914. Dichlorophenoxyacetic acid, 2,4- (Pesticide residues in food: 1996 evaluations Part II Toxicological)

2,4-DICHLOROPHENOXYACETIC ACID (2,4-D)

First draft prepared by
J. C. Rowland
Office of Pesticides Programs, US Environmental Protection Agency,
Washington DC, USA

Explanation
Evaluation for acceptable daily intake
Biochemical aspects
Absorption, distribution, and excretion
Biotransformation
Enzyme induction and other biochemical parameters
Toxicological studies
Acute toxicity
Short-term toxicity
Long-term toxicity and carcinogenicity
Reproductive toxicity
Developmental toxicity
Genotoxicity
Special studies
Dermal and ocular irritation and dermal sensitization
Dermal toxicity
Neurotoxicity
Canine malignant lymphoma
Observations in humans
Case-control studies
Soft-tissue sarcomas
Non-Hodgkin's lymphoma
Cohort studies
Overall assessments of epidemiological studies
Comments
Toxicological evaluation
References

Explanation

Data on the toxicity of 2,4-dichlorophenoxyacetic acid (2,4-D)
were reviewed by the JMPR in 1970, 1971, 1974, and 1975 (Annex I,
references 14, 16, 22, and 24). The 1970 Meeting did not establish an
ADI, owing to the absence of long-term studies. The 1971 Meeting
established an ADI of 0-0.3 mg/kg bw on the basis of a NOAEL of
31 mg/kg bw per day in a two-year dietary study in rats. The 1974
Meeting reviewed data on the use and residues of 2,4-D and concluded
that there was no need to modify the previously established ADI. The
1975 Meeting reviewed a study of tissue distribution in rats and a
study of teratologenicity in mice and reaffirmed the established ADI.
Since that meeting, studies have become available on acute toxicity;
short-term toxicity in mice, rats, and dogs; long-term toxicity in
mice, rats, and dogs; carcinogenicity in mice and rats; acute and

chronic neurotoxicity; developmental toxicity in rats and rabbits;
mutagenicity; and epidemiological studies of 2,4-D. These studies and
summaries from the previous monograph and monograph addenda (Annex 1,
references 15, 17, 23, and 25), with studies of the acute, subchronic,
and developmental toxicity and genotoxicity of the four amine salts,
diethanolamine (DEA), dimethylamine (DMA), isopropylamine (IPA), and
triisopropanolamine (TIPA) salts, and two esters, butoxyethylhexyl
(BEH) ester and 2-ethylhexyl (EH) ester, are summarized below.

Evaluation for acceptable daily intake

1. Biochemical aspects

(a) Absorption, distribution, and excretion

The pharmacokinetics of ¹⁴C-2,4-D (purity, 98%) was studied in
groups of 26 male B6C3F1 mice after a single oral dose at 5, 45, or
90 mg/kg bw and a single intravenous administration of 90 mg/kg bw. In
order to evaluate excretion balance, groups of five mice were given
the same single doses of ¹⁴C-2,4-D by gavage or an intravenous dose
of 5 or 90 mg/kg bw. Plasma, liver, and kidneys were analysed for
radiolabel at 0.083, 0.25, 0.5, 1, 2, 4, 6, 8, 12, 24, 36, 48, and
72 h after treatment. Urine was collected before treatment and at 0-6,
6-12, 12-24, 24-36, 36-48, 48-72 h, and every 24 h thereafter for
168 h after treatment. Faeces were collected before treatment and
0-12, 12-24, and every 24 h up to 168 h after treatment. At 168 h
after treatment, the animals were killed, and blood, liver, kidneys,
and residual carcasses were sampled for radiolabel.

Disappearance of the label from plasma in animals at each dose
was analysed by reiteratively weighted nonlinear regression analysis
to obtain the apparent pharmacokinetic parameters by both the oral and
intravenous routes. Because of the apparent lag in clearance at the
higher doses and the high levels of 2,4-D-derived radiolabel in
plasma during the first 4 h after administration of these doses, a
two-compartment model with Michaelis-Menten limited clearance was
chosen. The half-lives were calculated to be 28-45 h. At least 50% of
the administered dose was cleared within 12 h, suggesting that the
estimates are lower than the actual clearance constants.

After oral administration, the area under the curve (AUC) for
time vs concentration was 95 and 1087 µg × h/ml at the 5 and
45 mg/kg bw doses, respectively; with 90 mg/kg bw given orally and
intravenously, the AUCs were 2257 and 2548 µg × h/ml, respectively.
Therefore, the AUC increased with dose. The apparent volumes of
distribution were also found to increase with dose: after oral
administration, they were 143, 213, and 300 ml/kg at 5, 45, and 90
mg/kg bw, respectively; after intravenous administration of 90 mg/kg
bw, the volume of distribution was 263 ml/kg.

The main route of elimination of radiolabel was the urine,
accounting for 63, 84, 71, 53, and 65% of the dose excreted by animals
receiving 5 mg/kg bw orally, 5 mg/kg bw intravenously, 45 mg/kg bw
orally, 90 mg/kg bw orally, and 90 mg/kg bw intravenously,
respectively. Faecal elimination represented 7.6% of the dose in
animals receiving 5 mg/kg bw orally and 5.2% of the dose in those
given 5 mg/kg bw intravenously. A greater portion of the 2,4-D-derived
radiolabel appeared in the faeces in animals at the higher dose: 15%
in those at 45 mg/kg bw orally, 16% with 90 mg/kg bw orally, and 12%

with 90 mg/kg bw intravenously. Most of the radiolabel was eliminated
in urine collected during the first 0-6 h after treatment with 5 mg/kg
bw intravenously, within 0-12 h after treatment with 5 mg/kg bw
orally, and within 6-24 h after treatment with 45 or 90 mg/kg bw.

At 168 h after treatment, very little radiolabel was detected.
None was found in blood or plasma after the intravenous doses, and
only one animal had a detectable, low level of radiolabel in plasma
after oral administration. The liver and kidneys contained similar
microgram equivalents per gram of tissue of 2,4-D-derived radiolabel
at each dose. Less than 1.1% of the dose was retained in the animals
seven days after administration of ¹⁴C-2,4-D, independently of the
dose and route of administration. The urinary clearance of 2,4-D
appeared to be a saturable process in male mice at doses > 45 mg/kg
bw (Eiseman, 1984).

The pharmacokinetics of ¹⁴C-2,4-D (radiochemical purity, > 99%)
was examined after oral and intravenous administration to several
groups of male Fischer 344 rats. The objective of the study was to
investigate the dose-dependent fate of the compound and to identify
the approximate dose at which the kinetics of elimination of
¹⁴C-2,4-D begin to show evidence of saturation. In order to determine
the time course of disappearance of the compound from plasma and the
rate of excretion in urine, three groups of three rats with jugular
cannulae received an oral dose of 10, 50, or 150 mg/kg bw, and two
similar groups received intravenous doses of 5 or 90 mg/kg bw. The
concentrations of radiolabel were determined 1, 2, 3, 6, 9, 12, 15,
18, 24, 36, 48, 60, and 72 h after treatment; urinary ¹⁴C levels were
measured at 6-h intervals for the first 24 h of collection and at 12-h
intervals up to 72 h after treatment; faecal samples were collected at
24-h intervals. In order to determine the effect of dose on the ratio
of the concentrations of ¹⁴C in kidney and plasma, five groups of six
rats were given single oral doses of 10, 25, 50, 100 or 150 mg/kg bw
and were killed 6 h after treatment, when plasma, urine, and kidneys
were analysed for ¹⁴C activity.

Absorption of 2,4-D after oral administration was complete, as
urinary excretion represented > 85% of the dose within the first 12 h
after treatment, and 97% of the 10 mg/kg bw oral dose and 95% of the
150 mg/kg bw dose were recovered in urine. After intravenous
administration, 99 and 86% of the 5- and 90-mg/kg bw doses were
recovered within the first 12 h and 100 and 91% after 72 h,
respectively. The absorption rate constant in plasma was 1.4 h^-1.
Saturable clearance from the plasma was detected and was corroborated
by the disproportionate increase in the AUC with increasing dose. This
effect probably reflects saturable urinary excretion, in view of the
increasing ratio of plasma:kidney ¹⁴C concentrations with increasing
dose.

The mean half-lives for the alpha phase by the intravenous and
oral routes were 0.92 and 1.0 h, respectively. The mean half-lives for
the ß phase were 14 and 18 h for the intravenous and oral routes,
respectively. Doses > 50 mg/kg bw were required to bring the plasma
concentration above the K_m. At or above this dose, saturation of
clearance became evident. The rapid elimination of ¹⁴C-2,4-D in the
urine and the small contribution of the ß phase indicated low
potential accumulation of 2,4-D in rats (Smith et al., 1990).

The absorption, distribution, metabolism, and excretion of
¹⁴C-2,4-D were further examined after oral and intravenous
administration to Fischer 344 rats. Four groups of five male and five
female rats received either a single oral administration of ¹⁴C-2,4-D
by gavage at 100 mg/kg bw, ¹⁴C-2,4-D at 1 mg/kg bw as a single oral
dose, ¹⁴C-2,4-D at 1 mg/kg bw as a single intravenous dose, or 14
daily oral doses of non-radiolabelled 2,4-D at 1 mg/kg bw followed
by a single oral dose of ¹⁴C-2,4-D at 1 mg/kg bw on day 15. Two
additional groups of four male rats were given a single oral dose and
then 1 or 100 mg/kg bw through jugular cannulae in order to define the
concentration-time course in plasma. Plasma concentrations were
determined for 24 h after treatment.

In all groups, > 94% of the administered dose was recovered
within 48 h after treatment. The primary route of excretion was the
urine (85-94%), the faeces being a minor excretory pathway (2-11%). No
sex-related difference was seen, and repeated oral treatment did
not alter the excretory route. ¹⁴C-2,4-D was rapidly and almost
completely absorbed, as peak plasma levels were attained about 4 h
after treatment and 85-94% of each dose was excreted in the urine. The
non-proportional AUCs and the delayed urinary excretion of radiolabel
strongly imply, however, dose-dependent non-linear kinetics. Although
the elimination of radiolabel was saturated during the first few hours
after the high dose of 100 mg/kg bw, the excretion of radiolabel was
rapid, most of the administered dose having been excreted by 36 h
after treatment in all groups. Rapid excretion of ¹⁴C-2,4-D is also
corroborated by the approximate half-life of 5 h for urinary excretion
after oral administration. The rapid clearance of 2,4-D from plasma
and its rapid excretion in the urine indicate that it has little
potential to accumulate in rats. Analysis of all major tissues and
organs for residual ¹⁴C activity indicated that only a small
fraction of the dose was still present 48 h after treatment. Tissues
and organs from animals at the low dose contained < 0.7% of the
administered dose (Timchalk et al., 1990). These results indicate
that the fate of ¹⁴C-2,4-D in the rat is independent of dose and sex,
that the compound is rapidly and almost completely eliminated,
essentially by the urinary route, and that it has little potential to
accumulate. The main results on ¹⁴C-2,4-D metabolism in rats are
discussed below.

In a limited study of metabolism, male Fischer 344 rats were
given either unlabelled IPA salt of 2,4-D at 2.7 mg/kg bw, ¹⁴C-2,4-D
at 10 mg/kg bw, or both unlabelled IPA salt at 2.7 mg/kg bw and
¹⁴C-2,4-D at 10 mg/kg bw. The fate of the two compounds was
unaffected by their co-administration. After the single dose of
¹⁴C-2,4-D, alone or in combination with IPA salt, 2,4-D was rapidly
absorbed and excreted, primarily in the urine. IPA salt, administered
alone or in combination with ¹⁴C-2,4-D, was readily absorbed and
rapidly excreted as unchanged parent compound in the urine. For both
groups, > 90% of the administered dose was excreted as IPA salt
within the first 12 h after treatment (Dryzga et al., 1993).

The absorption, distribution, and excretion of ¹⁴C-TIPA salt of
2,4-D were also studied in male Fischer 344 rats, which received a
targeted dose of 10.7 mg/kg bw TIPA salt or 10 mg/kg bw 2,4-D and
20-30 µCi of ¹⁴C per animal. Blood was collected from each rat at
0.25, 0.5, 0.75, 1, 1.5, 2, 4, 6, 8, 12, 18, 24, 48, and 72 h after
treatment, and plasma was analysed for radiolabel. Urine was collected
at 6, 12, 24, 48 and 72 h after treatment, and the radiolabel in urine
and the cage rinse was combined for each collection interval and
expressed as radiolabel excreted in the urine. Faeces were collected
at 24-h intervals and analysed for radiolabel. Expired air was passed
through a charcoal trap to capture expired organic ¹⁴C and then
through a monoethanolamine:1-methoxy-2-propanol trap to capture
expired ¹⁴C-carbon dioxide; the latter traps were changed at 12, 24,
36, 48, and 72 h after treatment and analysed for radiolabel. Samples
of liver, kidneys, perirenal fat, skin, and remaining carcass were
collected from animals killed 72 h after treatment and analysed for
radiolabel. Metabolites were characterized in pooled urine samples
collected 0-6 and 6-12 h after treatment.

¹⁴C-TIPA salt was rapidly absorbed by the gastrointestinal tract
and excreted in the urine unchanged. The concentration of radiolabel
in the plasma peaked 0.25 h after treatment and then decreased in a
tri-exponential manner. Owing to its rapid elimination, ¹⁴C-TIPA
salt did not accumulate in the tissues: < 1% of the administered
radiolabel remained in the tissues and carcass in rats sacrificed 72 h
after treatment. ¹⁴C-TIPA salt did not undergo extensive metabolism,
as essentially all of the radiolabel excreted in the urine represented
unchanged TIPA salt. By the first 24 h after treatment, 80% had been
excreted in the urine. Faecal excretion accounted for 4-7% of the
dose, expired ¹⁴C-carbon dioxide for 3-4%, and the final cage rinse
for about 1% of the dose. ¹⁴C-TIPA salt was thus well absorbed and
rapidly excreted in the urine, primarily as unchanged compound; it
does not accumulate in rat tissues. Excretion of the parent acid,
2,4-D, was not affected by addition of the TIPA salt (Dryzga et al.,
1992a).

The absorption, metabolism, and excretion of the BEH ester of
2,4-D were studied in a group of four male Fischer 344 rats given a
single dose of 13.9 mg/kg bw ¹⁴C-BEH ester in corn oil by gavage.
Blood samples were collected 0.25, 0.5, 0.75, 1, 1.5, 2, 4, 6, 8,
12, 18, and 24 h after treatment; urine, cage rinse, and expired
¹⁴C-carbon dioxide were collected from rats 6, 12, 24, and 48 h after
treatment. The 0-6-, 6-12-, and 12-24-h pooled urine specimens were
analysed for unchanged BEH ester, 2,4-D, ethylene glycol, and
¹⁴C-2-butoxyethanol and its metabolites by gas chromatography-mass
spectrometry (GC-MS) and high-performance liquid chromatography (HPLC)
with radiochemical detection.

¹⁴C-BEH ester was rapidly absorbed and hydrolysed to 2,4-D and
¹⁴C-2-butoxyethanol. 2,4-D was eliminated unchanged in the urine. By
48 h after treatment, the mean recovery of radiolabel represented 78%
of the administered dose. The urine was the major route of elimination
(58% of the administered dose); expired ¹⁴C-carbon dioxide contained
17% and the faeces 2.4% of the administered dose. Elimination was
rapid, as indicated by a recovery of 49% in the urine 12 h after
treatment; the elimination half-life of radiolabel in urine was 4.6 h.
No unchanged parent compound was detected in blood or urine. The
radiolabelled metabolites identified in urine included 2-butoxy-
ethanol, 2-butoxyacetic acid, ethylene glycol, and their conjugates.
The major metabolite was 2-butoxyacetic acid (Dryzga et al., 1992b).

The absorption, distribution, excretion, and biotransformation of
¹⁴C-EH ester of 2,4-D were studied in male Fischer 344 rats that
received a single oral dose of 15 mg/kg bw ¹⁴C-EH ester. Blood was
collected from each rat 0.5, 1, 1.5, 2, 4, 6, 8, 12, 18, and 24 h
after treatment, and plasma was analysed for radiolabel; urine was
collected 6, 12, 24, and 48 h after treatment, and the radiolabel in
urine and the cage rinse was combined for each collection interval and
expressed as radiolabel excreted in the urine. Faeces were collected
at 24-h intervals and analysed for radiolabel; expired ¹⁴C-carbon
dioxide collected at 6, 12, 24, and 48 h was trapped in a solution
of monoethanolamine:1-methoxy-2-propanol, and the radiolabel was
quantified. No tissue samples were analysed. Metabolites were
characterized in pooled urine (0-6- and 6-12-h) and in faecal (0-24-h)
samples by GC-MS, and unchanged EH ester in pooled urine and faecal
extracts was determined by HPLC.

¹⁴C-EH ester was rapidly absorbed, with a peak plasma
concentration of 1.0 µg/g 4 h after treatment, decreasing with a
half-life of 9 h. Once absorbed, ¹⁴C-EH ester was extensively
metabolized and was eliminated in the urine, faeces, and as expired
¹⁴C-carbon dioxide. It was rapidly hydrolysed to 2,4-D and
¹⁴C-2-ethylhexanol, since no ¹⁴C-EH ester was found in blood, urine,
or faeces. The principal route of excretion was the urine (62-66%),
less being eliminated in the faeces (14-21%) and expired carbon
dioxide (9-12%). The metabolites found in both urine and faeces were

2-ethylhexanol, 2-ethylhexanoic acid, 2-ethyl-1,6-hexanedioic acid,
and 2,4-D. Metabolites found in the urine but not in the faeces were
2-ethyl-5-ketohexanoic acid, 2-ethyl-5-hydroxyhexanoic acid,
2-heptanone, and 4-heptanone. These metabolites were previously
reported as metabolites of ¹⁴C-2-ethylhexanol. The EH ester of 2,4-D
is thus converted rapidly to 2,4-D, which is then excreted in the
urine. Therefore, the EH ester is toxicologically equivalent to 2,4-D
itself (Dryzga et al., 1992c).

Dermal absorption of 2,4-D, the DMA salt, and the EH ester was
studied after topical application to male Sprague-Dawley rats,
New Zealand white rabbits, and rhesus monkeys. About 1 µCi of
¹⁴C-labelled compound dissolved in acetone was applied to the shaven
mid-dorsal regions of rats and rabbits and to the mid-dorsal forearm
or forehead of the monkeys. The area of application varied but
provided a constant dose rate of 4 µg/m³. Nonocclusive gauze patches
were used to protect the treated areas, except the monkey forehead.
Urine samples were collected after 4 and 8 h on the first day and then
at 24-h intervals for 14 days after treatment. Total dermal absorption
and excretion half-lives were calculated. The monkey forehead was more
permeable than the monkey forearm; the rate of urinary excretion of
radiolabel after dermal absorption was similar in all cases. Dermal
absorption of 2,4-D was 36% in rabbits, 15% in monkey forearm, and 29%
in monkey forehead; the half-lives were 2.1 days in rabbits, 1.9 days
for monkey forearm, and 1.5 days for monkey forehead. Dermal
absorption of the DMA salt was 14% in rats, 6% in monkey forearm, and
31% in monkey forehead; the half-lives were 1.4 days, 1.8 days, and
2.3 days in rats, monkey forearm, and forehead, respectively. Dermal
absorption of the EH ester was 50% in rabbits, 40% in monkey forearm,
and 56% in monkey forehead; the half-lives were 2.1 days in monkey
forearm and 2.0 days in monkey forehead; no half-life was determined
in rabbits (Moody et al., 1990).

A study was conducted to determine the extent to which dogs
absorb and excrete 2,4-D in urine after contact with treated lawns
under natural conditions. Concentrations of > 10.0 µg/litre were
found in the urine of 33 of 44 neighborhood mixed-breed dogs (75%)
potentially exposed to 2,4-D-treated lawns an average of 10.9 days
after application, and concentrations of > 50 µg/litre were found
in 17 (39%). Of 15 dogs with no known exposure to a 2,4-D-treated
lawn during the previous 42 days, 4 (27%) had 2,4-D in urine (1
at a concentration > 50 µg/litre). The odds ratio (OR) for an
association between exposure to a 2,4-D-treated lawn and the presence
of > 50 µg/litre 2,4-D in urine was 8.8 (95% confidence interval
[CI], 1.4-56). Dogs exposed to lawns treated within seven days before
urine collection were more than 50 times as likely to have 2,4-D at
concentrations > 50 µg/litre than dogs exposed to a lawn treated
more than one week previously (OR, 56; 95% CI, 10-312). The highest
mean concentration of 2,4-D in urine (21.3 mg/litre) was found in dogs
sampled within two days after application of the herbicide (Reynolds
et al., 1994).

