 1 -
ethanolamlne salt equivalent to 100 mg/kg 2*4-0 was given to male rats, peak
plasma concentrations of 200 yg/mi 2,4-0 w?re .reached by 7 hours. Pigs
that received 50 or 100 mg/kg of the compound had peak plasma levels of 120
and 210 vg/mi, respectively, by fi hours postexposure. When rats were
given single doses of the potassium-sodium s<1t of 2,4-D {equivalent to 100
mg/kg 2,4-0), absorption was similar to tha\ of the 2,4-0 amine. Similar
administration of 2,4-0 butyl ester (100 mg 2,4-D/kg) to rats resulted 1n
much lower plasma levels (-20 yg/rrl) than were reached at a later time
(-7 hours). Erne (1966a) suggested that Incomplete and delayed absorption
of 2,4-0 butyl ester accounted for the observed lower plasma levels, since
Its solubility In water Is poor. Only the arid form of 2(4-D, rather than
the Intact ester, could be found In plasma at any time after daslng; thus,
the author concluded that the ester underwent complete hydro* ys1s during
absorption.
Khanna and Fang (1966) reported that 93 96% of an oral duse of 3-30
mg/kg of iaC-2,4-0 (acid) to rats was excreted almost entlfely In the
urine within 24 hours of dosing. Smith et al. (1980) recovered 90% of an
administered dose 1n the urine of rats of tp to 150 mg/kg 2,4-0 as the
sodium salt 1n 12 hours and 95% In 24 hours, Indicating >95% absorption.
01420
III-l
02/15/87
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When 14C-2,4-D was administered to rats as a bolus of 10 ymole/kg (con-
taining 10 yCl/kg 1n a volume of 0.5 ml/kg) through the duodenal
cannula, -92% of the administered radioactivity was recovered In the urine
after 24 hours (Sleber, 1976).
The rapid absorption of 2,4-D observed In experimental animals has been
confirmed by studies that used human volunteers. Although Individual varia-
tion was appreciable, significant levels of 2,4-0 were detected In six men
as early as 1 hour following Ingestion of 5 mg/kg. Peak plasma levels were
obtained within 7-24 hours and averaged -35 yg/mi (Kohll et al., 1974;
Khanna and Kohll, 1977).
In a similar study by Sauerhoff et al* (1977), oral administration of
the same dose, 5 mg/kg 2„4-D, to three men also resulted 1n rapid absorption
with peak levels of between 9 and 25 yg 2,4-D/mi plasma, achieved within
4 hours after Ingestion. Values for absorption rate coefficients (k )
a
were calculated using a nonlinear parameter estimation program and were
reported as 0.165, 0.202 and 0.415 h"1 for the three subjects.
Oermal application of 4 yg/cm2 of 2,4-0 acid 1n acetone to the
forearms of six men resulted In total excretion of 5.8% of the administered
dose In the urine collected over 5 days. Measurable amounts were detected
In the urine as early as 4 hours after exposure, Indicating rapid percutan-
eous absorption (Feldman and Malbach, 1974).
An abstract of a study by Draper and Street (1982) reported dermal expo-
sure to 2,4-D (1.2-18 mg of 2,4-D was washed from the hands of men engaged
01420
III-2
02/15/87
-------
1n spray applications) resulted In maximum urinary excretion between 16 and
40 hours after the exposure. Inhalation exposure was m1n mal; further
details were not reported.
Distribution
Erne (1966a) conducted a study on the distribution of 2,4-1' as the tr1-
ethanolamlne salt using rats and pigs. The animals were giver single oral
doses of 2(4-D equivalent to 100 mg 2,4-D/kg and were killed by exsanqulna-
tlon. Tissues containing the highest levels of 2,4-D 1n both species at 6
hours were the I1ver( kidney, lungs and spleei. These levels declined after
24 hours. Brain levels of 2,4-D 1n the pics were relatively low, ~5% of
plasma levels, but could be Increased by rejeated administration of toxic
doses. Only trace amounts of 2,4-D *ere founl In fat. The but^l ester form
of 2,4-D was distributed In a similar manner, but lower tissue levels were
achieved. Apparent volumes of distribution (Vrf) of different 2,4-0 com-
pounds (amine, potassium-sodium salt, butyl ester), estimated from elimina-
tion data plotted on a sem1-logar1thulc scale, ranged between 25 and 50% of
the body weight, Intermediate between extracellular body volunre and total
body water. These estimates suggested that some of the compound entered
cells. The results of the in vitro study by Erne (1966a) support this
concept by reporting -10% of plasma levels of 2,4-0 were fouid In blood
cells.
Khanna and fang (1966) reported tissue lev?ls of 2,4-D In ra;s killed at
various times between 1 and 41 hours after receiving 1 or 80 mg of 14C
2,4-0 by gavage. Distribution was widespread; radioactivity was detected in
all of 12 tissues examined. Peak concentrations were reached In the tissues
01420
111-3
01/31/85
-------
between 6 and 8 hours at the low dose with no detectable radioactivity after
24 hours. At the high-dose level, peak concentrations were reached at 8
hours, remained fairly constant for 17 hours and then declined; however,
radioactivity was still detected after 41 hours. The hlghe-st levels of
radioactivity were found 1n blood, liver, kidney, heart, lungs and spleen,
with lower levels 1n muscle and brain. At 17 hours after the high dose and
at 1 hour after the low dose, the kidney level exceeded that of plasma. The
Intracellular distribution of 2,4-D was also Investigated In kidney, liver,
spleen, brain, heart and lunqs. Higher levels of radioactivity were found
in the soluble and nuclear fraction and relatively low levels were found 1n
the mitochondrial and microsomal fractions.
The observations of low accumulations of 2,4-D In adipose tissue (Erne,
1966a; Khanna and Fang, 1966) and the low level of absorption by Intestinal
lymphatics observed by Sleber (1976) are not surprising. Sleber (1976)
collected samples of perirenal or mesenteric fat from rats killed 24 hours
after administration of a U^lus dose of 14C-2,4-D (10 ymoles/kg) through
a duodenal cannula during Infusion with physiological salt solution. Only
1.0+1.7% of the administered radioactivity was found 1n body fat (estimated
as 12% of body weight). This finding was consistent with the low chloro-
form: buffer partition coeff1r(0.007+0.002) for 2,4-0, Indicating very
low 11p1d solubility.
Some studies (Erne, 1966a; Khanna and Fang, 1966) report that relatively
low levels of 2,4-0 were found 1n the brain. Erne (1966a) found that brain
levels of 2,4-D could be Increased by Increasing the dose. In support of
this observation are the results of Elo and YHtalo (1977, 1979), who demon-
01420
111-4
02/15/87
-------
strated that 2,4-0 levels In the brain and CSF of rats could be greatly
Increased If the animals were pretreated w< th high {250 mg/k(|) s«c. Injec-
tions of 2,4-0 sodium salt before 1.v administration of 8 vC1/kg
**C-2,4-0 with a specific activity of 0.9 wC1/mg. The measure of radio-
activity in tissues as a percentage of plasma levels 3.5 and 4.5 hours after
i4C-2,4~D administration showed plasma levels reduced to 67%, while liver
levels Increased -3.5 times, and testis, lung* heart and muscle levels
Increased -2-fold when compared with levels In sallne-pretreated controls.
Pretreatment greatly Increased the brain level by 6.5 times [2.3% In con-
trols, 15% 1n pretreated rats) and the CSI level by 23.5 t'mes (0.4% In
control, 9.4% 1n pretreated rats).
The low levels of 2,4-0 In brain tissue after a low dose is assumed to
be due to the functioning of the blood-brain barrier. Based on experiments
Investigating 2-chloro-2-methyl pherioxyacetk acid (MCPA) b1ndJng to plasma
protein .In vitro, the authors suggested that If plasma protein binding sites
are saturated by the high exposure levels, more unbound 14C-2,4-D Is free
to be distributed to tissues (Elo and Ylltal), 1979). The binding of 2,4-0
to serum protein has been confirmed by 1_n vitro studies (Mason, 1975; Haque
et al., 1975; Kuhne et al., 1979; Fang and Llndstrom, 1980). The greater
enhancement of brain and CSf accumulation of 2,4-0 at high doses may be due
to disruption (by high circulating levels o: unbound 2,4-0) of the blood-
brain barrier, with increased Influx or decreised efflux or a combination of
both processes (Elo and Ylltalo, 1979).
01420
III-5
02/15/87
-------
This hypothesis Is supported by a study by Prltchard (1980), who demon-
strated that 14C-2,4-D was capable of being actively transported by rabbit
choroid plexus preparations In vitro. The jptake of 2,4-0 by the choroid
plexus was shown to be energy dependent (Inhibited by metabolic inhibitors),
sa< ;>le, and specific for organic anions. 2,4-0 was also shown to Inhibit
the transport of 5-hydroxy-3-1ndo1e-acet1c add, a metabolite of the neuro-
transmitter, serotonin. Since 2,4-D has been found In brain tissues,
Prltchard (1980) suggested that 2,4-0 can be accumulated In the brain In
much the same way as the kidney accumulates this substance (see Elimination
Sections). Intracellular binding before efflux may account for the observed
brain levels of 2,4-0 and for the CNS toxicity.
The distribution of 2,4-D In pregnant animals has also been studied.
Undqulst and Ullberg (1971) subjected late stage pregnant mice (gestational
day not specified) to whole body autoradiography following l.v. Injection of
i«C-2,4-D (10 pCI = 0.05 mg/mouse) and killing at 5 minutes, 20 minutes,
1 hour, 4 hours and 24 hours. The autoradlograms showed that 2,4-D accumu-
lated 1n the visceral yolk sac epithelium to a small degree, entered the
fetus and was eliminated from all tissues within 24 hours.
The ability of 2,4-0 to cross the placenta was also Investigated by
Fedorova and Belova (1974), who administered a single Intragastric dose of
0.05 mg/kg **C-2,4-D to pregnant rats on the 19th day of gestation. The
rats were killed 24 hours later and th« levels of radioactivity present 1n
the uterus, placenta, fetus and Intrauterine fluid were 2.7( 3.5, 4.7 and
4.9% of the administered dose, respectively.
01420
111-6
01/31/85
-------
Erne (1966a) also found that 2,4-D crossed the placenta of a sow fed 500
ppm 2,4-0 during the entire term of pregnanry. Tissue levels of 2,4-0 found
In dead piglets (10 of 15 died) were VS yg/g of liver, 27 pg/kg of
kidney and 30 yg/g of lung. Upon delivery, the level of 2,4-0 In the
placenta was j<]/g.
Several forensic Investigations have determined levels of ?,4-D ^ human
tissue samples obtained at autopsy from people who had Ingestej fatally high
doses. Nielsen et al. (1965) determined he following tlssje levels (1n
micrograms of 2,4-0 per gram of tissue) 1ti an apparent suicide victim (a
23-year-old man who Ingested -62 g of 2,4-0): muscle, 70; spleen, 134;
liver, 183; blood, 669; kidney, 63; brain, 12.5; adipose tissue, 165.
Analysis of tissue samples taken at autopsy from an elderly man who died 6
days after ingesting an unknown quantity cf 2,4-0 revealed the following
tissue concentrations of 2,4-0 (1n yg/g tissue): blood, 57.6; brain, 93.4;
kidney, 193.4; liver, 407.9; and muscle, V7.5 (Dudley and Thapar, 1972).
Geldmacher et al. (1966) reported the following tissue levels of 2,4-0
(wQ/g tissue) In two fatalities: case one (33-year-old woman) - blood,
480; brain, 62; kidney and liver combined, 113; and case two (51-year-old
woman) - blood, 25; brain 164; liver, 116; lungs, 88; and heart, 63. Levels
of 2,4-0 determined In the tissues of a Temale suicide victim {In yg/g
tissue) were reported 1n an abstract (CoutseHnls et al., 1977): liver, 21;
spleen, 12; and kidney, 82.
Quantitative comparisons of the above Information are difficult to make
because of the unknown amounts of 2,4-0 Ingested and the uncertainty of the
time of death after Ingestion (the elderly man died 6 days afl;er Ingesting
01420
III-7
01/31/85
-------
2,4-D). There was also much variation between the victims with respect to
relative blood to kidney or liver to kidney ratios. These studies do,
however, confirm the findings 1n animal Investigations that 2,4-D 1s widely
distributed throughout the body tissues after oral administration of high
doses.
Sauerhoff et al. (1977) calculated apparent for three men who
ingested 5 mg/kg 2,4-0. The changes in plasma levels with time Indicated a
one-compartment model for subjects 2 and 3, while a two-compartment model
seemed more appropriate for the data of subject 1 (blphaslc plasma elimina-
tion curve). The Vd values were 238 and 294 ml/kg for subjects 2 and 3,
respectively. Vd-j (for the central compartment) was 83 and Vd2 (for the
slow exchange compartment) was 218 ml/kg for subject 1. These small
values Indicated that 2,4-D was not widely distributed to tissues. Kohll et
al. (1974) determined a Vd of -100 ml/kg by averaging the data from six
men who had Ingested 5 mg/kg of 2,4-D, which is additional evidence of
Uttle distribution Into the tissues of 2,4-D at low doses.
Metabolism
In studies to determine the extent of metabolism of 2,4-D, Erne (1966b)
administered the amine salt of the compound to pigs, either In 3 oral doses
of 50 mg/kg eachf 1n 23 oral doses of 50 mg/kg each, or In the feed at 500
ppm for 5 months. The percent conjugation of 2,4-D 1n the urine of 6 pigs
ranged from 0-18% conjugation as determined by differential add hydrolysis.
No correlation between the different exposure regimens and the amount of
conjugation was evident. No significant amount of conjugation was detected
1n plasma. When 2,4-D butyl ester was administered orally to pigs (either 3
01420 III-8 01/31/85
-------
or 23 doses of 50 mg/kg each) or to rats {single dose of 130 mg/kg), only
trace amounts of esterlfled compound could be detected In plasma, urine, red
blood cells or liver, Indicating complete fydrolysls of the ester linkage In
vivo. The report did not state ai; what tlrie after exposure these determina-
tions were made.
Khanna and fang (1966) found only unchanged 2,4-0 1n urine and tissue
extracts from rats given oral do<;es of -3-300 mg/kg 14C-2,4-0 when deter-
mined by paper chromatography. Counterc irrent separation of urine and
tissue extracts, however, Indicated the presence of a very small amount of a
metabolite of 2,4-D. This metabolte accounted for -0.25% o': the radioac-
tivity 1n urine samples, 0.7% In the lung extracts and 6.IX 1n the liver.
Paper chromatography of the metabolite using a 2-propanol-NH^0H-H20
solvent system gave an R^ of 0.67-0.69, c xnpared with an R^ of 0.55-0.59
for 2,4-0. Further characterization was not performed.
Whole body extracts of mice prepared after an s.c. Injection of 100
mg/kg 2,4-0, 2,4-0 butyl ester or 2,4-0 Isoictyl ester 1n dlmothylsulfoxlde
failed to show the presence of 2,4-d1chloro[>henol, suggesting that cleavage
of the ether linkage 1s not a major metabolic pathway 1n anlmcls (Zlellnksl
and F1shbe1n, 1967). 6runow and Boehme (1974) found primarily unchanged
2,4-0 In the urine of rats administered 200 mg/kg of 2,4-0; ~3% of the
administered dose was Identified a> the glycine and taurine ;onjugates of
the compound.
Limited human data also Indicate that 2 4-D does not undergo biotrans-
formation to any great extent. Of five men who Ingested 5 mg/kg of 2,4-0,
01420
111-9
07/31/84
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four excreted between 4.8 and 27.IX of the administered dose as conjugated
2,4-0. The rest of the 2,4-D excreted (82%) was detected as unchanged
compound (Sauerhoff et al., 1977). Similarly, -10% of the total 2,4-0
excreted In the urine of a man who had Ingested an herbicide containing
2,4-0 and mecoprop [I.e., 2-(4-chloro-2-m^ ^ ^phenoxy(propanoic acid], as
the amine salts, was In the form of acId-1 ab 11 e conjugates. The total dose
was unknown but was sufficient to result 1n unconsciousness and myotonia
(Park et al.# 1977). Urine samples, collected from six men who ingested 5
mg 2,4-0/kg, were analyzed by gas chromatography and found to contain no
metabolic products of the compound (KohH et al., 1974).
Elimination
The elimination of 2,4-0 (administered as the trlethanolamlne salt, the
potassium-sodium salt, or the butyl ester) from the tissues of rats and ptgs
was studied by Erne (1966a) following single oral doses equivalent to 100 mg
2,4-0/kg. Blood samples were collected at 2-3 hour Intervals for 12 hours
after dosing and then less frequently for up to 50 hours, and then were
analyzed for 2,4-D. Plasma half-Hfe values (Table III-l) were calculated
from semi 1ogar1thmlc dose-el1m1nat1on curves. The linearity of the terminal
curves Indicated first order elimination rates. As seen from Table III-l,
the elimination of 2,4-0 from the plasma 1n rats occurred at a slightly
slower rate after administration of the butyl ester than after administra-
tion of the amine salt or potassium-sodium salt. Intact butyl ester was
detected only In trace amounts In plasma; 2,4-0 acid was the predominant
form present In plasma (and tissues) after oral administration of the ester.
01420
111-10
01/31/85
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T/lBLE 111-1
Plasma Half-Life Values of 2,4-0 After Administration
of a Single Oral Oose of 2,4-0 Salt or Ester Equivalent
to IOC mg 2,4-D'kg*
Administered
Compound
Species
Plasma Half-Ufe (hours)
2,4-0 amine
Rat,
male
2.9 ~ 0.4
Rat,
female
3.3 ~ 0.5
Pig
12 t 2
2,4-D K-Na salt
R.st,
male
3.5 ~ 0.5
2,4-0 butyl ester
Rat,
male
6 + 1
Pig
10 ~ 0.8
•Source: Erne, 1966a
01420
11 [-11
07/31/84
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Erne (1966a) observed lower rates of elimination of 2,4-0 from tissue
(tissue half-life values for rats ranged between 5 and 10 hours, for pigs
between 10 and 30 hours) than from plasma. Elimination of 2,4-0 was
essentially complete within 72 hours. Thus, 2,4-0 did not accumulate In the
tissues examined (Erne, 1966a).
Zlellnskl and Flshbeln (1967) compared the differences In whole body
elimination from mice given single s.c. Injections of 100 mg/kg 2,4-0, 2,4-D
butyl ester, or 2,4-0 Isooctyl ester In dlmethylsulfoxlde. The mice were
killed various times after dosing, homogenized, and the extracts analyzed by
gas chromatography for each compound. The whole body half-life values
(assuming first order kinetics) were reported as 4.1 hours for 2,4-0, 1.1
hours for 2,4-0 butyl ester and 3.4 hours for 2,4-0 Isooctyl ester. The
esters did not appear to be hydrolyzed, as 2,4-0 was not detected upon
methylatlon of the whole mouse extracts. Differences between the results of
this experiment and the data of Erne (1966a) may be attributable to route of
administration and the use of dlmethylsulfoxlde as a vehicle by Zlellnskl
and Flshbeln (1967). Erne (1966a) administered the butyl ester of 2,4-0 as
an emulsion of the ester In petroleum solvent, with water.
Fedorova and Belova (1974) reported that, following oral administration
of 14C-2,4-0 to rats at a level of 0.05 mg/kg, 92.IX of the administered
dose was excreted In the urine within 3 days, while 6.1% of the radioactiv-
ity was detected In the feces In this time period. The study demonstrated
that 2,4-D was excreted In the milk of nursing rats given a single oral dose
of 100 mg/kg "C-2,4-0 immediately after delivery. Radioactivity was
01420
111-12
07/31/84
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detected In the GI tract of pups killed up io 7 days after birth, with the
maximum amount of 2,4-D excreted during the second or third day.
The excretion pattern of 14C-l-2,4-D or i«c-2-2,4-D -In rats was
studied by Khanna and Fang (1966i. No radioactivity was found In th?
expired carbon dioxide of rats thai, were gWen oral doses of 3-300 mg/kij,
although 93-96% and 94-98% of the radioactivity was detected In the urine
and feces within 24 and 48 hours, respectively, after dosing wHh 3-30 mg/kg
14C-2,4-D. Almost all of the 2,4-D had been excreted In the urine, with a
small unspecified amount in the "'eces. Js the dose of llC-2t4-D was
Increased (60-300 mg/kg), the percentage of radioactivity recovered In the
urine and feces declined In a linear fashion; excretion was not complete
even 144 hours after administration of 300 mg/kg.
Elimination of 2,4-D from tissues appeared to be complete within 24
hours after low doses were given (Khanna and ang, 1966). When the dose was
raised to -240 mg/kg 14C-2,4-D, radioactivity was still detected at 41
hours and the elimination appeared to be blphaslc, with the ;econd phase
becoming apparent -30 hours after dosing. The half-life values for the
Initial phase of elimination from blood, liver, kidney, heart, lungs and
spleen were averaged and reported as 3.1 hours (range: 3.0-3.5 hours) as
opposed to -0.58 hours when the dose was 3 mj/kg. These values were crude
estimates based upon levels of radioactivity determined from animals killed
sequentially at various times after dosing.
01420
111-13
02/15/87
-------
Smith et al. (I960) also observed a dose-dependent blphaslc urinary
excretion pattern of 2,4-0 In rats- A maximum urinary excretion rate of 7
mg/ltg/hour was determined for rats given single oral doses of 10-150 mg/kg
of the compound or Injected 1.v. with doses of 5 or 75 mg/kg of the 2,4-0
sodium salt. Urinary excretion showed a blphaslc pattern, with half-life
values of 2 and 21 hours estimated for the fast phase and for the slow
phase* respectively. Urinary excretion patterns became nonlinear at dose
levels >100 mg/kg 2,4-0. The observations of Khanna and Fang (1966) and
Smith et al., (1980) on delayed elimination of 2,4-0 at high doses suggest a
saturation mechanism.
Sauerhoff et al. (1977) studied the pharmacokinetic profile of 2,4-0
administered orally to five human male volunteers. Following Ingestion of c
mg/kg of the compound, the subjects excreted an average of 82% of the dost
as unchanged 2,4-0 1n the urine collected over 6 days (range: 48-97%). The
average urinary half-life of 2,4-0 was estimated to be 17.7 hours {range:
10.2-28.5 hours), using a one-compartment linear model for each Individual
set of data. Plasma clearance of the compound occurred by apparent first-
order process with an average half-life of 11.6 hours; however, one of the
three subjects Investigated showed plasma clearance kinetics that suggested
a two-compartment model rather than a one-compartment model. The researchers
did not establish the factors that accounted for the wide range of calcu-
lated kinetic values; the authors suggested that changes 1n levels of
protein binding and kidney function may be important. A delayed peak 1n
urine excretion was also observed 1n the subject for whom a two-compartment
model was Indicated (on the basis of blphaslc plasma elimination).
01420
111-14
07/31/84
-------
The elimination of 2,4-0 was s'mllarly studied by Kohll et al. (1974) 1n
six men who also Ingested 5 mg/kg of th; compound. Gas chromatography
analysis of urine samples detected 2,4-0 a; early as 2 hours after Inges-
tion. By 4 days, an average of 77% of the administered dose of 2,4-0 had
been excreted as unchanged compound. The calculated half-life for plasma
clearance was 33+3.1 hours, which represents the mean for the six subjects.
This half-Hfe value for plasma clearance 1s -3 times loncer than that
calculated by Sauerhoff et al. (1977); however, there were wide Individual
variations and small numbers of subjects 1i the two studies After l.v.
Injection of 1 yC1 of 14C-2,4-0 as a solitlon of 1 Cl/mi 1n propylene
glycol to six men, 100% of the adm'nlstered dose was recovered In the urine
after 5 days. The estimated half-life of elimination of 2,4-0 administered
by this route was 13 hours (Feldmann and Halbach, 1974).
Young and Haley (1977) analyzec data collected from a case In which a
young woman had survived the Ingestion of a nlxture of 2,4-0 anJ Dlcamba and
was being treated for the Intoxication. These Investigators made certain
assumptions: that the Initial dost?, Qq, was 12.29 g [20.1V. of 100 ml
mixture or 20.1 g x 0.9 (10% was lost becau;e of gastric lavage) x 0.6788
(67.88% of the 2,4-D salt 1s free «c1d)]; Uat by the time of lavage, all
2,4-0 had been absorbed; and an assumed time zero blood concertratlon (Cq)
of an arbitrary value higher than the fl st measured blooj level was
picked. This value, 1100 yg/mtf was used to eitlmate the V^.
0q/Zo = Vd * -12'29 9 * 11.2 %
u u 1100 yg/mt
Thus the data collected over 219 minutes fo blood and urinary levels of
2,4-0 were analyzed for best fit using an E.A.I. Pacer 500 Hybrid computer,
and a one-compartment model for 2,4-D with an "Interactive urlna'y excretion
01420
11 -15
03/27/85
-------
pathway" (kl blood-urine) to account for Dlcamba elimination. First-order
rate constants (hr~*) for the 2,4-D best-fit model were kK, , ,
blood-feces
°-01°: kblood-ur1ne = °-002: k'b1ood-uMne = °-030; ke = °"012 (wUh-
out ^'blood-urine'* ke = 0-042 (w1th k'b1ood-ur1ne)' The t1/2 was
16.7 hours when the Interactive urinary excretion pathway was not shared
with Dlcamba and 59 hours when Dlcamba used this pathway. When a "tissue
compartment" for the 2,4-D model was Included in the computer analysis, no
better fit was obtained. Even though many assumptions were made 1n the
analysis, the results supported the view that 2,4-D Is rapidly absorbed
following oral exposure, Is not accumulated to any great extent In tissues,
and Is rapidly eliminated from the body.
In urine collected over 5 days after the application of 4 mg/cm2 of
2t4-D In acetone to the forearms of six volunteers, 5.8% of the applied dose
was recovered (Feldmann and Halbach, 1974). The discussion of absorption of
2,4-D suggests that the exposure to humans engaged in 2,4-D spraying In
experiments simulating operations is mainly dermal. Draper and Street
(1982) reported in an abstract that maximum elimination of 2,4-D 1n the
urine of male ground spray applicators occurred 16-40 hours er termina-
tion of exposure. Taskav et al. (1982) reported a relatively ang retention
time for 2,4-D 1n some of 11 subjects who participated In spraying with the
herbicide, since an average of 5.05 yg of 2,4-D was detected 1n 12-hour
pooled urine samples 5 days after exposure. The results of this study were
widely variable and very poorly reported.
The half-life for elimination of 2,4-D calculated from data collected
from men engaged In agricultural spraying of 2,4-D ranged from 35-48 he
01420
111-16
02/15/87
-------
after a single exposure (Nash et al., 1982], One-time ground application of
2,4-D by 26 men resulted 1n mean 24-hour urinary excretion levels of 0.002
mg/kg for applicators, 0.003 mg/kg for mixer-loaders and 0.004 mg/kg for
mixer-load applicators. Maximum mean 24-hour urinary excretion of 2,4-0 by
17 men exposed Intermittently during aerial spraying were 0.006 mg/kg for
p1lots and 0.02 mg/kg for mixer-loaders.
Blood and urine levels of 2,4-0 have besn determined 1n humans who were
exposed to 2,4-0 during spraying operations, but exposure occurred by both
the Inhalation and dermal routes. The limited data available suggest that
exposure 1s mainly by the dermal route, but io not provide a basis for esti-
mating the contribution of the dermal route t:> total absorption.
A poorly reported study by Taskav et ill. (1982) reported mean serum
levels of 106.63 ng/ml, ranging from traci? amounts to 482 ng/mi, from
blood collected Immediately after spraying fi gallons (3.8 lb acid equiva-
lent/gal) of 2,4-0 for 3 hours b> 11 male volunteers. Exposures were
determined from denim patches on the* necks, chests and backs (dermal) and
from air filter monitors (inhalation!. Average amounts of residues on the
denim patches were 131.45 yg/sq ft, while air filter residues averaged
43.1-60.1 ppt.
Plasma levels of 2,4-D 1n four mun durlnc a workweek of spiaylng a 2%
emulsion of 2,4-0 1n kerosene ranged from undetectable amounts (<0.02
wg/mi) to 0.1-0.2 wg/ml. In this study, the exposures wiire Inter-
mittent and occurred by both dermal end 1nhalit1on routes (Kolmodln-Hedman
and Erne, 1980).
01420
111-17
01/31/85
-------
In contrast, Lavy et a 1. (1982) detected very little, 1f any, 2,4-D In
the urine of 18 men engaged 1n aerial spraying of the herbicide. Of 524
urine samples collected during the day of exposure and for 5 days afterward,
only -150 contained detectable amounts of 2,4-0. Those men with detectable
levels were crew members who actually performed the spraying. Denim patches
-..diyzed for dermal exposure and air filters analyzed for inhalation expo-
sure contained very little 2,4-0, Indicating minimal exposure by either
route.
Summary
2,4-0 trlethanolamlne and potassium-sodium salts are readily absorbed
from the GI tract of nonrumlnant mammals and reach peak plasma concentra-
tions as early as 7 hours In rats and 5 hours In pigs (Erne, 1966a). 2,4-0
butyl ester 1s less completely absorbed and appears to be hydrolyzed to the
free acid before absorption. That 2,4-0 Is rapidly and almost completely
absorbed from the gut Is suggested by reports of high urinary recoveries
(90-96%) of Intact 2,4-D within 24-48 hours In rats after oral administra-
tion (Khanna and Fang, 1966; Smith et al., 1980; Sleber, 1976). Studies
with limited numbers of human volunteers have confirmed that 2,4-0 1s
absorbed rapidly; significant levels have been detected In the plasma as
early as 1 hour and peak plasma levels have been reached as early as 4-7
hours after Ingestion (Koh11 et al., 1974; Khanna and Kohll, 1977; Sauerhoff
et al., 1977).
The presence of 2,4-0 1n the blood and urine of humans who were exposed
to 2,4-D during spraying Indicate that absorption can occur by dermal or
respiratory routes or both (Kolmodln-Hedman and Erne, 1980; Taskav et al..
