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    HALOXYFOP

    First draft prepared by
    E. Bosshard
    Federal Office of Public Health, Food Science Division,
    Schwerzenbach, Switzerland

    Explanation
    Evaluation for acceptable daily intake
         Biochemical aspects
              Absorption, distribution, and excretion
              Biotransformation
         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
                   Peroxisome proliferation
                   Stereochemical inversion
         Comments
         Toxicological evaluation
    References
    Appendix: Expert report

    Explanation

         Haloxyfop is a substituted phenoxypropionic acid derivative which
    has been developed as a selective herbicide for the control of grass
    weeds in broad-leaf crops. In the first formulations produced, the
    active substance was either racemic haloxyfop ethoxy ethyl ester or
    the racemic methyl ester. When applied to plants, the ester is rapidly
    hydrolysed to the acid which has herbicidal activity. As it has been
    demonstrated that the R-isomer of haloxyfop is the herbicidally active
    compound, and essentially no activity is associated with the S-isomer,
    a resolved methyl ester has been developed which is approximately 98%
    R-isomer. Haloxyfop was evaluated for the first time by the present
    Meeting.

    Evaluation for acceptable daily intake

    1.  Biochemical aspects

    (a)  Absorption, distribution, and excretion

         Several studies on the fate of haloxyfop and its esters were
    performed in rats, mice, dogs, monkeys, and also humans. All of the
    studies with radiolabelled material were conducted with haloxyfop acid
    or ester labelled with 14C in the phenyl ring.

         Male and female B6C3F1 mice were treated with a single oral dose
    of 5 mg/kg bw 14C-haloxyfop (acid), administered as the sodium salt,
    and its absorption, distribution, and elimination were investigated
    over 168 h. The absorption half-life was about 2 h, and the clearance
    half-lives in plasma, liver, kidney, and excreta were about two days
    in animals of each sex. At least 60% of the applied dose was excreted
    in the faeces and about 20% in the urine seven days after treatment.
    No sex difference in the excretion pattern of haloxyfop was observed.
    The bile was the major route of excretion in males and females
    (Smith  et al., 1984).

         In a range-finding study, Fischer 344 rats were given single
    intravenous or oral doses of 0.5 or 50 mg/kg bw (males) or 10 mg/kg bw
    (females) of 14C-labelled haloxyfop (acid) as the sodium salt.
    Plasma was sampled and the excreta were collected for 120 h after
    treatment. The animals were then killed, and the organs were analysed
    for 14C activity. During a collection period of five days, the mean
    urinary excretion in male rats was about 14% of the applied dose and
    mean faecal excretion was 29%. No meaningful difference was found
    between the two routes of application or between doses. Three days
    after the oral dose of 10 mg/kg bw, the urine of female rats contained
    about 45% and faeces, 21%. After intravenous injection, urinary
    excretion in females was 57% and faecal excretion about 16%,
    indicating biliary excretion. The plasma half-life in males was about
    four days after intravenous administration of either dose, and a
    similar plasma half-life of about three days was found after oral
    treatment. In females, a plasma half-life of one day was observed,
    regardless of the route of administration. In the definitive study,
    male and female Fischer 344 rats were given a single oral dose of
    0.1 mg/kg bw 14C haloxyfop (acid) applied as the sodium salt. Males
    excreted about 70% of the applied dose in faeces and about 19% in
    urine during a 28-day observation period; females excreted about 21%
    in faeces and 72% in urine during an observation period of 13 days.
    Most of the urinary radiolabel appeared within the first few days
    after treatment in females. The plasma half-lives were about six days

    for males and one day for females. The concentrations of radiolabel in
    selected tissues were highest in liver, with 0.6 ppm haloxyfop
    equivalents one day after treatment in males and 0.5 ppm in females.
    The average half-life in male tissues was six days and that in female
    tissues one day, corresponding to the plasma half-lives (Smith
     et al., 1982).