The excretion, tissue residues, and metabolism of ¹⁴C-2,4-D were
investigated in a lactating goat given an oral dose of 483 ppm for
three consecutive days in a capsule. On the basis of established
tolerances, this dose is equivalent to 68% of the maximal exposure of
dairy cattle to 2,4-D residues. Urine and faeces were collected daily
and milk each morning and evening. At the end of the study, selected
tissues were sampled for determination of radiolabel concentration.
About 90% of the dose was recovered in the urine and faeces; milk,
liver, kidneys, composite fat, and composite muscle accounted for
< 0.1% of the total dose received. The residues in the milk were
0.22-0.34 ppm at the morning milking and 0.04-0.06 ppm in the evening.
Kidneys accounted for the highest residue concentration, 1.4 ppm;
liver contained 0.22 ppm, fat, 0.09 ppm, and muscle, 0.04 ppm (Guo &
Stewart, 1993).

After a single oral dose of 5 mg/kg bw 2,4-D to a male human
subject, the plasma level was 35 µg/ml after 2 h, 25 µg/ml after 24 h,
and 3.5 µg/ml after 48 h. The levels in whole blood decreased from
21 µg/ml after 2 h to 2.1 µg/ml after 48 h. A total of 73% of the dose
was excreted in the urine within 48 h after treatment. On the basis of
this study, it was estimated that 1 mg/kg bw of 2,4-D can be eliminated
by humans within 24 h (Gehring & Gorden, personal communication, 1971).

The absorption and excretion of 2,4-D was investigated in six
healthy volunteers (age, 22-30 years) after ingestion of 5 mg/kg bw in
gelatin capsules. Blood samples were obtained 1, 2, 7, 12, 24, 32, 48,
56, and 168 h after ingestion; urine samples were collected during the
first 24 h. 2,4-D was absorbed fairly quickly, a significant amount of
the compound being detected 1 h after administration. The highest
concentration of 2,4-D in plasma was reached in 7-24 h, after which
the amount declined steadily. After absorption, 2,4-D was quickly
excreted; about 75% of the dose was found unchanged in the urine 96 h
after administration. No metabolites were detected in urine (Kohli
et al., 1974).

The pharmacokinetics of 2,4-D were studied in five male subjects
(29-40 years; 70-90 kg) given 5 mg/kg bw 2,4-D (analytical grade)
either as a slurry in milk or as the powder followed by water. Blood
samples were collected at 1, 4, 8, 12, 24, 36, 48, 72, 96, 120, and
144 h after treatment, and urine samples were collected at 12-h
intervals. The concentrations of 2,4-D were determined in the plasma
of three subjects and in the urine of all five subjects at intervals
after ingestion. The mean half-life for absorption of 2,4-D was 3.8 h
(range, 1.7-4.2 h). The average half-life for clearance from plasma
was 11.6 h, and that for urinary elimination was 17.1 h (range,
10.2-28.4 h). About 82% of the 2,4-D was excreted unchanged, and about
13% was excreted as conjugates. Plasma clearance followed first-order
kinetics in two subjects, with plasma half-lives of 7.3 and 11 h, and
biphasic kinetics in one subject, with half-lives of 4.3 and 16 h.

Despite these apparent differences in clearance pharmacokinetics, the
overall clearance rates were not markedly different. Urinary excretion
followed first-order kinetics in all three subjects (Saurehoff et
al., 1977).

The percutaneous absorption and urinary excretion of ¹⁴C-2,4-D
(purity unspecified) after dermal or intravenous administration were
studied in male subjects (age and weight unspecified). ¹⁴C-2,4-D
(1 µCi; 4 µg/cm²) was dissolved in acetone and applied to the
volunteers' forearms. The area of application was not covered, but the
subjects were asked not to wash the area for 24 h. Within 120 h after
dermal administration, 5.8 ± 2.4% of the dose was excreted in the
urine. After intravenous administration of a tracer dose of 1 µCi,
100% was excreted in the urine, with a half-life for excretion of 13 h
(Feldman & Maibach, 1974).

The percutaneous absorption of 2,4-D (purity, 99.5%) and the DMA
salt was examined in male subjects aged 27-48 years after dermal
application in acetone at a rate of 10 mg per 9 cm² on the back
of the hand. The area of skin was not protected, to reduce the
possibility of a covering dislodging the test material; subjects were
instructed to avoid contact of the application site with clothing or
other materials. In order to determine the amount of 2,4-D that could
be removed from the skin, the marked area was rinsed with distilled
water and scrubbed with a toothbrush 6 h after the application, and
the resulting wash water was analysed. Urine samples collected over
144 h after application were analysed for 2,4-D. An average of 4.5%
was recovered in the urine after application of 2,4-D, and 1.8% after
application of the DMA salt. More DMA salt (7.6%) than acid (5.4%) was
found in the hand wash, indicating that the amount of chemical
absorbed is related inversely to the amount washed off. Urinary
excretion of neither material was complete: 96 h after application,
averages of 85% of the total dose of the acid and 77% of the salt were
recovered in the urine; the approximate average half-lives for
excretion were 40 h for the acid and 59 h for the DMA salt (Harris &
Solomon, 1992).

The dermal absorption of the DMA salt and the EH ester was
studied after application to four male volunteers. A dose of 0.7 mCi
of ¹⁴C-labelled compound, dissolved in water or acetone, was applied
to a 45-cm² area of the forehead of each volunteer. The site was
washed with soap and water 24 h after application. Urine samples were
collected 4, 8, and 12 h daily for seven days. Dermal absorption
represented 58% of the total dose of DMA salt in water and 6% EH ester
in acetone (Moody et al., 1990).

(b) Biotransformation

The metabolism of 2,4-D in Fischer 344 rats was investigated
by analysing urine samples collected after oral and intravenous
administration of ¹⁴C-2,4-D at 1 or 100 mg/kg bw. The nature and
amounts of the 2,4-D metabolites excreted were determined in urine
samples by direct HPLC and/or GC-MS after an extraction step.
¹⁴C-2,4-D was eliminated primarily unchanged, representing > 97% of
urinary radiolabel, by both males and females. Two minor metabolites
were detected in urine from most groups, which accounted for 0.5-3.2%
of the radiolabel excreted in the first 12-h urine. Because of the
limited amount available, no attempt was made to identify these minor
metabolites; but their HPLC elution indicated that they might be 2,4-D
conjugates (Timchalk et al., 1990).

2,4-D metabolites were investigated in samples and tissues
collected from a lactating goat after oral administration of
¹⁴C-2,4-D. Urine was analysed directly by HPLC. The ¹⁴C residues in
various matrices and liver were extracted with organic and aqueous
solvents, then analysed by thin-layer chromatography, HPLC, and MS.
2,4-D was the major ¹⁴C component in urine, milk, and various
extracts. Some polar conjugates of the parent compound, which were
readily hydrolysed to 2,4-D under acidic conditions, were found in
milk. A non-polar ¹⁴C component detected in milk was identified as
2,4-dichloroanisole. Low levels of 2,4-dichlorophenol were tentatively
identified in milk and fat (Guo & Stewart, 1993).

In four of five human subjects given a single oral dose of 2,4-D
at 5 mg/kg bw, an acid-hydrolysable conjugate was detected in the
urine, representing 4.8-27% of the administered dose (Sauerhoff
et al., 1977).

No metabolites were found in the urine of male subjects given a
single oral dose of 5 mg/kg bw 2,4-D in gelatin capsules. About 75% of
the administered dose was detected unchanged in the urine after 96 h
(Kohli et al., 1974).

The acute effects of 2,4-D on the activities of lactate
dehydrogenase, alkaline phosphatase, aspartate transaminase, alanine
transaminase, amylase, creatinine, glucose, total protein, and albumin
were investigated in male Wistar rats given a single oral dose
of 0.6 g/kg bw, which is close to the lower limit of the LD₅₀
(0.6-1.3 g/kg). The serum levels of lactate dehydrogenase, alkaline
phosphatase, and creatinine increased by one- to fourfold 5, 8, and
24 h after treatment, whereas the activities of aspartate and alanine
transaminases were higher only at 8 and 24 h. Amylase activity was
increased only 8 h after administration of 2,4-D and then returned to
normal. In contrast, 2,4-D reduced the serum levels of glucose and

total protein 5, 8, and 24 h after treatment and serum albumin levels
by 5 h. Thus, an acute dose of 2,4-D disrupts the serum levels of
several entities considered to be indicators of tissue injury. The
authors speculated that these alterations reflected mainly hepatic and
muscular tissue damage, but they suggested that significant pancreatic
and kidney toxicity may also have occurred (Paulino & Palermo-Neto,
1991).

The effect of 2,4-D on the biogenesis of liver mitochondrial and
peroxisomal proteins was tested in male Fischer 344 rats fed a
diet containing 100 ppm (5 mg/kg bw) for 26 weeks. The parameters
investigated were somatic index, histochemistry, enzymatic activities
in purified peroxisomes and mitochondria, protein (by electrophoresis
and immunolabelling), and mRNA hybridization with specific DNA probes.
2,4-D was a peroxisomal proliferator, while mitochondria were weakly
affected. The compound modified mitochondrial protein patterns
(Cherkaoui Malki et al., 1991).

Metabolic alterations were investigated in hepatocytes from male
Wistar rats (200-250 g) treated with 2,4-D at 1-10 mmol/litre. Cell
viability was determined by measurement of cytosolic lactate
dehydrogenase leakage into the medium. Intracellular glutathione and
oxidized glutathione levels were determined and adenine (ATP, ADP and
AMP) and pyridine nucleotides (NADH, NAD⁺) were extracted and
analysed. 2,4-D was cytotoxic to the hepatocytes, as indicated by
increased leakage of lactate dehydrogenase, and it caused dose- and
time-dependent cell death accompanied by depletion of intracellular
glutathione, mirroring increases in oxidized glutathione. ATP and
NADH levels were also rapidly depleted by 2,4-D metabolism in the
millimolar range. 2,4-D completely depleted cellular ATP, resulting in
cell death. The compound appeared to be hepatotoxic and to initiate
the process of cell death by decreasing cellular glutathione. After
this primary disturbance, alteration of adenine and pyridine
nucleotide content is a critical event in the induction of
irreversible cell injury (Palmeira et al., 1994).

The inhibitory activity of several compounds, including 2,4-D,
on the Ca²⁺-transport-ATPase of human erythrocyte membranes was
determined to ascertain the usefulness of this screening test for
characterizing cellular toxicity. Some compounds, particularly those
with lipophilic properties, are incorporated into membranes, where
they disintegrate the structure. Since the Ca²⁺-transport-ATPase of
human erythrocytes is a membrane-bound enzyme, incorporation of such
compounds into membranes impairs membrane function and results in the
reduction of enzyme activity. Although several other compounds were
successful in inhibiting the enzyme, 2,4-D did not do so, probably
because it is negatively charged under physiological conditions
(pH 7.4 or 7.0 in this assay) and was therefore unable to penetrate
biological membranes (Janik & Wolf, 1992).

2. Toxicological studies

(a) Acute toxicity

The acute toxicity of 2,4-D, the DEA, DMA, IPA, and TIPA salts,
and the BEH and EH esters are summarized in Table 1. After their oral
administration, the clinical signs of toxicity observed consistently
were ataxia, myotonia, and decreased limb tone. No dermal or systemic
toxicity was seen in rabbits treated dermally, and no deaths were seen
after inhalation. Clinical signs of toxicity seen during exposure were
decreased activity and closed eyes; all rats had dried white material
on their fur (presumably test material). Signs seen at the end of and
during the week after exposure were salivation, lacrimation, mucoid
nasal discharge, laboured breathing, dried red or brown material
around the eyes and nose, matted fur, and staining of the fur in the
anogenital region. None of these signs was seen within three to seven
days of treatment. There was no significant finding post mortem
(Myer, 1981 a,b,c,d,e,f; Carreon et al., 1983; Squibb et al., 1983;
Streeter & Young, 1983; Auletta & Daly, 1986; Jeffrey et al., 1987a,b;
Streeter et al., 1987; Schults et al., 1990a,b; Cieszlak, 1992).

(b) Short-term toxicity

Mice

Groups of 20 male and 20 female B6C3F1 mice were fed diets
designed to provide technical-grade 2,4-D (purity, 96.1%) at doses of
0, 5, 15, 45, or 90 mg/kg bw per day for 13 weeks. No treatment-
related effects were seen on survival, clinical signs, or body weight,
or by ophthalmology, haematology, or gross pathology. Increases in the
weights of the pituitary and adrenals in animals of each sex and the
kidneys of females were observed, the latter effect being correlated
with histopathological lesions in that organ. Treatment-related
histopathological alterations were seen in the kidneys of 3, 9, 18,
and 20 males and 1, 4, 6, 12, and 14 females at 0, 5, 15, 45, and
90 mg/kg bw per day, respectively. The effects were characterized by
increased homogeneity and altered tinctorial properties of the
cytoplasm and decreased intracellular and intraluminal vacuolization
in the renal cortex of the males, and increased homogeneity and
altered tinctorial properties of the cytoplasm with or without
cytoplasmic swelling in the renal cortex of the females. Since renal
lesions occurred in a dose-related manner, including the lowest dose
tested (5 mg/kg bw per day), there was no NOAEL (Serota, 1983a).

Table 1. Acute toxicity of 2,4-D, amine salts, and esters in male and female animals

Compound Species Route LD₅₀ (mg/kg bw) Reference
or LC₅₀ (mg/litre)

2,4-D Rat Oral 699 Myer (1981a)
2,4-D Rat Oral 443 Squibb et al. (1983)
DEA salt Rat Oral 910 Schults et al. (1990a)
DMA salt Rat Oral 949 Myer (1989b)
IPA salt Rat Oral 2322 (m) Carreon et al. (1983)
1646 (f)
TIPA salt Rat Oral 1220 (m) Berdasco et al. (1989a)
1074 (f)
BEH ester Rat Oral 866 Jeffrey et al. (1987a)
EH ester Rat Oral 896 Myer (1981c)
2,4-D Rabbit Dermal > 2000 Myer (1981d)
DEA salt Rabbit Dermal > 2000 Shults et al. (1990b)
DMA salt Rabbit Dermal > 20000 Myer (1981e)
IPA salt Rabbit Dermal > 2000 Carreon et al. (1983)
TIPA salt Rabbit Dermal > 2000 Berdasco et al. (1989b)
BEH ester Rabbit Dermal > 2000 Jeffrey et al. (1987b)
EH ester Rabbit Dermal > 2000 Myer (1981f)
2,4-D Rat Inhalation > 1.8 Auletta & Daly (1986)
DEA salt Rat Inhalation > 3.5 Jackson & Hardy (1990)
DMA salt Rat Inhalation > 3.5 Streeter et al. (1990)
IPA salt Rat Inhalation > 3.2 Streeter et al. (1983)
TIPA salt Rat Inhalation > 0.84 Nitschke & Lomax (1990)
BEH ester Rat Inhalation 4.6 Streeter et al. (1987)
EH ester Rat Inhalation > 5.4 Cieszlak (1992)

DEA, diethanolamine; DMA, dimethylamine; IPA, isopropanolamine; TIPA, triisopropylamine;
BEH, butoxyethylhexyl; EH, 2-ethylhexyl
Groups of 10 male and 10 female B6C3F1 mice were fed diets
containing technical-grade 2,4-D (purity, 96.1%) at 0, 1, 15, 100, or
300 mg/kg bw per day for 13 weeks. Treatment at 1, 15, or 100 mg/kg bw
per day had no adverse effect on survival, body weight, body-weight
gain, or food consumption, or on ophthalmological, haematological,
clinical chemical, or gross pathological parameters. Treatment-related
changes at 100 mg/kg bw per day included significant (p < 0.05)
decreases in glucose and thyroxine levels in females and males,
increased mean absolute and relative kidney weights in females and a
liver lesion in one female. Treatment-related changes at 300 mg/kg bw
per day included transient decreases in food consumption (only up to
week 7); decreases in glucose and thyroxine levels in females and

males, respectively; a significant decrease in the kidney:brain weight
ratio in males; histopathological lesions in the kidneys of males
characterized as karyomegaly, loss of brush border, and decreased size
of tubular lining cells; and histopathological lesions in the livers
of animals of each sex, characterized as nuclear hyperchromatism, and
decreased glycogen in periportal hepatocytes. The NOAEL was 15 mg/kg
bw per day on the basis of renal toxicity (Schulze, 1991a).

Rats

Groups of 20 male and 20 female Fischer 344 rats were fed diets
designed to provide technical-grade 2,4-D (purity, 97.5%) at doses of
0, 1, 5, 15, or 45 mg/kg bw per day for 13 weeks. No treatment-related
effects were seen on survival, clinical signs, body weights, food
consumption, ophthalmoscopic findings, or haematological parameters.
Clinical chemistry indicated decreases in alanine and aspartate
transaminase and alkaline phosphatase activities and blood urea
nitrogen in animals of each sex at 15 and 45 mg/kg bw per day and
increased thyroxine values in males at 5 and 15 mg/kg bw per day. No
treatment-related gross pathological changes were seen. Both the
absolute and relative kidney weights were significantly (p < 0.05)
increased in animals of each sex at 45 mg/kg bw per day. The absolute
and relative thyroid weights were significantly increased in males at
all doses and in females at 5, 15, and 45 mg/kg bw per day; however,
there were no corroborative thyroid lesions. Histopathological
examination revealed renal lesions in animals of each sex at 5, 15,
and 45 mg/kg bw per day, characterized by increased homogeneity,
altered tinctorial properties, and fine vacuolization of the cytoplasm
in the renal cortex. The lesions were generally diffuse, most
frequent, and more severe at the high dose, with a multifocal, less
severe pattern at the lower doses. Renal lesions were seen in only one
female at 1 mg/kg bw per day; none of the males at this dose exhibited
this lesion. The NOAEL was 1 mg/kg bw per day on the basis of renal
toxicity (Serota, 1983b).

Groups of 20 male and 20 female Fischer 344 rats received
technical-grade 2,4-D (purity, 96.1%) in the diet to provide doses of
0, 1, 15, 100, or 300 mg/kg bw per day, for 13 weeks. Treatment at
1 or 15 mg/kg bw per day caused no adverse effects, but toxicity was
seen in animals of each sex at 100 and 300 mg/kg bw per day. At
termination, significant (p < 0.05) reductions in body-weight gain
were observed in males at 100 and 300 mg/kg bw per day and in females
at 300 mg/kg bw per day. Rats of each sex at 100 and 300 mg/kg bw per
day had alterations in some haematological and clinical chemical
parameters and changes in organ weights. Histopathological lesions in
the liver, adrenals, and kidneys were seen at 100 mg/kg bw per day and
in the eye, liver, testis, adrenals, kidneys, thymus, bone marrow,
spleen, thyroid, and lungs at 300 mg/kg bw per day. In some instances,
the histopathological changes correlated well with the alterations
seen on haematology, clinical chemistry, and organ weight measurement.

Increases in liver weight and in alanine and aspartate transaminase
activity were associated with centrilobular hepatocellular hypertrophy
in animals of each sex at 100 and 300 mg/kg bw per day. The decreased
thyroxine levels were correlated with follicular-cell hypertrophy of
the thyroid gland in females at 300 mg/kg bw per day. An increase in
adrenal weight may have been correlated with hypertrophy of cells of
the zona glomerulosa of the adrenal glands in animals of each sex at
100 and 300 mg/kg bw per day. The decreased mean thymic weight may
have been correlated with the atrophy seen in males and females at
300 mg/kg bw per day. Atrophy of mesenteric adipose tissue in the
peritoneal cavity may have been correlated with the mean decreases in
body weight seen in animals of each sex at 300 mg/kg bw per day.
Additional treatment-related histological lesions seen were bilateral
cataracts in females at 300 mg/kg bw per day, brush border loss in
proximal tubular cells of the kidneys in animals of each sex at
100 and 300 mg/kg bw per day, and alveolar macrophage accumulation and
hypocellularity of the bone marrow in animals of each sex at 300 mg/kg
bw per day. The NOAEL was 15 mg/kg bw per day, and the doses selected
for the two-year study of toxicity and carcinogenicity were 0, 5, 75,
and 150 mg/kg bw per day (Schultze, 1991b).