01420
111-18
02/15/87
-------
1982; Lavy et al.ff 1982; Draper and Street, 1982). Rapid percutaneous
absorption Is Indicated by the detection of 2,4-D In the blood is early as 4
hours after experimental dermal application (Feldmann and Nalbacf, 1974).
Animal J1es indicate that distribution of absorbed 2f4-D Is wide-
spread and rapid, with peak tissue concentrations occurring as iarly as 6-8
hours after oral exposure (Erne, 196fia; Khanna and Fang, 1966). The highest
concentrations are found in the liver, kidney, spleen, heart am! lungs, and
the lowest levels in the muscle, brain and fat. Distribution of 2,4-D to
tissues other than those involved with excretion of the compound Is enhanced
relative to plasma levels at high doses. 2,4-D can cross the placenta of
mice, rats and sows (Lindquist and Ullberg, 1971; Fedorova and Bilova, 1974;
Erne, 1966a), an observation that 1;; notable in view of the Ionization of
2,4-D (pK -3) at plasma pH (Erne, 1966a), Forensic investigators in humans
confirm the findings of animal studies that 2,4-D is widely distributed
throughout the body tissues after oral administration of high doses (Nielsen
et al., 1965; Geldmacher et al.v 196t»; Oudley and Thapar, 1972; Coutselinis
et al., 1977).
2,4-0 appears to be excreted essentially unchanged regardlejs of dose,
route of administration or animal species (Erne, 1966b; Khanna and Fang,
1966). More sensitive detection techniques have, however, provided evidence
of metabolism to 2,4-dichlorophenol (Zielinski and Flshbein, 1S67) and of
conjugation with amino adds (Grunow and Boehme, 1974). Limited data from
humans also indicate that 2,4-D is not biotransformed to a large extent,
although some conjugation may occur (Kohli et al., 1974; Sauerhoff et al.,
1977).
01420
[i:i-19
09/19/84
-------
Elimination of low levels (<100 mg/kg) of 2,4-0 from the plasma, tissues
and bodies of animals 1s rapid (generally complete 1n 24-72 hours) and
follows first-order kinetics (Erne, 1966a). 2,4-0 Is excreted almost
completely In the urine. As the concentration Increases, blphaslc patterns
are observed, 1nd1< ...? that the saturation of some process takes place
(Khanna and Fang, 1966; Smith et al.t 1980). Tox1cok1net1c studies of
humans who Ingested 5 mg/kg of 2,4-D have estimated (using one-compartment
assumptions) a urinary half-Hfe of -17.7 hours (Sauerhoff et a 1977) and
a plasma half-life of -33 hours (Kohll et al., 1974). An elimination half-
I1fe of 35-48 hours was calculated from data collected from men engaged 1n
agricultural spraying of 2,4-0 (Nash et al., 1982).
01420
II1-20
07/31/84
-------
IV. HUMAN EXPOSURE
Humans may be exposed to chemicals such as 2,4-0 from a variety of
sources, Including drinking water, food, ambient air, occupational settings
c.d consumer products. This analysis of human exposure to 2,<-0 Is limited
to drinking water, food and ambient air because those media are considered
to be sources common to all Individuals. Even In limiting the analysis to
these three sources, 1t must be recognized that Individual exposure will
vary widely based on many personal choices and on several factors over which
there Is little control. Where one lives, works and travels, *hat one eats,
and physiologic characteristics related to age, sex and health status can
all profoundly affect dally exposure and Intake. Individuals living In the
same neighborhood or even 1n the same household can experience vastly
different exposure patterns.
Detailed Information concerning the occurrence of and exposure to 2,4-0
1n the environment 1s presented 1n .mother document entitled "Occurrence of
Pesticides 1n Drinking Water, Food and A1r" (Johnston et al., 1984). This
chapter summarizes the pertinent Information presented 1n that document 1n
order to assess the relative source contribution from drinking water, food
and air.
In the Exposure Estimation section of this chapter, available informa-
tion 1s presented on the range of hjman exposure and Intake for 2,4-0 from
drinking water, food and ambient air for the 70 kg adult male It 1s not
possible to provide an estimate of the number of Individuals experiencing
specific combined exposures from those three sources. However, the Summary
section of this chapter provides some Insight Into the rang? of Intake
values suggested by the available data.
01430 1V-1 01/03/85
-------
Exposure Estimation
Drinking Water. Levels of 2,4-D 1n drinking water vary from one
location to another. The highest level of 2,4-D monitored 1n the available
5tudies was 50 yg/i in Oregon (Elliott, 1979), below the Maximum Contam-
inant 1 - i (MCL) of 100 vg/l. In the national studies, the highest
level of 2,4-D was 1.1 yg/l (Boland, 1981). However, levels of 2,4-D In
drinking water typically appear to be lower than these levels. Analysis of
the National Screening Program for Organlcs 1n Drinking Water (NSP) (Boland,
1981) and the Rural Water Survey (RWS) (U.S.. EPA, 1984) suggests that median
levels of 2,4-D In drinking water systems would be below 0.5 yg/l, and
possibly below 0.01 yg/l, because only 1 of 117 systems sampled 1n the
NSP contained a level of 2,4-D above 0.5 yg/l, and none of 92 systems
sampled In the RWS contained a level above 0.01 yg/l. 2,4-D may not be
present In drinking water in some areas. The available monitoring data are
not sufficient to determine regional variations In exposure levels for 2,4-D.
The dally Intake of 2,4-D from drinking water was estimated using the
assumptions presented In Table IV-1 and the values presented above. The
estimates In Table IV-1 Indicate that the dally Intake of 2,4-D from drink-
ing water ranges from 0.0-1.4 yg/kg/day. However, the values presented do
not account for variances 1n Individual exposure or uncertainties 1n the
assumptions used to estimate exposure.
A tolerance level for 2,4-D 1n potable water of 100 yg/l (negligible
residues) has been established from certain specific aquatic uses of 2,4-D
(21 CFR 193.100, April 1, 1979).
Diet. Data are limited on the dietary intake of 2,4-D 1n the United
States. Dietary exposure to 2,4-D appears to be low; there have been no
findings of 2,4-0 in FDA adult market basket surveys since 1973. The
01430 IV-2 01/03/85
-------
TAIJLE IV-I
Estimated Intake of 1!,4-D from Drinking Water*
Drinking Water Concentration
Ug/i)
Intake
Ug/kg/day)
0.0
0.0
0.01
0.0003
0.5
0.014
1.1
0.031
50
1.4
~Assumptions: 70 kg adult male consuming 2 I of water/day.
01430
IV-3
01/03/85
-------
average total dally Intakes of 2,4-0, based on detectable levels of 2,4-D 1n
market basket studies performed between 1965 and 1973, were calculated to
range from 0.0006-0.07 ng/kg/day (FDA, 1982).
Detectable residues of 2,4-D were found In the FY 76 market basket
survey for toddlers. The av>- ^ dally Intake, ;ed on the residue levels
detected 1n the toddler diet In that year, was calculated to be 0.0058
ug/kg/day (FDA, 1980).
It Is expected that dietary levels of 2,4-D vary somewhat with geograph-
ical location, with higher levels occurring 1n foods from areas near the
sources of 2,4-0 exposure. However, because of Insufficient data, no
estimates could be made of variations n Intake by geographical region.
Tolerance levels for residues of 2,4-D 1n foods and 1n and on raw
agricultural commodities are presented In Table IV-2.
Air. Data on levels of 2,4-D 1n ambient air are limited. A maximum
level of 4 ng/m3 (0.004 pg/m3) of 2,4-D was reported from atr monitor-
ing studies of 16 cities (Grover et al., 1976). Using a range of air levels
of 2,4-0 of 0.0-0.004 pg/ma, the respiratory Intake of 2,4-D was esti-
mated. Assuming that a 70 kg adult male Inhales 23 m3 of air/day (ICRP,
1975), a range of respiratory Intake of 0.0-0.0013 vg/kg/day was esti-
mated. The values presented do not account for variances 1n Individual
exposure or uncertainties In the assumptions used to estimate exposure.
Sunmary
Data on the Intake of 2,4-D from drinking water, food and ambient ai~
are Insufficient for use 1n determining which of the three sources is the
major contributor to total Intake. FA0/WH0 and EPA have established accept-
able dally Intakes (ADIs) for 2,4-D of 300 and 125 yg/kg/day, respectively
01430
IV-4
01/03/85
-------
TABLE IV-;'
Tolerances for 2,4-0 1n Foods and In and Cn Raw Agricultural Commodities3
Commodity Tolerance (yg/kg)
Food
Barley, milled fractions (exc. flour) 2,000
Oats, milled fractions (exc. flour) i?,000
Rye, milled fractions (exc. flour) i!,000
Sugarcane, molasses !i,000
Wheat, milled fractions (exc. flour) 11,000
Raw agricultural commodity
Apples '.1,000
Asparagus 5,000
Avocados 100 Nc
l,000d
Barley
forage 20,000
grain 500
8lueberr1es 100
Cattle
fat 200
kidney 2,000
meat byproducts (exc. kidney) 200
meat 200
CItrus fru1ts 100 Nc
1,000d
Including pre- and post-harvest 5,000
Corn
fodder 20,000
forage 20,000
fresh. Including sweet** 500
grain 500
Cottonseed 100 Nc
l,000d
Cranberries 500
01430
IV-5
01/03/85
-------
TABLE IV-2 (cont. )
Commodity Tolera.nce (yg/kg
Cucurbits 100 Nc
1,000^
Eggs 50
F1sh 1,OOOd
l,G00f
Fruits
pome 100 Nc
1,000^
small 100 NC
l,000d
stone 100 Nc
1,000d
Goats
fat 200
kidney 2,000
meat byproducts (exc. kidney) 200
meat 200
Grain crops 100 Nc
1 ,000<*
Grapes 500
Grasses
forage 100 Nc
l,000d
hay 300,000
pasture ^ ,000,000
rangeland 1,000,000
Hogs
fat 200
kidney 2,000
meat byproducts (exc. kidney)
200
meat 200
Hops
100 Nc
l,000d
01430
IV-6
01/03/85
-------
TABLE IV-2 (cont.)
Conmodlty Tolerance (yg/kg)
Horses
fat 200
kidney ;?,000
meat byproducts (exc. kidney) 200
meat 200
Legumes, forage 100 Nc
",000d
Lemons, post-harvest f>,000
M1Ik 100
Nuts 100 Nc
l.OOO*
Oats
forage 20,000
grain 500
Pears 5,000
Potatoes 200
Poultry 50
Quinces 5,000
Rice 100
straw 20,000
Rye
forage 20,000
grain 500
Sheep
fat 200
kidney 2,000
meat byproducts (exc. kidney) 200
meat 200
Shellfish l,000d
Sorghum 500
fodder 20,000
forage 20,000
01430 IV-7 01/03/85
-------
TABLE IV-2 (cant.)
Commodlty
Tolerance Ug/kg)
Strawberries
50
100 N<-
l,000d
Sugarcane
forage
2,000
20,000
Vegetables
fru1ting
100 Nc
1,000d
100 Nc
l,000d
100 Nc
l,000d
TOO Nc
1,000d
leafy
root crop
seed and pod
Wheat
forage
grain
20,000
500
aSources: 40 CFR 180.142, July 1, 1981; 21 CFR 193.100, April lf 1979.
^Kernel plus cob with husks removed
cFrom application to irrigation ditch banks in the Western United States
^From application to control water hyacinth
eFor 2,4-D and/or its metabolite 2,4-d1ch1orophenol
^From application for Eurasian water milfoil control In dams and reser-
voirs of the TVA system
N = Negligible residues
01430 1V-8 01/03/85
-------
(FDA, 1982). In addition, EPA has reported a maximum safe level of 2,4-0
(from all sources) of 16 yg/kg/da> (U.S. EPA, 1976). The Intake of 2,4-0
from drinking water, food and air appears to be below these levels.
References
Boland, P.A. 1981. National screening program for organics In drinking
water. Part II. Data. Prepared by SRI International, Menlo Park, CA, for
Office of Drinking Water, U.S. Environmental Protection Agency, Washington,
OC. EPA Contract No. 68-01-4666.
Elliott, k.M. 1979. Portland's public water supply -- pure and simple.
Proceedings of the National Conference on Environmental Engineering. ASCE,
New York.
FDA (Food and Drug Administration). 1900. Compliance program report of
findings. FY 77 total diet studies -- Infants and toddlers (7320.74), Food
and Orug Administration, U.S. Department of Health, Education and Welfare,
Washington, DC.
FDA (Food and Drug Administration). 1982. The FDA surveillance Index.
Bureau of Foods, Food and Drug Administration, Washington, 0C.
Grover, R.t L.A. Kerr, K. Wallace, K. Yoshlda and J. Mayfcank. 1976.
Residues of 2,4-D 1n air samples from Saskatchewan: 1966-1975. J. Environ.
Set. Health. SI 1 (4):331-347.
01430
JV-9
01/03/85
-------
ICRP (International Coimilsslon on Radiological Protection). 1975, Report
of the Task Group on Reference Man. Pergamon Press, New York. ICRP Publi-
cation 23.
Johnston, P., F. Letklewicz, 0. Borum, et al. 1984. Occurrence of pesti-
cides 1n drinking water, food and air. Interim draft report. Prepared by
JRB Associates, McLean, VAf for Office of Drinking Water, U.S. Environmental
Protection Agency, Washington, DC.
U.S. EPA. 1976. National Interim primary drinking water regulations.
Office of Water Supply, U.S. Environmental Protection Agency, Washington,
DC. EPA 570/9-76-003.
U.S. EPA. 1984. Rural water survey. Computer data provided by Department
of Sociology, Cornell University, Ithaca, NY.
01430
IV-10
01/03/85
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V. HEALTH EFFECTS IN ANIMALS
Acute Toxicity
Acute toxic levels of 2,4-D and certain of Its salts and esters by
different routes of administration are summarized 1n Table V-l. The LD—
Jv
range 1s generally betwee - and 1000 mg/kg; there do not appear to be
significant differences 1n toxicity between the free acid and the various
salt and ester derivatives. Hill and Carlisle (1947) determined oral
LD5Qs of 666, 375, 800 and 1000 for 2,4-0 sodium salt 1n rats, mice,
rabbits and guinea pigs, respectively; the maximum doses 1n these species
not causing death were 333, 125, 200 and 333 mg/kg, respectively. Individu-
al monkeys that were fed single doses of -286 or 428 mg/kg oF 2,4-0 sodium
salt or 286 mg/kg of 2,4-0 ammonium salt regurgitated a large portion of the
material, precluding determinations of lethal doses (H111 and Carlisle,
1947). Symptoms other than nausea were not described In these monkeys.
Approximately 214 mg/kg of 2,4-0 sodium salt was fed to another monkey with-
out development of vomiting or "serious Illness" (Hill and Carlisle, 1947).
Comparison of the species sensitivity to 2,4-0 Indicates that dogs may show
greater sensitivity to this compound (Rowe and Hymas, 1954). This greater
sensitivity observed 1n dogs may reflect an Inability of kidney processes In
this species to effectively clear phenoxyacetlc acids (Selter, "978).
H111 and Carlisle (1947) noted that fatal poisoning of several types of
laboratory animals (mice, rats, guinea pigs, rabbits and monkeys) with the
sodium and ammonium salts of 2,4-0 produced similar symptoms. Animals died
within several hours to 3 days following oral or 1,p. administration of the
salts. Progressive symptoms Incltded muscular Incoordination, lethargy,
paralysis of the hindquarters, stupor, coma and death. The authors noted
01440 V-l 01/31/85
-------
2.4-0 for*
Vehicle
Route
At Id
Acid
Acid
Ac Id
Acid
Sodium salt
Sodium salt
Sodlun slit
Sodium salt
Sodlun salt
Sodium salt
Sodium salt
Sodlun salt
Sodlun salt
Sodlun salt
Sodlun salt
Sodium salt
olive oil oral
olive oil oral
olive oil oral
gelatin oral
capsule oral
saline oral
saline oral
saline oral
saline oral
H?0 oral
H20 oral
saline l.p.
saline l.p.
saline l.p.
saline 1.p.
saline l.v.
saline s.c.
TABLE V-1
Acute Toxicity of 2,4-D Compounds
Species
Dose
Response
Reference
mice
368
Lt»50
Rowe and Hynas, 1954
guinea pigs
469
IO50
Rove and Hynas, 1954
rats
375
1050
Rowe and Hynas, 1954
rats
666
333
3/4 deaths
0/4 deaths
Hill and Carlisle, 1947
dogs
100
Ll>50
Drill and Hlratrka, 1953
mice
375
125
LDS0
tolerated dose*
Hill and Carlisle, 1947
guinea pigs
1000
333
I0«o
tolerated dose*
m«" and Carlisle, 1947
rabbits
000
200
">50
tolerated dose*
H1 and Carlisle, 1947
rats
666
333
iflso
tolerated dose*
Hill and Carlisle, 1947
rats
BOS
L&50
Rowe and Hynas, 1954
guinea pigs
551
l05O
Rowe and Hynas, 1954
mice
375
125
toferated dose*
HI 11 and Carlisle, 1947
guinea pigs
666
333
to50
tolerated dose*
Hill and Carlisle, 1947
rats
666
25
LDS0
tolerated dose*
Hill and Carlisle, 1947
rabbits
400
200
Lt>$0
tolerated dose*
Hill and Carlisle. 1947
rabbits
400
200
l»50
tolerated dose*
H111 and Carlisle, 1947
mice
2B0
lD5G
Bucher, 1946
-------
TABU V-1 (cont.)
2,4-0 For*
Vehicle
Route
Species
Oose
<*g/kg)
Response
Reference
AdviionluM sail
saline
oral
rats
333
0/6 deaths
Hill and Carlisle. 1947
salt
saline
oral
guinea pigs
33
0/6 deaths
Hill and Carlisle, 1947
kmanim salt
saline
oral
rabbits
200
0/4 deaths
Hill and Carlisle, 1947
Isopropyl ester
olive oil
oral
mice
541
ID50
Rowe and Hyiaas, 1954
Isopropyl ester
olive oil
oral
rats
700
L0S0
Rowe and Hyaas, 1954
Isopropyl ester
olive oil
oral
guinea pigs
550
lD50
Roue and Hyuas, 1954
Butyl esters
corn oil
oral
¦Ice
713
LO50
Rowe and Hyuas. 1954
Butyl esters
corn oil
oral
guinea pigs
B48
ID50
Rowe and Hyws, 1954
Butyl esters
corn oil
oral
rabbits
4?4
u>so
Rowe and My-e:,
Butyl esters
corn oil
* %
Vf
rats
620
LO50
Rowe and Hyaas, 1954
Butyl esters
NR
oral
cats
020
lo50
Konstantlnova, 1970
Butyl esters
MR
oral
rats
920
id50
Konstantlnova, 1970
Butyl esters
MR
oral
•Ice
380
LD50
* intlnova, 1970
PMC esters
corn oil
oral
rats
570
LO50
Rc. ¦ and Hyws, 1954
Crotyl ester
NR
oral
¦Ice
580
t°so
Fetlsov, 1966
Crotyl ester
NR
oral
rats
452
LD50
Fetlsov. 1966
'Tolerated dost - largest amount caus'ng no deaths
MR - Not reported; PfiBC - propylene glyol butyl ether
-------
that the skeletal muscle changes resembled those observed In congenital
myotonia. Pathological examination showed cloudy swelling and enlargement
of the kidneys In ?11 species; liver damage (centrllobular degeneration and
parenchymal damage) was noted only 1n dogs succumbing to massive doses of
2,4-D.
Drill and Hlratzka (1953) described myotonia with pathologic changes of
GI mucosa Irritation, moderate hepatic necrosis and mild renal tubular
degeneration In dogs that were lethally poisoned by acute oral administra-
tion of 2,4-D at doses of 100-400 mg/kg.
Bucher (1946) found that myotonia persisted for 8-24 hours In strain A
mice that were Injected 1.p. with sublethal doses (100-200 mg/kg) of 2,4-D.
No significant differences were found In the effects produced when 2,4-D was
administered s.c., I.p. or l.v.
Subchronlc Toxicity
In a Dchronlc exposure feeding study, 200 B6C3F^ mice were sorted
Into five groups of 40 animals, 20 of each sex per group, and fed the diet
chow mixed with 97.5% pure 2,4-D (Hazelton Laboratories, 1983). Mice were
fed 0.0 (controls), 5.0, 15.0, 45.0 or 90.0 mg/kg/day (calculated doses) of
the diet-chemical mixture for 91 days. Criteria used to determine toxicity
were survival, dally exam for clinical symptomology, weekly changes In body
weights, growth rates t food Intake, ophthalmological alterations, organ
weight changes, and clinical, gross and hlstopathologlcal alterations.
01440
V-4
02/14/87
-------
The results of the study demonstrated Increases 1n mean white blood cell
counts (both sexes) at 15.0 mg/kg/day, and Increased platelet and reticulo-
cyte counts {females only) at the 15-45 mg/kg/day dose levels. There were
also statistically significant reductions 1n absolute brain Weights {males
only) at the 15.0 mg/kg/day or higher dose. Alteration of relative brain-
to-body weight was found (female'; only) at the 15.0 mg/kg/day si higher
groups. Statistically significant reductions In kidney weights (absolute
and relative) were found In males (45.0 mg/kg/day or higher) and In females
at the 90.0 mg/kg/day dose level. Statistically significant reductions 1n
liver weights were found only 1n the 90.0 mg/kg/day groups of males. Pitui-
tary glands were hypertrophled (statistically significant) (absolute and
relative weights) In both sexes at 15.0 or 90.0 mg/kg/day groups. Adrenal
glands were also Increased (statistically significant) at the 15.0 mg/kg/day
dose In males and at the 5.0 mg/kg/day dose In females. Ovarian weights
were reduced (statistically significant) In the 15.0 mg/kg/day dose level.
One control female died at week 1? and one female In the high-dose group
died at week 1. These deaths were rot treatment-related.
Hematological* hepatic and renal toxicity were demonstrated 1n a sister
study In Fischer rats (strain #344) during a subchronlc exposure feeding
study performed at the Hazelton Laboratories (1983). 2,4-D (97.5% pure) was
added to the diet chow and fed to the rats for 91 days at doses calculated
to be 0.0 (controls), 1.0, 5.0, 15.0 or 45.0 mg/kg/day. In each of the five
groups were 20 animals of each sex, 40 animals per treatment group or a
total of 200 animals. Criteria examined to determine toxicity were surviv-
al, dally exam for clinical symptomology, weekly change In tody weights,
growth rates, food Intake, ophth
-------
The results of the study demonstrated statistically significantly reduc-
tions 1n mean hemoglobin (both sexes)> mean hematocrit and red blood cell
levels (both sexes), mean reticulocyte coun*s (both sexes), mean platelet
counts (females only) and mean leucocyte levels (males only) at the 5.0
mg/kg/day dose or higher after 7 weeks. There were also statistically
significant reductions 1n liver enzymes LDH, SGOT, S6PT and alkaline phos-
phatase at week 14 1n animals treated at the 15.0 mg/kg/day or higher doses.
Kidney weights (absolute and relative) showed statistically significant
Increases In all animals at the 15,0 mg/kg/day dose or higher at the end of
the experimental protocol, Hlstopathologlcal examination correlated well
with kidney organ weight changes showing cortical and subcortical pathology.
Increases In ovarian weights, T-4 levels and a decrease In BUN were reported
but not considered to be treatment related.
Rowe and Hymas (1954) administered doses of 0, 30, 100 or 300 mg/kg/day
2,4-0 to groups of 5 or 6 female rats (strain not specified) by Intubation 5
times/week for 4 weeks (Table V-2). The 2,4-0 was administered In olive oil
that was emulsified 1n 5-10% aqueous gum arable, and the controls were
vehicle treated. Rats that received 30 mg/kg/day or less reportedly showed
no adverse treatment-related clinical or pathological effects; but treatment
with 100 mg/kg/day elicited 61 Irritation, depressed growth rate and slight
cloudy swelling of the liver. Rats that received 300 mg/kg/day 2,4-D suc-
cumbed rapidly (additional details were not given) and died; severe GI
irritation was reportedly the principal adverse effect observed.
01440
V-6
02/14/87
-------
TABLE V 2
Subchronlc Toxicity of Orally Administered 2,4-0 Compounds
Specles/
Strain
Sex/No
Vehicle
Purity
Dosage/Exposure
Dose*
(mg/kg/day)
Response
Reference
R1ce/B6Cy j
?0 each sex
per group
diet chow
97.5*
NS
6.0, 15, 45 Significant reduction In
or 90 brain weight and Increased
white blood cell counts at
IS mg/kg bw/day or higher;
significant reductions In
liver weights at high doses;
pituitary gland hypertrophy
at 15 mg/kg bw/day or higher;
other effects also noted.
Hazelton
laboratories, 1983
Rats/fIscher
344
Rat/NS
young adult
Rats/
Long-Evans
?0 each sex
per group
diet chow
97.5%
NS
5-6 F/group
olive oil/ NS
gun arable
3. 30, 100 or 300
mg/kg/day by
gavage S days/week
for 4 weeks
No. ambiguous
-8 treated nr
control H/
treatment
perlod
diet
analytical
. i j
.» i«i«wa i u
grade
?000 ppm In diet
* 5, 5 or i weens
1.0, 5.0, 15 Significant reductions In
or 45 measured blood parameters
at 5.0 mg/kg bw/day or
higher; liver enzyme activi-
ties were reduced and 1n-
creA*»ft
occurred with corresponding
hlstopathology at 15 mg/kg
bw/day or higher.
?.14, 21.4, ?. 14 or 21.4 mg/kg had no
71.4, ? 14 adverse effects as judged
by growth, behavior, mortal-
ity, hematologic and BUN
values-, organ weights« and
gross and microscopic appear-
ance of tissues; 71.4 mg/kg
caused Gl Irritation, de-
pressed growth, slight cloudy
swelling In liver; 214 mg/kg
caused rapid deterioration
and death; GI Irritation
200 No effect on food
tton, no overt signs of
toxicity, slight Increase
in amount glycogen/1Iver,
slight decrease 1n amount
RNA/liver, slight decrease
In absolute and relative
liver weights
Hazelton
Laboratories,
1983
Rowe and Hymas,
1954
rt..»
-------
1ABU V ? |cont.)
Spec lei/
Strain
Sen/No.
Vehicle
Pur H y
Dosage/Exposure
Itats/NS
young adult
Rats/NS
young
Guinea
plgs/NS
Dogs/aongrel
7 1S kg
5 f/group
7 N/group
diet
NS
diet
pur If ted
comerc lal
fc/group
sex NS
Control, 7 f;
2 mq/kq, 1 ft,
1 f; 5 ag/kg,
1 H, U;
10 ag/kg, 3 «;
?0 ag/kg, 3 A.
1 f
saline
capsule
purified
cooaerclal
98. SX
coMRerclal
100. 300. 1000.
3000 or 5000 ppa
In diet x 113 days
0, ?00 or 400 pp*
In diet x 31 days,
100 ppa x ?! days,
then 1000 ppa for
the subsequent 10
days (390 ppa IMA)
SO or 100 ag/day
of 2.4-0 sodlua
salt by Intubation
10 dally doses In
1? days
0, Z. 5, 10 or ?0
ag/kg/day, 5 days/
week x 13 weeks
Dose*
(ag/kg/day)
Response
Reference
10. 30. 100,
NA. MA
0. 10. ?0 or 40
39b
139.8 or
?S?.6C
0. 1.4, 3.6.
1.1 or 14.3
No effect at ? lowest doses
as Judged by aortallty,
growth, food consuaptIon,
hematologic jnd BUN values,
organ weights, gross and
alcroscoplc appearance of
tissues; 100 ag/kg produced
•excessive" aortallty. de-
creased growth, slight
cloudy swelling In liver,
food consuaptIon NS at this
dose; at ? highest dosages,
rats refused to eat
No adverse effects as Judged
by food consuaptlon, growth,
aortallty or other charac-
teristic signs of Intoxica-
tion (I.e., auvcular signs
or paralysis)
No treataent-related signs
of Intoxication or aortallty
1.4, 3.6 and 7.1 ag/kg
caused no signs of toxicity,
no significant effects on
body weight, organ weight,
gross and histological
appearance of organs, and
heaatologtc values; 14.3
ag/kg caused death of 3 by
day 49, ataxia, stiffness
of hind legs, difficulty In
swallowing, no significant
lesions
ftowe and Hyaas,
1954
H111 and Carlisle,
1947
H111 and Carlisle
1947
Drill and
Hlratrka. 1953
aWhen the chealcal was given In the diet, the dose was calculated by assualng that a young rat or dog consuaes the equivalent of 0.1 or 0.0?9, respec-
tively, of Its body weight per day as food.
bllae weighted average dose
C?,4-D acid-equivalent dose
NS c Not specified; NA = not applicable because food was not eaten
-------
In a related study, groups of five young adult female rets (strain not
specified) were maintained on diets that contained 0, 100, 300 or 1000 ppm
2,4-0 In the diet for 113 days (Rowe and Hymas, 1954). If U 1s assumed
that young rats consume 10% of their weight In food per day, Jie correspond-
ing daily doses are 0, 10, 30 and 100 mg/kg/day. Rats that vere exposed at
the 1000 ppm level experienced excessive mortality (not quantified),
depressed growth rate and slight cloudy swelling of the liver. These
effects were not observed at the two lowest doses (see Table V-2). Groups
of five rats that were given diets that contained higher concentrations of
2,4-D (3000 or 5000 ppm) were sacrificed after 12 days because they were not
eating and were rapidly losing welijht; examinations revealed Increased liver
and kidney weights and slight but unspecified pathologic changes.
Chang et al. (1974) reported that dietary admlnlstratlori of 2,4-0 to
rats at levels of 2000 ppm In the diet (~200 mg/kg/day) :or 4-7 weeks
produced a slight Increase 1n liver glycogen content, a slight decrease In
Hver RNA content and slight decreases in absolute and relative liver
weights, but no overt signs of toxicity {see Table V-2).
