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    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 g  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 g  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  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 Km. 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  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 Ci 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 g/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 Ci 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 g/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 g/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 g/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 g/litre). The odds ratio (OR) for an
    association between exposure to a 2,4-D-treated lawn and the presence
    of > 50 g/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 g/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 g/ml after 2 h, 25 g/ml after 24 h,
    and 3.5 g/ml after 48 h. The levels in whole blood decreased from
    21 g/ml after 2 h to 2.1 g/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 Ci; 4 g/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 Ci,
    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).

    (c)  Effects on enzymes and other biochemical parameters

         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).

    (c)  Long-term toxicity and carcinogenicity

    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 F1a litters and the
    beginning of mating for the F1b litters and 30 days after weaning of
    the latter litters. The F1 generation, selected from the F1b 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 F2a litters. Dosing continued
    through a two-week rest period and during mating, gestation, and
    lactation of the F2b litters and for at least 30 days after weaning
    of the F2b 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 F1b
    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 F1a litter had significantly
    (p < 0.05) lower body weights on days 7, 13 and 20 of gestation,
    while those producing the F1b litter had significantly (p < 0.05)
    lower body weights only on day 20 of gestation. While the body weights
    of F1 dams producing the F2a 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 F2b 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 F1a 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 F1b 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 F2a and F2b pups were comparable to those of the
    respective controls. The viability of F1a and F1b pups was affected
    only by treatment at 80 mg/kg bw per day. Live litter sizes were
    reduced in the F1a (9%; 10.1% in controls) and F1b (5.1%; 0.5%
    in controls; p < 0.01) generations. There was a significant
    (p < 0.01) decrease in the sex ratio of the F1a 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 F1b generation (110 dead pups) in comparison with controls (5
    dead pups), but the viability of the F2a and F2b pups was not
    affected. Examination of the F1b 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 F1b 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 F1b 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 g/plate            96.1      Negative      Lawlor &
                                TA98, TA100,               with S9; 66.7-6670                                     Valentine (1990a)
                                TA1535, TA1537,            g/plate without S9
                                TA1538
    Reverse mutation            S. typhimurium             0-1000 g/plate                NR        Negative      Rashid et al.
                                TA98, TA100,                                                                      (1984)
                                TA1535, TA1537,
                                TA1538
    Reverse mutation            S. typhimurium             0-3333 g/plate                NR        Negative      Soler-Neidzieler
                                                                                                                  et al. (1988)
    Reverse mutation            S. typhimurium             0-1000 g/plate                NR        Negative      Kappas (1988)
                                TA97, TA98, TA100,
                                TA1535, TA1537,
                                TA1538
    Reverse mutation            S. typhimurium             0-2000 g/plate                NR        Negative      Rashid & Muma
                                TA1538, TA1535                                                                    (1986)
    Reverse mutation            S. typhimurium             < 5000 g/plate                NR        Negative      Simmon et al.
                                TA98, TA100,                                                                      (1977)
                                TA1535, TA1538
    DNA damage                  E. coli K12, WP2           0-2000 g/plate                NR        Negative      Rashid & Mumma
                                                                                                                  (1986)
    DNA damage                  E. coli PQ 37              0-200 g/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 g/ml                   NR        Positive      Pavlica et al.
                                fibroblasts, hprt                                                                 (1991)
                                locus
    Chromosomal aberration      Chinese hamster            500-920 g/ml without S9;      NR        Negative      Galloway et al.
    in vitro                    ovary cells                1900-5000 g/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                   Positivea     (1986)
    Chromosomal aberration      Human lymphocytes          0.03-0.04 mg/m3b               33.3      Negative      Mustonen et al.
    in vivo                                                                                                       (1986)
    Chromosomal aberration      Rat bone marrow            0-350 g/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        Negativec     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 g/ml without S9        NR        Positive      Galloway et al.
    in vitro                    ovary cells                500-4200 g/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 g/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 g/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 g/ml                   73.8      Negative      McKeon (1990)
                                rat hepatocytes

    DMA salt
    Reverse mutation            S. typhimurium             333-10 000 g/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 g/ml                 66.2      Negative      Cifone (1990b)
                                rat hepatocytes

    IPA salt
    Reverse mutation            S. typhimurium             10-10 000 g/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 g/ml                 50.2      Negative      Linscombe & Lick
                                ovary cells, hprt locus                                                           (1994a)
    Chromosomal aberration      Rat lymphocytes            96-6137 g/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 g/ml                    50.2      Negative      McClintock &
                                rat hepatocytes                                                                   Gollapudi (1990)

    TIPA salt
    Reverse mutation            S. typhimurium             1000-10 000 g/plate           72.2      Negative      Samson &
                                TA97, TA98, TA100,                                                                Gollapudi (1989b)
                                TA1535, TA1537
    Gene mutation               Chinese hamster ovary      78-5000 g/ml                  70.9      Negative      Linscombe & Lick
                                cells, hprt locus                                                                 (1994c)
    Chromosomal aberration      Rat lymphocytes            800-5000 g/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 g/plate with S9;       95.6      Negative      Samson &
                                TA97, TA98, TA100,         1.6-1667 g/plate without                              Gollapudi (1990)
                                TA1535, TA1537,            S9
                                TA1538
    Chromosomal aberration      Rat lymphocytes            87.5-1400 g/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 g/ml                    95.6      Negative      McLintock &
                                rat hepatocytes                                                                   Gollapudi (1990)

    EH ester
    Reverse mutation            S. typhimurium             333-10 000 g/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 g/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 Ume 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.

    (c)  Overall assessments of epidemiological studies

         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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    Kirsh, P. (1983) Report on the study of the irritation to the eye of
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    Kohli, J.D., Khanna, R.N., Gupta, B.N., Dhar, M.M., Tandon, J.S. &
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    Martin, T. (1991) Developmental toxicity (embryo-fetal toxicity and
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    Martin, T. (1992b) Developmental toxicity (embryo-fetal toxicity and
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    Mattsson, J.L., McGuirk, R.J. & Yano, B.L. (1994a) 2,4-D acute
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    McClintock, M.L. & Gollapudi, B.B. (1990a) Evaluation of a formulation
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    McClintock, M.L. & Gollapudi, B.B. (1990b) Evaluation of 2,4-dichloro-
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    Mizell, M.J (1990b) 2,4-D BEE: 21-Day dermal irritation and dermal
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    Myer, J.R. (1981d) 2,4-Dichlorophenoxyacetic acid technical.
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    Myer, J.R. (1981e) 2,4-Dichlorophenoxyacetic acid, dimethylamine salt.
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    Myer, J.R. (1981f) 2,4-Dichlorophenoxyacetic acid, isooctylester.
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    See Also:
       Toxicological Abbreviations