The toxicity of purified 2,4-D was evaluated in groups of 15
Fischer 344 rats of each sex, which received diets designed to provide
doses of 0, 15, 60, 100, or 150 mg/kg bw per day for 13 weeks. The
toxicity of the purified 2,4-D was similar to that of technical-grade
2,4-D. At 15 mg/kg bw per day, a minimal increase in epithelial
vacuolization of the renal convoluted tubules was seen in females but
not in males. Treatment at 60 mg/kg bw per day induced a slight
reduction in body-weight gain in females, a decrease in thyroxine
levels in females, increases in absolute kidney and relative liver
weights in females, and renal lesions in males and females. Treatment
at 100 or 150 mg/kg bw per day decreased body-weight gains, altered
alanine and aspartate transaminase and alkaline phosphatase
activities and thyroxine levels, increased absolute and/or relative
kidney weights, increased relative liver weights, and induced
histopathological lesions in the kidneys (renal tubular changes) and
liver (hepatocellular cytoplasmic swelling and homogeneity of the
liver cells) of animals of each sex. The NOAEL was 15 mg/kg bw per day
(Gorzinski et al., 1981).

Groups of 10 male and 10 female Fischer 344 rats were fed diets
containing the DEA salt of 2,4-D (purity, 73.8%) at doses of 0, 1.5,
27, 150, and 440 mg/kg bw per day (equivalent to 0, 1, 18, 100, or
300 mg/kg bw of the acid per day) for 13 weeks. The DEA salt had no
adverse effects at 1.5 or 27 mg/kg bw per day. Treatment-related
effects observed at higher doses included mortality; decreases in mean
body-weight gain and mean food consumption; alterations in some
haematological and clinical chemical parameters; changes in organ
weights; gross pathological changes; and histopathological changes in
the bone marrow, eyes, kidneys, liver, lungs, lymphoid tissues,
stomach, thyroid, thymus, testes, epididymides, seminal vesicles,

prostate, ovaries, and uterus. Histopathological alterations at the
two highest doses included diffuse regeneration of the renal tubular
epithelium of the kidneys; hypertrophy and necrosis of individual
centrilobular hepatocytes; bilateral retinal degeneration; foamy
macrophage accumulation in the lungs; follicular-cell hypertrophy in
the thyroid glands accompanied by decreased colloid; necrosis and
regeneration of the epithelium with submucosal oedema in the stomach;
decreased cellularity in the sternal bone marrow; lymphoid atrophy
of the spleen and cervical lymph node; atrophy of the thymus;
degeneration of seminiferous epithelium in the testes; decreased
spermatozoa in the epididymides; decreased secretory content in the
prostate and seminal vesicles; and atrophy of the ovaries and uterus.
The NOAEL was 27 mg/kg bw per day, equivalent to 18 mg/kg bw per day
of acid (Serrone et al., 1991).

Groups of 10 male and 10 female Fischer 344 rats received diets
containing the DMA salt of 2,4-D (purity, 66.2%) at doses of 0, 1.2,
18, 120, or 360 mg/kg bw per day (equivalent to 0, 1, 15, 100, or
300 mg/kg bw per day of the acid) for 13 weeks. No adverse effects
were seen at 1.2 or 18 mg/kg bw per day. There were decreases in mean
body weight, body-weight gain, and food consumption and alterations in
some haematological and clinical chemical parameters at 120 mg/kg bw
per day. The highest dose was associated with decreases in body-weight
gain, reduced food consumption, alterations in haematological and
clinical chemical parameters, changes in various organ weights, and
histopathological changes consisting of bilateral retinal degeneration
and cataract formation, centrilobular hepatocellular hypertrophy, and
hypoplasia of the spleen in females, atrophy of the testes in males,
and hypertrophy of thyroid follicular cells, brush border loss in
proximal tubular cells in the kidney, and hypoplasia of the bone
marrow in males and females. The NOAEL was 18 mg/kg bw per day,
equivalent to 15 mg/kg bw per day of the acid (Schultze, 1991c).

Groups of 10 male and 10 female Fischer 344 rats were fed diets
containing the IPA salt of 2,4-D (purity, 35.6%) at 0, 1, 19, 130, or
380 mg/kg bw per day (equivalent to 0, 1, 15, 100, or 300 mg/kg bw per
day of the acid) for 13 weeks. The IPA salt at 1 or 19 mg/kg bw per
day had no adverse effects. The treatment-related effects seen at
130 mg/kg bw per day were decreased mean body weight, body-weight
gain, and food consumption in animals of each sex; minor alterations
in haematological, clinical chemical, and urinary parameters in
animals of each sex; increased relative kidney weights in males and
females; and histopathological changes in the liver and kidneys of
males and the kidneys, adrenals, and thyroid glands of females.
Primary treatment-related effects observed at 380 mg/kg bw per day
included decreases in mean body weight, body-weight gain, and food
consumption in animals of each sex; alterations in haematological
parameters in females and in clinical chemical and urinary parameters
in animals of each sex; changes in organ weights; gross pathological

changes; and histopathological lesions in the eyes, kidneys, liver,
and thyroids of animals of each sex. Effects secondary to decreased
weight gain, the debilitated condition of the rats, and/or toxicity in
other organs occurred in the adrenals, bone marrow, mesenteric fat,
lungs, spleen, thymus, and testes. The NOAEL was 19 mg/kg bw per day,
equivalent to 15 mg/kg bw per day of the acid (Yano et al., 1991a).

Groups of 10 male and 10 female Fischer 344 rats received diets
designed to provide the TIPA salt of 2,4-D (purity, 72.2%) at doses of
0, 2, 28, 190, or 560 mg/kg bw per day (equivalent to 0, 1, 15, 100,
or 300 mg/kg bw per day of the acid) for 13 weeks. The TIPA salt had
no adverse effects at 2 or 28 mg/kg bw per day. The treatment-related
effects seen at 190 mg/kg bw per day were decreased body-weight gain
in females; minor alterations in haematological, clinical chemical,
and urinary parameters in animals of each sex; and histopathological
changes in the liver and kidneys of males and in the kidneys and
adrenal glands of females. Treatment-related effects seen in animals
of each sex at 560 mg/kg bw per day were decreased mean body weight,
body-weight gain, and food consumption, alterations in haematological,
clinical chemical, and urinary parameters, changes in organ weights,
gross pathological changes, and histopathological lesions in the eyes,
kidneys, liver, and thyroids. The NOAEL was 28 mg/kg bw per day,
equivalent to 15 mg/kg bw per day of the acid (Yano et al., 1991b).

Groups of 10 male and 10 female Fischer 344 rats received the BEH
ester of 2,4-D (purity, 94.6%) in their diet to provide doses of 0,
1.5, 22, 140, or 440 mg/kg bw per day (equivalent to 0, 1, 15, 100, or
300 mg/kg bw per day of the acid) for 13 weeks. No adverse effects
were seen at 1.5 or 22 mg/kg bw per day. Treatment at 140 mg/kg bw
per day decreased mean body weight, body-weight gain, and food
consumption; caused alterations in some haematological and clinical
chemical parameters; changed thyroid hormone concentrations; and
induced histopathological lesions in the thyroids. The high dose was
associated with decreased body-weight gain, reduced food consumption,
alterations in haematological and clinical chemical parameters,
changes in various organ weights, changes in thyroid hormone
concentrations, and histopathological lesions in the eye, liver,
kidneys, and thyroids. The NOAEL was 22 mg/kg bw per day, equivalent
to 15 mg/kg bw per day of 2,4-D (Szabo & Rachunek, 1991).

Groups of 10 male and 10 female Fischer 344 rats were fed diets
designed to provide the EH ester of 2,4-D (purity, 98%) at 0, 1.5,
23, 150, or 450 mg/kg bw per day (equivalent to 0, 1, 15, 100, or
300 mg/kg bw per day of the acid) for 13 weeks. The EH ester had no
adverse effects at 1.5 or 23 mg/kg bw per day. Treatment at 150 mg/kg
bw per day decreased mean body weight, body-weight gain, and food
consumption and induced alterations in some haematological and
clinical chemical parameters. The high dose was associated with
decreased body-weight gain and food consumption, alterations in
haematological and clinical chemical parameters, changes in various

organ weights, and histopathological lesions which included bilateral
retinal degeneration and cataract formation in females; lymphoid
hypoplasia of the thymus in females and of the spleen in animals of
each sex; centrilobular hepatocellular hypertrophy; hypoplasia of the
bone marrow; hypertrophy of thyroid follicular cells; atrophy of the
testes; vacuolization of the tubular cells of the kidney; and brush
border loss in proximal tubular cells in the kidneys of females. The
NOAEL was 23 mg/kg bw per day, equivalent to 15 mg/kg bw per day of
the acid (Schultze, 1991d).

Dogs

Groups of five male and five female beagle dogs were given
gelatin capsules containing 2,4-D (purity, 96.1%) at doses of 0, 0.3,
1, 3, or 10 mg/kg bw per day for 13 weeks. No treatment-related
effects were observed at 0.3 or 1 mg/kg bw per day. At 3 mg/kg bw per
day 2,4-D caused significant (p < 0.05) increases in blood urea
nitrogen and creatinine levels and renal lesions characterized as
cellular alterations in the proximal convoluted tubules in three male
dogs. Treatment-related changes at 10 mg/kg bw per day included
morbidity in two males and one female; clinical signs of toxicity
(thin and languid appearance, anorexia, emesis, and swollen testes);
decreased mean body weights (-8% in males and -14% in females) and
body-weight gains (-50% in males and -83% in females); alterations in
haematological (decreased haemoglobin, haematocrit, and platelet
counts) and clinical chemical (increased blood urea nitrogen and
creatinine levels) parameters in animals of each sex; decreased
absolute testicular weights; increased relative kidney weights in
females; and renal lesions in animals of each sex. The renal lesions
were characterized as cellular alterations in the proximal convoluted
tubules in three of three males and one of four females. The NOAEL was
1 mg/kg bw per day (Schultze, 1990a).

Groups of four male and four female beagle dogs received 2,4-D
(purity, 96.7%) in their diet to provide doses of 0, 0.5, 1, 3.8, or
7.5 mg/kg bw per day for 13 weeks. No treatment-related effects were
observed at 0.5 or 1 mg/kg bw per day. No mortality, clinical signs of
toxicity, ophthalmological changes, alterations in haematological or
urinary parameters, gross pathological changes, or changes in organ
weights were seen at 3.8 or 7.5 mg/kg bw per day. Body-weight gains
were decreased in males (-50%) and females (-47%) at 3.8 mg/kg bw per
day and in males (-39%) and females (-42%) at 75 mg/kg bw per day.
Food consumption was decreased (by about 15%) in animals of each
sex at 3.8 and 7.5 mg/kg bw per day. Clinical chemical analyses
showed significant (p < 0.05) increases in blood urea nitrogen,
creatinine, and alanine transaminase levels at four- and 13-week
intervals in animals at 3.8 and 7.5 mg/kg bw per day. The
toxicological significance of these increases is unknown as there were
no alterations in organ weights or corroborative histopathological
renal lesions. Except for a moderate, chronic, active perivascular

inflammation of the liver in one male and one female at 7.5 mg/kg bw
per day, no treatment-related histopathological lesions were seen.
There was no correlation between the severity of liver lesions and the
increased alanine transaminase activity. The NOAEL was 1 mg/kg bw per
day on the basis of the effects on body weight (Dalgard, 1993a;
Charles et al., 1996).

Groups of four male and four female beagle dogs were fed diets
containing the DMA salt of 2,4-D (purity, 55.45%) at doses equivalent
to 0, 1, 3.8, or 7.5 mg/kg bw of the acid per day for 13 weeks. The
DMA salt had effects similar to those of the acid. No treatment-
related effects were observed on survival, clinical signs of toxicity,
ophthalmological, haematological, or urinary parameters, gross
pathological appearance, organ weights, or histopathological
appearance in animals of either sex. At 3.8 mg/kg bw per day, there
was a nonsignificant decrease in body-weight gain (-40%) in females
and statistically significant (p < 0.05) increases in blood urea
nitrogen, alanine transaminase, and creatinine levels in animals
of each sex at four- and 13-week intervals. The toxicological
significance of these increases is unclear as no corroborative changes
in organ weights or histopathology were seen at this dose. The
treatment-related changes seen at 7.5 mg/kg bw per day included
decreased body-weight gains in males (-31%) and females (-67%;
p < 0.05); reduced food consumption (by about 15%); significant
(p < 0.05) increases in blood urea nitrogen, creatinine, and
alanine transaminase levels at four- and 13-week intervals in animals
of each sex; and decreases in absolute (-42%) and relative (-38%)
testicular weights. The toxicological significance of these increases
is unknown as no alterations in organ weights or corroborative
histopathological lesions were seen in the kidneys or testes. A
minimal increase in the average severity of perivascular, chronic,
active inflammation in the liver was seen in two males and one female;
however, there was no correlation between the severity of liver
lesions and the increase in alanine transaminase activity. The NOAEL
was 1 mg/kg bw per day on the basis of the decreases in body-weight
gain (Dalgard, 1993b; Charles et al., 1996).

Groups of four male and four female beagle dogs received diets
containing the EH ester of 2,4-D (purity, 62.7%) at doses equivalent
to 0, 1, 3.8, or 7.5 mg/kg bw of the acid per day for 13 weeks. The
effects were similar to those seen with the acid and the DMA salt. No
treatment-related effects were observed at 1 mg/kg bw per day, and
no mortality, clinical signs of toxicity, ophthalmological changes,
alterations in haematological or urinary parameters, gross
pathological changes, altered organ weights, or histopathological
alterations were seen at 3.8 or 7.5 mg/kg bw per day. Body-weight
gains were decreased in males (-48%) and females (-62%) at 3.8 mg/kg
bw per day and in males (-85%) and females (-50%) at 7.5 mg/kg bw per
day. Food consumption was decreased (by about 15%) in animals of each
sex at 3.8 and 7.5 mg/kg bw per day. Significant (p < 0.05)

increases in blood urea nitrogen, creatinine, and alanine transaminase
levels were seen at four- and 13-week intervals in animals of each sex
at 3.8 and 7.5 mg/kg bw per day. The toxicological significance of
these increases is unknown as no alterations in organ weights or
corroborative lesions were seen in the kidneys. Liver lesions,
characterized as perivascular, chronic, active inflammation, were seen
in two male dogs; no liver lesions were seen in females. There was
no correlation between the severity of the liver lesions and the
increased alanine transaminase activity. The NOAEL was 1 mg/kg bw per
day on the basis of the effects on body weight (Dalgard, 1993c;
Charles et al., 1996).

Mice

Groups of 50 male and 50 female B6C3F1 mice were fed diets
designed to provide 2,4-D (purity, 97.5%) at 0, 1, 15, or 45 mg/kg bw
per day for two years. Ten mice of each sex at each dose were killed
at 12 months. Survival, body weight, food consumption, and clinical
signs were noted; haematological parameters were evaluated at 12, 18,
and 24 months and organ weights and histopathological changes at 12
and 24 months. There were no treatment-related effects on survival,
clinical signs, body weights, haematological or urinary parameters, or
gross pathology. An increased relative kidney weight was seen in
females at 15 mg/kg bw per day, increased absolute kidney weight in
males at 45 mg/kg bw per day, and increased relative kidney weight in
animals of each sex at 45 mg/kg bw per day. Histopathology revealed
treatment-related kidney lesions only in male mice at 15 and 45 mg/kg
bw per day. Renal lesions, characterized as cytoplasmic homogeneity of
the renal tubule epithelium, were seen in 11/60 control males (18%),
15/60 at 1 mg/kg bw per day (25%), 48/60 at 15 mg/kg bw per day (80%,
p < 0.0001), and 58/59 at 45 mg/kg bw per day (98%, p < 0.0001).
This change was associated with a reduction in the cytoplasmic
vacuoles that are normally present in the renal tubular epithelium. No
evidence of carcinogenicity was seen; the tumour types and incidence
were similar in the treated and control groups. The NOAEL was 1 mg/kg
bw per day on the basis of the increase in kidney weights and renal
lesions (Serota, 1987).

2,4-D (purity, 96.4%) was administered in the diet of groups of
50 male B6C3F1 mice at doses of 0, 5, 62, or 120 mg/kg bw per day and
to groups of 50 female B6C3F1 mice at doses of 0, 5, 150, or 300 mg/kg
bw per day for two years. Ten mice of each sex at each dose were
killed at 12 months. Survival, body weight, food consumption, and
clinical signs were noted; haematological parameters were evaluated at
12, 18, and 24 months and organ weights and histopathological changes
at 12 and 24 months. No treatment-related effects were seen on
survival, body-weight gain, clinical signs, haematological parameters,
or gross pathological appearance in males at any dose. Except for a

transient decrease in body-weight gain (-14% at three months but
comparable to that of controls at termination) in females at 300 mg/kg
bw per day, treatment did not affect survival, induce clinical signs,
alter haematological parameters, or cause gross pathological changes
in females at any dose. Treatment-related changes in organ weights
were limited to the kidney. Dose-related increases in absolute (5 and
7%) and relative (6 and 10%) weights were seen in males at 62 and 120
mg/kg bw per day, respectively, only at 24 months. In females at 150
and 300 mg/kg bw per day, absolute kidney weights were increased by
14 and 17% and relative weights by 22 and 30% at the two doses,
respectively, at 12 months; at 24 months, the absolute weights were
increased by 14 and 22% and the relative weights by 12 and 20%,
respectively.

Histopathological examination revealed dose-related renal lesions
in males at 62 and 120 mg/kg bw per day, comprised of a constellation
of changes that involved five different diagnoses. Degeneration with
regeneration of the descending limb of the proximal tubule was seen in
25/50 (50%) and 48/50 (96%) animals at 62 and 120 mg/kg bw per day,
respectively, but not in the controls or in animals at 5 mg/kg bw per
day. Decreased vacuolization of the renal proximal tubule was seen in
39/50 (78%) and 48/50 (96%) animals at 62 and 120 mg/kg bw per day,
respectively. Both of these lesions were also seen in a dose-related
manner at the interim sacrifice. Mineralization of the tubules
occurred in 29/50 (58%) and 36/50 (72%) animals and multifocal
cortical cysts in 22/50 (44%) and 20/50 (40%) rats at 62 and 120 mg/kg
bw per day, respectively. In females at 150 and 300 mg/kg bw per day,
the renal lesions were characterized by hypercellularity in 32/50
(64%) and 25/50 (50%) animals and degeneration with regeneration of
the tubules in 38/50 (76%) and 34/50 (68%), respectively. A variety of
benign and malignant tumours at different sites was seen in both the
control and treated mice but were similar in number and type to those
commonly seen in this strain and age of mice. The NOAEL was 5 mg/kg bw
per day in animals of each sex on the basis of the renal lesions (Sott
et al., 1995).

Rats

Groups of 25 male and 25 female Osborne-Mendel rats were given
diets containing 2,4-D to provide doses of 0, 5, 125, 625, or 1250 ppm
(equal to 0, 0.25, 6.2, 31, and 62 mg/kg bw per day) for two years.
Treatment had no adverse effect on survival, clinical signs, body
weight, organ weights, or haematological parameters. Slight hepatitis
was seen in one rat each at 5 and 25 ppm and three rats each at 625
and 1250 ppm, with none in the controls. The authors reported that 15,
14, 18, 20, 23, and 22 rats were still alive after two years, but
the numbers were not given by sex. The total numbers of males with
malignant tumours were 1, 2, 4, 2, 5, and 6, and the numbers of
females were 5, 6, 3, 5, 3, and 8, respectively, at 0, 5, 25, 125,

625, and 1250 ppm. The NOAEL was 625 ppm, equal to 31 mg/kg bw per
day (Hansen et al., 1971). Two working groups convened by the IARC
considered this study to be inadequate for assessing carcinogenicity
(IARC, 1977, 1982).