Administration of 0, 100, 200 or 400 ppm dietary 2,4-D (-0, 10, 20 or 40
mg/kg/day, respectively) to groups of seven rats for 1 month did not
adversely affect food Intake or r.ite of growth, or elicit characteristic
signs of intoxication (skeletal muscular signs or paralysis) (Hill and
Carlisle, 1947). Dietary administration of 2,4-D at a level of 100 ppm for
21 days and subsequently 1000 ppm for 10 days (average total dose -39.0
mg/kg/day) was similarly nontoxic for rats. Groups of six guinea pigs that
01440
7-9
02/14/87
-------
were given 10 dally doses of 50 or 100 mg 2,4-D 1n 12 days (-88 or 177
mg/kg/day) by Intubation also did not develop characteristic evidence of
Intoxication.
Drill and Hlratzka (1953) administered 2,4-D orally in capsules to
groups of 2-4 dogs at doses of 0, 2, 5, 10 or 20 mg/kg/day, 5 days/week, for
13 weeks. When adjusted for a 7-day week, the respective dally doses were
0t 1.4, 3.6, 7.1 and 14.3 mg/kg/day. As detailed In Table V-2, toxic
effects were only observed at the high dose. Treatment at 20 mg/kg/day pro-
duced death 1n 3/4 dogs between days 18 and 49, and symptoms In the moribund
animals Included hind leg stiffness, ataxia, weakness, gum bleeding and
difficulty In chewing and swallowing. A terminal decrease 1n the percentage
of blood lymphocytes was noted 1n the three dogs that died, but significant
effects on the hemoglobin, red cell count or total white cell count were not
observed. The dog that survived 2,4-D treatment at the high dose, as well
as the dogs exposed to the lower levels of 2,4-D, showed no significant
hematologic, gross or histopathologic effects.
In a study of limited design, pigs (Lantras strain, 18-25 kg, 8-12 weeks
old) were administered 50 mg/kg commercial grade 2,4-D tMethanolamlne
(water solvent) or 2,4-D butyl ester (diluted 1n petroleum solvent and emul-
sified In water) by Intubation (Bjorklund and Erne, 1966). The compounds
were administered at different frequencies and durations, but only one pig
per exposure schedule was tested. Four pigs were exposed to the tMethanol-
amlne salt for 3 doses In 5 days or 15 doses 1n 20 days, and three pigs were
exposed to the butyl ester for 5 doses In 8 days or 12 doses in 17 days.
01440
V-10
04/07/88
-------
Effects (e.g., anorexia, diarrhea, catarrhal gastroenteritis, fatty degener-
ation 1n the kidneys) were noted 1n 3/4 pigs exposed to 2,<-0 trlethanol-
amlne and In 2/3 pigs exposed to 2,4-0 butyl ester. Single pigs exposed to
fifty-one 50 mg/kg doses of trlethanolamlne salt, to 100 rog/kg trlethanol-
ar' salt (3 doses In 7 days or 7 doses 1n 9 days), to twenty-three 50
mg/kg doses of butyl ester In 39 clays, or to 300 mg/kg butyl ester (2 doses
1n 4 days or 3 doses In 6 days) exhibited similar effects.
Repeated s.c. Injections of 93 mg/kg levels of 2,4-0 sod'um salt dally
for 90 days did not produce significant symptoms In treated mice and histo-
logical examination did not show abnormalities (Bucher, 1946). Dilated
lungs, liver and kidneys, however, were noted In moribund animals Injected
with 200 mg/kg levels of compound; the significance of these changes Is
unknown.
Effects of s.c. Injected 2,4-0 sodium salt on the thyroid gland of
treated rats have been reported (Florshelm and Velcoff, 1962; Florshelm et
al., 1963). These Investigators showed that thyroid weight was decreased
following seven dally Injections of 2,4-0 at a level of 100 mg/kg. Adminis-
tration of 2,4-0 at 80 mg/kg over this period Increased radioactive Iodine
uptake by the thyroid, lowered the binding of radiolabeled thyroxine by
serum proteins, and increased the amount of radiolabeled compound In the
liver of treated rats.
Des1 et al. (1962) described the toxic effects of 2,4-0 or the nervous
system of rats administered lethal doses of the compound 1.p. Animals
Injected dally with 200 mg/kg of 2„4-D (form not specified) died within 6
01440 V-11 09/19/84
-------
days. Progressively decreased conditioned reflex responses were observed
over this period, as well as the appearance of large slow waves In the EES.
Histological examination Indicated that demyellnlzatlon was present 1n the
dorsal portion of the spinal tract. Within 10-15 minutes following a single
1.p. Injec*4-' of the compound, EEG changes were observed (decreased
cerebral and reticular desynchronlzatlon); recovery was seen 1n -1 hour.
The authors postulate that the neurological effects produced by 2,4-0 1n
this study are due Initially to action of the compound on the reticular
formation, followed by later ejects on cerebral tissue. Histological
examination, however, failed to show any morphological changes 1n the cortex
or subcortical regions of treated animals. The demyellnlzatlon observed 1n
the spinal cord may be responsible for the hind limb paralysis noted by
other Investigators after poisoning of animals with 2,4-D.
The subchronlc dermal toxicity of the dlmethylamlne salt and the
Isooctyl and butyl esters of 2,4-0 has been studied In rabbits (Kay et al.,
1965). Solutions containing the salt or esters at levels corresponding to
0.6 and 3.IX 2,4-0 were applied to gauze patches, occluded, and left 1n
place for 7-hour periods, 5 times/week for 3 weeks. No nervous system
damage, body weight effects or hematological changes were observed following
these levels of treatment. Local skin Inflammation was noted 1n both
control and treated animals, but subepithelial fibrosis and mononuclear
Infiltration appeared to be Increased only In those animals treated with the
oil dilutions of either of the 2,4-D esters.
01440
V-12
09/19/84
-------
Chronic Toxicity
Hansen et al. (1971) conducted 2-year feeding studies with technical
grade (96.7% pure) 2,4-D In Osborne-Mendel rats. In the rat study, 25
animals of each sex were exposed to 0, 5, 25, 125, 625 or 1250 ppm 2,4-0 In
the diet (or 0, 0.25, 1.25, 6.25, 31.25 or 62.5 rog/kg bw//day, respectively,
assuming a rat consul - o% of Its body weight per day) from 3 weeks of age.
At the conclusion of treatment, all rats were autopsled, but comprehensive
histopathologic examinations were performed only on 6 rats/sex from the
high-dose and control groups; the liver, kidneys, spleen, ovaries or testes
and other tissues that contained gross lesions were histologically examined
In the remaining rats In the high exposure and control groups and In the
rats at the other dose levels. Significant differences 1n survival, mean
body weight and organ-to-body weight ratios (liver, kidney, heiirt, spleen or
testes) were not found between any of the treated groups an J the control
group during the 2-year treatment ar at the end of the study. Significant
treatment-related pathologic effects were not observed and, as detailed In
the Carcinogenicity Section, the Incidence of tumors did not differ signifi-
cantly between the groups. Several hematologic Indices (hemoglobin, hemato-
crit, total white cell count) were similar In the treated and control
groups, but the red blood cell count of the treated rats (1210, 625 and 5
ppm groups) showed a "tendency1' toward macrocytosls, "very slight to slight"
polychromasla, and "slight to moderate" hypochromasla. The teidency toward
macrocytosls was reportedly not present and the other red cell abnormalities
were of a "minor degree" 1n the control rats. The toxlcolog'cal signifi-
cance of these vaguely reported effects 1s unclear.
01440
V-13
04/07/88
-------
In a 2-generat1on reproduction study by Bjorklund and Erne (1966) that
1s also discussed In the Other Reproductive Effects Section, administration
of 1000 ppm 2,4-D In the drinking water (-50-100 mg/kg/day) of rats
(5/group) during pregnancy and for a further 10 months had no significant
effects on the maternal animals (not specified) or offspring (clinical signs
or malformations). Similar exp- . j of 22 weaned offspring (10 males, 12
females) for up to 2 years was also nontoxic as Judged by normal clinical
chemistry [Indices were hematocrit, hemoglobin, plasma GOT, plasma elimina-
tion rate of 2,4-D (3 hours)]t relative organ weights (heart, spleen, liver,
kidneys, lungs, testes, ovaries), or gross or microscopic pathology.
However, reduced food and water Intake and consequent growth retardation,
temporary diarrhea and poor general condition were observed. Other repro-
duction studies that are detailed In the Teratogenicity and Other Reproduc-
tive Effects Section reported that dietary exposure to 1500 ppm (-75 mg/kg
bw) 2,4-D for 2 years (Hansen et al., 1971) and dietary exposure to 1000 ppm
(-100 mg/kg) for 3 months (Gaines and Klmbrough, 1970) before mating and
during pregnancy and lactation caused an 'Increase 1n preweanllng mortality.
Hansen et al. (1971) also fed 6- to 8-month-old beagle dogs (3 of each
sex/group) 0, 10, 50, 100 or 500 ppm technical grade 2,4-D In the diet (-0,
0.29, 1.45, 2.9 or 14.5 mg/kg/day) for 2 years. Treatment-related effects
based on observations of mortality as well as gross and microscopic tissue
examinations were not Indicated In any of the treated groups or the control
group.
01440
V-14
04/07/88
-------
Santoluclto (1975) reported E£G changes In a group of six squirrel
monkeys (sex unspecified) that were exposed orally (method unspecified) to
0.2 2,4-D/kg bw/day for 3 years. EEG recordings were aDparently made
only once, at the end of 3 years, at which time the treated monkeys were
compared with seven concurrent controls. Conges during anaesthetized sleep
Included an Increased proportion of h ,gh-frequency EEG vaves and an
Increased number of zero potential crossovers per EEG recording. The data
were obtained by on-Hne computer-assisted Interval analysis ol: 5 minutes of
each recording, but the toxlcologlcal significance of these changes Is not
known.
Carcinogenicity
Several studies have Investigated the ability of 2,4-C and related
compounds to produce tumors In laboratory rats and mice.
B1onet1cs Research Laboratories (Blonetlcs, 1968a; Innes ?t al.f 1969)
conducted a broad survey of the potential carcinogenic activity of several
pesticides and Industrial chemicals, Including 2,4-D (90% pure) and Its
Isopropyl (99% pure), butyl (99% pure) and Isooctyl (97% pure) esters.
Carcinogenic effects following chronic oral administration or single s.c.
Injection were Investigated In two strains of C57B1/6 mice designated
B6C3F1 and B6AKF^.
The oral administration regime consisted of Intubation of the compound
suspended 1n 0.5% gelatin to groups of 18 male and 18 female mice from 7-28
days of age, followed by dietary administration for -10-24 months. Both
strains of mice were given 46.4 mg/kg Initial bw of the Isopropyl, butyl or
01440
V-15
02/14/87
-------
Isooctyl ester by Intubation, followed by dietary concentrations of 111, 149
and 130 ppm (-14.4, 19.4 and 16.9 mg/kg/day), respectively, for 73-83 weeks.
B6AKF.J mice received 2,4-D by Intubation at 46.4 mg/kg Initial body
weight, followed by 149 ppm (-19.4 mg/kg/day) 1n the feed ft»r 75 weeks.
Both strains received 2f4-D by Intubation at 100 mg/kg Initial body weight,
followed by 323 ppm (-42 mg/kg/day) In the feed for'1 -*G weeks. Groups of
36 male and 36 female mice of both species received 0.5% gelatin or no
treatment at all. These control groups were assigned randomly to rooms
housing treated animals.
Following the treatment period, all surviving mice were grossly examined
on dissection, and the tissues from livers, spleens, kidneys, adrenals,
stomachs, Intestines and genitalia, which had been fixed and stained, were
examined microscopically by a pathologist. In addition, mice that were
killed when moribund were given a gross pathologic examination, and tissues
were examined microscopically as deemed appropriate (criteria unspecified).
No statistically significant (p<0.05) Increase In tumor Incidence over
controls was found when any group or combination of groups was compared.
Because of the relatively small number of animals/group and the limited
nature of the histopathologic examinations, weak carcinogenic effects might
not have been detected.
Groups of 18 male and 18 female mice of both strains received a single
s.c. Injection (neck) of 215 or 464 mg 2,4-D/kg bw dissolved In dlmethyl-
sulfoxlde (DMSO) at age 28 days and were observed for 78 weeks. Similar
groups of mice received a single s,c. Injection of 100, 21.5 or 21.5 mg/kg
bw of the Isopropyl, isobutyl or isooctyl ester of 2,4-D (1 n corn oil),
01440
V -16
04/07/88
-------
group and the high dose level with respect to the number of wale rats with
malignant tumors (Table V-3). The tumors observed, however, were not
associated with any specific tissue, but were randomly distributed and were
of types usually observed 1n aging Osborne-Mendel rats.
In an unpublished evaluation of this study, Reuber (1979) reexamined the
original hlstopathology sections, and reported substantially more tumors
among dosed animals than had been reported by Hansen et at. (1)71). A more
detailed hlstopathologlcal examination of all tissues and especially of
those 1n the lower dose animals was apparently deemed necessary by this
author. Reuber (1979) reported n greater number of lymphosarcomas 1n
treated rats of both sexes and found a significant (Fisher vxact p<0.05)
Increase In the Incidence of this tumor among female rats at «11 five dose
levels. The differences In tumor Incidence reported by Hansen ot al. (1971)
and Reuber (1979) might be resolved If an Independent reexamlratlon of the
tissue sections were performed.
The amine salt of 2,4-0 has been tested for carcinogenic activity 1n
rats and mice following oral administration (Archlpov and Kozlova, 1974).
Rats, 120 males and 45 females, were fed 2,4-0 amine at one-tenth the LO^q
level (not specified) for life. A similar dietary level of confound was fed
to a group of 100 mice for their lifespan. Neither species of test animal
developed a significant Increase In tumors following oral treatment. The
only tumors Identified were a mammary fibroadenoma and a hemangioma of the
mesenterlum 1n two treated rats and a mammary fibroadenoma 1n one untreated
rat. These Investigators also reported that long-term application of 2
drops/week of a 10% acetone solution of compound to the shaved backs of 100
mice failed -to produce tumors. When this treatment with 2,4-0 ^as preceded
oi44o v-18 Preceding page biank 09/19/84
-------
TABLE V-3
Tumor Incidence 1n Rats Fed 2,4-Da
Oose
(ppm)
Ratsb with
Tumors
Ratsb with Mallqnant Tumors
Males
Females
Males
Females
0
3
12
1
5
5
5
9
2
6
25
5
13
4
3
125
6
14
2
5
625
6
17
5
3
1250
7
15
6C
8
aSource: Hansen et al., 1971
bRats/sex/dose = 25
cp<0.05
01440
V-19
09/19/84
-------
by dermal application of 1 drop of a 5% solution of 3-metl-ylcholanthrene
(MCA), an Increase (0-17.1%) 1n sicln papillomas was observed. The authors
concluded that 2,4-0 showed significant cocarclnogenlc activity; this proto-
col suggests that 2,4-0 was tested for tumor promoter activity. No treat-
ment of MCA-lnltlated control animals was carried out over the 20-month test
period. It Is unclear how long after 2,4-0 treatment began these papillomas
developed.
An additional animal bloassay (Industry Task Force. 1985/ In rats and
mice has been provided to EPA, although the bloassay 1s not Independently
evaluated by the ORD Carcinogen Assessment Group In this assessment, the
study will be reviewed prior to f1nal1zat1on of this document. On an
Interim basis therefore this document reports the assessment position.
According to EPA, 1988, (EPA Press Release, Tuesday March 15, 1988; 2,4-0)
"A rat bloassay (Industry Task Force, 1985) found an appareit treatment-
related Increased Incidence of brain tumors In males at the highest dose
level. However, the Increased Incidence of tumors seen In the male rats at
the highest dose level was not statistically significant wher compared to
control male rats, although a marginally statistically significant trend was
observed. No tumor response was seen 1n female rats or mice", The Office
of Pesticides tentatively concluded that the tumor induction from the rat
study provided limited evidence of carcinogenicity In animals. In June
1987, the FIFRA Scientific Advisory Panel reviewed the Office of Pesticides
classification (limited evidence) ar\d advised that the evidence should be
viewed as equivocal and recommended additional testing. The Office of
Pesticides has accepted the assessment of the animal data by the SAB.
01440
V-20
04/07/88
-------
Available data from laboratory animals have not provided a sufficient
demonstration of carcinogenicity of 2,4-D, although Increased tumor produc-
tion 1s suggested. This question cannot be adequately resolved until more
compelling evidence Is available.
Epidemiology studies have associated excess tumor Incidence 1n humans
with mixed exposures to chlorophenoxy herbicides, including 2,4,5-T (which
may be contaminated with 2,3,7,8-TCDD) and 2,4-D (which 1s not contaminated
with this dloxln Isomer), These studies do not specifically attribute
carcinogenic effects to 2,4-D alone, and are summarized In the Subchronlc
and Chronic Effects Section 1n Chapter VI.
Mutagenicity
The mutagenic activity of 2,4-D has been Investigated 1n a number of
organisms Including bacteria, yeast, Drosophlla melanoqaster. algae and
several species of plants (Tables V-4 and V-5). Mammalian studies relating
to the mutagenicity of 2,4-D have included the mlcronucleus assay, the
dominant lethal assay, Inhibition of testicular DNA synthesis and several Jji
vitro assays of peripheral blood lymphocytes or cell lines treated with
2,4-D.
Investigations of the mutagenicity using microorganisms have generally
failed to show activity of the compound. These negative results Include
testing with Saccharomyces cerevlslae (Fahr1g„ 1974), Salmonella typhlmurlum
(Andersen et al., 1972; Styles, 1973; Andersen and Styles, 1978; Ercegovlch
and Rashld, 1977; Commoner, 1976; Zetterberg et al., 1977), T^ bacterio-
phage (Anderson et al., 1972), Bad!lus subtIlls (Shlrasu et al., 1976) and
Escher1ch1a coll (F1scor and Piccolo, 1972; Ercegovlch and Rashld, 1977).
01440 V-21 04/06/88
-------
TABLE V-4
Mutagenicity Testing of 2,4-0
Assjy
Strain
Compound
Concentration
Manna 11 an
Activation
Application
Response Reference
Gene conversion
Saccharowyces cerevlslat
i
rv
Gene combination
SaccharoMycei cerevlslae
Reversion. SalMoneUa
typhlMurtuM
00
00
®4
*4
®4
05
»5
RAD IB
TA1535
TA1S38
TA1530
TA1531
Ms Mutants
TA9B
TA100
TA1535
2,4-0
2,4-0
2,4-0
2,4-0
2.4-0
2,4-0
2,4-0
2,4-0
2.4-0
2.4-0
2.4-0
2,4-0
2.4-0 acetate
2.4-0 acetate
2.4-D acetate
KR
2 mi of U-46
solution
0.? Mg/t
0.2 Mg/i
200 wg/kg,
oral Intubation
200 Mg/ltg,
oral Intubation
no
no
200 iRQ/kg. host-mediated,
oral Intubation CBA Mice
0.$ ng/l no
0.3 Mg/Mt no
0.3-0.6 Mg/Mt. no
0.3-0.8 Mg/Mt no
• v iyi • v(i
4-2500 ng/plate
4-2500 |ig/pTate
4-2S0G pg/plate
host-Mediated,
CBA Mice
host-Mediated,
CBA Mice
1-5 wt technical no
Aroclor Induced
rat S-9
Aroclor Induced
rat S-9
Aroclor Induced
rat S-9
liquid holding,
neutral pM
liquid, low pH
l.p. Injection of
bacteria
liquid, low pH
liquid, low pH
Uould. low oH
liquid, low pM
liquid
liquid
l.p. Injection of
bacteria
1.p. Injection of
bacteria
agar overlay
agar
agar
agar
fahrlg, 1974
Slebert and
Lenperle, 1974
Zetterberg
et al., 1977
letterberg
et al., 1977
Zetterberg
et al., 1977
ZeUerbers
et al., 1977
Zetterberg,
197B
Zetterberg
et al., 197?
Zetterberg
et al., 1977
Zetterberg
et al., 1977
Zetterberg
et al., 1977
Anderson
et al., 197?
Anderson and
Styles, 1978
Anderson and
Styles, 1978
Anderson and
Sijte*, »97o
-------
Assay
Strain
Compound
Reversion, Salmonella TA1S38 2.4-0 acetate
tifphlmurlum
his mutants 2,4-0
tils rntints
2,4-0
Reversion,
bacteriophage
Point mutation,
bacteriophage
DMA mollification,
Escherchla coll
his mutants
T4, r1I mutants
ONA polymerase
deficient
£ 3110 and p 3478
WP2 try
2.4-0
2,4-0
technical grade
2,4-0
technical grade
2,4-0
2,4-0
2,4-0
ONA cell binding,
tscherchla coll
DMA modification.
Bad Mus subtllls
Recombination,
Bacillus subtllls
Recessive lethals,
Drosophlla melanoqaster
S strains
Q13
HI? and H45
H17 Rec*
H45 Rec~
Berlln K Mies.
ln(l>SCSRSCflR*
S.SC^'SC^B
females
2,4-0 amine,
dlcamba,
dlmethylamlne
2,4-0
2,4-0
2,4-0
2,4-0
2,4-0
TABLE V-4 (cont.)
Concentration
HamMllan
Activation
Application
Response
Reference
4-2500 ng/plate
NR, oral
NR
10 yg/plate
SO vfl/plate
50 yg/plate
NR
5 mg/plate
20-25 vg/p1ate
NR
Arodor induced
rat S-9
modified host-
mediated, rat
NR
no
no
NR
no
NR
NR
agar
bacteria Incubated
In serum from
treated rats and
plated
agar overlay
tryptone plates
agar
disc
disc
disc
disc
Anderson and
Styles. 1978
Styles. 1973
CrcegovUh and
Rashld, 1977
Commoner, 1976
Anderson
et al., 1972
Anderson
et al., 1972
Rosenkrani and
lelfer, 1979
Simmon. 1979
Nagy and
Antonl, 1975
Mscor and
Piccolo, 1972
20-200 pm
5 mg/plate
0.02 mg/plate
0.02 mg/plate
9 1m
* rat S-9 or egg-
white lysozomes
no
no
no
no
liquid
disc
disc
disc
oral. 3 days
Kublnskl
et al., 1981
Simmon, 1979
SMrasu
et al.. 1976
Shlrasu
et al.. 1976
Vogel and
Chandler, 1974
-------
I AOL t V-4 (cont.)
Assay
Strain
Compound
Concentration
Hawnallan
Actuation
Application
Response Reference
Recess We lethal*,
Orosophlla iwUnoqasttr
2.4-0
dlethylamlne
i SomatU stations,
^ SrsivyHi U nc mumsitr
nondisjunction,
Orosoohlla melanooaster
Sex chromosome loss,
QrosopMla melanogaster
Ouabain resistant
mutation, hamster lung
cell cultures
Unscheduled DMA
| u iini i/l mw
human fibroblasts SY-40
o
o»
Ns
00
00
Cell transformation
human lung cell culture
2,4-0
stable uhlti locus 2,4-0
yvtn/ywsn i
pttn/y+Y
stable white locus 2,4-0
unstable white
locus
unstable white
locus
2,4-0
2,4-D
stable white locus 2,4-0
unstable white
locus
yv*f/yv*f*yv*f/
y*tl*
yw*f/yw*fKyw*f/
y*Y8«
V-79 cell line
VA-4 cell line
VA-4 cell line
Wl-38
Ml-38
2.4-0
2,4-D
2,4-0
2,4-0 fluid
2,4-0 fluid
2,4-0 fluid
2,4-0 acetate
?.4-D acetate
0.08-8 mg/mi MR
100 ppm no
100 ppm
$0 ppm
100 ppm
SO ppm
7* nna
25 ppm
100 ppm
100 ppm
10 pm
1-1000
1-1000 vm
0.08-250 pg/mt
0.08-250 Mg/«t
no
no
no
no
no
no
none added
hiim
rat S-9
none added
rat S-9
NR
larval feeding
larval feeding
larval feeding
larval feeding
larval feeding
itrvii feeoing
larval feeding
larval feeding
larval feeding
liquid
liquid
liquid
liquid
Serin et al.,
1973
Nagnusson
et al., 1977
Rasmusson and
Svahltn, 1978
Rasmusson and
Svahltn, 1978
Rasmusson and
Svahlln, 1978
Rasmusson and
Svahlln, 1978
Rasmusson and
Svahlln, 1978
Rasmusson and
Svahlln, 1978
Ramel and
Nagnusson,
1979
Ramel and
Hagnusson,
1979
Ahmed et al.,
1977
Ahmed et al.,
1977
Ahmed et al.,
1977
Styles, 1977
St«1e; 197?
-------
Assay
Strain
Compound
Cell transformation
Syrian hamster kidney
BKH-21
8HK-21
2,4-D acetate
2,4-0 acetate
1
WUronucleus assay,
mouse
male CBA
2,4-D
1
Dominant lethal assay,
mouse
ICR/Ha
Swiss
2,4-D
Chromosomal aberrations,
mouse bone marrow
linear
white
2,4-D
linear
white
2,4-D
Chromosomal aberrations,
human blood 1yiiq>hocytes
NR
MR
2,4-D
2,4-0
Sister chromatid
exchange, human blood
11 ymphocytes
NR
NR
2,4-D
2,4-D
Unscheduled ONA synthe-
sis. primary rat hepato-
cyte cultures
F344
2,4-0
Chromosomal aberrations,
human blood lymphocytes
NR
NR
2,4-0
2,4-0
1
Chromosomal aberrations,
fmbryonlc bovine kidney
cells
NR
2,4-0
i
Inhibition of thymidine
ilncorporatIon Into
testicular ONA, mice
NR
2,4-0
NR • Not reported
TABLE V4 (cont.)
Concentration
Ranwia Han
Activation
Application
Response Reference
0.00-250 yg/mi none added
0.08-250 vg/mt
100 mg/kg
125 mg/kg
300 mg/kg
50 mg/kg
20 iig/Mft
2 tiQ^
10-60 yg/rt
0.2 Mg/mt
0.5-1000 nm/mt
SO-60 vq/mi
0.2-40 ng/m
1-1000 yq/m
rat S-9
In vivo
In vivo
In vivo
In vivo
none added
none added
none added
none added
present In hepato-
cyte culture
none
none
none
liquid
liquid
1.p. Injection
1.p. Injection
l.p. Injection
l.p. Injection
medium
medium
medium
medium
medium
medium
Medium
medium
Styles. HIT
Styles, 1ST?
Jenssen and
Renberg, 1976
Epstein
et iK, 19T2
Plllnskaya
et al., 1976
Pillnskaya
et al., 1976
Plllnskaya
et al., 1976
Plllnskaya
et al., 1976
Korte and
Jala I, 1902
Korte and
Jalal, 1982
Probst et al.,
1981
Korte and
Jalal, 1982
Korte and
Jalil, 1982
Bongso and
Basrur, 1973
200 mg/kg
In vivo
oral
Seller, 1979
-------
TABLE ¥-5
Mutagenicity Testing of 2,4-0 In Plants
Test
Compound
Concentration
Application
Hutagenlclty
Assay
Reference
Barley
i
2.4-B
1.5 Ml
Injection Into
spike
~
phenotyplc Mutants
Denward, 1954
Barley
2.4-0
100 ppn
9-hour treatment
of seeds
~
chlorophyll Mutation
Khalatkar and
fthargava, 1962
Barley
2,4-0 nixed
butyl ester
200 ppM
(-hour treatment
of seeds
~
chlorophyll Mutation
lo'iandas and Grant,
*12
Wheat
2,4-0 Mint
B ounces
pre-boot and
tillering stages
f
phenotyplc Mutants
Sandhu, 19)7
Wheat, barley
2,4-0 ester
12 ounces acid/
acre
spray
~
chromosome aberrations
Onrau and Larter,
1951
5 species, weed
2*4-0 aarine
907 g acid/0.4 ha
spray
~
chroaosoMe aberrations
Tonkins and Grant,
19*6
Tobacco
2,4-0
0.4 ppa, 120 hours
medlun
~
chroMosoMe aberrations
Nutl-Ronchl et al.,
1976
Bean
2,4-0
0.39 ppa
spray
~
chroMosoMe aberrations
A»er and All, 1974
Pea
2,4-0
40 ppw. 8-12 hours
seedling
chroMosone aberrations
Ruhllng et al.,
I960
Geranlun
2,4-0
io~« n
liquid
~
somatic Mutation
PohlhelM et al.,
1917
Onion
2,4-0 Mixed
butyl esters
SO pp10 ppa
NR
~
chrOMO* ll»rrinfti»t
- . — ¦» • f
19B2
M • Hot reported
-------
Positive mutagenic effects of 2,4-D were reported by Slebert and Lemperle
(1974) In S. cerevlslae following treatment of cell suspensions with 2 ml
of commercial U-46 D-Fluld (2,4-D acid) at pH 4.5; this concentration of
2,4-0 produced toxicity In treated cells. Mutagenic effects have also been
reported by Simmon (1979) for PNA repa1r-defIclent strains of E. coll and B.
subtl11s treated with 2»4-0 .a mg/plate). Clarification of these varied
results has been provided by the work of Zetterberg et al. (1977), who
Illustrated a definite pH-dependency In obtaining mutagenic effects of 2,4-D
1n S. cerevlslae. At pH 4.5, cells showed a dose-dependent Increase In gene
conversion and cellular toxicity at 2,4-0 concentrations from O.l-O.b
mg/mfc, while at neutral pH, neither effect was observed. The authors
indicate that, at neutral pH, 2,4-D Is primarily 1n a dissociated (Ionized)
state and does not readily penetrate cell membranes. Zetterberg (1978) also
showed a pH-dependent Increase In S, cerevlslae revertants exposed to 0.2
mg/mt of 2,4-0. Kublnskl et al. (1981) reported that binding of 2,4-D to
I* col 1 Q13 DNA was enhanced at close to neutral pH (7.2-7.4) 1n the
presence of a cell membrane dlsruptor (egg-white lysozymes) or a metabolic
activator (rat liver enzymes).
Detection of sex-Hnked recessive lethals 1n Drosophlla melanoqaster
treated with 2,4-D has been used as a mutagenicity assay by several Investi-
gators (Vogel and Chandler, 1974; Magnusson et al., 1977; Rasmusson and
Svahlln, 1978). Vogel and Chandler (1974) were unable to show a statisti-
cally significant Increase In recessive lethals after larval feeding of 9 mM
2,4-D for 3 days; examination was for the generation. Positive muta-
genic effects were reported by Magnusson et al. (1977) following 15-day
larval feeding with 100 ppm 2,4-D. Lethals observed 1n F1 were not
significantly Increased, but pooled data from the F^ and F^ generations
01440 V-27 04/06/88
-------
showed a 2- to 3-fold Increase In lethals over controls. Th»se Investiga-
tors were unable to show chemically Induced nondisjunction or loss of sex
chromosomes In D. melanoqaster treated with the same level o: 2,4-D. This
assay, however, 1s less sensitive than the production of recessive lethals
for screening of mutagenic agents. Rasmusson and SvahUn (1978) compared
phenotyplc changes In eye color Induce uy 2,4-D In two strains of
melanoqaster. They observed that larval exposure to 25 ppm of 2,4-D
produced a significant Increase In mutations for an unstable 0. melanoqaster
strain (foreign DNA Inserted 1n the structural gene), but failed to do so
for a stable strain. The positive control, ethyl methane sulfonate, at 500
ppm levels produced mutations In both strains.
Studies involving 2,4-D and Its salts and esters applied to various
plant species have Indicated chromosomal effects of the compounds. Grant
(1978) has argued that the effects of pesticides 1n plants correlate well
with effects 1n cultured mammalian cells and, therefore, such effects 1n
plants should be considered to Indicate possible mutagenic act'vUy of 2,4-D
In mammals. Positive mutagenic effects 1n plants treated with 2,4-D have
been reported by many Investigators; these have Included experiments with
barley (Denward, 1954; Mohandas and Grant, 1972; Khalatkar and Bhargava,
1982), wheat (Sandhu, 1957), wheat and barley (Unrau and Larter, 1951),
tobacco (Nut1-Ronch1 et al.t 1976), beans (Amer and AH, 1974), carrots
(Bayllss, 1973), geranium (Pohlhelm et al.t 1977) and several weed species
(Tomklns and Grant, 1976). Denward (1954) recorded seven mutants In the
second generation progeny of barley plants that had been Injected near the
spikes with 1.5 ml of a 2,4-0 solution (concentration unknown). Spraying
of wheat and barley plants at various stages of growth with 2,H-D ester at a
level equivalent to 12 ounces of 2,4-D add/acre has been reported by Unrau
01440 V-28 04/06/88
-------
and Larter (1951) to produce a number of melotlc abnormalU1es. Chromosome
aberrations Included bridges, fragments, aneuploldy and polyploidy, chain
and ring formation, and sticky chromosomes. The Incidence of these chromo-
some effects at different times after emergence Indicated that sensitivity
to 2,4-D changes throughout the growth cycle. S^ndhu (1957) reported a
higher number of off-plants 1n progeny of barley -reated with 8 ounces of
2,4-0 amine solution. Khalatkar and Bhargava (1982) reported the Induction
of mitotic and melotlc chromosomal aberrations, pollen sterility, spike and
seed morphological alterations In the generation and chlorophyll-
deficient mutations 1n the generation grown from barley seeds soaked In
a 100 ppm solution of 2,4-D for 9 hours.
Treatment of germinated barley seeds with 200 ppm of a commercial herbi-
cide preparation of the mixed butyl esters of 2,4-D for 6 hours has been
reported to produce five chlorophyll mutants 1n seedlings during field
trials (Hohandas and Grant, 1972). These Investigators also noted chromo-
some effects similar to those described by Oenward (1954) In root tip cells
Allium cepa (onion) treated with 50-100 ppm of 2,4-0 for 6 hours. Data
on the scoring of these chromosome effects were not presented. Two other
species of plants also showed root tip effects after application of this
commercial 2,4-0 ester preparation, while three species tested failed to
show an Increase In abnormal cells. The types of aberrations described.
Including the finding of C-m1tos1sv suggest that 2,4-D may Interact with the
spindle apparatus during cell division. Seller (1978) points out that
cereals are generally Insensitive to the auxin-like activity of chloro-
phenoxy acetic acids, but whether the in vitro effects observed In plant
cells treated with 2,4-0 are the result of physiological effects on cell
growth or direct effects on chromosomal material 1s not clear.
01440 V-29 04/06/88
-------
Tomklns and Grant (1976) found increased chromosome aberrations In 5 of
12 weed species sprayed with a 2,4-0 amine solution at a dose of 907 g of
2,4-D acid equivalent per 0.4 ha. These Included chromosome fragments,
bridges and lagging chromosomes. Similar chromosome effects *ere observed
by Amer and A11 (1974) In bean plants following spraying with 0.39 ppm 2,4-0
for 5 days. These workers found that the growth stage if the plants
Influenced the sensitivity to 2,4-D treatment. Disturbed metaphases and
anaphases Indicated effects on the mitotic spindle. Slddlqul et al. (1982)
observed abnormalities In melotlc chromosomes of Hellanthus aniuus treated
with 10 or 20 ppm of 2,4-0, but not 5 ppm. These aberrations Included
chromosome pairing failure, stickiness, bridges, Irregular separation,
laggards and fragments. Huhllng et al. (1960) were unable to show Increases
In chromosome breaks following treatment of pea seedling roots with 40 ppm
levels of a 2,4-0 solution; however, colchlclne-llke effects observed In
mitotic preparations suggested that 2,4-D may have acted a: a spindle
poison. Bay 11ss (1973) noted similar chromosomal and mitotic abnormalities
In root tip preparations of carrots treated In vitro with 3.1 mg/l of
2,4-D. Increased chromosome breakage 1n tissue cultures of tobacco plant
cells following \n vitro exposure to 0.4 ppm 2,4-0 has also been reported by
Nut1-Ronch1 et al. (1976). These effects were also produced 1n this habitu-
ated line of plant cells by the addition of a synthetic auxin, klnetln.
NonhabUuated cells that required the presence of growth factor For survival
In culture showed no chromosome breakage from treatment with 2,4-0 or
klnetln.
Assays using in vivo mammalian metabolic activation have failed to show
mutagenic activity of 2,4-D, Inducing the host mediated assay with S.
typhlmurlum (Styles, 1973) and the host mediated assay with S. cerevlslae
01440 V-30 04/06/88
-------
(2etterberg et al., 1977). Similarly, two mammalian assays, the dominant
lethal assay (Epstein et al., 1972) and the mlcronucleus test (Jenssen and
Renberg, 1976), have not demonstrated genotoxlc effects. This lack of
activity In mammalian systems may correlate with the finding of Jensen and
Renberg (1976) that <5% of a 100 mg/kg dose of 2,4-D Injected l.p. Into ^--.e
was available for penetration Into bone marrow cells within 24 h^urs.
PlUnskaya et al. (1976), however, reported an Increase 1n chromosome
aberrations of bone marrow cells from mice treated with 100 or 300 mg/kg
oral doses of 2,4-D. No data on the scoring of these cells were presented,
but the authors stated that the chromosome aberrations were primarily single
fragments. The purity of the 2,4-D and the vehicle used In this study were
not described.
Conflicting results for the genotoxlclty of 2,4-D have been observed in
several in vitro assays using mammalian cells. Probst et al. (1981) report-
ed that 2,4-0 did not stimulate unscheduled DNA synthesis 1n primary rat
hepatocyte cultures that retain metabolic capability, Murakami and Fukaml
(1980, 1982) reported the absence of 2,4-D binding to human embryonic DNA In
cultured cells. Ahmed et al. (1977), however, found an Increase 1n oubaln
resistant mutants following treatment of cultured V-79 Chinese hamster lung
cells with a 2,4-D concentration of 10 pM. Further, they found Increased
unscheduled DNA synthesis and Increased bromodeoxyuHdlne photolysis In
SV-40 transformed human fibroblasts treated with or without a source of
metabolic activation using the same concentration range of 2,4-D. Calcula-
tions to determine the number of breaks produced In 2,4-D-treated cells In
the photolysis assay Indicated very little Increase as the dose of 2,4-D was
Increased from 10-100 yM. Styles (1977) was unable to show Increased
transformation of cultured baby hamster kidney or human lung cell lines
01440 V-31 04/06/88
-------
treated in vitro with or without a source of metabolic activation at concen-.
tratlons of 0.08-250 vg 2,4-D/ml.
Peripheral blood lymphocytes ha*e been cultured 1n the presence of 2,4-0
and scored for chromosome damage (Bongso and Basrur, 1973; Plllnskaya et
al.( 1976; Korte and Jalal, 1982). Plllnskaya et al. (19761 reported an
Increase In chromosome aberrations following treatment of cultured human
lymphocytes with 20 vg/roi- Data on controls were not presented, nor
were scoring data reported. Bovine peripheral blood cells exposed to
10-1000 ppm levels of 2,4-0 showed altered mitosis and an elevated mitotic
Index, but no chromosomal aberrations (Bongso and Basrur, 1973).
Korte and Jalal (1982) Incubated cultured human blood cell; with 0.2-60
vg 2,4-D/mi for 48 hours and examined the chromosomes for evidence of
aberration and sister chromatid exchange. They observed statistically
significant Increases In gaps and deletions 1n lymphocytes treated with 50
and 60 vg/mt (p=0.05), and In sister chromatid exchanges 1r lymphocytes
treated with 10, 20, 30, 40, 50 and 60 ng/ml (p=0.05) over untreated
controls.
A recent report has found thai; oral administration of 2»4-D to mice
Inhibited thymidine incorporation 1 rito testicular 0NA (Seller, 1979). This
Inhibition was observed when 200 mg/kg of 2,4-D was administered to mice 1
hour after an l.p. injection of l4C-thym1d1ne. The author noted that the
order of mutagenic activity (MCPA > 2,4,5-T > 2,4-D) suggested by the work
of other Investigators was also observed In this assay (which has not been
validated as a mutagenicity screening test).
01440
V-32
04/06/88
-------
These studies suggest that 2,4-D may have mutagenic activity In certain
systems; however, the general lack of positive genotoxlc effects ijn v1 vo for
mammalian assays may Indicate that sufficient levels of 2,4-D are not able
to reach the target tissues. No Information 1s available 'on mammalian
mutagenicity testing conducted with the esters of 2,4-D; these forms could
theoretically show higher levels of penetration into target cells.
Teratogenicity and Other Reproductive Effects
Teratogenicity. The teratogenic and embryotoxlc effects of 2,4-D and
several derivatives of 2,4-D have been Investigated 1n several species of
laboratory animals. Overall, 2,4-D and Its derivatives appear to be embryo-
toxic but only weakly teratogenic or nonteratogenlc. The teratogenic or
embryotoxlc (or both) effects observed following oral administration of
2,4-D and Its derivatives during gestation are summarized In Table V-6.
Courtney (1977) Investigated the ability of 2,4-D (no dloxlns detected)
and several derivatives of 2,4-D (no dloxlns detected). Including Isopropyl,
n-butyl, Isooctyl and propylene glycol butyl ether (PGBE) esters of 2,4-D,
and 2,4-D butyric add, to Induce cleft palates In CD-I mice. Dally gastric
Intubation of 2,4-D and the propylene glycol butyl ether (PGBE) and n-butyl
ester derivatives were given at levels of 124 mg/kg/day of 2,4-D on days
7-15 of gestation and 221 rng/kg/day of 2,4-D on days 12-15 of gestation.
The Isooctyl and Isopropyl esters and butyric acid derivatives were adminis-
tered at 124 mg/kg/day of 2,4-D on days 7-15 of gestation for the two esters
and on days 11-13 of gestation for butyric add. The day of detection of a
vaginal plug was taken as day 1 of gestation; the animals were killed on day
18 of gestation. Control animals were given dally gastric Intubations of
the vehicle, consisting of 0.1 ml corn olltacetone (1n a ratio of 9:1).
01440 V-33 04/06/88
-------
nui«. v -
Teratogenicity of Orally Administered 2,4-0 and Derivatives of 2,4-0
Species/ Dose as Haternotoxlc, Fetotoxlc and
Compound Strain No. 0ms at Start Vehicle Purity Dosage/Exposure mg/kg/day of Teratogenic Effects Reference
2,4-D
2.4-0
mlce/CD-1
vehicle controls:
4 groups, 7-16/
group; treated:
•/low dose,
14/hlgh dose
corn oil
acetone
90*
0.5* irt/kg on
days 7 through
15 of gestation
124
2% deft palate/litter as
compared with OK for con-
trols; no other effect on
fetal parameters; no effect
on maternal weight gain; In-
crease (p<0.05) In maternal
relative liver weight
Courtney, 1977
1.00 rfl/k) on
days 12 through
IS of gestation
221
6X cleft palate/Utter as
compared with OX for con-
trols; decreased (p<0.05)
fetal weight among litters;
no effect on maternal weight
gain; Increase (p<0.0S) In
maternal relative liver
PUE ester
of 2,4-0
m1ce/CD-1
vehicle controls
4 groups, 7-1S/
group; treated:
10/low dose,
7/hlgh dose
n-Butyl
ester of
2.4-0
mlce/CD-1
vehicle controls:
A iirAiin* 1
i- —r-» •
group; treated:
9/dose level
corn oil
acetone
99.9*
corn oil
98.4*
o
¦S
CD
0.56 rt/kg on
days 7 through
1$ of gestation
124
1.00 ril/kg on
days 12 through
IS of gestation
0.S6 nfl/kg on
oays f through
IS of gestation
1.00 rft/kg on
days 12 through
IS of gestation
221
124
221
81 cleft palate/Utter as
compared with OX for con*
trols; decreased (p<0.0S)
fetal weight among litters;
no effect on maternal weight
gain; Increase (p<0.0$) In
maternal relative liver
weight
16* cleft palate/litter as
compared with OX for con-
trols; decreased (p<0.05)
fetal weight among Utters;
no effect on maternal weight
gain or relative liver weight
No effect on
or on maternal weight gain
or relative liver weight
IS* cleft palate/litter as
compared with OK for con-
trols; decreased (p<0.0S)
fetal weight among Utters;
no effect on maternal weight
gain; Increase (p<0.0S) In
(Miernai relative liver
weight
Courtney, 1977
WW I HI«J ,
-------
TABLE V-6 |cont.)
Spectcs/
Confound Strain
No. OiM at Start
Vehicle
Purlty
Dosage/Exposure
Isopropyl mlce/CO-1
ester of i
2.4-0
Isooctyl
ester of
2.4-0
lutyrU
acid of
2,4-0
2.4-0
Isooctyl
ester of
2.4-0
¦tce/CD-1
»1ce/C0-l
rats/
Mlstar
rats/
MHtar
vehicle controls:
4 groups. 7-16/
group; treated: 10
vehicle controls:
4 groups. 7-16/
group; trotted: It
vehicle controls:
4 groups, 7-16/
group; treated: 8
6-14/dose/expt.
17 controls.
5-6 treated/dose
corn oil
acetone
corn o11
acetone
corn oil
acetone
corn oil
or aqueous
gelatin
99*
96.8*
99. b*
no dloxlns
detected
corn oil
m
0.56 ^1/kg on
days 7 through
15 of gestation
0.56 afl/kg on
days 7 through
15 of gestation
0.56 irfl/kg on
days 11 through
13 of gestation
0. 25. 50. 100.
150 mg/kg/day on
days 6 through
15 of gestation
50 mg/kg/day on
days 6 through
15 of gestation
150 mg/kg/day on
days 6 through
15 of gestation
Oose as
mg/kg/day of
2.4-0
Haternotoxlc, Fetotoxlc and
Teratogenic Effects
Reference
124 No Incidence of cleft palate; Courtney. 1977
decreased (p<0.05| fetal
weight among litters; no
effect on maternal weight
gain or relative liver weight
124 No Incidence of cleft palate; Courtney, 1977
decreased (p<0.05) fetal
weight among litters; no
effect on Maternal weight
gain or relative liver weight
124 No Incidence of cleft palate; Courtney, 1977
no effect on other fetal
parameters; no effect on
maternal weight gain; In-
crease (p<0.05) In maternal
relative liver weight
0. 25, 50. Dose-related Increased fetal Khera and
100. 150 mortality and decreased fetal McKlnley. 1972
weight significant (p<0.05)
at 100 and 150 mg/kg/day; In-
creased Incidence of skeletal
malformations* among litters
significant (p<0.05) at 25.
100 and 150 mg/kg/day as com-
pared with controls; no effect
on maternal body weights.
33.2 N0AEL as judged by fetal Khera and
mortality, fetal weights. RcMnley, 1972
occurrence of skeletal or
visceral anomalies*
99.5 Oecreased (p<0.05) fetal
weight among Utters; In-
creased (p<0.05) Incidence
of skeletal malformations*
among litters; no effect on
maternal body weights
-------
Specie*/
Compound Strain No. Dam at Start Vehicle Purity
duty1 rats/ 17 controls, corn oil Nft
ester of Mlstar 4-5 treated/dose
llutoxy- rats/
ethanol Wlstar
ejter of
;?,4-o
IS controls,
8*9 treated/tfose
corn oil NR
Dimethyl- rats/ IS controls. corn oil NR
amine salt Mlstar 7-10 treated/dose
of 2,4-0
2,4-0 rats/ controls: 2 corn oil commercial
Sprague- groups, 36 and grade
DjiwiIau fI; trtitcd:
13-21/dose
Oose as Naternotoxlc, Fetotoxlc and
Dosage/Exposure mg/kg/day of Teratogenic Effects Reference
2,4-D
SO mg/kg/day on
days 6 through
IS of gestation
40
NOAEl as Judged by fetal
mortality, fetal weights,
occurrence of skeletal or
visceral anomalies*
Khera and
AcKlnley, 1972
ISO mg/kg/day on
days 6 through
IS of gestation
120
SO mg/kg/day on
days 6 through
IS of gestation
34.4
Increased (p<0.0S) fetal
Mortality among litters; de-
creased (p<0.05) fetal weight
among litters; Increased
(p<0.0S) Incidence of skele-
tal malformations* among
litters; no effect on
maternal body weights
NOAEL as Judged by fetal
mortality, fetal weights,
occurrence of skeletal or
v1 cr*r A1 i*mi* !»«'•
Khera and
HtMnley, 1972
ISO mg/kg/day of
days 6 through
IS of gestation
100 mg/kg/day on
days 6 through
IS of gestation
103.2 Increased (p<0.0S) Incidence
of skeletal malformations*
among litters; no effect on
maternal body weights
41.0 NOAEL as Judged by fetal
mortality, fetal weights,
occurrence of skeletal or
visceral anomalies*
Khera and
fkKlnley, 1972
300 mg/kg/day on
days 6 through
IS of gestation
12.S mg/kg/day
2,4-0 on days 6
mrougn 15 of
gestation
2S mg/kg/day
2,4-0 on days 6
through IS of
gestation
12S.S
12.5
25
Increased (p<0.0S| Incidence
of skeletal malformations*
among litters; effect on
maternal body weights
Increased (p<0.0S) Incidence
of delaved o*s1Mr»ts®»
skull bones among fetuses
and Utters; no effect on
maternal body weight
No significant effect on
fetuses or on maternal body
weight
Schwetz
ii tali
-------
TABU V 6 (cont.)
Species/ Dote as JUternotoxIc, fetotoilc and
Compound Strain Ko. Oam at Start Vehicle Purity Oosage/Exposure mg/kg/day of Teratogenic Effects Reference
2.4-0
7.4-0
P&Bf ester
of 2,4-D
rats/
Sprague-
Oawley
controls: 2
groups, 36 and
41; treated:
13~21/dose
corn oil
commercial
grade
SO mg/kg/day
2,4-0 on days 6
through IS of
gestation
75 or 07.5 mg/
kg/day 2.4-0 on
days i through
15 of gestation
dose equlmolar
to 12.5 mg/kg/day
2,4-0 on days 6
through IS of
gestation
dose equlmolar to
25 mg/kg/day 2,4-0
on days 6 through
15 of gestation
dose equlmoUr to
SO mg/kg/day 2,4-0
on days 6 through
IS of gestation
SO
75 or 07.5
12.5
25
50
Intermediate respon- between Schwetz
2 lower dosage levels (12.5 et al.( 1971
and 25 mg/kg/day) and 2 higher
dosage levels (75 and 07.5
mg/kg/day)
Decreased (p
-------
TABLE V-6 (cont.)
Species/ Dost as Raternotoilc, FetotoxU and
Compound Strain No. Oams at Start Vehicle Purity Dosage/Exposure mg/kg/day of Teratogenic Effects Reference
2.4-0
PGBE ester
of 2,4-0 .
Isooctyl
ester of
2.4-0
rats/
Sprague-
Oawley
controls: 2
groups, 36 and
41; treated:
T3-21/dose
corn oil
commercial
grade
doses equlmolar to
75 or B7.5 mg/kg/
day 2,4-0 on days
6 through IS of
gestation
dose equlmolar
to 12.5 mg/kg/day
2,4-0 on days b
through IS of
IS or 87.5 Decreased (p<0.0S) fetal
weight among Utters; In-
creased (p<0.05) Incidences of
skeletal defects (Including
delayed ossificate- of
sternebrae and/or li, wavy
ribs, lumbar ribs, .sing
sternebrae) among ^ uses and
litters; Increased (p<0.05)
Incidence of subcutaneous edema
among fetuses and litters; no
effect on Maternal body weight
12.5 Increased (p<0.05) Incidence
of subcutaneous edema among
fetuses; no effect on maternal
body weight
Schweti
et al.. 1971
Schwetz
et al, 1971
dose equlmolar to
25 mg/kg/day 2,4-0
on days 6 through
15 of gestation
dose equlmolar to
50 ng/kg/day 2,4-0
on days ( through
15 of gestation
doses equlieolar
to 75 or B7.5
mn/kn/A*~ ) I n
on days 6 through
15 of gestation
25 Increased (p<0.05| Incidence
of sternebrae with split
centers of ossification among
litters and of delayed ossifi-
cation of skull bones among
fetuses; no effect on maternal
body weight
50 Intermediate response between
2 lower dosage levels (12.5
and 25 mg/kg/day) and 2 higher
dosage levels (75 and 07.5
mg/kg/day)
75 or B7.5 Decreased (p<0.05) fetal
weight among litters; In-
crCaj&v ifnv>vi/ iiii iwvncvs OT
skeletal defects (Including
delayed ossification of
sternebrae and/or skull, wavy
ribs, lumbar ribs, missing
sternebrae) among fetuses and
litters; Increased (p<0.05)
Incidence of subcutaneous
edema among fetuses and
litters; no effect on maternal
body weight
-------
TABU V-6 (coM.)
Species/
Compound Strain
P6B£ ester rats/CO
of 2.4-0
Isooctyl
ester of
2.4-0
rats/CD
2.40
rats/NR
2.4-0;
three
samples
golden
Syrian
hamsters
No. Dams at Start
Vehicle
Purity
Dosage/Exposure
-37/group for
vehicle controls
and 2 lower dose
groups; 28 at 25
mg/kg/day; M at
67.5 mg/kg/day
-35/group for
vehicle controls
and 2 lower dose
groups; 2B at 25
mg/kg/day; 21 at
87.5 mg/kg/day
NR
controls: 86;
treated: 7-12/
dose/2.4-0 sample
corn oil
97.15%
corn oil
96.6%
0P-7
NR
acetone,
corn oil.
car boxy-
methyl
cellulose
no dloxlns
detected
doses equlmolar
to 6.25, 12.5
or 25.0 mg/kg/day
2.4-0 on days 6
through 15 of
gestation
dose equlmolar
to 81.5 mg/kg/day
2.4-0 on days 6
through 15 of
gestation
doses equlmolar to
6.25. 12.5 or 25.0
mg/kg/day 2.4-0 on
days 6 through 15
of gestation
dose equlmolar
to 87.5 mg/kg/day
2.4-0 on days 6
through 15 of
gestation
50 mg/kg/day
2.4-0 on days
through 14 of
gestation
20. 40. 80 or 100
mg/kg/day 2,4-0
on days 6 through
10 of gestation; 3
or 4 dose levels/
2,4-0 sample
Oose as
mg/kg/day of
2.4-0
6.25. 12.5
or 25.0
87.5
6.25. 12.5
or 25.0
87.5
50
20. 40. 80
or 100
flaternotoxlc, fetotoxlc and
Teratogenic Effects
Reference
No adverse effects on fetuses Unger et al
or dams 1901
¦Nlnor embryo-toxicIty which
was not deleterious to growth
and survival". I.e.. statisti-
cally significantly Increased
Incidence of lumbar (14th) rib
buds; no adverse effects on
body weight or survival of dams
No adverse effec
or dams
or fetuses
Unger e t a 1.,
1981
•Nlnor embryo-tonlclty which
was not deleterious to growth
and survival*. I.e., statisti-
cally significantly Increased
Incidence of lumbar (14th) rib
buds; no adverse effects on
body weight or survival of dams
Increased hemorrhages In the
thoracic and abdominal cavi-
ties and In the liver and soft
tissues of fetuses; details
of maternal toxicity were not
reported
Decreased (p<0.05) fetal via-
bility among litters at 40
mg/kg/day with 1 of 3 samples
and at 80 and 100 mg/kg/day
with another sample; no other
significant effects or malfor-
mations; details of maternal
toxicity were not reported
Konstantlnova
et. al. 1*76
Collins and
Williams. 1971
*Khera and NcKlnley (1972) did not score minor growth retardation and delays In ossification as anomalous developmental patterns. Skeletal malformations
Included wavy ribs, additional ribs, fused ribs; retarded ossification of frontal and parietal bones; sternal defects; small distorted scapula laterally
convex or distorted humerus shaft, and bent radius or ulna, resulting in mlcromella of the forellmb.
NR - Hot rroorted ®
-------
No cleft palates were observed In control animals. In all high dose groups,
fetal weight among litters was significantly decreased (p<0.05). The
Ircldence of cleft palate In offspring of dams treated with 2,4-D was 2 and
6% cleft palate/Utter for the low and high doses, respectively. In both
groups of animals treated with 2,4-D, no effect on maternal weight gain was
noted, but significant Increases (p) In maternal relative liver weight
were observed. A NOAEL for the n-butyl ester derivative may he defined at
124 mg/kg/day# as no effect on fet.al parameters or on maternal weight gain
or relative liver weight was observed at this dose level. At the high dose,
the n-butyl ester Induced 15% cleft palate/Utter; a significant increase
(p<0.05) In maternal relative liver weight without any effect on maternal
weight gain was also observed. The PGBE ester of 2,4-D was tte most toxic
compound tested, Inducing 8 and 16% cleft palate/Utter at 124 and 221
mg/kg/day, respectively. In addition, treatment with 124 mg/kg/day of the
PGBE ester resulted 1n significantly decreased (p<0.05) fetal weight among
111ter5 and significantly Increased (p<0.05) maternal relative liver weight.
No Incidence of cleft palate or maternal toxicity was observed 1n animals
treated with 124 mg/kg/day of either the Isopropyl or Isooctyl ester; fetal
weight among litters was significantly decreased (p<0.05). 2,4-0 butyric
acid, administered at 124 mg/kg/day, did not Induce cleft palate but
significantly increased (p<0.05) maternal relative liver weight. Based on a
calculated prenatal development Index value that considered botn fetotoxlc-
Uy and developmental effects (cleft palate), Courtney (1977) observed that
the relative order of prenatal toxicity of these compounds was PGBE ester >
2,4-D > Isopropyl ester > Isooctyl ester > n-butyl ester for We low level
of compounds administered on days 7 through 15 of gestation.
01440
V-40
04/06/88
-------
Khera and McKlnley (1972) studied the teratogenic and postnatal effects
of 2,4-D; the Isooctyl, butyl, and butoxyethanol esters of 2,4-D; and the
dlmethylamlne salt of 2,4-D administered orally to rats on days 6 through 15
of gestation. Day 1 of gestation was designated as the day after mating for
females having sperm 1n a vaginal smear; the dams were killed on day 22 of
gestation. Three commercial preparations and o-s purified (recrystalUzed)
preparation of 2,4-D (no dloxlns detected) were tested at two or more dose
levels of 25, 50, 100 and 150 mg/kg/day. The ester derivatives were admin-
istered at 50 and 150 mg/kg/day, while the d1mett»ylam1ne salt derivative was
tested at 100 and 300 mg/kg/day. Appropriate vehicle (corn oil or aqueous
gelatin) controls were used for each compound tested. Neither administra-
tion of 2,4-D nor any of Its derivatives had an effect on maternal body
weight. For the purposes of this study, minor growth retardation and
delayed ossification were not classified as teratogenic effects by the
authors. Skeletal malformations Included wavy ribs, additional ribs, fused
ribs; retarded ossification of frontal and parietal bones; sternal defects;
and small, distorted scapula, laterally convex or distorted humerus shaft,
and bent radius or ulna, resulting In mlcromella of the forellmb. 2,4-D
iduced a significantly Increased Incidence (p<0.05) of skeletal malforma-
tions at 25, 100 and 150 mg/kg/day; the Incidence at 50 mg/kg/day was higher
than In controls but was not statistically significant. The authors
expressed reservations about the significance obtained at the 25 mg/kg/day
level, but two of the fetuses from one of two replicate groups treated with
this dosage had malformations of the forellmb (previously described). Such
malformations were not observed 1n any control fetuses. A dose-related
Increase In fetal mortality and a decrease 1n fetal weight were also signif-
icant (p
-------
offspring of dams fed 150 mg/kg/day of the Isooctyl, butyl, or outoxyethanol
ester of 2,4-0 or 300 mg/kg/day of the 2,4-D dlmethylamlne salt. Fetal body
weights were depressed at the 150 mg/kg/day level of the Isooctyl and butyl
esters. A N0AEL for the Isooctyl, butyl, and butoxyethanol esters may be
defined at 50 rag/kg/day, and for the dlmethylamlne salt at 100 ii*g/kg/day, as
Judged by no apparent adverse effect on fetal mortality, <"n.a 1 weights or
occurrence of skeletal or visceral anomalies. Postnatal survival was not
affected at levels up to (but not Including) 200 mg/kg/day oi: 2,4-0, 150
mg/kg/day of the ester derivatives or 300 mg/kg/day of the salt derivative,
leading the authors to conclude that the observed skeletal defects were not
Incompatible with survival of newborn pups.