Groups of 50 male and 50 female Fischer 344 rats were fed diets
containing technical-grade 2,4-D (purity, 97.5%) at doses of 0, 1, 5,
15, or 45 mg/kg bw per day for two years. Haematological, clinical
chemical, and urinary parameters were evaluated before treatment and
after 2, 52, and 78 weeks of treatment. Necropsies were conducted on
10 rats of each sex at each dose after 52 weeks and on all surviving
animals after two years. No treatment-related effects were seen on
survival, clinical signs, or gross pathological appearance. Body
weight gain was significantly (p < 0.05) decreased in females at
45 mg/kg bw per day at 12 months (-7%) and at 24 months (-9%); no
adverse effects were seen in females at the lower doses or in males
at any dose. Food consumption was decreased (-2.4%) in females at
45 mg/kg bw per day. No treatment-related effects were seen on
haematological parameters. Clinical chemistry revealed a significant
(p < 0.05) increase in alanine transaminase activity in males (50%)
and females (43%) and a decrease in thyroxine level in females (-18%)
at 45 mg/kg bw per day at termination. No treatment-related effects
were seen on urinary parameters. Males at 45 mg/kg bw per day had
significant (p < 0.05) increases in the absolute (9%) and relative
(13%) weights of the kidneys after 52 weeks, while females at this
dose had significant increases in absolute (10%) and relative (16%)
kidney weights after 104 weeks. Thyroid weights were significantly
increased at termination in males (absolute, 22%; relative, 26%) and
females (absolute, 24%; relative, 26%) at 15 mg/kg bw per day and in
males (absolute, 26%; relative, 29%) at 45 mg/kg bw per day; females
at 45 mg/kg bw per day also showed increases in absolute (2%)
and relative (16%) thyroid weights, but the increases were not
statistically significant. Histopathological examination revealed
renal lesions in males and females at 5, 15, and 45 mg/kg bw per day,
including an increased frequency of a brown tubular epithelial-cell
pigment, pelvic microcalculi, and transitional epithelial-cell
hyperplasia secondary to microcalculi. Brown tubular-cell pigment was
seen in 2/50 (4%), 1/50 (2%), 9/50 (18%) 18/50 (36%), and 19/59 (38%)
males and 8/50 (16%), 10/50 (20%), 23/50 (46%), 20/50 (40%), and 15/50
(30%) females at 0, 1, 5, 15, and 45 mg/kg bw per day, respectively.
The increases reached statistical significance (p < 0.05) at doses
> 5 mg/kg bw per day. Increased incidences of pelvic microcalculi
were seen in 2/50 (4%), 2/50 (4%), 4/50 (8%), 8/50 (16%), and 11/50
(22%) males and in 19/50 (38%), 11/50 (22%), 15/50 (30%), 23/50 (46%),
and 35/50 (70%) females at 0, 1, 5, 15, and 45 mg/kg bw per day,
respectively. A slight increase in transitional epithelial cell
hyperplasia was seen in females at 45 mg/kg bw per day (11/50; 22%) in
comparison with controls (0%).

No treatment-related neoplastic lesions were seen at any dose.
There was an increase in the incidence of brain astrocytomas in male
rats, with 1/50 (2%), 0/50, 0/50, 2/48 (4%), and 6/50 (12%) seen in
the controls and in rats at 1, 5, 15, and 45 mg/kg bw per day,
respectively. Although there was a positive trend (p = 0.002), a
pairwise test did not show statistical significance (p = 0.055) when
the incidence at the high dose (6/60) was compared with that of the
controls (1/50). The brain astrocytomas are not attributable to
treatment because they did not occur earlier in treated rats than in
controls (no decreased latency); there were no preneoplastic lesions
such as gliosis in treated rats, and all the tumours were solitary;
the tumours in the treated rats were no larger or more anaplastic than
generally seen in control rats (the largest and most lethal tumour was
seen in a control rat); and the tumours were seen only in animals of
one sex. In another study in Fischer 344 rats (Jeffris et al., 1995;
discussed below), no brain tumours or any evidence of carcinogenicity
was seen in the same strain of rats treated at more than three times
the dose (175 mg/kg bw per day) that was tested in this study. The
NOAEL was 1 mg/kg bw per day on the basis of the histopathological
lesions seen in the kidneys of animals of each sex (Serota, 1986).

Groups of 50 male and 50 female Fischer 344 rats were fed diets
designed to provide 2,4-D (purity, 96.4%) at doses of 0, 5, 75, or
150 mg/kg bw per day for up to two years. Ten animals of each sex at
each dose were killed at 12 months. Survival, body weight, food
consumption, and clinical signs were noted; haematological parameters
were evaluated at 12, 18, and 24 months and organ weights and
histopathological changes at 12 and 24 months. Treatment had no
adverse effect on survival, and there were no treatment-related
clinical signs of toxicity. At termination, the body weights were
lower than those of the respective controls for females at 75 mg/kg
bw per day (-14%) and males (-8%) and females (-26%) at 150 mg/kg bw
per day. The body-weight gains were also lower than those of the
respective controls for females at 75 mg/kg bw per day (-24%) and
males (-17%) and females (-48%) at 150 mg/kg bw per day. A concomitant
decrease in mean food consumption occurred in females at 75 mg/kg bw
per day (-4%) and in males (-5%) and females (-12%) at 150 mg/kg bw
per day. Statistically significant (p < 0.05) increases in the
plasma levels of alanine and aspartate transaminases, alkaline
phosphatase, and/or cholesterol were seen in females at 75 mg/kg bw
per day and in males and females at 150 mg/kg bw per day at various
times. These increases may be due to treatment as hepatic lesions were
observed at the interim sacrifice in females at 75 mg/kg bw per day
and at terminal sacrifice in males and females at 150 mg/kg bw per
day. It should be noted, however, that the hepatic lesions were
limited to altered tinctorial properties involving all hepatocytes
within the hepatic nodules and were not associated with hepatocellular
degeneration or necrosis. Thyroxine levels were decreased at 6, 12,
and 24 months in males and females at 75 and 150 mg/kg bw per day.

Increases in absolute and relative thyroid weights were seen, however,
only in females at 75 mg/kg bw per day and in animals of each sex
at 150 mg/kg bw per day at the interim and terminal sacrifices.
Histopathological lesions of the thyroid glands were seen only in
females at 150 mg/kg bw per day at the interim sacrifice. Gross
pathological examination revealed opacity of the lens and a general
decrease in fat in females and pale foci in the lungs of animals of
each sex at 150 mg/kg bw per day. The only treatment-related effects
on organ weights were the increases in thyroid weight.

After 12 months of treatment, the non-neoplastic lesions seen
were decreased haematopoiesis in the bone marrow of females at
150 mg/kg bw per day; altered tinctorial properties in the livers of
females at 75 mg/kg bw per day and in animals of each sex at 150 mg/kg
bw per day; bilateral retinal degeneration in females at 150 mg/kg bw
per day; multifocal alveolar histiocytosis of the lungs in females at
75 mg/kg bw per day and animals of each sex at 150 mg/kg bw per day;
degeneration of the descending portion of the proximal convoluted
tubules of the kidneys in animals of each sex at 75 and 150 mg/kg bw
per day; atrophy of the adipose tissue in females at 75 and 150 mg/kg
bw per day; atrophy of the testes at 150 mg/kg bw per day; and
decreased secretory material in the thyroid follicles of females at
150 mg/kg bw per day.

After 24 months of treatment, the non-neoplastic lesions were
limited to the eyes, liver, lung, and mesenteric fat. The eye lesions
were characterized as slight to severe bilateral retinal degeneration
and lenticular cataracts in animals of each sex at 150 mg/kg bw
per day. Liver lesions manifested as enlarged hepatocytes, often
accompanied by altered tinctorial properties that involved all
hepatocytes within the hepatic lobule of animals of each sex at
150 mg/kg bw per day. Lesions of the respiratory system included
subacute to chronic inflammation of the lungs in females at 75 mg/kg
bw per day and animals of each sex at 150 mg/kg bw per day. Atrophy of
the adipose tissue was increased in animals of each sex at 150 mg/kg
bw per day. It should be noted that the lesions seen in the spleen,
kidneys, testes, and thyroid glands of rats killed at 12 months were
not seen in those killed at 24 months. A variety of benign and
malignant tumours at different sites was seen in both the control and
treated mice, but they were similar in number and type to those
commonly seen in this strain and age of rats. The NOAEL was 75 mg/kg
bw per day in males and 5 mg/kg bw per day in females on the basis of
the decreases in body weights, body-weight gain, and food consumption,
increases in liver enzymes, decrease in thyroxine concentration,
increases in absolute and relative thyroid weights, and
histopathological lesions (Jeffries et al., 1995).

Dogs

Groups of three beagle dogs of each sex were fed diets containing
2,4-D at concentrations providing doses of 0, 10, 50, 100, or 500 ppm
(equal to 0, 0.25, 1.2, 2.5, or 12 mg/kg bw per day) for two years. No
gross or microscopic lesions were seen in any major organ (Hansen et
al., 1971).

Groups of five beagle dogs of each sex were fed diets containing
2,4-D (purity, 96.5%) at doses of 0, 1, 5, or 7.5 mg/kg bw per day for
52 weeks. No treatment-related effects were seen on survival, clinical
signs, ophthalmological, haematological, or urinary parameters, organ
weights, or gross pathological appearance at any dose. The body-weight
gains of dogs at 1 mg/kg bw per day were comparable to those of the
controls; the weight gains of animals of each sex at 5 and 7.5 mg/kg
bw per day were decreased, the effect being most pronounced in females
at the high dose. Increased blood urea nitrogen, creatinine, total
cholesterol, and alanine transaminase levels were seen in dogs at 5
and 7.5 mg/kg bw per day, and these alterations were corroborated by
histopathological changes in the livers and kidneys of these dogs. The
increases in blood urea nitrogen and creatinine are compatible with
either dehydration or mild compromise of the renal tubular epithelium,
while the elevations in alanine transaminase activity are indicative
of hepatocellular injury. The increases in total cholesterol are
nonspecific but are typically seen with alterations in lipid
metabolism by the liver. Histopathological examination revealed a
minimal increase in the frequency and average severity of sinusoidal
lining cells of the livers in females at 5 and 7.5 mg/kg bw per
day; minimal increases in the frequency and average severity of
perivascular, chronic, active inflammation of the liver; and an
increase in pigment in the tubular epithelium of the kidneys of
animals of each sex at 5 and 7.5 mg/kg bw per day. The NOAEL was
1 mg/kg bw per day on the basis of alterations in serum chemical
parameters and histopathological lesions in the liver and kidneys
(Dalgard, 1993d).

(d) Reproductive toxicity

Rats

In a two-generation study, groups of 30 male and 30 female
Fischer 344 rats were fed diets containing 2,4-D (purity, 97.5%) at
doses of 0, 5, 20, or 80 mg/kg bw per day for 105 days before mating
(F₀ generation). The rats were dosed in an analogous manner during
each mating, each gestation, and each lactation. The total and
continuous dosing of the F₀ rats lasted 40 weeks, which included two
weeks of rest between the end of lactation of the F_1a litters and the
beginning of mating for the F_1b litters and 30 days after weaning of
the latter litters. The F₁ generation, selected from the F_1b pups,
was exposed to 2,4-D in utero and continuously via the milk or the

feed for 125 days postnatally as well as prior to and throughout
mating, gestation, and lactation of the F_2a litters. Dosing continued
through a two-week rest period and during mating, gestation, and
lactation of the F_2b litters and for at least 30 days after weaning
of the F_2b litters. The dose of 80 mg/kg bw per day caused excessive
toxicity in the F₁ generation and was deleted, leaving groups dosed
at 5 and 20 mg/kg bw per day.

No adverse effects on fertility were seen in males or females at
any dose or in any generation. The length of gestation was prolonged
by one day in F₀ females at 80 mg/kg bw per day producing the F_1b
pups. This effect may have been the result of delayed implantation,
hormonal imbalance, or problematic parturition. The mean body weights
of F₀ males and females at 80 mg/kg bw per day were significantly
(p < 0.05) lower than those of the controls. The F₀ dams fed 80
mg/kg bw per day and producing the F_1a litter had significantly
(p < 0.05) lower body weights on days 7, 13 and 20 of gestation,
while those producing the F_1b litter had significantly (p < 0.05)
lower body weights only on day 20 of gestation. While the body weights
of F₁ dams producing the F_2a litters were comparable to those of the
controls at all doses during gestation, the F₁ dams fed 20 mg/kg bw
per day and producing the F_2b litters had significantly (p < 0.05)
lower body weights on days 7, 13, and 20 of gestation. The weights of
pups of each sex of the F_1a generation at 80 mg/kg bw per day were
significantly (p < 0.05) decreased during days 1-28 of lactation,
as were those of the F_1b generation at 20 mg/kg bw per day on day
28 of lactation and those at 80 mg/kg bw per day on days 1-28. The
weights of the F_2a and F_2b pups were comparable to those of the
respective controls. The viability of F_1a and F_1b pups was affected
only by treatment at 80 mg/kg bw per day. Live litter sizes were
reduced in the F_1a (9%; 10.1% in controls) and F_1b (5.1%; 0.5%
in controls; p < 0.01) generations. There was a significant
(p < 0.01) decrease in the sex ratio of the F_1a pups (109 males
and 71 females) when compared with controls (99 males and 114
females). Pup mortality was significantly (p < 0.01) increased in
the F_1b generation (110 dead pups) in comparison with controls (5
dead pups), but the viability of the F_2a and F_2b pups was not
affected. Examination of the F_1b pups at 80 mg/kg bw per day that
died before lactation on day 28 revealed bent ribs in 30 fetuses in
six litters, with none in the control fetuses; 14th rudimentary ribs
in 12 fetuses in six litters, with none in the control fetuses; and
slight or moderately malaligned sternebrae in 23 fetuses in nine
litters, and in one fetus per litter in the control pups. None
of these increases, however, showed statistical significance.
Histopathological examination revealed increased focal nuclear density
in the medullary renal tubules in animals of the F₀ generation at
20 mg/kg bw per day (7/30; 23%) and at 80 mg/kg bw per day (73%) and
in the F₁ adults at 20 mg/kg bw per day (4/29; 14%) when compared
with controls (0%). These lesions are indicative of degenerative or

atrophic change of the epithelial cells. No changes were seen at
5 mg/kg bw per day or in any of the F_1b weanlings. The NOAEL for
systemic parental toxicity in the F₀ and F₁ generations and for
reproductive and developmental toxicity was 5 mg/kg bw per day
(Rodwell, 1985).

(e) Developmental toxicity

Mice

The teratogenic potential of 2,4-D and its esters was investigated
in AKR, C57B1/6, C3H, and A/Ha mice by subcutaneous injection of doses
of 24-106 mg/kg bw per day on days 6-14 or 15 of gestation. Groups of
positive and negative controls were used periodically throughout the
study, but they were not matched with respect to either route or time
of administration. 2,4-D increased the proportion of abnormal litters
only in the AKR strain, in some tests but not others, depending on when
the tests were conducted. No significant increase in the incidence of
anomalies was noted with 2,4-D in C57, C3H, or hybrid C57 × AKR mice;
with the isooctyl ester of 2,4-D in C3H, A/Ha, or AKR mice; with the
butyl ester of 2,4-D in C57 or AKR mice; with the isopropyl ester of
2,4-D in C57 or AKR mice; with the methyl ester of 2,4-D in AKR mice in
a hybrid fetus resulting from mating a C57B1/6 female with an AKR male;
or with the EH ester of 2,4-D in C57 or AKR mice (Bage et al., 1973).

Rats

Groups of 15-19 pregnant Sprague-Dawley rats were given 2,4-D
(purity, 98.7%) in corn oil by gavage at doses of 12.5, 25, 50, 75, or
88 mg/kg bw per day on days 6-15 of gestation. Two control groups were
used: one for the animals at 88 mg/kg bw per day and another for those
at the lower doses. Fetuses were delivered by caesarean section on day
20 of gestation and were examined grossly, measured, weighed, and
examined for soft tissue and skeletal anomalies. There were no deaths,
and the body-weight gains of treated dams were comparable to those of
the controls. Statistically significant (p < 0.05) decreases in
fetal body weights were observed at doses > 50 mg/kg bw per day.
Fetal anomalies, such as subcutaneous oedema, lumbar and wavy ribs,
and delayed ossification of bones including the skull were observed
with increasing doses; however, these anomalies were also seen in both
control groups. Significant (p < 0.05) increases were observed in
both the fetal (8/119; 7%) and litter incidences (5/19; 26%) in
comparison with controls (fetal, 2/205, 1%; litter, 2/25, 6%). The
NOAELs were 88 mg/kg bw per day for maternal toxicity and 25 mg/kg bw
per day for developmental toxicity (Schwetz et al., 1971).

Groups of 35 pregnant Fischer 344 rats were given technical-grade
2,4-D (purity, 97.5%) in corn oil by gavage at doses of 8, 25, or
75 mg/kg bw per day during days 6-15 of gestation. The control group
received the vehicle alone by the same schedule. Dams were sacrificed
on day 20 of gestation; postmortem examination included gross
macroscopic examination of all internal organs with emphasis on the
uterus, uterine contents, position of fetuses in the uterus, and the
number of corpora lutea. Fetuses were weighed, sexed, and examined for
gross external abnormalities; they were prepared by Wilson's slicing
technique for visceral examination, after which they were stained with
alizarin red S for skeletal examination. Treatment did not alter
survival or induce clinical signs. Maternal toxicity was limited to
decreased body-weight gain in dams at 75 mg/kg bw per day during
treatment, which reached -43% during days 6-10 and -21% during days
6-15. No treatment-related effects were observed on the numbers of
viable fetuses, early or late resorptions, pre-implantation losses, or
corpora lutea or on the fetal sex distribution, fetal weights, or
fetal crown-rump length. No gross external or visceral anomalies
(malformations or variations) were seen at any dose. The incidence of
skeletal variations was increased in fetuses at 75 mg/kg bw per day
and included 7th cervical ribs in 4/127 (3%) fetuses and 3/26 (12%)
litters, 14th rudimentary ribs in 4/127 (3%) fetuses and 3/26 (12%)
litters, and missing sternebrae in 15/26 (12%) fetuses and 10/26 (38%)
litters; none were seen in the controls. Although these increases were
not statistically significant, they are attributable to treatment
since the same skeletal variations were also found at a high incidence
in the F_1b pups of dams fed 80 mg/kg bw per day 2,4-D in a study
in the same strain of rats (Rodwel, 1985) and in the fetuses of
Sprague-Dawley dams fed 87.5 mg/kg bw per day (Schwetz et al., 1971).
Thus, the weight of the evidence from the two-generation study of
reproductive toxicity and the studies of developmental toxicity in
two strains of rats indicates that the lowest observed effect level
for developmental toxicity was 75 mg/kg bw per day. The NOAEL was
25 mg/kg bw per day for maternal and developmental toxicity (Rodwell,
1983).

The salts and esters of 2,4-D were tested for developmental
toxicity in rats in a series of experiments with similar protocols:
The compounds were given by gavage on days 6-15 of gestation. The
control groups received the vehicle by the same schedule. The dams
were killed on day 20 of gestation, and post-mortem examination
included gross macroscopic examination of all internal organs with
emphasis on the uterus, uterine contents, position of fetuses in the
uterus, and the number of corpora lutea. Fetuses were weighed, sexed,
examined for gross external abnormalities, and prepared by Wilson's
slicing technique for visceral examinations, after which they were
stained with alizarin red S for skeletal examination.