Teratogenic effects following oral administration of 2,1-0 and the
Isooctyl and PG8E esters of 2,4-0 to Sprague-Dawley rats were Investigated
In a three-part study by Schwet2 et al. (1971). In the first part of the
study, pregnant rats were treated with commercial grades of compound 1n corn
oil suspension or solution at level* of 12.5, 25.0, 50.0, 75.C or 87.5 mg
2,4-D/kg/day or molar equivalents of the esters on days 6 through 15 of
gestation. The day sperm were first observed In a vaginal smear was consid-
ered to be day 0 of gestation; dams were killed on day 20 of gestation. The
maximum tolerated dose of 2,4-0 was found to be 07.5 mg/kg/day for Sprague-
Oawley rats. Control animals were administered 2.5 ml con oll/kg/day
orally. No effect on maternal bod) weight was observed with any of the
three compounds at the levels test-ad. At the 12.5 mg 2,4-0/kg/day dose
level, treatment with 2,4-D resulted 1n a significantly Increased Incidence
(p<0.05) of delayed ossification of skull bones among fetuses
-------
significantly Increased Incidence (p<0.05) of subcutaneous edema among
fetuses. The NOAEL 1n Wlstar rats of 50 mg/kg/day of isooctyl ester (equiv-
alent to 33.2 mg/kg/day of 2,4-0} derived from the Khera and HcKlnley (1972)
study may be Indicative of strain differences, as a LOAEL of 12.5 mg/kg/day
of 2,4-0 as the Isooctyl ester may be Inferred from this study for Sp-^gue-
Dawley rats. At the 25 mg 2,4-D/kg/day level, treatment with 2,4-D had no
effect on fetuses; treatment with the PGBE ester resulted In a significantly
Increased Incidence (p<0.G5) of delayed ossification of skull bones among
fetuses and Utters; and the Isooctyl ester resulted 1n significantly In-
creased Incidences (p<0.05) of sternebrae with split centers of ossification
among Utters and of delayed ossification of skull bones among fetuses.
Schwetz et al. (1971) pointed out that the Incidences of these effects
varied considerably between the two vehicle control groups. Treatment with
50 mg/kg/day of 2,4-0 on equlmolar doses of the esters gave an Intermediate
response between the two lower dosage levels (12.5 and 25 mg/kg/day) and the
two higher dosage levels (75 and 87.5 mg/kg/day) for all three compounds
tested. Treatment with 75 or 87.5 mg/kg/day of 2,4-0, or equlmolar doses of
PGBE ester, or the Isooctyl ester yielded decreased fetal weight among
Utters, significantly increased Incidences (p<0.05) of skeletal defects
(Including delayed ossification of sternebrae or skull (or both), wavy ribs,
lumbar ribs and missing sternebrae) among fetuses and Utters, and a signif-
icantly Increased Incidence (p<0.05) of subcutaneous edema among fetuses and
Utters. Schwetz et al. (1971) stated that, In their opinion, the "dose
level essentially without effect" was 25 mg 2,4-D/kg/day for 2,4-D and Its
PGBE and Isooctyl esters. They classified all of the anomalies as embryo-
toxic or fetotoxlc effects rather than as teratogenic responses, because
none of these anomalies adversely affected either fetal or neonatal develop-
ment and survival.
01440 V-43 04/06/88
-------
In the second part of the study, Schwetz et al. (1971) evaluated the
effect on Implantation of the PGBE and Isooctyl esters of 2,4-D, adminis-
tered orally on days 5 through 8 of gestation at levels constituting the
molar equivalent of 87.5 mg 2,4-0/kg/day. Neither administration of the
PGBE ester nor the Isooctyl ester affected the percentage of pregnancies or
the number of implantations. In the third part of the study, the Isooctyl
ester was administered orally at a level constituting the molar equivalent
of 87.5 mg 2,4-D/kg/day on days 8 through 11 or on days 12 through 15 to
differentiate between effects observed 1n early and late organogenesis. An
Increased Incidence of resorptions was seen In early but not late organo-
genesis. Isooctyl ester had no effect on fetal body measurements during
either stage of organogenesis. Thi? Incidence and magnitude of sternebral
anomalies was similar following treatment with Isooctyl ester during either
stage of organogenesis; however, subcutaneous edema was obse-ved only In
fetuses of dams treated during early organogenesis.
In a study similar to the first part of the Schwetz et al. [1971) study,
Unger et al. (1981) Investigated the teratogenic and postnatal effects of
the PGBE and Isooctyl esters of 2,4-0 administered orally to CD rats.
Groups of pregnant rats were dosed with the PGBE or Isooctyl ester at doses
equivalent to 0, 6.25, 12.5, 25.0 or 87.5 mg 2,4-D/kg/day on d
-------
reduced fetal survival or teratogenic effects were not observed. Unlike the
previous study of Schwetz et al. (1971), no adverse effects were produced at
any of the lower dose levels of either ester.
Konstantlnova et al. (1976) have studied the possible teratogenic
effects of 2»4-D» 2,4-dlchlorophenol and the combination of these two com-
pounds given orally to rats. Compounds were administered Intragastrlcally
as aqueous emulsions 1n OP-7, a mixture of oxyethylated alkylphenols.
Increased hemorrhages In the thoracic and abdominal cavities and In the
liver and soft tissues were observed In fetuses taken from animals treated
with 50 mg/kg/day 2,4-0 on days 7 through 14 of gestation or 1 mg/kg/day
2,4-dlchlorophenol on days 1 through 20 of gestation. No other gross
anatomical effects were found, and Increased embryolethallty was not ob-
served. The combination of 0.1 mg/kg/day 2,4-0 and 0.1 mg/kg/day 2,4-dl-
chlorophenol administered on days 7 through 14 of gestation also produced an
Increased hemorrhaging 1n internal organs. The authors expressed concern
about the positive effect seen with the combination of 2,4-D and a metabo-
lite (2,4-d1chlorophenol) occurring at a level at which neither compound
alone shows toxicity.
Aleksasklna et al. (1973) Investigated the embryotoxUlty of the
dlethylamlne salt of 2,4-D administered orally to rats. Administration of
0.5 mg/kg of the compound throughout pregnancy produced decreases In fetal
weight and length. When single doses of the 2,4-D salt were administered on
day 4, 6, 9 or 13 of gestation at one-half the LD^q level (-400-600
mg/kg), an Increase 1n fetal abdominal hemorrhages was observed. The nature
of these lesions was not defined In the available abstract. This level of
compound given orally on day 5, 9, 10 or 13 produced increased fetal deaths,
01440 V-45 04/06/88
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and on day 5, 9 or 13, produced decreased fetal weights. Ho Information was
available on the strain of animals tested, the purity of the compound, or
the type of vehicle used. No skeletal examinations were conducted during
this Investigation,
Further work by this group (Buslovlch et al.v 1976} comparec the embryo-
toxic effects of the dlethylamlne salt with those of the sodium salt, butyl
ester, and amine salt of 2,4-D following oral administration to rats.
Administration of the butyl ester at a single dose of one-half the L05Q
value produced Increased fetal deaths and resorptions when the compound was
given as a single dose on day 4, 5, 6, 9, 10, 11 or 13 of gestation. The
sodium and amine salts given at this dose did not produce embryolethal
effects; however, when administered on either day 10 or 14 o,: gestation,
they produced a decrease In fetal weight and length. The authors indicated
that the butyl ester of 2,4-0 produced the most severe embryotoxlc effects,
followed by the dlethylamlne salt, and then the sodium and emlne salts.
This conclusion appears to be based on comparisons of effects produced by
one-half the level, which does not necessarily represent equltoxlc
doses. Complete data from this study were not available for review. The
purity of the compounds and the vehicles used for administration were not
identified in the available abstract.
The teratogenic potential of 2,4-0 administered orally to hamsters was
Investigated by Collins and Williams (1971). Three commercial 2,4-D samples
(no dloxlns detected) were fed by Intubation at levels of 20-1C0 mg/kg/day
to hamsters on days 6 through 10 of gestation. The day after mating was
designated day 0 of gestation; the dams were killed on day 14 of gestation.
01440
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Controls were fed the vehicle containing acetone, corn oil and carboxymethyl
cellulose. No significant teratogenic or other effects were noted with any
of the preparations. Decreased fetal viability was noted with feeding of
one 2,4-0 sample at 80 and 100 mg/kg/day doses, while another 2,4-0 sample
showed this effect at 40 mg/kg/day; the authors did not consider this a
clear dose-related response.
B1one tics Research Laboratories (1968b) Initiated a large-scale screen-
ing program In 1964 to Investigate the teratogenic potential of a number of
herbicides and other chemicals under a contract with the National Cancer
Institute. Included In this study were 2,4-D, the butyl, Isooctyl, 1so-
propyl, methyl, and ethyl esters of 2,4-D, and the metabolite, 2,4-d1chloro-
phenol. All compounds were tested by dally s.c, Injections 1n several
strains of mice, usually 1n doses of 46-150 mg compound/kg/day, 1n DMSO on
days 6 through 14 of gestation (days 6-15 of gestation In AKR mice); dams
were killed on day 18 (or day 19 for AKR mice). Day 0 of gestation was the
day of detection of a vaginal plug. 2,4-D (100 mg/kg/day In 50% honey 1n
water, 0.1 ml) was also administered by gavage. Fetotoxlc and teratogenic
effects were observed for certain groups of mice administered 2,4-D orally
or s.c., and for the butyl, isooctyl, and Isopropyl esters of 2,4-D, and for
2,4-d1chlorophenol Injected subcutaneously. These effects were generally
seen at a dose of compound corresponding to 100 mg/kg/day of 2,4-D. The
methyl and ethyl esters of 2,4-D produced some decreases In fetal weights at
this level of administration but failed to produce teratogenic effects. The
majority of teratogenic effects observed were 1n one strain of mice, 8L-6,
and included such defects as mlcrothalmla, agnathla and anophthalmia. This
strain of mice showed Inconsistent responses to the administered compound;
results with BL-6 mice were therefore divided to Include those animals
01440 V-47 04/07/88
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treated with compounds from September through November, 1%6, and those
animals that were treated after November, 1966. Even responses of control
BL-6 mice to s.c. Injection with DMSO varied widely during these two time
periods. Data on maternal toxicity were not presented for animals treated
with the chlorophenoxy compounds. Because these studies were conducted as
part of a large-scale screening program, studies using other doses of
compounds were not generally don? and, thus, dose-response relationships
cannot be evaluated. The s.c. route of administration further complicates
the Interpretation of these results when applied to the more likely routes
of exposure (Inhalation and oral).
Teratogenic and embryotoxlc effects have been observed 1n nice following
s.c. injection of Hormoslyr 64, a commercial preparation that contains 2t4-D
at 330 g/i and 2,4,5-T at 170 g/u (Bage et al., 1973). QH>0, used as a
vehicle for test solutions, contained an unspecified mixture of petroleum
distillates. Animals were Injected on days 6 through 14 of pregnancy and
killed on day 18. When this preparation was Injected at a dose of 110
mg/kg, an Increase was observed In fetal cleft palate Inclderce, and fetal
weight and fetal survival were decreased. Injection of 50 mg/kg doses pro-
duced a small Increase In cleft palate Incidence and no significant embryo-
toxic effects. Other skeletal and Internal malformations notec after admin-
istration of the high level of the 2,4-0/2,4,5-T mixture Indited Increased
rib and vertebral abnormalities and Increased renal and subcutaneous hemor-
rhages. The authors noted that cystic kidneys and dilation of the renal
pelvis, defects reported by other Investigators after administration of
2,4,5-T to rats, were not seen 1n this Investigation. As noted previously
1n the B1onet1cs (1968b) study, the significance of this rout? of admlnVs-
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compounds from September through November, 1966, and those animals that were
treated after November, 1966. Even responses of control BL-6 mice to s.c.
Injection with DHSO varied widely during these two time periods. Data on
maternal toxicity were not presented for animals treated with' the chloro-
phenoxy compounds. Because these studies were conducted as part of a large-
sca . screening program, studies using other doses of compounds were not
generally done and, thus, dose-response relationships cannot be evaluated.
The s.c. route of administration further complicates the Interpretation of
these results when applied to the more likely routes of exposure (Inhalation
and oral).
Teratogenic and embryotoxlc effects have been observed In mice following
s.c. Injection of Hormoslyr 64, a commercial preparation that contains 2,4-D
at 330 g/i and 2,4,5-T at 170 g/a (Bage et al», 1973). DHSO, used as a
vehicle for test solutions, contained an unspecified mixture of petroleum
distillates. Animals were Injected on days 6 through 14 of pregnancy and
killed on day 18. When this preparation was Injected at a dose of 110
mg/kg, an Increase was observed 1n fetal cleft palate Incidence, and fetal
weight and fetal survival were decreased. Injection of 50 mg/kg doses pro-
duced a small Increase 1n cleft palate Incidence and no significant embryo-
toxic effects. Other skeletal and Internal malformations noted after admin-
istration of the high level of the 2,4-D/2#4,5-T mixture Included increased
rib and vertebral abnormalities and Increased renal and subcutaneous hemor-
rhages. The authors noted that cystic kidneys and dilation of the renal
pelvis, defects reported by other Investigators after administration of
2,4,5-T to rats, were not seen 1n this Investigation. As noted previously
In the 81onet1cs (1968b) study, the significance of this route of admlnls-
01440
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tratlon 1s questionable. Some malformations and hemorrhaging were observed
In vehicle-treated animals; the complicating effect of OMSG 1s therefore
added to the combination of the two herbicides.
Lutz-Ostert«c and Lutz (1970) reported embryotoxlc an! teratogenic
effects after spraying 2t4-D amine on pheasant and partridge eggs. A
commercial preparation of the herbicide was sprayed at a level of 1.1 kg/ha
after eggs had been Incubated for :3.5 days. These Investigates found high
mortality (43-77%) of embryos by days 20-22 of development. Surviving
embryos showed paralysis and sterility. Morphological examinations showed
fused vertebrae, curled toes, and testicular and ovarian anomalies. The
type of vehicle used and impurities present In the 2,4-0 preparation were
not described, nor were data on controls presented. No embryonic effects
were found following spraying on chicken eggs at a level of 11.2 kg/ha with
a commercial 2,4-D P6BE ester formulation (Somers et al.( 1970), while
immersion of chicken eggs In IX solutions of commercial 2,4-0 amine prepara-
tions has also been reported to produce no significant toxic effects (Gyrd-
Hansen and Dalgaard-Mlkkelsen, 197*). The findings of Lutz-Ostertag and
Lutz (1970) appear to be unique and suggest an effect not attributable to
the 2,4-D component. Gyrd-Hansen and Dalgaard-Hlkkelsen (1914), however,
did find Increased mortality, malformed beak and gastroschl.;ls in chick
embryos following direct Injection of the yolk sac with 2- 0 mg/egg of
commercial 2,4-D amine. The significance of embryonic effects produced by
direct injection of chicken eggs for determining the teratogerlc potential
of chlorophenoxys 1s uncertain.
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Other Reproductive Effects. Hansen et al. (1971) Investigated the
effects of long-term dietary administration of technical grade 2,4-D in a
3-generat1on reproduction study In rats. Starting at 3 weeks of age, rats
were maintained on diets containing 100, 500 or 1500 ppm {~5-7$ mg/kg bw) of
2,4-D for 2 years. Data from the F^ and generations Indicated
an Increase 1n preweanlV _ .Mortality at the 1500 ppm feeding, determined as
the percentage of pups weaned. At this maximum dose of compound, a signifi-
cant loss In weight of weanlings was also observed, but there was no effect
on parental fertility or Utter size. No deleterious effect was noted at
the lower dose levels (100 and 500 ppm) on fertility, mean litter size or
viability of pups during the first 21 days of age. Liver allesterase and
liver acylamldase activity did not differ between selected F^ rats
{10/sex) at 90 days of age. These authors cite unpublished work by Gaines
and Klmbrough (1970), who found Increased mortality of weanlings following
administration of 1000 or 2000 ppm 2,4-0 to groups of 10 female rats In the
diet for 3 months before mating and throughout pregnancy and lactation. At
2000 ppm, the pups were small at birth and 94% died before weaning. At 1000
ppm, more deaths were seen among offspring of treated dams than were seen
among offspring of control dams.
Bjorklund and Erne (1966) observed toxic effects 1n the offspring of a
single pig fed 500 mg/kg 2,4-0 trlethanolamlne 1n the diet throughout preg-
nancy and for 6 weeks after delivery (-25-50 mg/kg/day). Underdevelopment
and Increased mortality were observed 1n newborn piglets 1n the first 24
hours after parturition. "Anorexia" was observed 1n the sow throughout the
experiment; she died -6 weeks after delivery.
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As part of a two-generation 'study, Bjorklund and Erne (1966) adminis-
tered 0 or 1000 ppm (equivalent 1.0 0 or ~50-100 mg/kg/day, based on water
consumption) of 2,4-0 In the drinking water of pregnant rats (N=5/group)
throughout gestation and for 10 months following parturition,' and continued
with offspring for up to 2 years aft?" weaning. Although a reduced food and
water intake, with consequent growl.;, retardation, was observed 1n the treat-
ed offspring, no clinical signs, malformations or distinct morphological
changes were seen 1n treated offspring. This study 1s also discussed 1n
Chapter VII.
In a reproduction and fertility study, Lamb et al. (1980) iidmlnlstered a
mixture of 2,4-0, 2,4,5-T and 2,3,1,8-TCDO (simulated Agent Orange) to male
C57B1/6 mice. Groups of animals were given feed that contained various
concentrations of the three compounds, so that the dally doses of 40 mg/kg
2,4-0, 40 mg/kg 2,4t5-T, and 2.4 yg/kg 2,3,7,B-TCDD (Group II; or 40 mg/kg
2,4-0, 40 mg/kg 2,4,5-T, and 0.16 vg/kg 2,3,7,8-TCOD (Group III) or 20
mg/kg 2,4-0, 20 mg/kg 2,4t5-T, and 1.2 vg/kg 2,3,7,8-TCDO (Group IV) would
be achieved. Another group of animals, used as controls (Group I), were
given feed with only the corn oil (2%) vehicle added. At the end of B
weeks, the treated animals were reported to have dose-related liver and
thymus toxicity and significantly reduced body weight gain; however, the
liver and thymus recovered to normal or near normal weights following termi-
nation of treatment. No significant effect was noted on sperm abnormalities
either during or following the dosing period. After 8 weeks of treatment,
each treated male was mated to thre<; untreated virgin females, for a total
of 24 matlngs for each treated male. Exposure to the various mixtures of
simulated Agent Orange did not resuU In a significant decrement 1n fertil-
ity or reproduction, as evidenced by no effect on mating frequency, average
01440
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fertility, percent Implantation and resorption sites, and percent fetal
malformations. Furthermore, germ cell toxicity was not observed, and the
offspring of treated males were not affected by Increased lethality or
abnormal neonatal development.
Summary
Acute exposure to high levels of 2,4-0 results In progressive symptoms
of muscular incoordination, lethargy, hlndquarter paralysis, stupor, coma
and death in animals (Hill and Carlisle, 1947). These symptoms have been
observed consistently 1n a variety of species regardless of route of admin-
istration, and myotonia appears to be a dominant effect of exposure (Bucher,
1946; H111 and Carlisle, 1947; Drill and Hlratzka, 1953). Pathological
examinations have shown that acute exposure to high levels of 2,4-0 resulted
In kidney damage and skeletal muscle changes In a variety of species, but
hepatic damage has been described only 1n dogs (H111 and Carlisle, 1947;
Drill and Hlratzka, 1953). Acute oral LD^s In the range of 350-500 mg/kg
2,4-D have been reported for rodents, but significant differences In tox-
icity are not apparent between 2,4-D and Its salts and esters. Inhalation
toxicity data are not available.
Subchronlc oral toxicity studies have been conducted with rats that were
exposed to 2,4-D at levels of 3-300 mg/kg/day, 5 days/week for 4 weeks (Rowe
and Hymas, 1954), 2000 ppm 1n the diet for 4-7 weeks (Chang et al.» 1974);
100-5000 ppm 1n the diet for 113 days (Rowe and Hymas, 1954) and 200-400 ppm
In the diet for 31 days (H111 and Carlisle, 1947). In rats doses of 5.0
rag/kg bw/day or higher resulted 1n significant reductions 1n blood Indices
at all doses; liver enzyme activities were reduced at higher doses; kidney
toxicity was also evident at higher doses. Effects of higher doses Included
01440 V-52 04/06/88
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GI Irritation and mild liver effects (e.g., cloudy swelling. Increased
weights), as well as characteristic overt signs of toxicity
-------
mg/kg/day) (Hansen et al., 1971). EEG changes were reported In monkeys that
were exposed orally to 0.2 mg/kg/day 2,4-0 for 3 years, but the toxlcologl-
cal significance of the changes 1s unknown (Santoluclto, 1975).
Administration of 2,4-0 to mice by Intubation from days 7-28 of age at a
level of 100 mg/kg Initial bw and subsequently 1n the diet at a level of 323
ppra (-42 mg/kg bw/day) for 81-90 weeks was not tumorlgenlc (Blonetlcs
Research Lab., 1968a; Innes et al., 1969). Similar administration of 2,4-D
Isopropyl ester, butyl ester or Isooctyl ester [46.4 mg/kg by Intubation,
111-130 ppm 1n the diet (-14.4-16.9 mg/kg bw/day) for the subsequent 73-83
weeks] was also nontumor1gen1c. Hansen et al. (1971) reported that admin-
istration of 2,4-D at levels of 5-1250 ppm 1n the diet (-0.25-62.5 mg/kg
bw/day) for 2 years did not Induce treatment-related tumors In rats, but
reexamination of the hlstopathology sections by Reuber (1979) found a sig-
nificant increase 1n the Incidence of lymphosarcomas In females at all dose
levels; the differences In tumor incidences have not been resolved. Rats or
mice that were fed 2,4-0 amine salt at one-tenth the LD^g level for life
did not develop a significant Increase 1n tumors (Archlpov and Kozlova,
1974). Single s.c. Injections of 2,4-D (215 or 464 mg/kg), 2,4-D Isopropyl
ester (100 mg/kg) or 2,4-D Isobutyl ester (21.5 mg/kg) at age 28 days were
not tumorlgenlc to mice after 78 weeks, but similar Injection of 2,4-D
Isooctyl ester (21.5 mg/kg) Induced a statistically significant Increase In
reticulum cell carcinomas (Blonetlcs Research Lab., 1968a). Repeated dermal
application of 2,4-D reportedly-produced skin papillomas 1n mice only when
2,4-D treatment was preceded by a single dermal application of the Initiator
3-methylcholanthrene (Archlpov and Kozlova, 1974). A rat bloassay (Industry
Task Force, 1985) Is not evaluated 1n this assessment. Note 1s made that
EPA's Office of Pesticide Programs report tumor elevation at the high dose
01440 V-54 . 04/13/88
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In male rats and a marginally significant dose-response trend 1n the males.
The female rats and mice were negative. Taken as a whole the animal
evidence has been proposed by the Office of Pesticides (EPA 1988, Federal
Register) to be Inadequate.
2,4-0 has not produced mutagenic effects 1n assays with Salmonella or
bacteriophage T^, although positive responses were reported by Simmon
(1979) for DNA repair-deficient strains of E. coll and B. sul>t1Hs. Gene
conversion/combination tests with the yeast. S. cerevlslae. Mere positive
only when the pH of the system was lowered Into the acid range (Slebert and
Lemperle, 1974; Zetterberg et al., 1977; Zetterberg, 1978), where 2,4-0
would be nonlonlzed. 2,4-0 was weakly mutagenic for 0. melanoqaster In
recessive lethal and somatic mutation assays (Magnusson et al., 1977;
Rasmusson and SvahUn, 1978). 2,4-1) Induced ouabain resistance 1n Chinese
hamster V-79 lung cells (Ahmed et al., 1977), Induced unscheduled DNA
synthesis In cultured human fibroblasts (Ahmed et al., 1977), and Induced
chromosome aberrations and sister chromatid exchanges An cultured human
lymphocytes (PHlnskaya et al., 1976; Korte and Jalal, 1982), but was
Inactive 1n other In vitro mammalian assays (cell transformation In human
lung or hamster kidney cells, unscheduled DNA synthesis In rat hepatocytes,
chromosome aberrations 1n embryonic bovine kidney cells). Intraperitoneal
injection of 2,4-D Induced bone marrcw chromosome aberrations (FlUnskaya et
al., 1976) and oral administration of 2,4-D Inhibited thymidine Incorpora-
tion Into testicular DNA In mice (Seller, 1979), but other In vivo mammalian
assays with mice (mlcronudeus assay and dominant lethal assay) were
negative. Information regarding the mutagenicity of 2,4-0 esters 1n animal
systems was not located, but these compounds would theoretically penetrate
cells more readily than 2,4-0 at physiological pH.
01440 V-55 . 04/13/88
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Teratogenicity testing has been conducted with 2,4-D, several of Its
esters (n-butyl, Isopropyl, Isoctyl, PGBE, butoxyethanol)( the dlmethylamlne
salt, and 2,4-D butyric acid In mice, rats and hamsters (Courtney, 1977;
Khera and McKlnley, 1972; Schwetz et al., 1971; Unger et al., 1981;
Konstantlnova et al., 1976; Collins and Williams, 1971). Overall, these
studies Indicate that 2,4-D and Its derivatives are embryotoxlc but only
weakly teratogenic or nonteratogenlc. Oral doses (expressed as 2,4-D) of
124 mg/kg/day In mice (Courtney, 1977), 75-125.5 mg/kg/day 1n rats (Schwetz
et al., 1971; Unger et al., 1981; Khera and HcKlnley, 1972) and 40-100
mg/kg/day 1n hamsters (Collins and Williams, 1971) produced fetotoxlc
effects or malformations (cleft palates and other skeletal malformations).
Increased preweanllng mortality and weight loss were observed 1n the
offspring of rats that were exposed to 1500 ppm levels of 2,4-D 1n the diet
in a 3-generat1on reproduction study, but adverse effects on litter size or
fertility were not found (Hansen et al., 1971).
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VI. HEALTH EFFECTS IN HUMANS
Acute Effects
Clinical reports have described fatal poisoning In humans resulting from
Ingestion of 2,4-D solutions. Nielsen et al. (1965) described a suicide
case Involving a male agricultural student who ingested at least 6 g of a
commercial herbicide preparation of the dlmethylamlne salt of 2 4-0 {50%, by
weight). Death appeared to have beon preceded by vomiting and convulsions.
Pathological examination revealed acute congestion In most orgars as well as
acute pulmonary edema, and histological examination showed degenerative
ganglion cell changes 1n the brain. Geldmacher et al. (1966) reported two
cases of poisoning with 2,4-0. Vomiting and loss of consclousress occurred
1n a 33-year-old woman following ingestion of an unknown quantity of 2,4-D.
Terminal symptoms developed after 1 day, and Included weak pulse, tachy-
cardia and deep breathing. These authors described another case reported by
Herblch and Hachata (1963) of a 46-year-old man who died within 14 hours of
swallowing at least 13,5 g of an unciaracterlzed 2,4-0 solution. Constricted
pupils and respiratory paralysis wen? noted among the terminal symptoms. At
autopsy, both patients had generalised hyperemia of the organs as well as
edema of the brain and the lungs. Oudley and Thapar (1972) reported the
fatal poisoning of a 76-year-old m*ile who Ingested (assumedly) a pint of
2,4-D solution (in kerosene). Early symptoms Included vomiting and loss of
consciousness. Death occurred 6 days after the Ingestion of 2,1-D. During
medical treatment, the patient received pentobarbital, amplclllVi and qulnl-
dlne. Pathological examination Indicated edema of the lungs, mid-zonal
hepatic necrosis and pyelonephritis. The pulmonary effects noted may have
been due to the kerosene vehicle. Multifocal perivascular plaques of
demyellnlzatlon were observed during microscopic examination of the brain.
The
01450
VJ-1
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authors attributed death to atrial fibrillation; however, In this case, as
1n the other fatal poisonings described, the exact cause of death 1s unknown.
Berwick (1970) reported an Incident of nonfatal poisoning, 1n which a
farmer swallowed a mouthful of concentrated weed killer containing 2,4-0.
Initial symptoms Included acute gastritis and vomiting. Eighteen hours
later, Intense aching of the chest, painful and tender muscles, and a tender
abdomen were reported. Within the next 6 hours, the patient lost use of his
Intercostal muscles, and the muscles of his upper extremities exhibited
spontaneous fibrillary twltchlngs. Hyperactive biceps and triceps reflexes
were seen, but other reflexes were normal. The blood levels of several
enzymes, Including lactate dehydrogenase, SGOT, SGPT, aldolase and creatine
phosphoklnase, were Increased from days 4-7; myoglobinuria was observed,
Indicating probable skeletal muscle damage. Although this commercial formu-
lation of weed killer contained 49% Eptam, 35.5% 2,4-0 Isooctyl ester, 0.5%
eplchlorohydrIn and 5% emulslflers, the authors found no evidence that the
Eptam produced toxlcologlcal effects or cholInesterase Inhibition. Loss of
sexual potency was observed in this patient and persisted for -4 months;
however, other symptoms subsided within -2 week.s.
Neurological symptoms have been described 1n other reports of illness
related to human exposure to 2,4-0. In most of the cases of occupational
exposure to 2,4-0, the subjects had previously been exposed to a variety of
pesticides; however, the neurological symptoms observed 1n 2,4-0 exposed
experimental animals (see Acute Toxicity Section In Chapter V) suggest that
the compound has the potential to produce neurological effects. Honarca and
01V1to (1961) reported symptoms 1n a farmer who became 111 after applying a
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40% aqueous solution of 2,4-0 against the wind. The symptoms Included
ataxia, reflex disorders such as abolished Achilles tendon reflex and
reduced patellary reflexes, and a positive Romberg's sign (damage to the
dorsal column of the spinal cord). Symptoms persisted for 2-3 months and
subsided slowly. Goldstein et al. (1959) described three cases of periph-
eral neuropathy following exposure to an ester of 2,4-0. The First patient
had two dermal exposures within 2 months to spills of a 10% solution of
2,4-D ester, and experienced nausea, vomiting and diarrhea after each
exposure. He experienced numbness and aching of the digits 1 w;ek after the
second exposure. During the following 5 weeks, he showed the development of
peripheral neuropathy. The second patient also developed neurological
symptoms after two exposures, -1 year apart, to dermal wetting with an ester
of 2,4-0. This patient developed flaccid paraparesis 5 months after the
second exposure. The third patient reported wetting of his clothes with a
spray solution of 2,4-0 ester during application. Within 24 hours, he
developed malaise, headache, nausea and vomiting, and, within 48 hours,
severe vertigo. Paresthesia In the limbs appeared within 4-5 days, followed
by faslculatlons that became generalized. Ir all three cases, the symptoms
of peripheral neuropathy were of prolonged duration. The authors obtained
the label from one container that had been used for spraying and found that
the applied material was 44% Isopropyl ester and 56% Inert Ingredients.
Another clinical report of long-term (-2 years) peripheral neuropathy In a
farmer exposed to 2,4-0 ester while spraying has been reported by Todd
(1962). Berkley and Hagee (1963) described what appeared to te a primary
sensory neuropathy of the upper extremities 1n a farmer exposed to the
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cMmethylamlne salt of 2,4-0 while spraying a corn field. Bordas et al.
(1958) also noted weight loss and sensory and motor neuropathies In workers
exposed to 2,4-0 during spraying operations.
Polyneuritis has been described 1n a farmer who became 111 after spray-
ing 2,4-0 solutions of 235 and 410 g/i for several days In an open cab
tractor (Folssac-Gegoux et al., 1962). The patient developed facial anaes-
thesia and paresthesia. He subsequently lost feeling In his legs and was
forced to walk with a cane. Neurological examination showed Increased knee
reflex and decreased Achilles tendon reflexes at 2 months; 3 months after
exposure, motor and sensory symptoms of the face and legs had Improved, but
an elec^.romyelogram still showed abnormalities. In these reports of agri-
cultural exposures, the precise formulations of compounds used Is not known,
nor Is the amount of compound present In the air or on the skin of workers.
Subchrontc and Chronic Effects
Koncarclnoqenlc Effects. Reports on subjective clinical symptoms 1n
workers exposed to 2,4-0 during Us manufacture or use have been published.
Assouly (1951) reported that symptoms In workers employed In the fabrication
of 2,4-0 esters Included gastralgla, anorexia, somnolence, a sweet taste In
the mouth, Increased hearing sensitivity, a sensation of drunkenness and
heaviness of the legs. Bashlrov (1969) examined 292 workers (2*8 men and 44
women) engaged In the manufacture of 2,4-0 amine and butyl ester. He
reported a high frequency (63%) of symptoms of rapid fatigue, weakness,
headache or vertigo. Approximately 20% of these workers experienced hypo-
tension, bradycardia, jyspepsla and gastritis. Another Russian study
(Fetlsov, 1966) reported rapid fatigue, headache, loss of appetite, pains 1n
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the region of the liver and stomach and lowered acuity of taste and smell in
workers using preparations of the butyl ester, crotyl ester or amine salt of
2,4-0. Health effects of exposure to chlorophenoxys In a 2,4-D and 2,4,5-
trlchlorophenoxyacetlc acid (2#4,5-T) production plant were reported by
Poland et al. (1971). A study of health records Included 73 male workers
with an average duration of employment of 8.3 years. Symptoms reported for
these workers Included chloracne, hyperpigmentation and hirsutism; these
correlated together and were most probably related to exposure to 2,4,S-T
and dloxln Impurities. Other symptoms noted Included various gastro-
intestinal disturbances and decreased hearing acuity. One case reported
diminished proprioception and two workers failed to demonstrate ankle jerk
reflexes. None of these subjective clinical reports Indicate levels of
exposure, nor do they indicate other possible chemicals to which these
workers may have been exposed.
Kephart (1945) reported that an Individual who voluntarily Ingested 500
mg of 2,4-0 dally for 21 days denwnstrated no 111 effects. Additional
Information regarding this observation 1s not available.
WalUs et al. (1970) described neurological changes In a wcrker exposed
to 2,4-0 over a period of 1 year while spraying sugar cane fields. Over a
period of 2 days, this worker developed painful paresthesias In the hands
and feet. Ourlng the next 3 days, he developed painful muscular stiffness
1n all four limbs; this condition progressed over the next 2 years, Impair-
ing his gait and his manual dexterity. Movement was with deliberate slow-
ness and great effort. Medical evaluation showed faslculatlom; of facial,
rnasseter, trunk and extremity muscles. Electromyography Indicated normal-
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appearing motor units and periodic outbursts of repetitive short durations.
A biopsy of the right sural nerve showed that some fibers had undergone
degenerative changes. No detectable 2,4-D was found In a urine sample from
the patient. Treatment with dlphenylhydantoln relieved the muscular rigid-
ity In this patient as long as blood levels of drug were maintained.
Seabury (1963) administered a total of 12,712 mg of 2,4-D sodium salt,
369 mg of lndole-3-butyrlc acid and 38.3 mg of a-naphthalene acetic acid
to a patient that had terminal coccidioidomycosis during a period of 33 days
without observable toxicity. The 2,4-0 salt was administered dally 1n a
total of 23 doses; four of the first five doses were given via Intramuscular
Injection (8-24 mg/dose), doses 6 through 21 were given Intravenously (doses
11-12 were 960 mg/dose and doses 13-21 were 800 mg/dose), and dose 22 was
2000 mg. Although administration of the 2,4-0 until this point had been
without apparent adverse effects, a final Intravenous dose of 3600 mg 2,4-D
sodium salt 2 days after the 2000 mg dose, Infused over a 2-hour period,
elicited a lapse Into a semlstuporous state, fibrillary muscle movements In
the mouth and both hands that persisted for several hours, and hyporeflexla
In the knees, ankles and biceps that persisted for 24 hours. The patient
still complained of profound muscle weakness 24 hours after the Infusion.
Within the next 24 hours, recovery was observed, and no subsequent abnormal-
ities 1n neurological or muscular parameters were noted In the following 2
weeks.
Johnson (1971) summarized an unpublished Dow Chemical study on the
health effects of exposure to 2,4-D 1n a production facility. In this
study, 220 men exposed for 0.5-22 years to 2,4-D 1n a range of 30-40 mg/day
01450
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were reported to show no significant clinical effects when compared with a
control population of 4600 men not engaged 1n 2,4-0 or 2,4,5-V manufacture.
A battery of *at least 20 laboratory tests" was conducted, but additional
details of this unpublished study were not presented. The auUor noted that
karyotypes of peripheral blood lymphocytes from 10 of these workers showed
no chromosome aberrations.
Singer et al. (1982) assessed the nerve conduction velocities of the
median motor, median sensory and sural nerves of 56 employees (mean age of
35 years) engaged In the manufacture of 2,4,5-T and 2,4-0 (exposure levels
not determined) for an average of 7 years. The control group consisted of
25 subjects without previous exposure to neurotoxic agents, history of
diabetes, stroke, other neurological disease or excessive alcohol use.
Slowed nerve conduction velocities In one or more of the three bested nerves
were seen In 46% of the study group, as compared with 5% of the control
group. The most dramatic change occurred with sural sensory velocity, which
was significantly correlated with duration of employment. The significance
of these findings In relation to 2*4-0 exposure to humans wa:; not deter-
mined. Singer et al. (1982) Indicated that another study of a group of
workers exposed to 2,4,5-T contaminated with dloxlns but not exposed to
2,4-0 has been Initiated. Comparison of these two studies miiy shed some
light on the adverse effects In humans resulting from chronic exposure to
2,4-0.
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Carcinogenic Effects.* Several Swedish epidemiological studies of
workers exposed to chlorophenoxyacetlc acids and derivatives have been pub-
lished {Axelson and Sundell, 1974, 1977; Axelson et al.( 1979; Harden.
1977; Hardell et al., 1979, 1980; Hardell and Sandstrom, 1979; Eriksson et
a 1., 1977). Axelson and Sundell (1974) studied tumor Incidence and mortali-
ty In 730 Swedish railroad workers exposed for at least 45 days to various
herbicides Including chlorophenoxys, amlnotrlazole and monuron during spray-
ing operations. Exposed workers were divided Into four cohorts, based on
the type of herbicide exposure, and mortality and tumor Incidence were com-
pared with national average age and sex specific values. Hortallty was
reported In the following categories; 1) all causes, 2) all tumors, and
3) lung cancers. Latency periods of 0, 3 and 5 years were Included 1n cal-
culations (I.e., workers exposed within the last 3 or 5 years were excluded
from calculations). No excess for any of these three mortality causes was
found for the cohort exposed to chlorophenoxys and combinations. Data from
this study were recomblned and reanalyzed by Axelson and Sundell (1977) to
eliminate effects from combined exposure to chlorophenoxys and other agents
(pr1mar1ly amlnotrlazole). These Investigators concluded that an excess
tumor Incidence can be positively associated with chlorophenoxy exposure
alone, or with amlnotrlazole alone, and that the combination of the two
herbicide types may potentiate the tumorlgenlc effect.
Axelson et al. (1979) considered this same group of railroad workers In
a new study and extended the observation period from 1972 through October,
1978. Workers were divided Into three cohorts: those exposed only to
*For additional Indepth analysis of the cancer epidemiology studies for
phenoxy acetic acids and chlorinated phenols see U.S. EPA (1984).
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amlnotrlazole, those exposed only to chlorophenoxys, and those exposed to
both herbicide classes. Mortality evaluation Included the following:
1) total mortality, 2) mortality produced by all tumors, 3) mortality pro-
duced by tumors of the stomach, o' 4) mortality produced by tumors of the
lungs. When a 10-year latency period was used 1n calculations, the cohort
with combined herbicide exposure showed excesses In total mortality and
mortality produced by all tumors (7 cases vs. 1.78 expected). Those workers
who were exposed to chlorophenoxys showed only a small bu: significant
excess of tumors of the stomach (2 vs. 0.33 expected, p<0.05). This excess
in observed mortalities was associated with exposure to herbicides before
1962. The authors suggest that either the formulations used In that period
were more toxic, or that work practices may have been more lax during the
earliest period covered by this study (1957-1962). No estimates of the
levels of exposure to chlorophenoxys or the duration of those exposures
could be made. The 2,3,7,8-TCDD content of 2,4,5-T used by these workers
was not determined. This extremely toxic contaminant may produre effects In
the microgram range.
The discovery that 7 of 87 patients diagnosed with malignant mesenchymal
tumors had a history of exposure lo chlorophenoxy herbicides 10-20 years
earlier led Hardell (1977) to conduct a case-referent study of 52 cases of
soft-tissue sarcoma (STS) treated at Umea, Sweden, from 1970-977 (Hardell
and Sandstrom, 1979). The cases represented 21 living and 31 deceased male
patients, ranging 1n age from 26-80 years. Control subjects were matched
for sex, age, place of residence, and vital status. Deceased cases and
controls were matched for year of death. Cohorts were defined as those
exposed to chlorophenoxys only, those exposed to chlorophenoIs only, and
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those exposed to both classes of compounds. Exposure Information was
obtained by questionnaires and supplementary Interviews. These Investi-
gators calculated a relative risk of 5.3 (I.e., 5.3 x greater than the con-
trol risk) for developing STS In workers exposed to chlorophenoxys. This
cohort had been exposed primarily to 2,4,5-T, 2.4-D and HCPA. The authors
noted that 2,3,7,8-TCDD contamination of 2,4,5-T may have led to significant
exposure. The number of patients with exposure to only chlorophenoxys used
for the relative risk calculation was small (13 cases).
Another case-referent study on malignant mesenchymal tumors was Initi-
ated by this group for workers 1n a southern farming area of Sweden
(Eriksson et a!., 1977). Cases of STS were taken from the Swedish Cancer
Registry of reports made from 1974-1978. Patients were males, 72 living and
38 deceased, ranging 1n age from 25-75 years. The design of the earlier
study was retained for this Investigation (I.e., two control subjects were
selected for each case) and exposed workers were divided Into three
cohorts. The workers exposed to chlorophenoxys only showed a relative risk
of 6.8 for developing STS. Workers exposed to phenoxy herbicides not known
to be contaminated with polychlorlnated d1benzo-£-d1ox1ns (HCPA, 2,4-D,
mecoprop, dlchloroprop) showed a relative risk of 4,2. This subgroup,
however, represented only 7 of the 25 cases of sarcoma considered by
Eriksson et al. (1977). The authors suggest that this Increased risk found
In workers exposed to "nondloxln" herbicides alone could Indicate carcino-
genic effects produced by the chlorophenoxy herbicides themselves; however,
materials to which this group was exposed were not available for analysis.
In a later update of this work by Eriksson et al. (1981), he reiterated his
earlier finding of a roughly 6-fold Increase 1n the risk of STS from
exposure to phenoxy adds or chlorophenols. Again, he found that the risk
01450 " VI-10 04/07/88
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ratio given exposure to phenoxy acids free of 2,3,7,8-TCDD and dlbenzofurans
was 4.2. When consideration was given to persons exposed only to phenoxy
adds that contain such Impurities, the relative risk equalled 17.0 This
case-referent study was similar to that of Hardell and Sandstrom (1979) and
subject to the same cMtlslms. Recent work by Cochrane et al. (1980) Indi-
cates that 2,4-D may be contaminated with a small amount of chlorinated
d1benzo-f>-d1ox1ns other than the 2,3 (7(8-1somer.
Hardell et al. (1980) conducted a fourth case-referent study correlating
the Incidence of malignant lymphoma (both Hodgkln's and non-Hcdgk1n's lym-
phomas) with exposure to herbicides. The cases Involved were 117 living and
62 deceased male patients treated In Umea, Sweden, between 1974 and 1978.
The ages of patients ranged from 2!>-85 years. Workers exposej to chloro-
phenoxys alone showed a relative risk ratio of 4.8 for developing malignant
lymphomas. The nature of the herbicides Included 1n the category "chloro-
phenoxys" was not known, but probably Included 2,4-D, 2,4,5-T, MCPA,
plcloram and amlnotrlazole (these latter two are not chlorophsnoxy herbi-
cides). Of the 41 cases of mallgnan1: lymphoma with histories of exposure to
chlorophenoxys( seven cases were considered to have had primarily 2,4-D
exposure. The authors suggest that by dividing the 41 lymphoma cases Into
two groups, based on 90 days or more estimated exposure to chlorophenoxys,
an Increase In relative risk (7.0 versus 4.3) 1s seen with a longer duration
of exposure; however, this difference Is not statistically significant.
Methodology used In this study was the same as that used 1n the two earlier
case-referent studies conducted by this group. Because exposure was deter-
mined on the basis of questionnaires and telephone Interviews, very little
detail on the nature and duration of exposure could be determined*
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No increase 1n total mortality or deaths caused by malignant neoplasms
was found by Ott et al. (1980) In a study of 204 employees engaged 1n the
production of 2,4,5-T. Workers Included In the study had been employed for
1 or more months between 1950 and 1971 In 1 of 4 Jobs that Involved exposure
to chlorophenoxys and probably other agents such as chlorophenols and
styrene-butadlene latex. An Industrial hygiene survey conducted In 1969
found airborne exposure estimates of 0.2-0.8 mg/m3 for 2,4,5-T; <0.4
mg/m3 for 2,4-D; and 1.6-9.7 mg/m3 for 2,4,5-trlchlorophenol. These
levels, however, reflect only a single monitoring study and may have little
relationship to levels 10-1S years earlier. Comparisons of worker mortality
with average values for the total United States white male population using
5-year age Intervals found no significant differences for the exposed worker
population. The majority of the workers considered In this study had occu-
pational exposure of <12 months total to chlorophenoxys.
Lynge (1985), 1n an Incidence study of 3,390 males employed in two
factories manufacturing phenoxyacetlc acid herbicides, chiefly 2,4-0 and
MCPA, found a nonsignificant excess risk of STS In male employees. The
author stated that these results supported the Swedish observation of an
Increased risk of STS following exposure to phenoxyacetlc acid herbicides,
including 2,4-D, "unlikely to be contaminated with 2,3,7,8-TCDD." However,
after a 10-year latency, the excess of STS was significant (4 observed vs.
1.00 expected; p<0.05) In male employees of the single factory where 2,4(5-T
had been produced and used and where all five STS's arose. However, the
author cautions that because of the limited amount of 2,4,5-T processed at
that factory exposure 1s unlikely although not Impossible. The Lynge (1985)
study noted only a slight excess of lymphomas In males after a lapse of 10
01450
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years from initial exposure (4 observed vs. 3.04 expected). However,
sensitivity was somewhat reduced.
Hoar et al. (1986), In a population based case-referent study found
significantly high rates of non-Hodgk1n's lymphoma (NHL) In farmers In
Kansas who use herbicides, particularly 2,4-D and trlazlnes.
All newly diagnosed cases of STS, Hodgklns disease (HO) and non-Hodgk1ns
lymphoma between 1976 and 1982 among white male residents were Included In
the study Identified from the University of Kansas Cancer data service* a
population-based registry for the state of Kansas. There were '
-------
risk of NHL (OR = 6.0; 95% C.I. = 1.9, 19.5) relative to nonfarmers.
Frequent users who mixed or applied the herbicides themselves had an OR of
8.0 (95% C.I. = 2.3, 27.9) for NHL. Dose response excesses were associated
with use of phenoxyacetlc acid herbicides, essentially synonymous with use
of 2,4-0 (OR = 2.3; 95% CI = 1.3, 4.3), since only three patients and 18
controls had used 2,4,5-T and all but two of these controls had also used
2,4-D. Use of 2,4-0 only, I.e., eliminating 2,4,5-T users, was associated
with an OR of 2.6 (95% CI = 1.4, 5.0). Neither STS nor HD was associated
with herbicide or pesticide exposure. The authors concluded "this study
confirms the reports from Sweden and several U.S. studies that NHL 1s
associated with farm herbicide use, especially phenoxyacetlc acids. It does
not confirm the case-control studies or the cohort studies of pesticide
manufacturers and Vietnam veterans linking herbicides to STS or HD".
However, It Is not contradictory to the hypothesis that 2,3,7,B-TCD0 Is the
contaminant responsible for the development of STS, since 2,4-0 does not
contain 2,3,7,8-TCOD.
Few respondents could remember exposure to 2,4,5-T( which contains
2,3,7,8-TCOO. 2,4-D is not believed to contain 2,3,7,8-TCOO but does
contain other polychlorInated d1benzo-£-d1ox1n Impurities. The risk was
found to Increase with Increasing frequency and duration of herbicide
usage. Although "herbicide usage" could mean any of the herbicides
Identified by Hoar, she wrote that this Is "essentially synonymous" with use
of 2,4-0. The next most used herbicides I.e., trlazlnes and uracils are
nonsignificant when exposure to phenoxyacetlc acids are controlled for In
her analyses. However, this study has problems similar to the Hardell et
al. (1981) study In that there 1s a lack of substantiation of exposure, and
the Information 1s based on questionnaire responses that are subject to some
01450 VI-14 04/07/88
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recall bias. Moreover, there Is a statistically significant risk associated
with the use of other herbicides as well. I.e., trlazlnes, amides, and
tMfluralln. These uncertainties, while raising concerns, do not discount
the observed dose-response relationship or the observed Increased
Incidence. A more detailed critical analysis of this study nwy be provided
in a later version of this document.
Summary
Case reports of Individuals who acutely Ingested 2,4-D solutions Indi-
cate that early symptoms of exposure Include gastritis, vomiting and loss of
consciousness, and that muscular paralysis precedes death. Autopsies of
fatal poisoning cases have shown widespread pathologic effect; (e.g., con-
gestion and hyperemia of most organs, hepatic necrosis). Reports of human
poisoning from industrial or agricultural exposure to 2,4-D formulations
(dermal and Inhalation exposures) commonly described neurologlral signs and
symptoms (e.g., fatigue, nausea, reflex disorders, paresthesia). Most of
the clinical reports did not Identify other possible chemicals to which the
subjects may have been exposed, and did not Indicate levels of 2,4-0
exposure.
The epidemiologic studies of Ho<*r et al. (1986), Lynge (19B5), Eriksson
et al. (1961), Harden et al. (1981), and Hardell and San
-------
This carefully designed and conducted study provides evidence of a dose-
related excess risk of NHL with exposure to 2,4,-D. Because confidence In
Inferring a causal association from epidemiologic data Is Increased when
several Independent studies are concordant and with knowledge that NCI
presently plans to release the results of two additional 2(4-D studies
within 12 months, the CAG will withhold a welght-of-evldence Judgement on
the 2,4-0 human data.
01450
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VII. MECHANISMS OF TOXICITY
The myotonic syndrome produced 1n rats by the administration of 2,4-0
was studied by Ezyagulre et al. (1948). Intraperitoneal Injection of
100-250 mg/kg of the 2,4-0 sodium salt or Intra-arteMal administration of 2
mg of compound per animal produced neuromuscular effects that resembled
those observed In congenital myotonia or those produced by poisoning with
veratMne alkaloids. Muscles showed Increased sensitivity to stimulating
agents; a single spike was followel by a silent period and then a burst of
repetitive firing. Increases 1n both twitch tension and Ultch duration
were noted. Constant activity of the affected muscles caused :hese myotonic
symptoms to diminish, but following rest, the syndrome reappear.
Iyer et al. (1976) investlgatec the neural factors contributing to this
effect 1n a further study of the myotonic syndrome Induced by Intraperi-
toneal injection of 2,4-0 1n rats (dose unspecified). These Investigators
found that myotonic discharges appeared within 2 hours of a single Injection
and disappeared after 24 hours. Nerve block or nerve section produced after
the Initiation of myotonia had no effect on the condition; however, denerva-
tion of the muscle before treatment with 2,4-0 produced a progressive loss
of Its myotonic response.
Iyer et al. (1977a.b# 1981) and Ranlsh et al. (1977 ) reported In later
studies that rat skeletal muscle did not become myotonic following \n vivo
or iD. vitro exposure to 2,4-0 if the muscle was previously denorvated. They
concluded that 2,4-0 directly affects the muscle membrane (sarcolemrna) and
that the resting Ionic conductance of sarcolemrna Is influsnced by the
presence of neural factors. Further evidence supporting this mechanism of
01460
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01/31/85
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2,4-D toxicity was reported by Rudel and Senges (1972), following a study of
Intracellular recordings of membrane potential In rat diaphragm muscle
exposed to 2,4-D; the sarcolemma was altered by decreased resting membrane
conductance as a result of exposure to 2,4-D. Also, 1n a literature summary
on myotonia In mammalian skeletal muscle, Kw1ec1nsk1 (1981) noted the simi-
larity between the conditions following the direct and selective activity of
compounds such as 2,4-D on the sarcolemma of rats and those resulting from
hereditary myotonia In humans. In humans, hereditary myotonia is generally
thought to result from a genetic change In the structure and function of the
sarcolemma.
Hlstochemlcal examination of skeletal muscle from rats 1n which myotonia
had been Induced by the intraperitoneal Injection of 300 mg/kg 2,4-D Indi-
cated an Inhibition of phosphorylase activity (Heene, 1966a). further
experiments by Heene (1966b) demonstrated that addition of 2xl0~4 to
5x10"* H concentrations of 2,4-D to 10 >im tissue sections of skeletal
muscle would also Inhibit phosphorylase and transglycosldase activities.
Dux et al. (1977) reported changes In the membrane of skeletal muscles
from myotonic rats Induced by Intraperitoneal Injection of 2,4-D (50
mg/kg/day for 21 days). Using a pyroantlmonate precipitation technique to
localize calcium 1n dystrophic muscle sections, these researchers were able
to show lowered calcium In muscle triads and Increased calcium associated
with troponin C. They suggested that this shift In muscle calcium may lead
to alterations In the actlnomyosln contraction system. Seller (1978)
reported -60% In, vitro inhibition of both sodium-potassium and magnesium
stimulated ATPases 1n sarcolemma extracts of normal rats following the
01460
VI1-2
02/15/87
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addition of 2.5 mH concentrations of 2,4-0. Direct Injection of 100-500
mg/kg 2,4-0 Into the pectoralls muscle has been reported to Increase muscle
levels of glucose-6-phosphate wltMn 30-45 minutes, but alteration 1n levels
of ADPases and ATPases was not found (Kuhn and Stein, 1964) They further
reported an Inhibition of *5Ca uptake by a sarcolemna preparation Ijn vitro
following the addition of 2,4-0.
t Using embryonic chick fibroblasts prepared from primary muscle cultures,
Emmons et al. (1980) Investigated the relationship between perturbed sterol
metabolism In the sarcolemma and experimentally Induced myotonia. Applica-
tion of 10 vg/mi of 2#4-0 to the fibroblast culture:; resulted 1n
decreased cholesterol blosynthesis and an accompanying accumulation of
acetate derivatives in desmosterol and related sterols. The authors con-
cluded that the results obtained In this In vitro assay are consistent with
the hypothesis that experimentally Induced myotonia Is preceded by a pre-
requisite change 1n steroid composition of the sarcolenma.
In vitro effects of 2,4-0 on lipid biosynthesis In rat liver homogenates
were reported by Olson et al. (1974). The addition of 4.5 and 9.0
concentrations of 2,4-0 to liver preparations Inhibited the Incorporation of
"C-mevalonate Into nonsaponlflable lipids and, at the sime concentra-
tions, Inhibited 14C-acetate Incorporation Into cholesterol The authors
Indicated that 2,4-0 may produce hypolipidemic effects In a manner similar
to that of chlorophenoxylsobutyrate, a clinically used agent. Kolberg et
al. (1971) noted that L cell cultures exposed to 500 mg/ml 2,4-0 for a
24-hour period showed Increased Incorporation of ®H-)alm1tate and
1*C-acetate Into total cell lipids. The Increased *H-palin1tate Incorp-
01460
ViI-3
07/31/84
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oration was primarily In the triglyceride fraction, suggesting that In-
creased fatty acid uptake from the medium was produced by 2,4-0 at this
level.
Brody (1952) reported that 2,4-0 added in vitro to rat liver mitochon-
dria was an uncoupling agent for oxidative phosphorylation. At concentra-
tions as low as 5x10"* H, 2,4-0 began to decrease the phosphate/oxygen
ratio In mitochondria without significantly affecting respiration, while at
1x10"* M, more than BOX Inhibition of phosphorylation was observed.
Effects of 2(4-D on the oxidative phosphorylation of rat liver mitochondria
were also studied by Whltehouse (1964), who reported no uncoupling effects
at 2,4-0 levels of 1x10"* H; however, at 2.5x10"* H, decreases 1n the
phosphate/oxygen ratio were demonstrated. The differences 1n the Inhibitory
levels of 2,4-0 reported by these two Investigators are apparently due to
the nature of the rat mitochondrial preparations tested, because Brody
(1952) reports an almost 2-fold higher control value for phosphate/oxygen
ratio than that reported by Whltehouse (1964). Welnbach and Garbus (1965)
compared the uncoupling activity of several phenols 1n rat liver mito-
chondria and showed that complete uncoupling of oxidative phosphorylation In
their assay system could be produced by 2xl0~4 H dlchlorophenol; dlnltro-
phenol was effective 1n producing complete uncoupling at a 15-fold lower
level. The 2,4-0 uncoupling effects noted are therefore occurring at rela-
tively high In vitro levels.
Cytotoxic effects of 2,4-D on cultured cells were observed by Haag et
al. (1975). Exposure of cultured chick muscle cells to 2.5 nfl 2,4-0 for 44
hours produced moderate cytotoxicity that Increased when the concentration
01460
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07/31/84
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was raised to 5 mH. Morphological changes 1n cells treated at these 2,4-0
levels Included partial lack of polar orientation, diminished fiber forma-
tion and an Increased nuclear/cytoplasmlc ratio. Murakami and Fukaml (1978)
noted Inhibition of cell growth 1n a human embryonic lung cull line when
4x10~* M 2,4-0 was present In cultures for 48 hours. Very little of the
2,4-0 present 1n the medium was taken up by these cells (2.6-5.0 pmol/wg
cell protein). Complete growth Inhibition of I cells exposed for 24 hours
to 350 mg/mt of 2,4-0 has been reported by Kolberg et al. (1971].
Several Investigations have deiw>nstrated effects of 2,4-0 on 0NA syn-
thesis, mitosis and cell-cycle parameters when added to cultured cells.
Haag et al. (1975) observed that the S phase of the cell cycle was prolonged
In cultured chicken muscle cells exposed to 2.5x10"" H 2,4-0 for 44 hours.
Bongso and Basrur (1973) found that embryonic bovine kidney cells treated
with 10 ppm of 2,4-0 for 24 hours showed an elevated mitotic Irdex caused by
an Increase In the number of prophase cells. Treated cells at 48 hours had
Increases In nucleolar size, nuclear lobulation, polyploid ml to tic figures
and multipolar spindles. The authors suggest that these changes may be
produced by interaction of 2,4-0 with mitotic spindle proteins. Increased
mitotic activity has also been reported by Weiss and Beckert (1975) In
monkey kidney cells and Glrardl heart cells treated with 10 or 50 ppm of
2,4-0 for up to 12 hours. Seller (1979) found that oral administration of
2,4-0 to mice can Inhibit testicular DNA synthesis. One hour following
Intraperitoneal Injection of l4C-thym1d1ne, mice were administered 200
mg/kg 2,4-0 orally; 30 minutes later they were sacrificed and testicular OKA
was extracted. Treated mice showed a 29% decrease In the Incorporation of
labeled thymidine Into 0NA. Inhibition of DNA synthesis ^_n vitro, as
01460 VI1-5 09/19/84
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measured by the Incorporation of *H-labe1ed deoxyhucleoslde triphosphates
Into DNA by DNA polymerase, has been reported by Schwlmmer (1968) when
1.5xlQ"4 H 2,4-0 was added to the Incubation mixture.