The DEA salt of 2,4-D (purity, 73.1%) was administered at doses
of 15, 75, or 150 mg/kg bw per day (equivalent to 11, 55, or 110 mg/kg
bw per day of the acid) in distilled water to groups of 25 pregnant
Sprague-Dawley Crl:CD rats. Maternal toxicity at 75 mg/kg bw per
day was limited to a significant (p < 0.05) decrease in mean
body-weight gain during days 6-9 of gestation. At 150 mg/kg bw
per day, maternal toxicity consisted of significant (p < 0.05)
decreases in mean body-weight gain during days 6-9 of gestation and
reductions in mean food consumption during days 6-9 and 6-15 of
gestation. No effects attributable to treatment were observed on the
mean number of viable fetuses, early or late resorptions, pre- or
post-implantation losses, or corpora lutea or on the fetal sex
distribution or fetal crown-rump length. Singular fetotoxicity
observed at 150 mg/kg bw per day was a significant (p < 0.01)
reduction (-8%) in fetal body weight. Skeletal examination revealed
fetal variations in animals at 75 and 150 mg/kg bw per day. At
75 mg/kg bw per day, there was a significant (p < 0.05) increase in
the incidence of reduced ossification of the skull in 14/21 litters
(67%) in comparison with the control (5/23 litters; 22%) and an
increase in the incidence of bent ribs (6/21 litters, 29%; control,
0%). At 150 mg/kg bw per day, there was a nonsignificant increase in
the incidence of reduced ossification of the skull (12/23 litters,
52%; control, 5/23 litters, 22%). Significant (p < 0.05) increases
were seen in the incidences of 14th rudimentary ribs (15/21 litters,
65%; control, 4/23 litters, 17%) and of 7th cervical ribs (7/23
litters, 30%; control, 1/23 litters, 4%). These fetal anomalies
were due to treatment because the incidences were statistically
significantly higher than those in concurrent controls and exceeded
the historical control range of the testing laboratory. The NOAEL was
15 mg/kg bw per day, equivalent to 11 mg/kg bw per day of the acid,
for both maternal and developmental toxicity (Siglin et al., 1990).

Groups of 25 pregnant Sprague-Dawley Crl:CD rats received the
DMA salt of 2,4-D (purity, 66.2%) at doses equivalent to 12, 50, or
100 mg/kg bw per day of the acid in deionized water. Maternal toxicity
was seen at 50 mg/kg bw per day as a decrease in body-weight gain
(-8%) during treatment. Dams at 100 mg/kg bw per day had decreased
motor activity and ataxia and a significant (p < 0.01) decrease in
body-weight gain (-14%) during treatment. No effects attributable to
treatment were observed on the mean numbers of viable fetuses, early
or late resorptions, pre- or post-implantation losses, or corpora
lutea or on the fetal sex distribution or fetal crown-rump length.
Fetoxicity was limited to significant (p < 0.01) decreases in male
(-7%) and female (-8%) fetal body weights at 100 mg/kg bw. No external
or visceral malformations or variations were observed at any dose.
The treatment-related skeletal variation observed was a significant
(p < 0.01) increase in the incidence of wavy and/or incompletely
ossified ribs at 100 mg/kg bw (5/225 fetuses, 2.2%; 4/25 litters, 16%)

in comparison with the vehicle controls (0%) and historical controls
(range, 0-4%). The NOAEL was 12 mg/kg bw per day acid for maternal
toxicity and 50 mg/kg bw per day acid for developmental toxicity
(Lochry, 1990).

The IPA salt of 2,4-D (purity, 50.2%) was administered at doses
of 22, 65, or 190 mg/kg bw per day (equivalent to 9, 25, and 74 mg/kg
bw per day of the acid) in deionized water to groups of 30 pregnant
Sprague-Dawley rats. Maternal toxicity at 190 mg/kg bw per day was
seen as a significant (p < 0.05) decrease in body-weight gain
(-57%) and reductions in food consumption (-9%) on days 6-11 of
gestation. No treatment-related effects were observed on the mean
numbers of viable fetuses, early or late resorptions, pre- or
postimplantation losses, or corpora lutea or on the fetal sex
distribution, fetal body weight, or fetal crown-rump length. No gross
external, visceral, or skeletal malformations or variations were
observed at any dose. The NOAEL was 65 mg/kg bw per day (9 mg/kg bw
per day acid) for maternal toxicity; the NOAEL for developmental
toxicity was 190 mg/kg bw per day (equivalent to 74 mg/kg bw per day
of the acid), the highest dose tested (Schroeder, 1990a).

The TIPA salt of 2,4-D (purity, 72.2%) was given to groups of 30
pregnant Sprague-Dawley rats at doses of 32, 100, or 320 mg/kg bw per
day (equivalent to 12, 37, or 120 mg/kg bw per day of the acid) in
deionized water. Maternal toxicity included deaths, clinical signs,
and decreases in body-weight gain and food consumption at 320 mg/kg bw
per day: 4/30 dams (13.3%) died, 29 exhibited stiffness of the limbs,
10 had excessive salivation; body-weight gain was significantly
(p < 0.05) decreased (-42%) throughout treatment, and food consumption
was significantly (p < 0.05) reduced (-6.8%) during days 0-20 of
gestation. Embryotoxicity at 320 mg/kg bw per day included increases
in preimplantation loss (9.6%; controls, 5.4%; 78% increase); increases
in the number of resorptions per dam (2.6; controls, 0.8); and
increases in postimplantation loss (16.6%; controls, 5.3%; 217%
increase). Fetotoxicity at 320 mg/kg bw per day included decreases in
the number of males per litter (-38%) and in fetal body weights of
males (-11%) and females (-17%). Teratogenicity at 320 mg/kg bw per
day was evidenced by external and visceral malformations and skeletal
malformations and variations; the external and visceral variations
were comparable to those of the controls. The incidence of external
malformations was significantly (p < 0.05) increased for both
fetuses (5/272; 1.8%) and litters (5/23; 22%) in comparison with
controls (0%) and included filamentous tail (two fetuses in two
litters) and small bulging eyes (one fetus). The incidence of
visceral malformations was significantly (p < 0.05) increased for
both fetuses (7/144; 4.9%) and litters (6/23; 26%) in comparison
with controls (0%) and included microphthalmia (two fetuses in
two litters), anophthalmia (two fetuses in two litters), and
cardiovascular defects (one fetus). The incidence of skeletal
malformations was significantly (p < 0.05) increased for both

fetuses (16/131; 12%) and litters (9/23; 39%) in comparison with
controls (0%) and included defects of vertebrae, vertebral transverse
processes, sternebrae, and ribs. The incidence of skeletal variations
was increased significantly (p < 0.05; 127/131; 97%) in comparison
with controls (179/203; 88%) and included wavy ribs in five fetuses in
four litters each at 100 and 320 mg/kg bw per day, and fused ribs in
five fetuses in four litters at 320 mg/kg bw per day. The NOAEL for
maternal and developmental toxicity was 100 mg/kg bw per day,
equivalent to 37 mg/kg bw per day of the acid (Schroeder, 1990b).

Groups of 30 pregnant Sprague-Dawley CD rats were given the BEH
ester of 2,4-D (purity, 95.6%) in corn oil at doses of 25, 75, or
180 mg/kg bw per day (equivalent to 17, 50, 120 mg/kg bw per day of
the acid). Maternal toxicity at 180 mg/kg bw per day was shown by
decreased body-weight gain during days 6-9 (-33.3%) and 9-12 (-19%) of
gestation. No treatment-related effects were seen on the mean numbers
of viable fetuses, early or late resorptions, pre- or postimplantation
losses, or corpora lutea or on the fetal sex distribution, fetal body
weight, or fetal crown-rump length. No gross external or visceral
malformations or variations or skeletal malformations were observed at
any dose. The total incidences of fetuses with skeletal variations
were significantly (p < 0.05) increased at the high dose (162;
89.5%) when compared with the controls (138; 79.3%). Fetuses at
180 mg/kg bw per day had non-statistically significant increases in
incompletely ossified supraoccipital (29 fetal variations in 181
fetuses, 16%; controls, 14/174, 8%), squamosal (26/181, 12%; controls,
7/174, 4%), maxilla (6/181, 3%; controls, 0%), and 4th sternebrae
(20/184, 11%; controls, 12/174, 7%). The litter incidences were:
supraoccipital (12/27, 44%; controls, 6/25, 24%), squamosal (14/27,
52%; controls, 28%), maxilla (4/27, 15%; controls, 0%), and 4th
sternebrae (11/27, 41%; controls, 6/25, 24%). The NOAEL for maternal
and developmental toxicity was 75 mg/kg bw per day, equivalent to
50 mg/kg bw per day of the acid (Schroeder, 1990c).

The EH ester of 2,4-D (purity, 95%) was administered to groups
of 20 pregnant Sprague-Dawley rats at doses equivalent to 10, 30, or
90 mg/kg bw per day of the acid in 1% aqueous carboxymethylcellulose.
Maternal toxicity seen at 90 mg/kg bw per day consisted of clinical
signs (ataxia, decreased motor activity, and bradypnoea), significant
(p < 0.05) decreases in body-weight gain during days 6-9 of gestation
(-28%), and decreased food consumption throughout treatment. No
treatment-related effects were observed on the mean numbers of
viable fetuses, early or late resorptions, pre- or postimplantation
losses, or corpora lutea or on the fetal sex distribution, fetal body
weight, or fetal crown-rump distance. No treatment-related external or
visceral malformation or variations or skeletal malformations were
seen at any dose. There was a significant (p < 0.05) increase in
the incidence of incomplete or unossified sternebrae at 90 mg/kg bw

per day (18/207, 9%; controls, 8/182, 4%); the litter incidences
were comparable to those of controls. The NOAEL for maternal and
developmental toxicity was 30 mg/kg bw per day of the acid (Martin,
1992a).

Rabbits

2,4-D and its salts and esters were tested for developmental
toxicity in groups of 20 New Zealand white rabbits in a series of
experiments with similar protocols. The compounds were given orally or
by gavage on days 6-18 of gestation. The control groups received the
vehicle by the same schedule. The does were killed on day 29 of
gestation. The thoracic, abdominal, and pelvic cavities were examined
for gross lesions; if any were seen, the tissues were preserved in 10%
formalin. The uterus was removed, examined externally, weighed, and
then opened for internal examination. Uteri that appeared to be
from nonpregnant rabbits were stained with 10% ammonium sulfide to
determine pregnancy status. Corpora lutea were counted, and the
numbers and placements of implantations, early and late resorptions,
and live and dead fetuses were recorded. Fetuses were weighed, sexed,
examined for gross external abnormalities, and prepared by Wilson's
slicing technique for visceral examination, after which they were
stained with alizarin red S for skeletal examination. No treatment-
related effects were observed on the mean numbers of viable fetuses,
early or late resorptions, pre- or postimplantation losses, or corpora
lutea or on the fetal sex distribution, fetal body weight or fetal
crown-rump length.

Rabbits were given 2,4-D (purity, 96.1%) in 0.5% carboxymethyl-
cellulose orally at 10, 30 or 90 mg/kg bw per day. Maternal toxicity
at 90 mg/kg bw per day was shown by clinical signs such as ataxia,
decreased motor activity, loss of righting reflex, and cold
extremities in the two does that aborted; a decrease in body-weight
gain during (-27%) and after the treatment period (-16%); and a
nonsignificant reduction in corrected body-weight gain during the
entire period (-23%). No gross external, visceral, or skeletal
malformations or variations were seen at any dose. The NOAEL for
maternal toxicity was 30 mg/kg bw per day; the NOAEL for developmental
toxicity was 90 mg/kg bw per day, the highest dose tested (Hoberman,
1990).

Rabbits received the DEA salt of 2,4-D (purity, 73.09%) in
distilled water by oral administration at doses equivalent to 15, 30,
or 60 mg/kg bw per day of the acid. Maternal toxicity at 30 mg/kg bw
per day was characterized by significant (p < 0.01) decreases in
body-weight gain (-41% and -26%) and food consumption (-15% and-13%)
during days 6-19 and 0-29 of gestation, respectively. In animals at
60 mg/kg bw per day, maternal toxicity was seen, manifested as deaths
(one doe on day 19), abortion (one doe on day 23), and decreases in
body-weight gain (-51% and -24%) and food consumption (-28% and -17%)
during days 6-19 and 0-29 of gestation, respectively. No gross

external, visceral, or skeletal malformations were seen at any dose.
Skeletal variations seen at 60 mg/kg bw per day included a significant
(p < 0.05) increase in the number of litters with 7th cervical ribs
(4/17, 24%; controls, 0%). This increase was outside the historical
control range (0-6.7%) of the testing laboratory. This anomaly was
also observed in 30% of pregnant rats given DEA at 150 mg/kg bw per
day. The NOAEL was 15 mg/kg bw per day of the acid for maternal
toxicity and 30 mg/kg bw per day of the acid for developmental
toxicity (Rodwell, 1991).

Rabbits were given the DMA salt of 2,4-D (purity, 66.2%) in
deionized water orally at doses equivalent to 10, 30, or 90 mg/kg bw
per day of the acid. Maternal toxicity at 90 mg/kg bw per day included
the deaths of four does (two on day 10 and two on day 18), clinical
signs of toxicity such as decreased motor activity, myotonia,
ataxia, and impaired or lost righting reflexes, and a significant
(p < 0.05) decrease in food consumption on days 6-9 of gestation.
Except for the body weight loss in the does that died, no treatment-
related effects were observed on body-weight gain. No gross external,
visceral, or skeletal malformations or variations were seen at any
dose. The skeletal variation (wavy and/or incompletely ossified ribs)
observed in pregnant rats given 100 mg/kg bw per day was not seen in
rabbits. The NOAEL for maternal toxicity was 30 mg/kg bw per day of
the acid; the NOAEL for developmental toxicity was 90 mg/kg bw per day
of the acid, the highest dose tested (Martin, 1991).

Does received the IPA salt of 2,4-D (purity, 50.2%) in deionized
water orally at 13, 38 or 95 mg/kg bw per day (equivalent to 10, 30,
or 75 mg/kg bw per day of the acid). Maternal toxicity at 38 and
95 mg/kg bw per day included the deaths of two does at 38 mg/kg bw per
day and three at 95 mg/kg bw per day, morbidity in four does at
95 mg/kg bw per day, clinical signs of toxicity (decreased faeces
and myotonia at 38 and 95 mg/kg bw per day and lateral recumbency
at 95 mg/kg bw per day), significant (p < 0.05) decreases in
body-weight gain (-44% at 38 mg/kg bw per day and -56% at 95 mg/kg bw
per day) on days 7-20 of gestation, and a significant (p < 0.05)
increase (13%) in relative kidney weights. No gross external,
visceral, or skeletal malformations or variations were seen at any
dose. The NOAEL for maternal toxicity was 13 mg/kg bw per day
(equivalent to 10 mg/kg bw per day of the acid); the NOAEL for
developmental toxicity was 95 mg/kg bw per day (equivalent to 75 mg/kg
bw per day of the acid), the highest dose tested (Breslin et al.,
1991).

Does were given the TIPA salt of 2,4-D (purity, 73.1%) in
deionized water by oral administration at 19, 56, or 140 mg/kg bw per
day (equivalent to 10, 30, or 75 mg/kg bw per day of the acid).
Maternal toxicity at 56 and 140 mg/kg bw per day included the death of
one doe at 56 mg/kg bw per day, morbidity in three does at 140 mg/kg
bw per day, clinical signs of toxicity (decreased faeces, myotonia,

and lateral recumbency) at 56 and 140 mg/kg bw per day, and
significant (p < 0.05) decreases in body-weight gain (-46% at
56 mg/kg bw per day and -59% at 140 mg/kg bw per day) on days 7-20 of
gestation. No gross external, visceral, or skeletal malformations or
variations were seen at any dose. The NOAEL for maternal toxicity was
19 mg/kg bw per day, equivalent to 10 mg/kg bw per day of the acid;
the NOAEL for developmental toxicity was 140 mg/kg bw (equivalent to
75 mg/kg bw per day of the acid), the highest dose tested (Liberacki
et al., 1991).

Groups of 20 artificially impregnated New Zealand rabbits were
given the BEH ester of 2,4-D (purity, 95.6%) in corn oil by gavage at
15, 45, or 110 mg/kg bw per day (equivalent to 10, 30, or 75 mg/kg bw
per day of the acid). Maternal toxicity at 45 and 110 mg/kg bw per day
included the deaths of one doe at 45 and four at 110 mg/kg bw per day,
morbidity in one doe at 45 and four at 110 mg/kg bw per day, clinical
signs (decreased activity, myotonia, lateral recumbency, and
prostration), and decreases in the body weights of does that died or
were killed at these doses. No fetal gross external, visceral, or
skeletal malformations or variations were seen at any dose. The NOAEL
for maternal toxicity was 15 mg/kg bw per day, equivalent to 10 mg/kg
bw per day of the acid; the NOAEL for developmental toxicity was
110 mg/kg bw per day (equivalent to 75 mg/kg bw per day of the acid),
the highest dose tested (Zablotny et al., 1991).

Pregnant does were given the EH ester of 2,4-D (purity, 95.6%) in
1% methylcellulose by oral administration at doses equivalent to 10,
30, or 75 mg/kg bw per day of the acid. Maternal toxicity at 75 mg/kg
bw per day consisted of the deaths of two does, morbidity in two does,
and abortion by one doe; clinical signs of toxicity (decreased
activity, ataxia, impaired righting reflexes, loss of righting reflex
and bradypnoea); and decreases in body weight (-19%) on days 6-19 of
gestation. No gross external, visceral, or skeletal malformations or
variations were seen in fetuses at any dose. The NOAEL for maternal
toxicity was 30 mg/kg bw per day of the acid; the NOAEL for
developmental toxicity was 75 mg/kg bw per day of the acid, the
highest dose tested (Martin, 1992b).

(f) Genotoxicity

The mutagenic potential of 2,4-D, the DEA, DMA, IPA, and TIPA
salts and the BEH and EH esters has been evaluated in numerous assays.
The results are presented in Table 2.

Table 2. Results of tests for the genotoxicity of 2,4-D, its salts and its esters

End-point Test system Concentration Purity Results Reference
(%)

2,4-D
Reverse mutation S. typhimurium 100-10 000 µg/plate 96.1 Negative Lawlor &
TA98, TA100, with S9; 66.7-6670 Valentine (1990a)
TA1535, TA1537, µg/plate without S9
TA1538
Reverse mutation S. typhimurium 0-1000 µg/plate NR Negative Rashid et al.
TA98, TA100, (1984)
TA1535, TA1537,
TA1538
Reverse mutation S. typhimurium 0-3333 µg/plate NR Negative Soler-Neidzieler
et al. (1988)
Reverse mutation S. typhimurium 0-1000 µg/plate NR Negative Kappas (1988)
TA97, TA98, TA100,
TA1535, TA1537,
TA1538
Reverse mutation S. typhimurium 0-2000 µg/plate NR Negative Rashid & Muma
TA1538, TA1535 (1986)
Reverse mutation S. typhimurium < 5000 µg/plate NR Negative Simmon et al.
TA98, TA100, (1977)
TA1535, TA1538
DNA damage E. coli K12, WP2 0-2000 µg/plate NR Negative Rashid & Mumma
(1986)
DNA damage E. coli PQ 37 0-200 µg/plate NR Negative Sundermann et
al. (1989)
Sex-linked recessive Drosophila 0.1-10 000 ppm NR Positive Kale et al. (1995)
lethal mutation melanogaster larvae

Table 2. (Cont'd)

End-point Test system Concentration Purity Results Reference
(%)

Sex-linked recessive Drosophila 1000-10 000 ppm (feeding), > 99 Negative Zimmering et al.
lethal mutation melanogaster adults 10 000 ppm (injection) (1985)
Gene mutation Hamster V79 10-100 µg/ml NR Positive Pavlica et al.
fibroblasts, hprt (1991)
locus
Chromosomal aberration Chinese hamster 500-920 µg/ml without S9; NR Negative Galloway et al.
in vitro ovary cells 1900-5000 µg/ml with S9 Equivocal (1987)
Chromosomal aberration Embryonic bovine 1-1000 ppm NR Negative Bongso & Basrur
in vitro kidney and peripheral (1973)
lymphocytes
Chromosomal aberration Human lymphocytes 0.125-0.35 mmol/litre 55 Negative Mustonen et al.
in vitro 0.125-1.250 mmol/litre Positive^a (1986)
Chromosomal aberration Human lymphocytes 0.03-0.04 mg/m^3b 33.3 Negative Mustonen et al.
in vivo (1986)
Chromosomal aberration Rat bone marrow 0-350 µg/kg bw NR Negative Turkula & Jalal
in vivo intraperitoneally per 4 or (1987)
24 h; three replicates
Chromosomal aberration Rat bone marrow 0, 17.5, 35, or 70 mg/kg bw NR Negative^c Adhikari &
in vivo per day intraperitoneally Grover (1988)
twice
Sister chromatid exchange Rat lymphocytes 100 mg/kg bw NR Negative Mustonen et al.
in vivo (1989)
Sister chromatid exchange Chinese hamster 50-299 µg/ml without S9 NR Positive Galloway et al.
in vitro ovary cells 500-4200 µg/ml with S9 Negative (1987)
Sister chromatid exchange Human lymphocytes NR 33.3 Negative Linnainmaa (1983)
in vivo
Micronucleus formation ICR mouse bone 40-400 mg/kg bw 96.1 Negative Ivett (1990a)
in vivo marrow

Table 2. (Cont'd)

End-point Test system Concentration Purity Results Reference
(%)

Unscheduled DNA synthesis Primary Fischer 344 0.969-2890 µg/ml 96.1 Negative Cifone (1990a)
rat hepatocytes
DNA damage Human fibroblasts 0-100 nmol/litre NR Negative Clausen et al.
(PM2 DNA) (1990)

DEA salt
Reverse mutation S. typhimurium 500-14 000 µg/plate 73.8 Negative Lawlor &
TA98, TA100, with and without S9 Holloway (1990)
TA1535, TA1537,
TA1538
Chromosomal aberration ICR mouse bone 60-600 mg/kg bw 73.8 Negative Ivett (1990b)
in vivo marrow
Unscheduled DNA synthesis Primary Fischer 344 10-500 µg/ml 73.8 Negative McKeon (1990)
rat hepatocytes

DMA salt
Reverse mutation S. typhimurium 333-10 000 µg/plate 66.2 Negative Lawlor &
TA98, TA100, Valentine (1990b)
TA1535, TA1537,
TA1538
Micronucleus formation ICR mouse bone 60-600 mg/kg bw 66.2 Negative Ivett (1990b)
in vivo marrow
Unscheduled DNA synthesis Primary Fischer 344 70.5-100 µg/ml 66.2 Negative Cifone (1990b)
rat hepatocytes

IPA salt
Reverse mutation S. typhimurium 10-10 000 µg/plate 50.1 Negative Samson &
TA98, TA100, Gollapudi (1989a)
TA1535, TA1537

Table 2. (Cont'd)

End-point Test system Concentration Purity Results Reference
(%)

Gene mutation Chinese hamster 500-3000 µg/ml 50.2 Negative Linscombe & Lick
ovary cells, hprt locus (1994a)
Chromosomal aberration Rat lymphocytes 96-6137 µg/ml with and 50.2 Negative Linscombe & Lick
without S9 (1994b)
Micronucleus formation ICR mouse bone 75-750 mg/kg bw 50.2 Negative Gollapudi et al.
in vivo marrow (1990a)
Unscheduled DNA synthesis Primary Fischer 344 5-500 µg/ml 50.2 Negative McClintock &
rat hepatocytes Gollapudi (1990)

TIPA salt
Reverse mutation S. typhimurium 1000-10 000 µg/plate 72.2 Negative Samson &
TA97, TA98, TA100, Gollapudi (1989b)
TA1535, TA1537
Gene mutation Chinese hamster ovary 78-5000 µg/ml 70.9 Negative Linscombe & Lick
cells, hprt locus (1994c)
Chromosomal aberration Rat lymphocytes 800-5000 µg/ml 70.9 Negative Linscombe & Lick
(1994d)
Micronucleus formation ICR mouse bone 75-750 mg/kg bw 70.9 Negative Gollapudi et al.
in vivo marrow (1990b)

BEH ester
Reverse mutation S. typhimurium 5-5000 µg/plate with S9; 95.6 Negative Samson &
TA97, TA98, TA100, 1.6-1667 µg/plate without Gollapudi (1990)
TA1535, TA1537, S9
TA1538
Chromosomal aberration Rat lymphocytes 87.5-1400 µg/litre 94.6 Negative Linscombe & Lick
(1994e)

Table 2. (Cont'd)

End-point Test system Concentration Purity Results Reference
(%)

Micronucleus formation ICR mouse bone 37.5-375 mg/kg bw 95.6 Negative Gollapudi et al.
in vivo marrow (1990c)
Unscheduled DNA synthesis Primary Fischer 344 5-500 µg/ml 95.6 Negative McLintock &
rat hepatocytes Gollapudi (1990)

EH ester
Reverse mutation S. typhimurium 333-10 000 µg/plate 98 Negative Lawlor &
TA97, TA98, TA100, with and without S9 Valentine (1990c)
TA1535, TA1537,
TA1538
Micronucleus formation ICR mouse bone 50-500 mg/kg bw 98 Negative Ivett (1990c)
in vivo marrow
Unscheduled DNA synthesis Primary Fischer 344 0.5-25 µg/ml 98 Negative Cifone (1990c)
rat hepatocytes

^a Attributed to 'unidentified clastogens' (i.e. contaminants) by investigators
^b Occupational exposure. Present in urine at 0.09-1.14 mg/litre in nonsmokers and 0.11-1.56 in smokers
^c Positive at two highest doses, but results were similar with dimethyl sulfoxide solvent as a control
(g) Special studies

(i) Dermal and ocular irritation and dermal sensitization

No skin irritation was observed in rabbits after dermal
applications of 2,4-D, the amines DMA, DEA, IPA, and TIPA or the BEH
and EH esters for 4 h (Keller et al., 1977; Myer, 1981e; Carreon
et al., 1983; Jeffrey, 1987b; Mizell et al., 1989; Schults et al.,
1990d; Berdasco, 1992).

2,4-D, the DMA, DEA, IPA, and TIPA salts, and the BEH and EH
esters were shown to be severe eye irritants when instilled into the
conjunctival sac of rabbits. Consistent eye lesions observed in these
studies were corneal opacity, chemosis, redness of the conjunctivae,
and ocular discharge. Iridial inflammation was also seen. No evidence
of amelioration was evident three days later (Keller et al., 1977;
Kirsh, 1983; Carreon et al., 1983; Carreon & Rao, 1986; Jeffrey,
1987a; Berdasco & Mizell, 1989; Schults et al., 1990c)

The sensitization potential of 2,4-D, the DMA, DEA, IPA, and TIPA
salts, and the BEH and EH esters has been assessed in guinea-pigs by
the Buehler method of dermal induction. No evidence of delayed contact
hypersensitivity was seen in any of the studies (Keller et al., 1977;
Carreon et al., 1983; Carreon & Rao, 1985; Gargus, 1986; Jeffrey, 1986;
Jeffrey & Rao, 1986; Schults et al., 1990e).

(ii) Dermal toxicity

In a series of studies with 2,4-D and its salts and esters,
groups of five male and five female New Zealand white rabbits received
15 repeated dermal applications for 6 h/day, on five days per week for
21 days.

When 2,4-D (purity, 96.1%) was applied at 0, 10, 100, or
1000 mg/kg bw per day, no systemic toxicity was seen. 2,4-D was mildly
irritating to the skin, but no skin lesions were seen (Schultze,
1990b). Dermal applications of the DEA salt (purity, 73.9%) in
distilled water at 0, 15, 150, or 440 mg/kg bw per day induced hepatic
toxicity only at the high dose, seen as an elevation in the serum
activities of alanine and aspartate transaminases and alkaline
phosphatase, increases in absolute and relative liver weights, and
corroborative liver lesions consisting of hypertrophy of hepatocytes
and the presence of hyaline droplets within the hepatocytes. The DEA
salt at 150 and 440 mg/kg bw per day induced dermal toxicity
characterized by histopathological lesions of the skin, including
acanthosis, hyperkeratosis, and chronic dermatitis; acute dermatitis,
surface exudate, dermal haemorrhage, and vesiculation of the epidermis
were also seen at the highest dose. The NOAEL was 150 mg/kg bw per day
for systemic toxicity and 15 mg/kg bw per day for dermal toxicity
(Siglin, 1991).

The DMA salt (purity, 66.18%) in distilled water at 0, 18, 180,
or 540 mg/kg bw per day did not induce systemic toxicity. Dermal
toxicity induced by doses of 180 and 540 mg/kg bw per day was
characterized by histopathological lesions of the skin, including
acanthosis, hyperkeratosis, oedema, superficial crusting (inspissated
serum, necrotic cells, and debris on the epidermal surface) and
chronic active inflammation. The NOAEL was 540 mg/kg bw per day for
systemic toxicity and 18 mg/kg bw per day for dermal toxicity
(Schultze, 1990c).

Applications of the IPA salt (purity, 50.2%) in distilled water
at 0, 50, 125, or 350 mg/kg bw per day also induced no systemic
toxicity. Dermal toxicity at 125 and 350 mg/kg bw per day was
characterized by skin lesions including focal and multifocal
irritation (inflammation and epidermal hyperplasia). The NOAEL was
350 mg/kg bw per day for systemic toxicity and 50 mg/kg bw per day for
dermal toxicity (Mizell, 1990a).

The TIPA salt (purity, 72.2%) was applied in distilled water at
0, 100, 350, or 1000 mg/kg bw per day. No systemic or dermal toxicity
was seen (Mizell et al., 1990).

The BEH ester (purity, 94.6%) in corn oil at 0, 50, 150, or
500 mg/kg bw per day did not induce systemic or dermal toxicity
(Mizell, 1990b).

The EH ester (purity, 98%) at 0, 16, 160, or 1600 mg/kg bw per
day did not induce systemic toxicity, but the two highest doses
induced dermal toxicity characterized by histopathological lesions of
the skin, including acanthosis, hyperkeratosis, and necrotic cellular
debris on the epidermal surface. The NOAEL for dermal toxicity was
16 mg/kg bw per day (Schultze, 1990d).

(iii) Neurotoxicity

No polyneuropathy was seen in male Fischer rats given single
intraperitoneal injections of 2,4-D at 100 mg/kg bw per day on six
days per week for three weeks or 80 mg/kg bw per day on three days per
week for 12 weeks (Toyoshima et al., 1985).

Groups of 10 male and 10 female Fischer 344 rats received 2,4-D
in corn oil by gavage as single doses of 0, 15, 75, or 250 mg/kg bw.
Neurobehavioural evaluations consisting of a functional observational
battery and tests for motor activity were conducted one day before
treatment, 6 h after dosing (at the time of peak effect), and on days
8 and 15. Neuropathological examination of the central and peripheral
nervous tissues was conducted at termination. The functional
observational battery on day 1 showed that animals of each sex at
250 mg/kg bw had increased incidences of incoordination and slight
gait abnormalities, described as forepaw flexing or knuckling. The
incidence of incoordination had decreased to control levels by day 4

in males and day 5 in females. There were no treatment-related gross
or neuropathological alterations. The NOAEL for neurotoxicity was
75 mg/kg bw (Mattsson et al., 1994a).

Groups of male Fischer 344 rats were given oral doses of 2,4-D
in corn oil at 20, 40, or 80 mg/kg bw twice weekly for five weeks.
Significant increases were observed in grip strength (both fore- and
hind-limb) at all doses, but the effect appeared to dissipate with
time after cessation of treatment. The effect was confirmed in a
separate experiment in which male Fisher rats received 2,4-D at 10,
20, or 40 mg/kg bw per day on five days per week for four weeks. Both
fore- and hind-limb grip strengths were increased in rats receiving 20
or 40 mg/kg bw per day. In animals at 40 mg/kg bw, the increase
persisted only two weeks after the end of treatment and was absent
six weeks after dosing. The authors suggested that the increase
in grip strength was linked to the observed myotonia, a condition
characterized by difficulty in relaxing skeletal musculature after
forceful contraction (Squibb et al., 1983).

Groups of 15 male and 15 female Fisher 344 rats were fed diets
containing 2,4-D at 0, 5, 75, or 150 mg/kg bw per day for 12 months.
Functional observational battery and motor activity evaluations were
conducted at 3, 6, 9, and 12 months. The NOAEL was 75 mg/kg bw per day
on the basis of increased relative forelimb grip strength in animals
of each sex (Mattsson et al., 1994b).

Clinical signs indicative of neurotoxicity, such as ataxia,
decreased motor activity, myotonia, prostration, lateral recumbency,
and impairment or loss of righting reflexes, were observed in pregnant
rabbits after oral administration of 2,4-D, the DEA, DMA, IPA, and
TIPA salts and the BEH and EH esters at doses equivalent to 30 mg/kg
bw per day of the acid on days 6-18 of gestation (Hoberman, 1990;
Rodwell, 1991; Breslin et al., 1991; Liberacki et al., 1991; Martin,
1991; Zablotny et al., 1991).

Groups of four adult female mongrel dogs were given gelatin
capsules containing single doses of 2,4-D at 0, 25, 50, 75, 100, or
120 mg/kg bw. Clinical neurological examinations, electromyography,
and measurements of motor nerve conduction velocity were conducted
before treatment and on days 1, 3, 7, 14, 21, and 28 after treatment.
Histopathological examination was conducted on two dogs at each dose
killed on day 7 and at termination on day 28. Transient, generalized
myotonic discharges were observed in the skeletal muscles of dogs
given doses > 50 mg/kg bw (Steiss et al., 1987).

Electroencephalographic activity was evaluated in single English
pointer dogs given gelatin capsules containing the DMA salt of 2,4-D
at 0, 1.3, 8.8, 44, 180, or 220 mg/kg bw. By 24 h after treatment, the
dog given 180 mg/kg bw showed mild sedation accompanied by excessive
slowing in the electroencephalogram (EEG), with loss of low-voltage

activity. In the dog given 220 mg/kg bw, nonspecific alterations in
the EEG were suggestive of irritation, and mild seizure activity was
detected 7 h after treatment. The EEG had returned to normal 24 h
after treatment. No changes in the EEG were seen at the lower doses
(Arnold et al., 1991).

Groups of three male and three female English pointer dogs were
given gelatin capsules containing the DMA salt of 2,4-D at 0, 1, 1.3,
8.8, 44, 87, 180, or 220 mg/kg bw. An electromyogram (EMG) was taken
before and at various times after treatment. Dogs at 8.8, 44, and
87 mg/kg bw developed clinical manifestations of myotonia detectable
only with the EMG; however, dogs at 180 and 220 mg/kg bw rapidly
developed clinical and EMG manifestations consistent with a diagnosis
of myotonia or pseudomyotonia. No changes in the EMG were seen at the
lower doses (Beasley et al., 1991).

(iv) Canine malignant lymphoma

As a model for human non-Hodgkin's lymphoma, the association
between exposure to 2,4-D and the development of malignant lymphoma
was investigated in pet dogs in a veterinary hospital-based
case-control study. Dogs with histopathologically confirmed malignant
lymphoma were identified, and two types of controls were selected,
comprising dogs diagnosed with other malignancies and dogs in a
veterinary hospital for other reasons. The animals were matched by
age, year of hospital visit, and hospital. Information on exposure and
possible confounders were solicited by mailed questionnaires to the
owners. Information was obtained from the questionnaire and/or a
telephone interview for 491 cases, 466 nontumour controls, and 479
tumour controls. A modest association was found between malignant
lymphoma in dogs and application by their owner of 2,4-D on lawns
and/or use of a commercial lawn-care service, with an odds ratio (OR)
of 1.3 (95% confidence interval [CI], 1.0-1.7). The OR was not raised
for application of 2,4-D only (OR, 1.3; 95% CI, 0.9-1.8) or for sole
use of commercial lawn treatments (OR, 1.3; 95% CI, 0.96-1.7). The OR
for application by the owner plus use of a commercial lawn service was
1.9 (95% CI, 0.9-4.1). A positive trend (p < 0.02) was found for the
frequency of use by the owner (number of applications per year), but
not for the duration of use (number of years of application). No
significant trends were found for use of commercial lawn-care service.
The major weakness of this study was the lack of precise data on
exposure to herbicides (Hayes et al., 1991).

3. Observations in humans

Epidemiological studies have suggested an association between
exposure to chlorophenoxyacetic acid herbicides, including 2,4-D, and
two forms of cancer in humans: soft-tissue sarcomas and non-Hodgkin's
lymphoma. The results of these studies are not consistent, however,
the associations found are weak, and conflicting conclusions have been

reached by the investigators. In addition, most of these studies did
not provide information on exposure specifically to 2,4-D, and the
risk was related to the general category of phenoxy herbicides, which
might include 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) and
substances contaminated with dioxins, specifically 2,3,7,8-TCDD. While
some of the studies have shown a relationship between exposure to
2,4-D and non-Hodgkin's lymphoma, others (including those with
positive results) have produced inconsistent findings, raising doubts
about whether the relationship is causal.

(a) Case-control studies

(i) Soft-tissue sarcoma

Six case-control studies addressed the association between
exposure to phenoxyacetic acid herbicides and chlorophenols and the
development of soft-tissue sarcoma in humans. A positive association
was reported in patients with exposure to either group of compounds in
Sweden (Hardell & Sandstrom, 1979; Eriksson et al., 1981) and in
female rice weeders in northern Italy (Vineis et al., 1986). None of
these studies, however, reported an OR for exposure to 2,4-D. In
contrast, a number of case-control studies in New Zealand and the USA
failed to find an association between use of phenoxyacetic acid
herbicides and the development of soft-tissue sarcoma (Smith et al.,
1983, 1984; Hoar et al., 1986; Woods et al., 1987). The specific
findings are described below.

Hardell and Sandstrom (1979) studied 21 living and 31 deceased
male patients with soft-tissue sarcoma in northern Sweden who had been
exposed to phenoxyacetic acids or chlorophenols; 220 controls were
chosen from the general population. The cases of soft-tissue sarcoma
were identified from the records of the Department of Oncology of the
University Hospital of Umeå between 1970 and 1977. Information on
patterns of use of herbicides and chlorophenols was obtained from
questionnaires for 36.5% of the cases and 9.2% of the controls who
recalled exposure to these compounds. There was a significant
(p < 0.001), sixfold increase in risk for soft-tissue sarcoma (OR,
5.3; 95% CI, 2.4-11), with 13 cases who had been exposed to
phenoxyacetic acids. Of these 13 cases, nine had been exposed to 2,4-D
and 2,4,5-T combined, two to 2,4,5-T alone, one to MCPA alone, and one
possibly to 2,4-D only. The authors noted that the effects of the
individual chemical substances could not be evaluated, as nearly all
of the exposed subjects were also exposed to chlorinated dioxins,
including 2,3,7,8-TCDD.

Eriksson et al. (1981) confirmed the finding of Hardell and
Sandstrom of an association between soft-tissue sarcoma and
phenoxyacetic acids in southern Sweden, where MCPA and 2,4-D have been
widely used. The study involved 110 cases of soft-tissue sarcoma

reported in 1974-78 and 220 controls from the general population. The
ORs were 6.8 (95% CI, 2.6-17) for exposure to any phenoxyacetic acid
herbicide and 4.2 for exposure to chlorphenoxyacetic acid herbicides
other than 2,4,5-T.

Vineis et al. (1986) studied cases of soft-tissue sarcoma among
female rice weeders in northern Italy, where phenoxyacetic acid
herbicides have been used since the beginning of the 1950s. Interviews
were carried out with 68 persons (31 women) with histologically
confirmed soft-tissue sarcoma and 158 controls (73 women) who had been
exposed to 2,4-D, MCPA, and 2,4,5,-T. For live women who had been
exposed to phenoxyacetic acid herbicides at any time, the OR was 2.7
(90% CI, 0.59-12). For women < 75 years old at the time of interview
and who had been exposed in 1950-55, the age-adjusted OR was 15 (90%
CI, 1.3-180).

Smith et al. (1983, 1984) investigated the association between
soft-tissue sarcoma and exposure to phenoxyacetic acid herbicides in
New Zealand. The authors selected 82 subjects with soft-tissue sarcoma
and 92 controls with other types of cancer from the National Cancer
Registry for the years 1976-80. The study failed to show any
statistically significant association between use of the herbicides
and soft-tissue sarcoma. The OR was 1.3 (90% CI, 0.7-2.5) for those
potentially exposed and 1.6 (90% CI, 0.7-3.3) for those probably or
definitely exposed for more than one day before the five years prior
to cancer registration.