Enzyme Inhibition by 2,4-0 has also been reported 1n several other
studies. Wedding and Black (1963) showed that porcine heart malic dehydro-
genase, lactic dehydrogenase (rabbit muscle) and alcohol dehydrogenase
(yeast) were all Inhibited by 2,4-0 at levels of -10"* H. Inhibition was
competitive relative to the pyridine nucleotide cofactors; the authors
suggest that other pyridine nucleotlde-requlrlng enzymes may be Inhibited by
2,4-0. Increased activities of several placental enzymes have been observed
In guinea pigs following maternal administration of 2,4-D (Humlczewska and
Stanosz, 1971). Subcutaneous Injection of 30 mg/kg of 2,4-D for 6 days/week
throughout gestation Increased succinate dehydrogenase, alkaline phosphatase
and acid phosphatase activities as determined by hlstochemlcal methods.
Summary
Observations that rat skeletal muscle did not become myotonic following
iH v^v0 oi" iEL iMllL exposure to 2,4-0 1f the muscle was previously denervat-
ed (Iyer et al., 1976, 1977a,b, 1981; Ranlsh et al., 1977) and intracellular
recordings of membrane potential In rat muscle exposed to 2,4-0 (Rudel and
Senges, 1972) Indicate that 2,4-0 directly affects the sarcolemma. Changes
1n sarcolemma calcium (Dux et al., 1977 ; Kuhn and Stein, 1964), steroid
(Emmons et al., 1980), and Inhibition of muscle phosphorylase and trans-
glycosldase activities (Heene, 1966a,b) were also observed In Vn vitro and
iQ vivo experiments.
01460
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Addition of 2,4-D to rat liver preparations Inhibited lipid biosynthesis
(Kolberg et al., 1971; Olson et al., 1974) and oxidative phosphorylation
(Brody, 1952; Whltehouse, 1964). Cytotoxicity (Kolberg et al., 1971; Haag
et al., 1975; Murakami and Fukaml, 1978), Increased mitotic activity (Bongso
and Basrur, 1973; Haag et al., 1975; Weiss and Beckert, 1975), and inhibi-
tion of ONA synthesis (Schwlmmer, 1968; Seller, 1979) and nuc"eotlde-requlr-
Ing enzymes (Wedding and Black, 1363) have also been observed In mammalian
cells that were exposed to 2,4-0 Hi vitro and in vivo.
01460
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VIII. QUANTIFICATION OF TOPOLOGICAL EFFECTS
Introduction
The quantification of toxlcologlcal effects of a chemical' consists of
separate assessments of noncarclnogenlc and carcinogenic health effects.
Chemicals that do not produce carcinogenic effects are believed to have a
threshold dose below which no adverse, noncarclnogenlc health effects occur,
while carcinogens are assumed to act without a threshold.
In the quantification of noncarclnogenlc effects, a Reference Dose
(RfD), [formerly termed the Acceptable Dally Intake (AD1)] Is calculated.
The RfD Is an estimate (with uncertainty spanning perhaps an order magni-
tude) of a dally exposure to the human population (Including sensitive
subgroups) that Is likely to be without an appreciable risk of deleterious
health effects during a lifetime. The RfD Is derived from a no-observed-
adverse-effeet level (NOAEL) , or 1 owest-observed-adverse-effeet level
(LOAEL), Identified from a subchronlc or chronic study, and divided by an
uncertainty factor(s) times a modifying factor. The RfD 1s calculated as
follows:
RfD - _ mg/kg bw/day
[Uncertainty Factor(s) x Modifying Factor]
Selection of the uncertainty factor to be employed 1n the calculation of
the RfD Is based upon professional Judgment, while considering the entire
data base of toxlcologlcal effects for the chemical. In order to ensure
that uncertainty factors are selected and applied 1n a consistent manner,
01470
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the U.S. EPA (1986) employs a modification to the guidelines proposed by the
National Academy of Sciences (NAS, '977, 1980) as follows:
Standard Uncertainty Factors (UFs)
Use a 10-fold factor when extrapolating from valid experimental
results from studies using prolonged exposure to average healthy
humans. This factor Is intended to account for the variation
In sensitivity among the members of the human population [10H]
• Use an additional 10-fold factor when extrapolating from valid
results of long-term studies on experimental animals when
results of studies of human exposure are not available or are
Inadequate. This factor Is Intended to account for the uncer-
tainty In extrapolating animal data to the case of humans.
[10A]
Use an additional 10-fold factor when extrapolating from less
than chronic results on experimental animals when there is no
useful long-term human data. This factor Is Intended to
account for the uncertainty In extrapolating from less than
chronic NOAELs to chronic NOAELs. [10S]
Use an additional 10-fold factor when deriving an RfO from a
LOAEL instead of a NOAEL. This factor 1s intended to account
for the uncertainty In extrapolating from LOAELs to NOAELs.
[10L]
Modifylng Factor (MF)
Use professional judgment to determine another uncertainty
factor (MF) that 1s greater than zero and less than or equal to
10. The magnitude of the MF depends upon the professional
assessment of scientific uncertainties of the study and data
base not explicitly treated above, e.g., the completeness of
the overall data base and the number of species tested. The
default value for the MF Is 1.
The uncertainty factor used for a specific risk assessment 1$ based
principally upon scientific Judgment rather than sclentlf'c fact and
accounts for possible Intra- and Interspecies differences. Additional
considerations not Incorporated In the NAS/ODW guidelines for selection of
an uncertainty factor Include the use of a less than I1fet1r>e study for
deriving an RfO, the significance of the adverse health effects and the
counterbalancing of beneficial effects.
01470
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From the RfD, a Drinking Water Equivalent Level (OWEL) can be calcu-
lated1. The DWEL represents a medium specific (I.e., drinking water)
lifetime exposure at which adverse, noncardnogenlc health effects are not
anticipated to occur. The DWEL assumes 100% exposure from drinking water.
The DWEL provides the noncardnogenlc health effects basis for establishing
a drinking water standard. For Ingestion data, the DWEL Is derived as
follows:
DWEL = (Rf°) * = mg/l
Drinking Water Volume 1n l/day
where;
Body weight * assumed to be 70 kg for an adult
Drinking water volume = assumed to be 2 t/day for an adult
In addition to the RfD and the DWEL, Health Advisories (HAs) for expo-
sures of shorter duration (1-day, 10-day and longer-term) are determined.
The HA values are used as Informal guidance to municipalities and other
organizations when emergency spills or contamination situations occur. The
HAs are calculated using an equation similar to the RfD and DWEL; however,
the NOAELs or LOAELs are Identified from acute or subchronlc studies. The
HAs are derived as follows:
HA - {"OAEL or LOAEL) x (bw) _
(UF) x ( l/day)
Using the above equation, the following drinking water HAs are developed
for noncardnogenlc effects:
1. 1-day HA for a 10 kg child Ingesting 1 l water per day.
2. 10-day HA for a 10 kg child Ingesting 1 t water per day.
3. Longer-term HA for a 10 kg child Ingesting 1 l water per day.
4. Longer-term HA for a 70 kg adult Ingesting 2 t water per day.
01470
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The 1-day HA calculated for a 10 kg child assumes a single acute
exposure to the chemical and Is generally derived from a stucy of <7 days
duration. The 10-day HA assumes a limited exposure period of 1-2 weeks and
1s generally derived from a study of <30 days duration. The longer-term HA
Is derived for both the 10 kg child and a 70 kg adult anj assumes an
exposure period of -7 years (or 10% of an Individual's lifetime). The
longer-term HA 1s generally derived from a study of subchrcnlc duration
(exposure for 10% of animal's lifetime).
The U.S. EPA categorizes the carcinogenic potential of a chomlcal, based
on the overall we1ght-of-ev1dence, according to the following scheme:
Group A: Human Carcinogen. Sufficient evidence exists from
epidemiology studies to support a causal association between
exposure to the chemical and human cancer.
Group B: Probable Human Carcinogen. Sufficient evidence of
carcinogenicity 1n animals with limited (Group B1) or inade-
quate (Group B2) evidence In humans.
Group C: Possible Human Carcinogen. Limited evidence of
carcinogenicity 1n animals In the absence of human data.
Group D: Hot Classified as to Human Carcinogenicity. Inade-
quate human and animal evidence of carcinogenicity or for which
no data are available.
Group E: Evidence of Noncarclnoqenlclty for Humans. No
evidence of carcinogenicity In at least two adequate "animal
tests 1n different species or In both adequate epidemiologic
and animal studies.
If toxlcologlcal evidence leads to the classification of the contaminant
as a known, probable or possible human carcinogen, mathematical models are
used to calculate the estimated excess cancer risk associated with the
Ingestion of the contaminant in drinking water. The data usi?d In these
01470
VIII -4
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estimates usually come from lifetime exposure studies using animals. In
order to predict the risk for humans from animal data, animal doses must be
converted to equivalent human doses. This conversion Includes correction
for noncontlnuous exposure, less than lifetime studies and for differences
1n size. The factor that compensates for the size difference Is the cube
root of the ratio of the animal and human body weights. It 1s assumed that
the average adult human body weight 1s 70 kg and that the average water
consumption of an adult human 1s 2 I of water per day.
For contaminants with a carcinogenic potential, chemical levels are
correlated with a carcinogenic risk estimate by employing a cancer potency
(unit risk) value together with the assumption for lifetime exposure from
Ingestion of water. The cancer unit risk is usually derived from a linear-
ized multistage model with a 95% upper confidence limit providing a low dose
estimate; that 1s, the true risk to humans, while not Identifiable, is not
likely to exceed the upper 11m1t estimate and. In fact, may be lower.
Excess cancer risk estimates may also be calculated using other models such
as the one-h1t» Welbull, loglt and problt. There Is little basis In the
current understanding of the biological mechanisms Involved In cancer to
suggest that any one of these models Is able to predict risk more accurately
than any other. Because each model 1s based upon differing assumptions, the
estimates derived for each model can differ by several orders of magnitude.
The scientific data base used to calculate and support the setting of
cancer risk rate levels has an Inherent uncertainty that Is due to the
systematic and random errors 1n scientific measurement. In most cases, only
studies using experimental animals have been performed. Thus, there 1s
01470
VIII-5
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uncertainty when the data are extrapolated to humans. Wtv?n developing
cancer risk rate levels, several other areas of uncertainty exist, such as
the Incomplete knowledge concerning the health effects of contaminants In
drinking water, the Impact of tie experimental animal's age, sex and
species, the nature of the target organ system(s) examined and the actual
rate of exposure of the Internal t«irgets In experimental animals or humans.
Dose-response data usually are available only for high level* of exposure
and not for the lower levels of exposure closer to where a standard may be
set. When there Is exposure to more than one contaminant, additional
uncertainty results from a lack of information about possible synergistic or
antagonistic effects.
Noncardnoqenlc Effects
The different forms of 2,4-D (a:1ds, salts, esters) have bsen discussed
together In this document. Sufficient toxlcoklnetlc data are not available
to determine whether the esters should be considered separately from the
adds and salts. There are no data to Indicate how rapidly the esters of
2,4-0 are hydrolyzed by mammals; however, at physiological pH, 2,4-0 exists
In the Ionized form, which does not readily pass through biological mem-
branes, as compared with the esters that would (Zetterberg, 1977). In
humans, the rate of plasma uptake of orally administered 2,4-C, the degree
of conjugation of the compound and the rate of elimination may vary con-
siderably between Individuals (Sauerhoff et al.t 1977; Kohl 1 et al., 1974);
further characterization of this interlndlvldual variation In the human
population Is needed.
01470
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The LD^q range of 2,4-D 1s generally between 300 and 1000 mg/kg; there
does not appear to be significant differences In toxicity between the free
acid and the various salt and ester derivatives. H111 and Carlisle (1947)
determined oral LD^s of &&&. 375, 800 and 1000 for 2,4-D sodium salt 1n
rats, mice, rabbits and guinea pigs, respectively; the maximum doses in
these species not causing death were 333, 125, 200 and 333 mg/kg, respec-
tively. Individual monkeys that were fed single doses of -286 or 428 mg/kg
of 2,4-D sodium salt or 286 mg/kg of 2,4-D ammonium salt regurgitated a
large portion of the material, precluding determinations of lethal doses
(Hill and Carlisle, 1947). Symptoms other than nausea were not described 1n
these monkeys. Approximately 214 mg/kg of 2,4-D sodium salt was fed to
another monkey without development of vomiting or "serious Illness" (H111
and Carlisle, 1947). Comparison of the species sensitivity to 2,4-D Indi-
cates that dogs may show greater sensitivity to this compound (Rowe and
Hymas, 1954). The higher toxicity observed 1n dogs may reflect an Inability
of kidney processes 1n this species to effectively clear phenoxyacetlc acids
(Seller, 1978).
Drill and Hlratzka (1953) described myotonia with pathologic changes of
G1 mucosa Irritation, moderate hepatic necrosis and mild renal tubular
degeneration 1n dogs that were lethal 1y poisoned by acute oral administra-
tion of 2,4-D at doses of 100-400 mg/kg.
Bucher (1946) found that myotonia persisted for 8-24 hours 1n strain A
mice that were Injected Intraperitoneal^ with sublethal doses (100-200
mg/kg of 2,4-0). Ho significant differences were found In the effects
produced when 2,4-D was administered subcutaneously, Intraperitoneal^ or
Intravenously.
01470 VI11-7 02/25/87
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In a summary report, Rowe and Hymas (1954) described an experiment where
doses of 0, 30, 100 or 300 mg/kg/day 2%4-D to groups of 5 or 6 female rats
(strain not specified) by Intubation 5 times/week for 4 weeks were utilized.
The 2,4-0 was administered In olive oil that was emulsified In 5-20% aqueous
gum arable, and the controls were vehicle treated. Rats that received 30
mg/kg/day or less reportedly showed no adverse treatment-related clinical or
pathological effects, but treatment with 100 mg/kg/day elicited GI irrita-
tion, depressed growth rate and slight cloudy swelling of th<; liver. Rats
that received 100 mg/kg/day 2,4-C succumbed rapidly (not elaborated) and
died; severe GI Irritation was 'eportedly the principal adverse effect
observed.
In addition, groups of five young adult female rats (str«1n not speci-
fied) were maintained on diets tha: contained 0, 100, 300 or 000 ppm 2,4-0
in the diet for 113 days (Rowe and Hyrnas, 1954). If it Is assumed that
young rats consume 10% of their weight In food per day, the corresponding
dally doses are 0, 10, 30 and 50 mg/kg/day. Rats that were exposed at the
1000 ppm level experienced excessive mortality (not quantified), depressed
growth rate, excessive mortality and slight cloudy swelling of the liver.
These effects were not observed al: the two lowest doses. Groups of five
rats that wore given diets that contained higher concentratJons of 2,4-0
(3000 or 5000 ppm) were sacrificed after 12 days because l.hey were not
eating and were rapidly losing weigit;.examinations revealed Ircreased liver
and kidney weights and slight but unspecified pathologic changes.
Chang et al. (1974) reported Wat dietary administration of 2,4-0 to
rats at levels of 2000 ppm In the diet for 4-7 (~200 mg/kg/day) weeks
01470
VIII-8
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produced a slight Increase In liver glycogen content* a slight decrease in
liver RNA content and slight decreases 1n absolute and relative liver
weights, but no overt signs of toxicity.
Administration of 0, 200 or 400 ppm dietary 2,4-0 (-0, 10 or 20 mg/kg/
day, respectively) to groups of seven rats for 1 month did not adversely
affect food Intake or rate of growth, or elicit characteristic signs of
Intoxication (skeletal muscular signs or paralysis) (Hill and Carlisle,
1947). Oletary administration of 2,4-0 at a level of 100 ppm for 21 days
and subsequently 1000 ppm for 10 days (average total dose -39.0 mg/kg/day)
was similarly non-toxic for rats. Groups of six guinea pigs that were given
10 dally doses of 50 or 100 mg 2,4-0 1n 12 days (-88 or 177 mg/kg/day) by
Intubation also did not develop characteristic evidence of Intoxication.
Drill and Hlratzka (1953) administered 2,4-0 orally In capsules to
groups of 2-4 dogs at doses of 0, 2, 5t 10 or 20 mg/kg/day, 5 days/week, for
13 weeks. When adjusted for a 7-day week, the respective dally doses are 0,
1.4, 3.6, 7.1 and 14.3 mg/kg/day. Toxic effects were only observed at the
high dose. Treatment at 20 mg/kg/day produced death In 3 or 4 dogs between
days 18 and 49, and symptoms In the moribund animals Included hind leg
stiffness, ataxia, weakness, gum bleeding and difficulty In chewing and
swallowing. A terminal decrease 1n the percentage of blood lymphocytes was
noted 1n the 3 dogs that died, but significant effects on the hemoglobin,
red cell count or total white cell count were not observed. The dog that
survived 2,4-D treatment at the high dose, as well as the dogs exposed to
the lower levels of 2,4-D, showed no significant hematologic, gross or
histopathologic effects.
01470
V1I1-9
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Effects of subcutaneously Injected 2,4-0 (sodium salt) on the thyroid
gland ' of treated rats have been reported (Florshelm and Velcoff, 1962;
Florshelm et a")., 1963). These Investigators showed that, following seven
dally Injections of 2,4-D at a level of 100 mg/kg, thyroid weight was
decreased. Administration of 2,4-0 at 80 mg/kg over this period Increased
radioactive Iodine uptake by the thyroid, lowered the blnd'ng of radio-
labeled thyroxine by serum proteins, and Increased the amount of radio-
labeled compound In the liver of treated rats.
Toxic effects of 2,4-0 on the nervous system of rats admin1stered lethal
doses of the compound 1ntraper1ton*?ally have been described by Dcsl et al.
(1962). Animals injected dally with 200 mg/kg of 2,4-0 (form not specified)
died within 6 days. Progressively decreased conditioned reflex responses
were observed over this period, a; well as the appearance of large slow
waves 1n the E£G. Histological examination Indicated that dtmyellnlzatlon
was present In the dorsal portion oF the spinal tract. Within 10-15 minutes
following a single Intraperitoneal Injection of the compound, EEG changes
were observed (decreased cerebral and reticular desynchronlzatlon); recovery
was seen In -1 hour. The authors postulate that the neurological effects
produced by 2,4-0 In this study are due Initially to action of the compound
on the reticular formation, followed by later effects on cerjbral tissue.
Histological examination, however, Failed to show any morphological changes
In the cortex or subcortical regions of treated animals. The demyellnlza-
tlon observed In the spinal cord may be responsible for the hind limb para-
lysis noted by other investigators after poisoning of animals with 2,4-D.
01470
V1II-10
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Hansen et al. (1971) conducted 2-year feeding studies with technical
grade (96.7% pure) 2,4-D in Osborne-Mendel rats. In the rat study, 25
animals of each sex were exposed to 0, 5, 25, 125, 625 or 1250 ppm 2,4-D In
the diet (-0, 0.25, 1.25, 6.25, 31.25 or 62.5 mg/kg/day) frdm 3 weeks of
age. At the conclusion of treatment, all rats were autopsied, but compre-
hensive histopathologic examinations were performed only on 6 rats/sex from
the high-dose and control groups; the liver, kidneys, spleen ovaries or
testes and other tissues that contained gross lesions were histologically
examined In the remaining rats in the high exposure and control groups and
In the rats at the other dose levels. Significant differences 1n survival,
mean bw and organ/bw ratios (liver, kidney, heart, spleen or testes) were
not found between any of the treated groups and the control group during the
2-year treatment or at the end of the study. Significant treatment-related
pathologic effects were not observed. The incidence of tumors did not
differ significantly between the groups. Several hematologic Indices (hemo-
globin, hematocrit, total white cell count) were similar In the treated and
control groups, but It was noted that the red blood cell count of the
treated rats (1250, 625 and 5 ppm groups) showed a "tendency" toward macro-
cytosls, "very slight to slight" polychromasls, and "slight to moderate"
hypochromasla. The tendency toward macrocytosls was reportedly not present
and the other red cell abnormalities were of a "minor degree" 1n the control
rats. The toxlcologlcal significance of these vaguely reported effects Is
unclear.
In a two-generation reproduction study with rats that Is also discussed
1n Other Reproductive Effects 1n Chapter V (Bjorklund and Erne, 1966),
administration of 1000 ppm 2,4-D 1n the drinking water (-50-100 mg/kg/day)
01470
VIII-11
02/25/87
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of rats (5/group) during pregnancy and for a further 10 months had no sig-
nificant effects on the maternal animals (not specified) or offspring (cllr-
1cal signs or malformations). Similar exposure of 22 weaned offspring (10
males, 12 females) for up to 2 years was, with the exception 'of reduced food
and water Intake and consequent growth retardation, temporary diarrhea and
poor general condition, also nontoxic as Judged by normal clinical chemistry
Indices, hematocrit, hemoglobin, plasma GOT, plasma elimination rate of
2,4-0 (3 hours), relative organ weights (heart, spleen, liver, kidneys,
lungs, testes, ovaries), or gross or microscopic pathology. Other reproduc-
tion studies that are detailed In Teratogenicity and Other Reproductive
Effects In Chapter V reported that dietary exposure to 1500 ppm (-75 mg/kg
bw) 2,4-D for 2 years (Hansen et al., 1971) and dietary exposure to 1000 ppm
(-100 mg/kg) for 3 months (Gaines and Klmbrough, 1970) prior to mating and
during pregnancy and lactation caused an Increase In preweanllrg mortality,
Hansen et al. (1971) also fed 6- to B-month-old beagle dogs (3 of each
sex/group) 0, 10, 50, 100 or 500 ppm technical grade 2,4-D In the diet (~0,
0.29, 1.45, 2.9 or 14.5 mg/kg/day) for 2 years. Treatment-related effects
were not Indicated based on observations of mortality as well as gross and
microscopic tissue examinations In any of the treated groups or control
group.
Quantification of Noncarclnoqenlc Effects
Derivation of 1-Day HA, Clinical reports have descrlted cases of
human Ingestion of 2,4-D and Its derivatives (see Acute Effects In Chapter
VI), but there 1s only one report of nonfatal poisoning In wheh quantita-
tive exposure data were provided. Berwick (1970) described a farmer who
01470
VI11-12
02/25/87
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swallowed a mouthful of weed killer (49% Eptam, 35.5% 2,4-D Isooctyl ester,
0.5% eplchlorohydrIn and 5% emulslflers) and exhibited characteristic
symptoms of 2,4-0 exposure (e.g., acute gastritis, vomiting, skeletal muscle
damage) that subsided within -2 weeks. There was no evidence of chollnes-
terase Inhibition, further Indicating that the toxlcologlcal effects were
not Induced by the Eptam. If 1t 1s conservatively assumed that the volume
of a mouthful of liquid Is 25 ml and that the density of 2,4-D Isooctyl
ester 1s similar to that of 2,4-0 n-butyl ester (1.2 g/mt), the quantity
of Isooctyl ester Ingested can be estimated to be -11 g (25 ml x 0.355 x
1.2 g/ml); the equivalent quantity of 2,4-0 add would be -6.1 g (-96.7
mg/kg, assuming a body weight of 70 kg). Although factors such as the crude
quantitation of the quantity Ingested and the unknown pharmacokinetic
properties of 2,4-0 Isooctyl ester could preclude the Identification of a
human single-dose nonlethal level from this study, a more serious problem 1s
a lack of corroborating data. In other case reports, deaths were described
In a male agricultural student who Ingested at least 6 g of a commercial
herbicide preparation of (50% w/w) 2,4-0 dlmethylamlne salt (>2.5 g 2,4-0
acid equivalent) (Nlelson et al., 1965) and In a 46-year-old man who died
within 14 hours of swallowing at least 13.5 g of an uncharacterlzed 2,4-0
solution (Herblch and Hachata, 1963). It Is apparent from examination of
these data that the exposures In the above reports are too poorly character-
ized to be used to Identify an unequivocal nonlethal dose that could be used
to derive a 1-day HA; the estimated nonlethal adverse effect level In the
Berwick (1970) report (-6.7 g) appears to be bracketed by the apparent
lethal doses of Nlelson et al. (1965) (>2.5 g) and Herblch and Hachata
(1963) (>13.5 g).
01470
VIII-13
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Most of the information on the effects of single, oral exposures to 2,4-D
and derivatives In laboratory animals Is related to lethality (see Table
VI-1). Hill and Carlisle (1947), however, reported the results of toxlco-
loglcal studies 1n which both lethal doses and tolerated doser (the largest
dose that caused no deaths) for 2,4-D sodium salt were determined for four
species of rodents. The lethal and tolerated oral doses for 2,4-0 sodium
salt 1n rats, mice, guinea pigs an4 rabbits were 666 and 333 mg/kg bw, 375
and 125 mg/kg bw, 1000 and 333 mg/lcg bw, and 800 and 200 mg/kg bw, respec-
tively. These data Indicate that mice are the most sensitive species, but
It must be noted that a tolerated dose, as defined In this study, does not
Imply a N0AEL. Although the tolerated doses caused no deaths, effects other
than survival were not mentioned; symptoms and pathological changes were
only specifically described In the animals that died (I.e., the lethal dose
groups). Since Hill and Carlisle (1947) noted that small ranges of doses
wore tested (i.e., groups of 10 mice were administered 125, 25C, 375 and 500
mg/kg bw of 2,4-D sodium salt). It appears probable that some of the char-
acteristic signs or symptoms of Intoxication described at the lethal doses
(e.g., muscular effects and possible histological damage) woild also have
been evident In those animals given tolerated doses. A 1-day HA can be cal-
culated from the tolerated single dese for mice (125 mg 2,4-D sodium salt/kg
bw, equivalent to about 114 mg 2,4-D/kg bw) using an uncertainty factor (UF)
of 1000. This factor, as per previous guidelines (U.S. EPA, I960), repre-
sents 10-fold for both intra- and interspecies variability to the toxicity
of a chemical when specific data are lacking and an additional 10-fold
because the tolerated single dose Is assumed to have caused unreported
adverse effects and, therefore, 1s considered a L0AEL rather thai a N0AEL.
01470
VIII-14
02/25/07
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Thus. for a child;
1-day HA (child) « (114 mg/kg bw/day x 10 kg bw} * (1000 x 1 t/day)
-1.1 mg/i
This HA 1s equivalent to 1.1 mg/day or 0.1 mg/kg bw/day.
A 1-day HA can alternatively be derived from other data of H111 and
Carlisle (1947). In this experiment, groups of six guinea pigs that were
administered 10 doses of SO or 100 mg/day 2,4-0 sodium salt by gavage In 12
days did not develop characteristic evidence of Intoxication (I.e., muscular
signs) or mortality. If 1t Is assumed that the guinea pigs weighed 0.3 kg
(the reported approximate weight 1n the single dose studies), the lowest
reported no effect dose of 50 mg/day corresponds to a dally dose of about
139 mg/kg bw/day (50 mg/day + 0.3 kg bw x 10/12); the equivalent dose of
2,4-0 acid Is about 126 mg/kg bw/day. Although symptoms or signs of
Intoxication were not specifically associated with this exposure, these
criteria of toxicity are still too Insensitive to Justify using 126 mg/kg
bw/day as an animal NOAEL with an uncertainty factor of 100. If 126 mg/kg
bw/day Is regarded as a L0AEL (reasoning similar to representing the single
tolerated doses as LOAELs) and an uncertainty factor of 1000 1s used, the
1-day HA value for a child 1s approximately the same as the 1-day HA derived
from the single tolerated dose for mice:
1-day HA (child) ¦ (126 mg/kg bw/day x 10 kg bw) * (1000 x 1 t/day)
-1.3 mg/i
This HA Is equivalent to 1.3 mg/day or 0.13 mg/kg bw/day.
Either one of the above cited experiments can be used to estimate the
1-day HA. They are both of equal value and the calculated concentrations
are essentially the same.
01470
VIII-15
04/07/B8
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Derivation of 10-Day HA. Subchronlc or shorter duration studies may
be ustfd to calculate a 10-day health advisory. The Health Effects 8ranch of
the Office of Drinking Water has assessed the available data wJ th the objec-
tive of estimating a 10-day health advisory and discovered several defi-
ciencies. The National Academy of Sciences (NAS, 1977) alio concluded,
"There are substantial disagreements 1n the results of subchronlc and
chronic toxicity studies with 2,4-D( perhaps reflecting the use of different
formulations or preparations". This section provides critical evaluation of
data and attempts to generate a 10-day health advisory.
Limited human Information Is available that provides quantitative short-
term exposure data. Kephart (1943) moderated a discussion In which an
Individual reported that he had taken (presumably orally) 500 mg of 2,4-0/
day for 21 days with no demonstrable 111-effects. Additional information
was not available, but this exposure corresponds to a dally dose of ~7 mg/kg
bw 1f it Is assumed that the person weighed 70 kg. Seabury (1J63) reported
a case In which a patient was administered 18 Intravenous doses of 2,4-D
(with Indole butyric acid and naphthalene acetic acid) over a 33-day period
for the treatment of cocddlodomycosls. Infusion of a total of 10.7 g did
not produce observed side effects [the dosage was 800 mg/day for doses 9
through 17, and the 18th dose was Increased to 2000 mg). A final 19th dose
of 3.6 g that was Infused over 2 hours (-67 mg/kg) elicited fibrillary
muscle twitching and general hyporeflexla that completely subsided within 48
hours. Although these reports may provide a crude Indication of a human
tolerated dose, numerous factors preclude their use in deriving a 10-day HA
[e.g., anecdotal nature of the Inadequately reported Kephart (1945) data,
the Inappropriate route of administration In the Seabury (1963) study, the
lack of sensitive Indicators of toxicity].