Hoar et al. (1986) conducted a population-based case-control
study in Kansas, USA, where 2,4-D was the most commonly used
herbicide; 2,4,5-T was also used, 'along with myriad other chemicals'.
The study comprised 113 soft-tissue sarcoma cases identified through
the University of Kansas Cancer Data Service for the years 1976-82 and
948 controls from the general population of the state. No consistent
pattern of excess risk for soft-tissue sarcoma was seen for farmers
when compared with non-farmers (OR, 1.0; 95% CI, 0.7-1.6), for
herbicide use (OR, 0.9; 95% CI, 0.5-1.6) or for duration and frequency
of herbicide use (OR, 1.1; 95% CI, 0.7-1.7).

In a population-based case-control study, Woods et al. (1987)
evaluated the relationship between occupational exposure of men
in Washington State, USA, to phenoxyacetic acid herbicides and
chlorinated phenols and the risk of developing soft-tissue sarcoma.
The study comprised 128 cases of soft-tissue sarcoma and 694 randomly
selected controls without cancer. No statistically significant
association was seen with exposure to phenoxyacetic acid herbicides
(OR, 0.89; 95% CI, 0.4-1.9).

(ii) Non-Hodgkin's lymphoma

The association between exposure to phenoxyacetic acid herbicides
and the development of non-Hodgkin's lymphoma has been studied in
Sweden, New Zealand, and Kansas, Washington, Nebraska, Iowa, and
Minnesota, USA. The overall results of these studies suggest an
association, although the evidence is not entirely consistent.
Less clear, but still suggestive, is the evidence for a specific
association between non-Hodgkin's lymphoma and exposure to 2,4-D.
These studies must be interpreted with caution, however, because it is
difficult to isolate the specific herbicide (or other factor) that is
responsible for the association, which may be due to other chemicals
that farmers mix with 2,4-D or with impurities in the 2,4-D that was
sold commercially. The association with 2,4-D has not been replicated;
and use of 2,4-D may serve as a surrogate for some other, unknown
confounding factors. The specific findings are described below.

Hardell (1981) examined the association between exposure to
phenoxyacetic acids or chlorophenols and malignant lymphoma in Sweden.
The study comprised 60 hospitalized patients with Hodgkin's disease,
109 with non-Hodgkin's lymphoma, and 338 controls from the general
population. The questionnaire method used was similar to that of
Hardell and Sandstrom (1979). A significantly increased risk was found
with exposure to phenoxyacetic acid herbicides (OR, 4.8; 95% CI,
2.9-8.1). Although risk estimates were not reported separately for
Hodgkin's disease and non-Hodgkin's lymphoma, the authors reported no
meaningful difference.

Hoar et al. (1986) conducted a population-based case-control
study in Kansas, USA, that comprised 121 cases of Hodgkin's disease
and 170 of non-Hodgkin's lymphoma identified through the University of
Kansas Cancer Data Service for the years 1976-82, and 948 controls
from the general population of the State. No association was seen
between use of phenoxyacetic acid herbicides and Hodgkin's disease.
When the rates of non-Hodgkin's lymphoma for non-farmers were used
for comparison, associations of borderline significance were found
for farming (OR, 1.4; 95% CI, 0.9-2.1) and for phenoxyacetic acid
herbicide use (OR, 2.2; 95% CI, 1.2-4.1). The OR for use of herbicides
on wheat, corn, sorghum, or pasture was 1.6 (95% CI, 0.9-2.6). The
relative risk (RR) for non-Hodgkin's lymphoma was significantly
increased when evaluated by number of days of exposure to herbicides
per year and latency. Farmers exposed for more than 20 days per year
had a sixfold increase in risk for non-Hodgkin's lymphoma relative to
non-farmers (OR, 6.0; 95% CI, 1.9-20). When exposure was restricted to
users exposed only to 2,4-D (i.e. eliminating 2,4,5-T), the RR was
increased (OR, 2.6; 95% CI, 1.4-5.0). In men exposed only to 2,4-D for
> 20 days per year, the OR was 7.6 (95% CI, 1.8-32). The authors had
reservations about the accuracy of this determination because of the
way in which the questionnaire elicited dates and frequency of
herbicide use. Frequent users who mixed or applied the herbicide

themselves had an elevated risk (OR, 1.9; 95% CI, 1.1-3.3), and the
risk was even higher (OR, 8.0; 95% CI, 2.3-28) for men who mixed or
applied the herbicides and who were exposed for more than 20 days
per year. An association was also found between the occurrence of
non-Hodgkin's lymphoma and failure to use protective equipment, such
as robber gloves and masks (OR, 2.1; 95% CI, 1.0-4.2), in comparison
with those who protected themselves (OR, 1.5; 95% CI, 0.7-3.1). The
results were difficult to interpret, because the information on
exposure was gleaned exclusively from interviews with subjects or
their next-of-kin. There is reasonable doubt about whether the
next-of-kin would be knowledgeable about the subject's daily
weed-control practices or be able to recall with precision such
practices 15-20 years later. Furthermore, as no data were collected on
the frequency or duration of 2,4-D use per se, it was not possible
to estimate directly an association between the amount of exposure to
2,4-D and non-Hodgkin's lymphoma.

In another population-based case-control study, Woods et al.
(1987) evaluated the relationship between occupational exposure of men
in western Washington State, USA, to phenoxyacetic acid herbicides and
chlorinated phenols and the risk of developing non-Hodgkin's lymphoma.
The study comprised 576 cases of non-Hodgkin's lymphoma and 694
randomly selected controls with cancer. An association was found
between non-Hodgkin's lymphoma and application of herbicides in
farming (OR, 1.3; 95% CI, 1.0-1.7) or forestry (OR, 4.8; 95% CI,
1.2-19); however, the forestry sprayers reported combined use of 2,4-D
and 2,4,5-T and use of commercial preparations containing other
chemicals. The risk for developing non-Hodgkin's lymphoma was also
increased for workers potentially exposed to phenoxyacetic acid
herbicides in any occupation for a period of 15 years or longer during
the 15 years before cancer diagnosis (OR, 1.7, 95% CI, 1.0-2.8). No
statistically significant association was seen between non-Hodgkin's
lymphoma and exposure to phenoxyacetic acid herbicides (OR, 1.2; 95%
CI, 0.8-1.9), even at high levels. Men who reported using 2,4-D
specifically had an OR of 0.73 (95% CI, 0.43-1.3), although it was
difficult to determine if this OR was controlled for other exposures.
In a later report, Woods and Polissar (1989) concluded that
phenoxyacetic acid herbicide preparations (e.g 2,4-D and 2,4,5-T)
per se do not independently increase the risk but may enhance the
risks associated with use of various pesticides and other chemicals
in agriculture.

Pearce et al. (1986, 1987) and Pearce (1989) studied non-
Hodgkin's lymphoma and exposure to phenoxyacetic acid herbicides
in New Zealand. In contrast to the USA, where the herbicide evaluated
was 2,4-D, the compound used predominantly in New Zealand in 1950-80
was 2,4,5-T. These studies comprised 183 men with non-Hodgkin's
lymphoma and 338 male controls obtained from the New Zealand Cancer
Registry for the years 1977-81. No excess risk was found (OR, 1.0;
90% CI, 0.7-1.5). When the risk for non-Hodgkin's lymphoma was

examined by the number of days of use by year, the trend was not
significant, but the risk did increase with use for 10-19 days per
year (OR, 2.2; 95% CI, 0.4-13) and then decreased (OR, 1.1; 95% CI,
0.3-4.1) with use for > 20 days per year.

Zahm et al. (1990) examined the association between exposure
to 2,4-D and the development of non-Hodgkin's lymphoma in eastern
Nebraska, USA, in a population-based case-control study that comprised
201 white men with non-Hodgkin's lymphoma and 725 controls. The
distinctive feature of this study was that specific information was
obtained on the duration and frequency of 2,4-D use. No excess risk
for non-Hodgkin's lymphoma was found in subjects with a history of
ever having worked or lived on a farm (OR, 0.9; CI, 0.6-1.4), but men
who mixed or applied 2,4-D had a 50% increased risk (OR, 1.5; 95% CI,
0.0-2.5). The risk was even higher for farmers who had handled (mixed
or applied) 2,4-D for > 21 days per year (OR, 3.3; 95% CI, 0.5-22;
p = 0.051). No association was seen, however, with the number of years
2,4-D was used on the farm (p = 0.274). The risk was also raised with
the time that farmers wore their application work clothes before
changing into clean clothes: the OR was 1.1 (95% CI, 0.4-3.1) when the
clothes were changed immediately after handling and 1.5 (95% CI,
0.8-2.6) for those who changed clothes at the end of the work day or
4.7 (95% CI, 1.1-21) for those who waited until the following day or
later to change their clothes. As in the study in Kansas, information
on exposure was gleaned exclusively from interviews with subjects or
their next-of-kin. The suggestion of an increased risk was based on
only three patients with non-Hodgkin's lymphoma who reported use
of 2,4-D for > 21 days per year, derived almost entirely from
responses of next-of-kin. No trend of increasing risk with increasing
days of use was seen when the patients themselves reported on their
past exposure.

Cantor et al. (1992) studied pesticides and other agricultural
risk factors for non-Hodgkin's lymphoma among men in Iowa and
Minnesota, USA. The study comprised 622 white men with non-Hodgkin's
lymphoma and 1245 white controls. In comparison with the rates for
non-farmers, there was a small increase in risk for non-Hodgkin's
lymphoma among men who had ever lived or worked on a farm as an adult
(OR, 1.2; 95% CI, 1.0-1.5). No significant increase in risk was seen
for subjects who had ever handled, mixed, or applied specific
herbicides. Use of 2,4-D resulted in similar ORs in the following
analyses: for those who had ever mixed or applied 2,4-D (OR, 1.2;
95% CI, 0.9-1.8); for those who had handled 2,4-D with protective
equipment (OR, 1.2; 95% CI, 0.9-1.6) or without protective equipment
(OR, 1.2; 95% CI, 0.9-1.7); and for those who first used 2,4-D before
1965 in Iowa (OR, 1.2; 95% CI, 0.9-1.9) and Minnesota (OR, 1.4;
95% CI, 0.9-2.3). The authors reported only limited information
relevant to the hypothesis of an association between exposure to 2,4-D
and cancer. Specific information on the frequency of use of 2,4-D was
not reported.

(b) Cohort studies

Cohort studies conducted among workers with occupational exposure
to phenoxyacetic acid herbicides have not confirmed the initial
hypothesis of an association between exposure to 2,4-D and either
soft-tissue sarcoma or non-Hodgkin's lymphoma. While the cohort
studies conducted in Sweden, Finland, and the USA failed to show an
association (Riihimaki et al., 1983; Wiklund & Holm, 1986; Wiklund
et al., 1987; Bond et al., 1988; Wiklund et al., 1988; Wigle et al.,
1990), positive results were seen in four further studies (Lynge, 1985;
Coggon et al., 1991; Saracci et al., 1991 ), which, however,
provide conflicting results, each showing an increase in the risk for
only one of the two cancers of concern (a different cancer in each
cohort). There was an increased risk for soft-tissue sarcoma among
Danish workers employed in the manufacture of phenoxyacetic acid
herbicides, principally MCPA (Lynge, 1985), but there was an
insignificant increase in risk for soft-tissue sarcoma among workers
exposed to multiple phenoxyacetic acid herbicides and chlorophenol
(Saracci et al., 1991). A slight increase in the risk for
non-Hodgkin's lymphoma was seen among British cohorts exposed to
2,4-D, MCPA, 2,4,5-T, and other phenoxyacetic acids in a manufacturing
plant (Coggon et al., 1991). The specific findings are described
below.

Lynge (1985) examined cancer incidence among Danish chemical
workers involved in the manufacture of phenoxyacetic acid herbicides
in two plants, with 3844 workers in one and 615 in the other. The
phenoxyacetic acid herbicides manufactured in these two plants were
2,4-D, dichlorprop, and 2,4,5-T. Cancer cases were identified by
linkage with the Danish National Cancer Registry, and the expected
numbers of cancer cases were calculated from the incidence rates in
the general Danish population. An excess of soft-tissue sarcoma was
found, with five cases among male workers and 1.8 expected (relative
risk [RR], 2.7; 95% CI, 0.88-6.3); no cases occurred in female workers
(0.75 expected). When the latency exceeded 10 years, four cases of
soft-tissue sarcoma were observed, with 1.1 expected (RR, 3.7; 95% CI,
1.0-9.4). It should be noted that the chemical plants in which the
workers were employed manufactured diverse products, and three of the
four patients with soft-tissue sarcoma had been employed for three
months or less; only one had been assigned to chlorophenoxyacetic acid
operations (0.26 expected). Malignant lymphomas occurred in seven men,
with 5.3 cases expected (RR, 1.3; 95% CI, 0.52-2.7), and in one woman,
with 1.2 expected (RR, 0.83). None of the seven cases of malignant
lymphoma occurred in the department producing phenoxyacetic acid
herbicides. The author did not estimate the RR specifically for
exposure to 2,4-D.

Wiklund and Holm (1986) and Wiklund et al. (1988) studied
354 620 male Swedish agricultural or forestry workers, dividing the
cohort into six subcohorts with different presumed exposure to
phenoxyacetic acid herbicides. These workers were compared with a
reference population of 1 725 845 workers who were not involved in
agriculture or forestry. The study did not show a significant excess
risk for soft-tissue sarcoma or non-Hodgkin's lymphoma in agricultural
or forestry workers in comparison with other groups. Between 1961 and
1979, 331 cases of soft-tissue sarcoma were observed in the study
cohort and 1508 in the reference group (RR, 0.9; 95% CI, 0.8-1.0).
Non-Hodgkin's lymphoma occurred in 861 men in the study cohort. The RR
was not significantly increased in any subcohort, did not differ
significantly between the subcohorts, and showed no time-related
increase in the total cohort or any subcohort.

Bond et al. (1988) investigated the mortality of 878 workers
potentially exposed to 2,4-D and its derivatives during their
manufacture, formulation, or packaging between 1945 and 1983. Exposure
was estimated by establishing an 8-h time-weighted average for each
task, and the workers were categorized into three exposure groups:
< 0.5, 0.5-4.9, and > 5.0 mg/m³ per year. Special attention was
given to deaths from brain neoplasms because of the brain astrocytomas
seen in male rats fed 2,4-D in the diet; however, none of the 111
deaths in the cohort was due to a brain neoplasm. There were two
deaths from non-Hodgkin's lymphoma (one with generalized lymphosarcoma
and the other with reticulum-cell sarcoma) among a subset of workers
with potential additional exposure to dioxins (two observed; 0.5
expected; RR, 3.9; 95% CI, 0.4-14). The authors concluded that the
results did not support a cause-effect relationship between exposure
to 2,4-D and mortality from all causes or from any specific cancer.
Bloemen et al. (1993) reported the results of four years of
additional follow-up, through 1986, for the cohort studied by Bond
et al. No new deaths from non-Hodgkin's lymphoma were observed.

Wigle et al. (1990) studied the mortality of almost 70 000 male
farmers in Saskatchewan, Canada, identified in the 1971 Census of
Agriculture. No excess mortality was seen for any cause of death,
including non-Hodgkin's lymphoma, but a correlation was found between
non-Hodgkin's lymphoma and area sprayed with herbicides. Among farmers
with < 1000 acres (approx. 400 ha), the RR rose with the area sprayed
with herbicides: < 100 acres (approx. 40 ha), RR, 1.3 (95% CI,
0.7-2.4); 100-249 acres (approx. 40-100 ha), RR, 1.9 (95% CI,
1.2-3.3), and > 250 acres (approx. 100 ha), RR, 2.2 (95% CI,
1.0-4.6). The authors reported that the cholorophenoxy compound in
general use in the area was 2,4-D (90% by weight throughout the 1960s
and 75% in the 1970s), but the exposure was not directly related to
cases of the disease.

Coggon et al. (1991) examined cancer mortality and incidence at
four factories in England that produced phenoxyacetic acid herbicides.
The four cohorts comprised 2239 men employed during 1964-85 who were
exposed not only to 2,4-D, but also to MCPA, 2,4,5-T, and other
phenoxyacetic acid herbicides. The subjects were traced through the
National Health Service Central Registrar and the National Insurance
Index, and their mortality was compared with that in the national
population. No cases of soft-tissue sarcoma or Hodgkin's disease were
identified, but there were two deaths from non-Hodgkin's lymphoma with
0.87 expected (RR, 2.3; 95% CI, 0.3-8.3), both of which occurred > 10
years after first exposure to phenoxyacetic acid compounds.

Green (1991) studied the mortality of forestry workers who had
been employed for six months or more in forestry work at a Canadian
public utility during the period of 1950-82. The cohort consisted of
1222 men exposed to 2,4-D and other phenoxyacetic acid herbicides. No
overall excess mortality due to soft-tissue sarcoma or non-Hodgkin's
lymphoma was seen. The only statistically significant finding was for
suicide, with 11 cases observed and 5.2 expected.

Saracci et al. (1991) surveyed a population of 16 863 male and
1527 female production workers or sprayers in 10 countries, identified
through the International Registry of Workers Exposed to Phenoxy
Herbicides and their Contaminants, established by the International
Agency for Research on Cancer and the US National Institute of
Environmental Health Sciences. The cohorts of Lynge (1985), Coggon
et al. (1991), and Green (1991), described above, were included. The
workers were thus exposed to 2,4-D, dichlorprop, 2,4,5-T, MCPA, other
phenoxyacetic acids, and a number of chlorinated phenols. There was no
overall increase in mortality from any cause. Four deaths due to
soft-tissue sarcoma were seen, with 2.0 expected (RR, 2.0; 95% CI,
0.53-5.0); three occurred in sprayers (RR, 8.8; 95% CI, 1.8-26), all
occurred 10-19 years after first exposure, and two of the cases arose
after exposure of less than one year. Since the workers were exposed
to a number of chlorophenoxyacetic acid herbicides and chlorinated
phenols, it could not be determined which, if any, of these chemicals
was responsible for the reported increase in risk for soft-tissue
sarcoma.

Riihimaki et al. (1983) studied 1926 Finnish farm workers who
had been exposed to phenoxyacetic acid herbicides for at least two
weeks between 1951 and 1971. There was no excess cancer risk, and no
cases of soft-tissue sarcoma or non-Hodgkin's lymphoma were observed.

Wiklund et al. (1987) studied cases of non-Hodgkin's lymphoma
and Hodgkin's disease among 20 245 Swedish pesticide applicators, 72%
of whom were estimated to have been exposed to phenoxyacetic acid
herbicides. The most commonly used pesticide was MCPA, but 2,4-D was

also used. Lymphomas did not occur in excess: 11 cases of Hodgkin's
disease (RR, 1.2; 95% CI, 0.6-2.2) and 21 cases of non-Hodgkin's
lymphoma (RR, 1.0; 95% CI, 0.63-1.5) were observed, with 9.1 and 21
expected, respectively.

Asp et al. (1994) conducted an 18-year follow-up for cancer
mortality and morbidity in a cohort of 1909 men who had sprayed
chlorophenoxyacetic acid herbicides (a mixture of 2,4-D and 2,4,5-T)
in 1955-71. Overall mortality from cancer was slightly less than that
in the general population (SMR, 0.83; 95% CI, 0.65-1.0), and none of
the deaths was due to soft-tissue sarcoma or non-Hodgkin's lymphoma.
One case of non-Hodgkin's lymphoma was found, with 2.8 expected; no
cases of soft-tissue sarcoma were seen.

Over the past eight years, a number of scientific panels,
convened under the auspices of various groups, have evaluated the
epidemiological studies that addressed the possible association
between use of phenoxyacetic acid herbicides, 2,4-D in particular, and
the occurrence of soft-tissue sarcoma, non-Hodgkin's lymphoma, and
Hodgkin's disease. Their conclusions are summarized below.

A working group convened by the International Agency for Research
on Cancer (IARC, 1987) concluded that there was limited evidence that
chlorophenoxy herbicides are carcinogenic to humans. 2,4-D could not
be clearly distinguished from other chlorophenoxy herbicides, some of
which contain dioxins.

The Ontario Pesticide Advisory Committee of the Ontario Ministry
of the Environment (Anders et al., 1987), using IARC terminology,
concluded that '...there is limited evidence of carcinogenicity in man
from exposure to phenoxyacetic acid herbicides. In terms of exposure
to 2,4-D specifically, the evidence must still be regarded as
inadequate to classify it as a carcinogen.'

A panel at the Harvard School of Public Health (1990; Ibrahim
et al., 1991) concluded: 'Although a cause-effect relationship is
far from being established, the epidemiological evidence for an
association between exposure to 2,4-D and non-Hodgkin's lymphoma is
suggestive and requires further investigation. There is little
evidence of an association between use of 2,4-D and soft-tissue
sarcoma or Hodgkin's disease, and no evidence of an association
between 2,4-D use and any other form of cancer.'

Munro et al. (1992) concluded: 'The case-control epidemiological
studies that have been the source of the cancer risk hypothesis are
inconclusive. Problems in assessing exposure based on patient's
memories make these studies difficult to interpret. Cohort studies

of exposed workers do not generally support the specific hypothesis
that 2,4-D causes cancer. Taken together, the epidemiological studies
provide, at best, only weak evidence of an association between 2,4-D
and the risk of cancer.'

The Joint Committee of the Science Advisory Board/Scientific
Advisory Panel (US Environmental Protection Agency, 1994) concluded
'...that while there is some evidence that non-Hodgkin's lymphoma may
occur in excess in populations which are likely to be exposed to
2,4-D, the data are not sufficient to conclude that there is a cause
and effect relationship between exposure to 2,4-D and non-Hodgkin's
lymphoma. The data are, however, sufficient to require continued
examination of the issue through further studies.'

Comments

2,4-D was rapidly absorbed, distributed, and excreted after oral
administration to mice, rats, and goats. At least 86-94% of an oral
dose was absorbed from the gastrointestinal tract in rats. Once
absorbed, 2,4-D was widely distributed throughout the body but did not
accumulate because of its rapid clearance from the plasma and rapid
urinary excretion. 2,4-D was excreted rapidly and almost exclusively
(85-94%) in urine by 48 h after treatment, primarily as unchanged
2,4-D. No metabolites have been reported other than conjugates.
Pharmacokinetic studies with salts and esters of 2,4-D have shown that
the salts dissociate and esters are rapidly hydrolysed to 2,4-D, after
which their fate was indistinguishable from that of the acid. The
similarity in the fate of 2,4-D and its salts and esters explains
their similar toxicity.

In humans who ingested 2,4-D, it was quickly absorbed and
excreted rapidly in the urine; about 73% of the administered dose was
found in the urine after 48 h. No metabolites were detected.

After dermal applications of 2,4-D to volunteers, < 5.8%
of the dose was absorbed within 120 h. When the acid and its
dimethylamine (DMA) salt were applied, about 4.5% of the acid and 1.8%
of the salt were absorbed, and, of this, about 85% of the acid and 77%
of the salt were recovered in the urine 96 h after application.

2,4-D, its amine salts, and its esters are slightly toxic
when administered orally or dermally, the oral LD₅₀ values being
400-2000 mg/kg bw and the dermal LD₅₀ value generally exceeding
2000 mg/kg bw. In rats exposed to 2,4-D at the maximum attainable
concentration (up to 5.39 mg/litre) by inhalation for 4 h, no deaths
were seen. While 2,4-D and its amine salts and esters do not induce
dermal irritation in rabbits or dermal sensitization in guinea-pigs,
they cause severe eye irritation in rabbits. WHO has classified 2,4-D
as 'moderately hazardous' (WHO, 1996).

In mice fed diets that provided 2,4-D at doses of 0, 5, 15, 45,
or 90 mg/kg bw per day for three months, renal lesions were observed
in animals of each sex at all doses. An NOAEL was not identified.

In mice fed diets providing 2,4-D at doses of 0, 1, 15, 100, or
300 mg/kg bw per day for 90 days, treatment-related changes were
observed in animals of each sex at doses > 100 mg/kg bw per day.
These effects included decreases in glucose level in females,
decreases in thyroxine activity in males, and increases in absolute
and/or relative kidney weights in males. The NOAEL was 15 mg/kg bw per
day.

In rats fed diets providing 2,4-D at doses of 0, 1, 5, 15, or
45 mg/kg bw per day for 90 days, renal lesions were observed at doses
> 5 mg/kg bw per day. The NOAEL was 1 mg/kg bw per day.

In rats fed diets providing 2,4-D at doses of 0, 1, 15, 100, or
300 mg/kg bw per day for 90 days, treatment-related changes were
observed in animals of each sex at doses > 100 mg/kg bw per day.
These effects included decreases in body-weight gain, haematological
and clinical chemical alterations, changes in organ weights, and
histopathological lesions in the adrenals, liver, and kidneys. The
NOAEL was 15 mg/kg bw per day.

In six studies of toxicity, rats fed diets containing the
diethanolamine (DEA), DMA, isopropylamine (IPA), or triisopropanolamine
(TIPA) salts or the butoxyethylhexyl (BEH) or 2-ethylhexyl (EH) esters
at acid-equivalent doses of 0, 1, 15, 100, or 300 mg/kg bw per day for
13 weeks, the results demonstrated the comparable toxicity of the acid,
salts, and esters. The NOAEL was 15 mg acid-equivalent per kg bw per
day for all six compounds.

Dogs were given gelatin capsules containing 2,4-D at 0, 0.03, 1,
3, or 10 mg/kg bw per day or diets containing 2,4-D, the DMA salt, or
the EH ester at acid-equivalent doses of 0, 0.5, 1, 3.75, or 7.5 mg/kg
bw per day for 13 weeks. Treatment-related findings were observed in
the three studies at doses > 3.0 mg/kg bw per day. The NOAEL was
1.0 mg acid-equivalent per kg bw per day in all three studies.

In a two-year study of toxicity and carcinogenicity, mice were
fed diets providing 2,4-D at doses of 1, 15, or 45 mg/kg bw per day.
Increases in absolute and/or relative kidney weights and renal lesions
were observed at 15 and 45 mg/kg bw per day. There was no evidence of
carcinogenicity. The NOAEL was 1 mg/kg bw per day.

In another two-year study of toxicity and carcinogenicity, mice
were fed diets providing 2,4-D at doses of 0, 5, 62.5, or 125 mg/kg bw
per day (males) or 0, 5, 150, or 300 mg/kg bw per day (females).
Dose-related increases in absolute and/or relative kidney weights and
renal lesions were seen in animals of each sex at doses > 62 mg/kg
bw per day. There was no evidence of carcinogenicity. The NOAEL was
5 mg/kg bw per day.

In another two-year study, rats received diets providing 2,4-D at
doses of 0, 1, 5, 15, or 45 mg/kg bw per day. Renal lesions were seen
in animals of each sex at doses > 5 mg/kg bw per day. There was no
evidence of carcinogenicity. The NOAEL was 1 mg/kg bw per day.

In a further two-year study, rats were fed diets providing 2,4-D
at doses of 0, 5, 75, or 150 mg/kg bw per day. Treatment-related
effects were observed in animals of each sex at doses > 75 mg/kg bw
per day. The effects included decreases in body-weight gains and food
consumption, increases in serum alanine and aspartate aminotransferase
activities, decreased thyroxine concentrations, increases in absolute

and relative thyroid weights, and histopathological lesions in the
eyes, kidneys, liver, lungs, and mesenteric fat. There was no evidence
of carcinogenicity. The NOAEL was 75 mg/kg bw per day in males and
5 mg/kg bw per day in females.

Dogs were fed diets providing 2,4-D at doses of 0, 1, 5, or
7.5 mg/kg bw per day for 52 weeks. At 5 and 7.5 mg/kg bw per day,
body-weight gains were decreased, increases were seen in blood urea
nitrogen, creatinine, alanine aminotransferase activity, and
cholesterol, and histopathological lesions were seen in the kidneys
and liver. The NOAEL was 1 mg/kg bw per day.

In a two-generation study of reproductive toxicity, rats received
dietary doses of 2,4-D of 0, 5, 20, or 80 mg/kg bw per day. Reduced
body weights of F₁ dams and renal lesions in F₀ and F₁ adults were
observed at 20 and 80 mg/kg bw per day. The NOAEL for parental and
reproductive toxicity was 5 mg/kg bw per day.

In order to evaluate the dermal toxicity of 2,4-D and its salts
and esters, rabbits received 15 dermal applications of the acid, the
DEA, DMA, IPA, or TIPA salt, or the BEH or EH ester at acid-equivalent
doses of 0, 10, 100, or 1000 mg/kg bw per day for 6 h per day on five
days per week for 21 days. No systemic toxicity was seen at any dose,
and no dermal toxicity was seen with the acid, the TIPA salt, or the
BEH ester. Dermal lesions were observed in rabbits treated with the
DEA, DMA, or IPA salt or the EH ester at doses > 100 mg/kg bw per
day. The lesions were characterized as acanthosis, hyperkeratosis,
oedema, inflammation, and epidermal hyperplasia. The NOAEL was 10 mg
acid-equivalent per kg bw per day for dermal toxicity and 1000 mg
acid-equivalent per kg bw per day (the highest dose tested) for
systemic toxicity.

In a study of developmental toxicity, pregnant Sprague Dawley
rats were given 2,4-D in corn oil by gavage at doses of 12.5, 25, 50,
75, or 88 mg/kg bw per day during days 6-15 of gestation. There was no
maternal toxicity. Fetotoxicity was manifested as decreased fetal body
weights at doses > 50 mg/kg bw per day. The NOAELs were 88 mg/kg bw
per day for maternal toxicity and 25 mg/kg bw per day for
developmental toxicity.

In a further study, pregnant Fischer 344 rats received 2,4-D in
corn oil by gavage at doses of 8, 25, or 75 mg/kg bw per day during
days 6-15 of gestation. Decreased body-weight gain of dams at the high
dose during the treatment period and increased incidences of skeletal
variations (7th cervical and 14th rudimentary ribs and missing
sternebrae) were observed at 75 mg/kg bw per day. The NOAEL was
25 mg/kg bw per day for both maternal and developmental toxicity.

The developmental toxicity of the DEA, DMA, IPA, and TIPA salts
and the BEH and EH esters was evaluated in pregnant rats after oral
administration during days 6-15 of gestation. The acid-equivalent
doses tested were 11, 55, or 110 mg/kg bw per day for DEA; 12.5, 50,
or 100 mg/kg bw per day for the DMA salt; 9, 25, or 74 mg/kg bw per
day for the IPA salt; 12, 37, or 120 mg/kg bw per day for the TIPA
salt; 17, 50, or 120 mg/kg bw per day for the BEH ester; and 10,
30, or 90 mg/kg bw per day for the EH ester. The maternal and
developmental toxicity of the salts and esters of 2,4-D was comparable
to that of the acid. Maternal toxicity, as evidenced by reduced
body-weight gain during treatment, was seen in all dams at the high
dose of each compound; in addition, mortality, clinical signs, and
reduced food consumption were seen in dams given 120 mg/kg bw per day
TIPA salt. Although embryo- and fetotoxicity and teratogenicity were
observed with the high dose of the TIPA salt, this may be attributed
to maternal toxicity; none of the other compounds had such effects. No
external gross or visceral anomalies (malformations or variations)
were observed in any of the fetuses, but skeletal variations were seen
at the high dose of each compound except the IPA salt which were
similar to those seen in the fetuses of dams given the acid. The
overall NOAELs were approximately 10 mg acid-equivalent per kg bw per
day for maternal toxicity and 50 mg acid-equivalent per kg bw per day
for developmental toxicity.

In a study of developmental toxicity, pregnant rabbits were given
2,4-D orally at 0, 10, 30, or 90 mg/kg bw per day during days 6-18
of gestation. Maternal toxicity, which included clinical signs,
abortions, and reduced body-weight gain during and after the treatment
period, was seen only at the high dose. No gross, visceral, or
skeletal malformations or variations were seen in fetuses at any dose.
The NOAELs were 30 mg/kg bw per day for maternal toxicity and 90 mg/kg
bw per day (the highest dose tested) for developmental toxicity.

The developmental toxicity of the DEA, DMA, IPA, and TIPA
salts and the BEH and EH esters was evaluated in rabbits after oral
administration during days 6-18 of gestation. The acid-equivalent
doses tested were 10, 30, or 60 mg/kg bw per day for the DEA salt; 10,
30, or 90 mg/kg bw per day for the DMA salt; 10, 30, or 75 mg/kg bw
per day for the IPA salt; and 10, 30, or 75 mg/kg bw per day for the
TIPA salt and for the BEH and EH esters. Unlike 2,4-D, which produced
maternal toxicity only at the high dose, most of the amine salts and
esters were maternally toxic at the middle and high doses, as
evidenced by mortality, clinical signs of neurotoxicity, abortions,
and decreases in body-weight gain. No gross, visceral, or skeletal
malformations or variations were seen in fetuses at any dose. The
overall NOAELs were approximately 10 mg acid-equivalent per kg bw per
day for maternal toxicity and 90 mg acid-equivalent per kg bw per day
(the highest dose tested) for developmental toxicity.

In summary, of the four salts tested for developmental toxicity,
only the TIPA salt had developmental toxicity in rats and only at a
maternally toxic dose; no developmental toxicity was seen in rabbits
with this or the other salts. Consequently, the Meeting concluded that
the developmental toxicity of the TIPA salt is of little concern.

The genotoxic potential of 2,4-D has been adequately evaluated
in a range of assays in vivo and in vitro. Overall, the responses
observed indicate that 2,4-D is not genotoxic, although conflicting
results were obtained for mutation in Drosophila. In a more limited
range of assays, the DEA, DMA, IPA, and TIPA salts and the BEH and
EH esters were also not genotoxic in vivo or in vitro. The Meeting
concluded that 2,4-D and its salts and esters are not genotoxic.

In rats given single doses of 2,4-D at 0, 15, 75, or 250 mg/kg bw
by gavage, there were no treatment-related gross or neuropathological
changes at any dose. Animals of each sex at the highest dose exhibited
incoordination and gait abnormalities on day 1, but the signs had
disappeared by day 5. The NOAEL was 75 mg/kg bw. When rats were fed
diets containing 2,4-D at doses of 0, 5, 75, or 150 mg/kg bw per day
for 12 months, neurotoxicity, manifested as increased relative
forelimb grip strength, was seen in animals of each sex at 150 mg/kg
bw per day. The NOAEL was 75 mg/kg bw per day.

Epidemiological studies have suggested an association between the
development of soft-tissue sarcoma and non-Hodgkin's lymphoma and
exposure to chlorophenoxy herbicides, including 2,4-D. The results of
these studies are not, however, consistent; the associations found
are weak, and conflicting conclusions have been reached by the
investigators. Most of the studies did not provide information on
exposure specifically to 2,4-D, and the risk was related to the
general category of phenoxyacetic acid herbicides, a group that
includes 2,4,5-T, which can be contaminated with dioxins. Case-control
studies provide little evidence of an association between the use of
2,4-D and soft-tissue sarcomas. Although some case-control studies
have shown a relationship with non-Hodgkin's lymphoma, others (even
the positive studies) have produced inconsistent results, raising
doubt about the causality of the relationship. Cohort studies of
exposed workers have not confirmed the hypothesis that 2,4-D causes
either neoplasm.

The Meeting was informed of the on-going 'Agricultural Health
Study' initiated in North Carolina and Iowa, USA, and of a study of
pesticide applicators in Finland. The Agricultural Health Study
addresses both cancer and non-cancer risks in men and women directly
exposed to pesticides and other agricultural agents, including
neurotoxicity, reproductive effects, immunological effects, kidney
disease, non-malignant respiratory disease, and the growth and
development of their children.

The Meeting concluded that the toxicity of the salts and esters
of 2,4-D was comparable to that of the acid. An ADI was therefore
established for the sum of 2,4-D and its salts and esters, expressed
as 2,4-D. An ADI of 0-0.01 mg/kg bw was established on the basis of
the NOAEL of 1 mg/kg bw per day in the one-year study of toxicity in
dogs and the two-year study in rats and using a safety factor of 100.

Toxicological evaluation

Levels that cause no toxic effect

Mouse: 15 mg/kg bw per day (13-week study of toxicity)

5 mg/kg bw per day (two-year study of toxicity and
carcinogenicity)

Rat: 1 mg/kg bw per day (two-year study of toxicity and
carcinogenicity)

5 mg/kg bw per day (two-generation study of
reproductive toxicity)

10 mg acid-equivalent/kg bw per day (maternal toxicity
in a series of studies of developmental toxicity with
salts and esters)

15 mg acid-equivalent/kg bw per day (series of 13-week
studies of toxicity with salts and esters)

25 mg/kg bw per day (maternal and developmental
toxicity in a study of developmental toxicity)

Rabbit: 10 mg acid-equivalent/kg bw per day (maternal toxicity
in a series of studies of developmental toxicity with
salts and esters)

30 mg/kg bw per day (maternal toxicity in a study of
developmental toxicity)

90 mg/kg bw per day (highest dose tested in studies of
developmental toxicity with the acid and its salts and
esters)

Dog: 1 mg/kg bw per day (13-week and one-year studies of
toxicity)

Estimate of acceptable daily intake for humans

0-0.01 mg/kg bw (sum of 2,4-D and its salts and esters expressed
as 2,4-D)

Studies that would provide information useful for continued evaluation
of the compound

1. Follow-up on the Agricultural Health Study in North Carolina
and Iowa in the USA

2. Follow-up on the study of pesticide applicators in Finland

Toxicological criteria for estimating guidance values for dietary and non-dietary exposure to 2,4-dichlorophenoxyacetic acid
(2,4-D) and its amine salts and esters

Exposure Relevant route, study type, species Results, remarks

Short-term (1-7 days) Oral, toxicity, rat, acid, salts, and esters LD₅₀ = 400-2000 mg/kg bw
Dermal, toxicity, rabbit, acid, salts, and esters LD₅₀ > 2000 mg/kg bw
Inhalation, toxicity, rat, acid, salts, and esters LC₅₀ > 0.84-5.4 mg/litre
Dermal, irritation, rabbit, acid, salts, and esters Not irritating
Ocular, irritation, rabbit, acid, salts, and esters Severely irritating
Dermal, sensitization, guinea-pig, acid, salts, Not sensitizing
and esters
Oral, single dose, neurotoxicity, rat, acid NOAEL = 75 mg/kg bw

Medium-term (1-26 weeks) Dietary, 3 months, toxicity, mouse NOAEL = 15 mg/kg bw per day, renal
toxicity
Dietary, 3 months, toxicity, rat NOAEL = 1 mg/kg bw per day, renal lesions
Dietary, 3 months, toxicity, rat, salts and esters NOAEL = 15 mg acid-equivalent/kg bw
per day, renal toxicity
Dietary or capsule, 3 months, toxicity, dog NOAEL = 1 mg acid-equivalent/kg bw per day,
reduced body-weight gain and other systemic
toxicity
Dermal, 21 days, repeated dose, rabbit, acid, NOAEL = 1000 mg acid-equivalent/kg
salts and esters bw per day, highest dose tested
Dietary, 2 generations reproductive toxicity, rat NOAEL = 5 mg/kg bw per day, reduced body
weights in F₁ dams and renal lesions in
F₀ and F₁ adults
Oral (gavage), developmental toxicity, rat NOAEL = 25 mg/kg bw per day, maternal and
developmental toxicity
Oral (gavage), developmental toxicity, rat, salts NOAEL = 10 mg acid-equivalent/kg bw
and esters per day for maternal toxicity; 50 mg
acid-equivalent/kg bw per day for
developmental toxicity

Exposure Relevant route, study type, species Results, remarks

Oral (gavage), developmental toxicity, rabbit NOAEL = 30 mg/kg bw per day for maternal
toxicity; > 90 mg/kg bw per day for
developmental toxicity
Oral (gavage), developmental toxicity, rabbit, salts NOAEL = 10 mg acid-equivalent/kg bw
and esters per day for maternal toxicity; > 90 mg
acid-equivalent/kg bw per day (highest
dose tested) for developmental toxicity

Long-term (> 1 year) Dietary, 2 years, toxicity and carcinogenicity, NOAEL = 5 mg/kg bw per day, renal
mouse effects; no evidence of carcinogenicity
Dietary, 2 years, toxicity and carcinogenicity, rat NOAEL = 1 mg/kg bw per day, renal
lesions; no evidence of carcinogenicity
Dietary, 1 year, toxicity, dog NOAEL = 1 mg/kg bw per day, changes in
serum chemistry and lesions in kidneys
and liver

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    See Also:
       Toxicological Abbreviations