01470 VI11-16 02/25/87
-------
Rowe and Hymas (1954) administered 2,4-D (purity unspecified In olive
oil/gum arable vehicle) by gavage to groups of 5 or 6 young adult female
rats at doses of 0, 3, 30. 100 or 300 mg/kg bw for 5 days/week for 4 weeks
(0, 2.14, 21.4, 71.4 and 214 mg/kg bw/day, respectively). Adverse effects
as Judged by gross appearance and behavior, growth, hematological values
{not elaborated), blood urea-nitrogen concentrations, organ weights, gross
and hlstopathologlcal examinations (tissues not reported) and mortality were
not observed at doses of 2.14 or 21.4 mg/kg bw/day. Gastrointestinal Irri-
tation, slight cloudy swelling of the liver and depressed growth rate were
apparent at 71.4 mg/kg bw/day, and rats that were administered 214 mg/kg
bw/day 2,4-D died; the time to death was not reported, but severe GI Irrita-
tion was the principal effect observed (other pathological effects were not
discussed). In another study of longer duration, the same investigators
(Rowe and Hymas, 1954) administered 0, 100, 300 or 1000 ppm 2,4-D 1n the
diet to groups of five young female rats for 114 days. If It Is assumed
that young rats consume 10% of their body weight 1n food per day, the corre-
sponding dally doses would be 0, 10, 30 and 100 mg/kg bw/day. No effects
(same Indices as In the 4-week gavage study) were found at 10 or 30 mg/kg
bw/day, but 100 mg/kg bw/day produced "excessive mortality" with depressed
growth rate, slightly Increased liver weights and slight cloudy swelling of
the liver. Rats exposed to higher levels of 2,4-D in the diet (3000 and
5000 ppm) were not evaluated because they refused food and consequently lost
weight and the experiment was terminated. Both of the above Dow Chemical
Company studies used small groups of animals and were not reported In
detail, but multiple dose levels were tested and a number of toxicity
Indices were evaluated.
01470
VI11-17
02/25/87
-------
In another briefly reported dietary study with rats (Hill and Carlisle,
1947>^ adverse effects as Judged t>y signs of Intoxication, decreased food
consumption, growth Impairment or mortality were not observed in groups of
seven rats that were exposed to purified commercial 2,4-D at cally doses as
high as -40 mg/kg bw/day (200 or 400 ppm In the diet for 31 days, 100 ppm In
the diet for 21 days and 1000 ppm In the diet for the subsequent 10 days).
Although this study did not examine sensitive toxicity Indices (e.g.,
clinical chemistry or hematology Indices, hlstopathology), thi» reported no
effect level of -40 mg/kg bw/day Is consistent with that reported by Rowe
and Hymas (1954) after 4 weeks of tjavage exposure {21.4 mg/kg bw/day) or 16
weeks of dietary exposure (30 mg/kg bw/day). Chang et al. (1974) found in a
single dose study with an unspecified number of rats (~8) that dietary expo-
sure to 2000 ppm analytical grade 2,4-0 (-200 mg/kg bw/day) for 4-7 weeks
produced mild liver effects (slight Increase 1n glycogen, slight decrease In
RNA, slight decrease 1n absolute ancl relative weights) but no effect on food
consumption or overt signs of toxicity. The results of this study are some-
what supportive of the 71.4 mg/kg bw/day L0AEL 1n the Rowe and Hymas (1954)
4-week gavage study (cloudy swelling In liver, GI Irritation, depressed
growth) but are not consistent with the Rowe and Hymas (1954) 16-wcek diet
study that found excessive mortality at 100 mg/kg bw/day and food refusal at
higher levels.
In another short-term oral study with a limited number of animals (Drill
and Hlratzka, 1953), dogs were given commercial grade 2,4-D 1r capsules at
doses of 0 (2 females), 2 (1 male, 1 female), 5 (1 male, 1 female), 10 (3
males) or 20 mg/kg bw (3 males, 1 f«?male) 5 days/week for 13 wteks (0, 1.4,
3.6, 7.1 or 14.3 mg/kg bw/day, respectively). The dogs survived exposure to
01470
VIII-10
02/25/87
-------
<7.1 mg/kg bw/day without symptoms or changes 1n body weighty organ weights,
hemoglobin content, blood count, or gross or microscopic tissue structure,
but 3 of the 4 dogs that received 14.3 mg/kg/day died with neuromuscular
symptoms and terminal lymphocytosis (but no significant, pathological
lesions). The FEL 1n this study (14.3 mg/kg bw/day) Is therefore lower than
the highest NOAELs reported In the short-term rat studies (Rowe and Hymas,
1954; Hill and Carlisle, 1947). Although corroborating short-term exposure
data In dogs are not available, both acute oral lethal (Drill and Hlratzka,
1953) and longer-term oral (Hansen et al., 1971) studies also Indicate that
dogs are more sensitive than rats; however, 14.5 mg/kg bw/day was reported
by Hansen et al. (1971) to be a 2-year oral NOAEL for dogs (see Assessment
of Chronic Oral Data and Derivation of a Lifetime Adjusted Acceptable Dally
Intake 1n this chapter).
Teratogenicity testing with rats, mice and hamsters has shown that oral
administration of 2#4-D during gestation may produce fetotoxlc and develop-
mental effects at dally doses that are In the range of the rat and dog sub-
chronic NOAELs. Khera and HcKlnley (1972) found Increased fetal mortality
and an Increased Incidence of skeletal malformations In rats following oral
administration of 2,4-0 and 2,4-D esters and salts at 100 mg/kg bw/day (or
higher levels) on days 6 through 15 of gestation. Fetal mortality was not
elevated at lower dosages but the incidence of skeletal malformations was
slightly elevated by 2,4-D at 25 or 50 mg/kg bw/day. This increase was
significant (p<0.05) at the 25 but not at the 50 mg/kg/day level. Schwetz
et al. (1971) reported similar types of effects 1n rats after administration
of 2,4-D or Hs PGBE or Isooctyl ester at 75 or 87.5 mg 2,4-D/kg on days 6
through 15 of gestation. Lower doses of 12.5 or 25 mg/kg on days 6 through
01470
VIII-19
04/07/88
-------
15 of gestation produced statistically significant Increases in the inci-
dences of some developmental effects (e.g., delayed osslf1c< tlon, massing
sternebrae, and subcutaneous edema), although the Incidences of these
effects among different control groups were variable. For I't4-D specifi-
cally, a significant Increase 1n the Incidence of delayed ossification of
skull bones was reported at doses of 12.5, 50, 75 and 87.5 mg/kg bw/day but
not at 25 mg/kg bw/day. The author reported that the Incidence of this
response at 12.5 mg/kg bw/day was lower than the spontaneous Incidence In
the second control group of this study. Furthermore, at thuse levels of
treatment, generalized fetotoxlc effects were not seen. Jnger et al.
(1981), In a similar study with rats, detected slight fetotoxlclty at 87.5
mg 2,4-D/kg, administered as the PG3E or Isooctyl ester, but no effects were
detected at lower doses (<25 mg/kg). Courtney (1977) observed an Increased
Incidence of cleft palate formation and Increased fetotoxlclty 1n CD-I mice
after oral administration of 221 mg/kg 2,4-0 or equlmolar levels of the P6BE
or n-butyl esters on days 12 throucih 15 of gestation. The most toxic 2,4-D
ester 1n this assay, PGBE ester, produced cleft palates
-------
These teratogenicity tests for 2,4-D and Its esters Indicate that oral
doses (expressed as 2t4-D) of 40, 60 or 100 mg/kg bw/day In hamsters on days
6 through 10 of gestation (Collins and Williams, 1971), of 75-125.5 mg/kg
bw/day In rats on days 6 through 15 of gestation (Schwetz et al., 1971;
linger et al., 1981; Khera and McKlnley, 1972} and of 124 mg/kg bw/day In
mice on days 7 through 15 of gestation (Courtney, 1977) produced fetotoxlc
effects or malformations. The threshold for adverse effects on the fetus 1s
not clearly defined: sporadic evidence of mild fetotoxlclty was reported 1n
rats at doses as low as 12.5 and 25 mg 2,4-D/kg bw/day (Schwetz et al.,
1971; Khera and McKlnley, 1972) for both 2,4-0 and 2,4-D esters, but these
effects were also seen in controls. Furthermore, generalized fetotoxlc
effects were not seen at these levels of treatment.
Because these teratogenicity studies have shown evidence of adverse
fetal effects at dally doses that are higher than the subchronlc NOAELs, one
of these latter NOAELs would be the most appropriate basis for derivation of
a 10-day HA. The NOAEL chosen here 1s 30 mg/kg bw/day (Rowe and Hymas,
1954). This NOAEL Is the highest available based on several toxicity end-
points Including hlstopathologlcal analysis. The lower FEL of 14.3 mg/kg
bw/day In a limited number of dogs (Drill and Hlratzka, 1953) 1s not con-
sidered relevant to this analysis, because It 1s contradicted by a ?-year
feeding study with a large number of dogs (Hansen et al., 1971).
01470
VIII-21
02/25/87
-------
Using the same assumptions as In the 1-day HA calculation, a 100-foM
uncertainty factor for an animal NOAEL, and an additional 10-fold safetv
factor for deficiencies 1n the ciosen study, a 10-day HA Is derived for a
child as follows:
10-day HA (child) » (30 mg/kg bw/day x 10 kg bw) * (100 x 10 x 1 i/day)
-0.30 rog/i
This HA 1s equivalent to 0.30 mcj/dity or 0.030 mg/kg bw/day.
Derivation of Longer-term HA. A longer-term HA has not been calcu-
lated because of the lack of appropriate data.
Assessment of Lifetime Exposure and Derivation of a (ML. Lifetime
OWELs are normally derived from ?-year feeding studies In animals. The
animal species that 1s most sensitive to the toxic effects or the species
that metabolizes the compound in a manner similar to that 1n nwm Is selected
for estimating DWELs for humans. In these studies, a no aJverse health
effects level Is Identified. This level Is divided by an uncertainty factor
which may vary from 10-1000 based on the overall scientific Judgment to
determine an DWEL.
In 1976, the U.S. EPA, Office of Drinking Water, established an Interim
primary drinking water standard of 0.1 mg 2,4-D/i. This was developed
from an article -- Summaries of Pesticide Toxicity by Lehman. A lowest
long-term level of 8 mg/kg bw/day with minimal or no effects 1n dogs was
Identified* A safety factor of 500, an average dally Intake of 2 I of
water by man and 20% of the total acceptable dally Intake were the other
factors taken Into consideration to derive the final standard.
01470
VIII-22
02/25/87
-------
The National Academy of Sciences (NAS, 1977) recommended a concentration
of 0.09 mg 2,4-0/1 In drinking water, assigning 20% of the total RfD to
the drinking water source. This "ecommendatIon was based on a study In dogs
by Hansen et al. (1971). The Academy applied an uncertainty factor of 1000
to the NQAEL 1n dogs, recognizing the deficiencies In the study. The
Academy's calculations are given below;
12.5 rag/kg x 70 kg x 0.2 = q.09 mq/l
2 i x 1000
where: 12.5 mg/kg = no adverse effect level
70 kg = assumed average body weight of an adult
0.2 = factor representing 20% of total Intake from water
2l= assumed average dally Intake of water for man
1000 * uncertainty factor due to Inter- and Intraspecles varia-
tions and deficiencies In the studies
Johnson (1971) Indicated that 220 Dow Chemical Company production
workers who were exposed to 2,4-D In the range of 30-40 mg'day (*0.4-0.6
mg/kg bw/day, assuming a weight of 70 kg) over a period ol 0.5-22 years
showed no significant clinical effects when compared with an unexposed
population. These data are from an unpublished study and are inadequate as
reported for derivation of a lifetime DWEL. Particularly, additional infor-
mation is needed regarding the exposure estimate (which presumably reflects
inhalation and dermal exposures) and the effects (1t was reported only that
a battery of "at least 10 laboratory tests" was conducted). Singer et al.
(1982) reported that nerve conduction velocities were slowed In workers who
were engaged In the manufacture o1: 2,4-0 and 2,4,5-T for an average of 7
years, but exposure levels were not determined.
01470
VIII-23
02/25/87
-------
Hansen et al. (1971) fed technical grade 2,4-0 to groups of 25 Qsborne-
Mendel rats of each sex at levels of 0, 5, 125, 625 and 1250 ppm (0, -0.25.
6.25, 31.25 and 62.5 mg/kg bw/day respectively, assuming corsumpt1 on of 5%
of body weight 1n food/day) for ;? years. Adverse effects on growth, sur-
vival, organ weights, tissue histology or hematological values were not
attributed to exposure at any of the treatment levels up to and Including
-62.5 mg/kg bw/day. Although all rats were autopsled at tie end of the
2-year test, comprehensive histological examinations were performed only on
six rats of each sex from the hJigh-dose and control groups; histological
examinations 1n the remaining rats from the high-dose and control groups and
In the rats 1n the other groups w?re limited to the liver, kidney, spleen,
ovary or testis and other tissues that contained gross lesions.
In a similarly designed study with dogs, 6- to 8-month-old beagles (3 of
each sex/group) were fed either 0, 10, 50, 100 or 500 ppm In .he diet for 2
years (Hansen et al., 1971). If 1t Is assumed that dogs ccnsume 2.9% of
their weight In food/day, the corresponding dally doses woulj be 0t 0.29,
1.49, 2.9 and 14.5 mg/kg bw/day, respectively. Treatment-related gross or
hlstopathologlcal effects were not associated with any of the exposures.
Twenty-eight of the 30 treated dogs survived the test period end wore clin-
ically normal, but the report did not state 1f hematological analyses were
performed as 1n the rat study.
A 3-generatlon, 6-lltter reproduction study was also conducted by Hansen
et al. (1971) In Osborne-Mendel rats, A decrease 1n averag? preweanllng
weight and In the survival of offspring during the first 3 weeks after birth
was observed when test animals were maintained on 1500 ppm 2,4-D In the diet
01470
VI11-24
02/25/07
-------
(-75 mg/kg bw/day) for 2 years. These effects were not obser/ed at 500 ppm
(-25 mg/kg bw/day) or 100 ppm (-5 mg/kg bw/day). These authors also
reported an unpublished study by Gaines and Klmbrough (1970) In which feed-
ing 1000 or 2000 ppm 2,4-0 In the diet (-50 or 100 mg/kg bw/day, respec-
tively) to rats for 3 months before and during pregnancy and lactation
resulted In Increased mortality among the offspring. Bjorkiund and Erne
(1966), however, found no significant effects on dams or their offspring
following administration of 1000 ppm 2,4-D In drinking water (50-100 mg/kg
bw/day) to pregnant rats throughout gestation and for 10 months beyond
parturition, and to the offspring for up to 2 years.
The chronic toxicity and reproduction studies of 2,4-D Indicate no
adverse effects at dietary levels up to 500 ppm in dogs (-14.5 mg/kg
bw/day), up to 1250 ppm In rats (~i2.5 mg/kg bw/day) (Hansen i>t al.# 1971)
or at levels of 1000 ppm 1n drlnklmj water (50-100 mg/kg bw/day) In pregnant
rats (exposed throughout gestation .and for 10 months following parturition)
or their offspring (exposed for up to 2 years after weaning) (IJJorklund and
Erne, 1966). As previously discussed, however, a secondary reference to
another study reported an Increase 'm mortality among young rals whose dams
received -50 mg/kg bw/day of 2,4-D in the diet for 3 months before mating
and throughout gestation and lactation (Gaines and Klmbrough, 1970). More-
over, the subchronlc study of Haze!ton Laboratories (1983) Indicates that
these chronic studies may not be the most appropriate basis for the deriva-
tion of an RFD. Hazelton Laboratories (1983) report multiple adverse effect
at subchronlc doses of 5.0 or 15 mg/kg bw/day, or higher in both mice and
rats. A dose of 1.0 mg/kg bw/day was reported as a N0AEL In rats. In
addition, preliminary results from a ,2-year bloassay Indicate ro change In
this latter N0AEL.
01470
VI11-25
02/25/87
-------
Giving consideration to all cf these studies, 1t seems reasonable to
estimate a lifetime DWEL for a 70 kg human from the rat NOAEL of 1.0 mg/kg
bw/day of Hazelton Laboratories (1983) using an uncertainty :actor of 100.
This factor represents a 10-fold decrease in dose for both Intra- and Inter-
species variability to the toxicity of a chemical when specific data are
lacking.
Lifetime DWEL = (1.0 mg/kg hw/day x 70 kg bw) * (100 x <'? t/day)
* 0.35 mg/l
This HA 1$ equivalent to an RfD of 0.70 mg/day or 0.010 mg/kg brf/day.
There are discrepancies In th Inadequacies of the short-term studies
as previously discussed (e.g., small numbers of animals per dose, Inadequate
reporting of data). These differences In 2,4-D toxicity could also possibly
be due to a sharp break In the no~ei:fect, effect dose region. These differ-
ences may be resolved by additional testing.
01470 VIII-26 02/25/87
-------
Conclusions, A summary of the data used to calculate the HAs and the
lifetime DWEL Is provided In Table VI11-1. The values derived for the HAs
and the DWEL represent estimates of the concentrations of 2,4-D In drinking
water that will not cause adverse effects after 1 day, 10 days or lifetime
exposures.
Carcinogenic Effects
Quantification of Carcinogenic Effects
The available animal and epidemiology carcinogenicity studies have not
conclusively shown that 2,4-D alone 1s carcinogenic. Administration of
2,4-0 or the butyl, Isopropyl or Isooctyl esters of 2,4-D by Intubation
(46-100 mg/kg bw/day) on days 7 through 28 of age and subsequently In the
diet (111-323 ppm, -14-42 mg/kg bw/day) for up to 90 weeks was not tumorl-
genlc for mice (Blonetlcs Research Lab., 1968b). Administration of 2,4-D In
the diet at levels as high as 1250 ppm (-62.5 mg/kg/day) for 2 years was
originally reportedly not carcinogenic for rats (Hansen et al., 1971), but
later examination of the histology sections by Reuber (1979) found a signi-
ficant Increase In the Incidence of lymphosarcomas in females at all dose
levels; histopathologic reevaluation and consideration of the spontaneous
Incidence of lymphoid tumors In Osborne-Mendel rats are needed to resolve
the discrepancy. A Russian study reported that administration of 2,4-D
amine salt In the diet for life at one-tenth the LD^p (level not
specified) was not tumorlgenlc for rats or mice (Archlpov and Kozlova,
1974). Single subcutaneous Injections of 2,4-D (215 or 464 mg/kg bw), 2,4-D
Isopropyl ester (100 mg/kg bw) or 2,4-D Isobutyl ester (21.5 mg/kg bw) were
not tumorlgenlc for mice after 78 weeks, but similar Injection of 2,4-D
Isooctyl ester (21.5 mg/kg bw) Induced a significant Increase 1n reticulum
01470
VIII-27
04/07/88
-------
TABLE Vlll-1
2 Summary of Data Used to Derive HA and DUEL
o
Criteria Animal Oose
Duration
Effect
Value of HA or AAD1
Adult
Child
Reference
l-Oay HA
10 Day HA
114 mg/kg
(assumed
mouse LOAEL)
30 mg/kg
(rat NOAEL)
i Longer-term HA
CD
Lifetime
DUEL
1.0 mg/kg
(rat NOAEL)
single exposure
113 days
subchronlc
exposure*
highest dose NA
not causing
death
NOAELt higher NA
doses caused
liver toxicity
umu «cpr fs jcu
growth rate
1.3 mg/t
0.30 mg/t
NOAEL, higher
doses caused a
variety of
effects In
blood, liver
and kidney
indices.
Insufficient Data
0.35 mg/t NA
HU1 and Carlisle,
1947
Rowe and Hymas,
1954
Hazelton
Laboratorles, 1983
*Hnwpvpr rpr*ni frost t he 2 yCu" study CuiiT 11 m iiic subthronic viuAtL or I.u mg/kg/day
uncertainty factor of 100 Is used to estimate the DUEL rather than the usual 1000.
o NA — Not applicable
Thus, an
CD
CD
-------
we i i idiiMiuma* ^diuucikj nticai tn udu . , i?ood j . nepedieQ dppi nauuii) ui
2,4-0 to the skin of mice only produced papillomas when treatment was
preceded by a single dermal application of the Initiator
3-methylcholanthrene (Archlpov and Kozlova, 1974).
m
An addUonal animal bloassay (Industry Task Force, 19B5) Conducted by
Hazelton Labs In Virginia 1s available although It has not been critically
evaluated by ORO's Carcinogen Assessment 6roup 1n this document.
Epidemiology studies (see Subchronlc and Chronic Effects 1n Chapter VI)
have associated excess tumor Incidence (primarily soft-tissue sarcomas) 1n
humans with mixed exposures to chlorophenoxy herbicides that contain 2,4,5-T
(which may be contaminated with 2,3,7,8-TCOO) and 2,4-0 (which 1s not con-
taminated with this dloxln Isomer). Prior to 1986, IARC and EPA have Judged
the epidemiologic evidence for chlorophenoxy herbicides to be "limited*,
i.e. providing evidence of causality but not without the possibility of
alternative explanations such as chance, bias or confounding factors. The
EPA overall welght-of-evidence classification for the chlorophenoxy herbi-
cides 1s Group 61; IARC (1967) 1s 26. While 2,4-D Is a member of the
chlorophenoxy class and Is known to be present as one constituent 1n some of
the chlorophenoxy studies, 1t Is not possible from the studies prl^- to 1986
to Isolate 2,4-D and conclude that It Is or Is not a causative agent. Thus,
prior to 1986, the epidemiology data for 2,4-0 alone was Judged to be
Inadequate.
01470
VI11-29
04/13/88
-------
A new case-controi stuay (Hoar et at., lytibj, nowever, 1:. more focused
on 2,4-D than the earlier data base. In a carefully designed and well-
conducted study, Hoar et al. (1986) found a statistically significant
association between exposure to 2,t-D (controlling for the otHer herbicides
present) and an excess risk of n:>n-Hodgk1ns lymphoma (NHL).. The authors
also found a dose-related Increase of NHL with exposure to 2,4-3."
The CAG believes that the Hoar et al. (1986) study provides evidence of
a causal association of 2,4-D exposure with NHL; however, CAG believes that
alternative explanations for this association such as chance, bias or
confounding factors cannot be excljded. The CAG Is aware that the authors
of the Hoar et al. (1986) study are currently conducting two additional
case-control studies of NHL cases In populations different fron those of the
Hoar et al. (1986) study. Results from these studies should be available
within 12 months. As the Guidelines for Carcinogen R1:;k Assessment
Indicate, confidence 1n Inferring a causal association from epidemiologic
data 1s Increased when several Independent studies are concordant 1n showing
the association. Because the results of the two additional studies will
likely be available within 12 months, the CAG has decided to withhold Its
weight of evidence evaluation of the human data as well as classification of
the overall weight of evidence for 2,4-D. CAG's evaluation of the weight of
evidence of this chemical will t><* made pending receipt of the expected
epidemiologic studies and a critical evaluation of the Industry Task Force
animal bloassay.
Note Is made that OPP, by March 23, 1988 Federal Register Notice, has
proposed a we1ght-of-evidence for ttie cancer data base that takes account of
01470
VIII-30
04/13/88
-------
the Industry Task Force ( 1985) animal stuay and the Hoar et al. (1986)
epidemiologic data. OPP has proposed that the animal and human data, be
viewed as Inadequate for 2,4-0, resulting 1n an over-all welght-of-evidence
for 2,4-D of Group D, I.e. data Is Inadequate to refute or demonstrate a
human carcinogenic potential. OPP proposes that additional animal studies
be conducted and that a reevaluatlon of the data base could be Initiated at
a later date. Public comment on this proposal carries through May 23, 1988.
2,4-D as a commercial product has been shown to contain chlorinated
dlbenzodloxlns as an Impurity. A rigorous characterization of the
impurities 1s beyond the scope of this document; however, 2,3,7,8-TCDO and
the hexa- Isomer have not been detected, while ppb amounts (5-900) have been
dted of d1-, tr 1 - and 1,3,6,9- or 1,3,6,8-tetra- Isomers, depending upon
2,4-D acid %. From a risk characterization perspective, the role, If any,
of the Impurities warrants recognition as It may pertain to toxicologic
observations as well as to the description of the human exposure to 2,4-0,
e.g., exposure via drinking water pathway may be to an altered 2,4-0 mixture
rather ttian the original commercial preparation, which might not be the case
for a pesticide formulator/appHcator, for instance.
2,4-0 has been tested for mutagenicity 1n a variety of systems, includ-
ing microorganisms, plants, fruit flies, cultured mammalian cells, and in
vivo mammalian assays (see Mutagenicity In Chapter V). 2,4-D Induced
mitotic gene conversion and recombination In Saccharomyces cerevlslae
(Simmon, 1979); Induced recessive lethal and somatic mutations (weakly
mutagenic) 1n Drosophlla melanoqaster (Magnusson et al., 1977; Rasmusson and
SvahUn, 1978); Induced mutation to ouabain resistance In cultured Chinese
hamster V-79 lung cells and Induced unscheduled DNA synthesis In cultured
01470 VIII-31 04/14/88
-------
human fibroblasts tAhmed et a 1., 1977); Induced chromosome aberrations and
sister chromatid exchanges In cultured human lymphocytes (Pll'nskaya et al.,
1976; Korte and Jalal, 1982); Induced bone marrow chromosome aberrations
(PlUnskaya et al., 1976); and Inhibited thymidine Incorporation Into
testicular OKA (Seller, 1979) In mice exposed ^In vivo. Mutagenicity testing
of 2,4-0 In plants was almost universally positive (see Mutagenicity 1n
Chapter V). A preponderance of negative responses 1n Minimal assays,
however. Indicates that pH may be a critical factor; unless the pH Is In the
acid range, 2,4-0 will be Ionized and may not readily cross cell membranes
or reach the target tissues. Mutagenicity testing of 2,4-0 esters has not
been performed, but theoretically these compounds could show higher levels
of penetration Into target cells. Thus, 1t may be prudent to expect that
these chemicals are mutagenic.
Existing Guidelines. Recommendations and Standards
The national Academy of Sciences has suggested an acceptable level 1n
drinking water of 0.09 mg/l (0.09 ppm) for 2,4-0 1n drinking water, assum-
ing that 20% of exposure 1s attributable to drinking water (NAS, 1977).
This level was calculated from a U0EI from the Hansen et al. (1971) 2-year
feeding study with dogs. The Interim primary drinking water standard for
2,4-0 Is 0.1 mg/l (federal Register, 1975).
The American Conference of Governmental Industrial Hyglenlsts currently
recommends an 8-hour TWA-TLV of 10 mg/m# for occupational exposure to
2,4-0 (ACGIH, 1980). ACG1H also recommends a STEL of 20 mg/m* for any
15-m1nute exposure period. These recommendations are Intended to protect
against local and systemic effects by Inhalation and are derived from
unspecified Ingestion studies.
01470 " VII1-32 04/13/88
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Occupational exposure limits for 2,4-0 have not been recommended by the
National Institute for Occupational Safety and Health (NIOSH) or promulgated
by the Occupational Safety and Health Administration (OSHA).
The U.S. EPA (1982) has established the following toleran'ces for 2,4-D
residues In or on raw agricultural commodities: 5 ppm for apples, pears,
quinces, apricots (includes residues from the preharvest of the dlroethy-
lamlne salts) and citrus fruits (Includes residues from the preharvest
application of the Isopropyl and butoxyethyl esters and the postharvest
application of the alkanolamlne salts or the Isopropyl ester); and 0.2 ppm
for potatoes. Tolerances are established for residues of 2,4-0 1n acid
form, or In the form of several salts and esters on the following commodi-
ties: 1000 ppm for rangeland and pasture grasses; 300 ppm for grass hay; 20
ppm for barley, corn, millet straw, oats, rice straw, rye, sorghum, sugar-
cane and wheat used for forage and fodder; 2 ppm for sugarcane; 0.5 ppm for
various grains, cranberries and grapes; and 0.1 ppm for blueberries and rke
(U.S. EPA, 1902).
In Instances where 2,4-0 dlmethylamlne salt is applied to Irrigation
ditch banks In the western United States under various Federal programs, the
established tolerance for 2,4-D residues Is 0.1 ppm for the following com-
modities: avocadoes, various fruits and vegetables, grain crops, hops,
forage grasses and legumes, curcurblts, cottonseed and nuts (U.S. EPA, 1982).
A tolerance for residues of 2.4-D sodium, ethanolamlne and Isopropano-
1amine salts calculated as 2,4-0 has been established at 5 ppm for asparagus
and 0.05 ppm for strawberries (U.S. EPA, 1982).
01470
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Established tolerances for residues of 2,4-D are 1 ppm from application
of Us dlmethylamlne salt for water hyacinth control 1n slow novlng aquatic
media.(e.g., western United States Irrigation ditch banks) ani In fish and
shellfish (U.S. EPA, 1982).
Tolerances are established for 2,4-D or Us metabolite, 2",4-d1chloro-
phenol, 1n the following animal food commodities: 2 ppm for cattle, goat,
hog, horse and sheep kidney; 0.2 pt>m for cattle, goat, hog, horse and sheep
meats, meat by-products or fat; 0.1 ppm for milk; and 0.05 ppm In eggs and
poultry. A tolerance of 1.0 ppm 2,4-0 for residues of Us dlmethylamlne
salt or butoxyethanol ester Is established for fish In Tennessee Valley
Authority dams and reservoirs being controlled for Eurasian Watermllfoll
(U.S. EPA, 1982).
A maximum ADI level of 2,4-D for man has been recommended as 0.3 mg/kg
by the Joint Meeting of the FA0 Working Party of Experts on Pesticide
Residues and the WHO Expert CommHtee on Pesticide Residues (WHO, 1976),
after considering published experimental data and national tolerances
established by several countries. An odor threshold for 2,4-0 In water was
reported by Slgworth (1965) as 3.13 mg/t.
Special Sroups at Risk
People who are occupatlonally exposed to 2,4-D (I.e., agricultural
workers and those Involved In the manufacture and distribution of the chemi-
cal) should be regarded as a special group at risk because they may be
exposed to high levels of this chemical. Particularly, therj is evidence
that humans exposed to chlorophenoxy herbicide formulations containing
01470
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04/13/88
-------
mixtures of 2,4-D and 2,4fb-T may develop cancer. Toxlcoklnetlc studies
ylth humans Indicates that considerable Inter individual variation occurs In
the rates of absorption and excretion and In the amount of 2,4-D conjugated;
these differences might result In a wide range of sensitivity to 2,4-D among
Individuals. Pregnant yomen should also be regarded as a sensitive popula-
tlon because 2,4-0 and some of Hs salts and esters have produced fetotoxlc
and developmental effects In experimental animals. Because 2,4-D is
excreted primarily In the urine and has some toxicity to the kidneys,
persons with renal disease would also be a special group at risk.
01470
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04/13/88
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