         Male and female Fischer 344 rats were given a single dose of
    0.104 mg/kg bw 14C-haloxyfop methyl ester by gavage. The 14C
    activity was excreted primarily (60% of the applied dose) in the
    faeces of males and mainly (67%) in the urine of females; about 20%
    was excreted in the urine and faeces of males and females. The
    elimination half-lives in liver and kidney were consistent with the
    plasma half-lives: about five days in males and about one day in
    females. After oral administration of haloxyfop methyl ester, the
    plasma contained the highest 14C concentration, followed by liver,
    kidney, erythrocytes, and fat. The 14C concentration in fat varied
    between 0.009 and 0.067 ppm haloxyfop methyl ester equivalents-
    typically five to thirty times lower than the plasma concentration.
    Thus, the propensity for deposition and accumulation of the methyl
    ester in adipose tissue is low (Waechter  et al., 1982).

         Male Fischer 344 rats, animals were given a single dose of
    0.12 mg/kg bw of labelled haloxyfop ethoxy ethyl ester in corn oil by
    gavage, and the kinetic profiles in blood and tissues were determined
    over 10 days. The plasma concentration of radiolabel peaked about 4 h
    after treatment. The half-life for absorption was 2 h and that for
    elimination about four days. The administered label was excreted
    primarily in the faeces, to about 50% of the applied dose over 10
    days; about 15% was excreted in the urine over the same period. The
    elimination half-lives in plasma, liver, kidney, and excreta were
    similar, averaging five days (Smith  et al., 1983).

         Male beagle dogs received a single oral dose of 2.4 mg/kg bw
    14C-haloxyfop (acid) as the sodium salt. The compound was rapidly
    absorbed, with a half-life of 9 min. Plasma elimination was
    bi-exponential; the half-life of the initial, rapid phase was about
    1-2 h and that of the terminal phase about 34 h. Within eight days of
    treatment, about 77% of the dose had been eliminated in the faeces and
    about 10% in the urine. A high concentration of radiolabel was also
    found in bile (40 times the concentration in plasma), indicating
    biliary excretion (Nolan  et al., 1987).

         In a range-finding study, unlabelled haloxyfop (acid) was
    administered as the sodium salt to male cynomolgus monkeys by
    nasogastric intubation, either as single oral doses of 40, 80, or
    120 mg/kg bw or as multiple doses of 20, 40, or 60 mg/kg bw per day
    over five consecutive days. The doses of 80 and 120 mg/kg bw resulted
    in loss of appetite and decreased activity. Repeated administration of

    40 mg/kg bw per day was associated with slight loss of appetite and
    weight: loss and the dose of 60 mg/kg bw per day with emesis and
    severe loss of appetite. On the basis of these results, doses of 5 and
    20 mg/kg bw per day were selected for a four-week study of peroxisome
    stimulation, summarized in section ( g)(i) (Gerbig  et al., 1985).

         One male monkey was given a nasogastric dose of 1 mg/kg bw
    unlabelled haloxyfop as the sodium salt, and blood, urine, and faeces
    were collected over 21 days. After a 10-week recovery, the monkey was
    given 1 mg/kg bw of 14C-haloxyfop, and blood and the excreta were
    analysed over six days. The compound was rapidly absorbed from the
    gastrointestinal tract, with a half-life of < 30 min, mainly in the
    urine (about 84% of the applied dose), with only 0.5% in the faeces.
    Plasma elimination was bi-exponential; the half-life of the initial,
    rapid phase was 2.5 h and that of the slower, terminal phase, three
    days (Gerbig  et al., 1985).

         Laying hens were given 14C-haloxyfop (acid) in gelatin capsules
    for 11 days at a level equivalent to 12 ppm. The excreta contained 87%
    of the radiolabel, mainly as unchanged haloxyfop; 2 ppm was present in
    liver and 4 ppm in kidney, primarily as the parent haloxyfop and polar
    conjugates, while the residues in body fat and egg yolks consisted
    primarily of non-polar lipids, which in egg yolks were found to be
    incorporated into triacylglycerols (Yackovich & Miller, 1983a).

         Two lactating goats were given 14C haloxyfop (acid) twice daily
    in gelatin capsules for 10 days at a rate of 16 ppm. About 90% of the
    applied dose was excreted in urine as unchanged haloxyfop. The
    residues in tissues 12 h after the last dose were 0.4 ppm in liver,
    1.3 ppm in kidney, and < 0.1 ppm in fat and other tissues. The
    residue levels in whole milk, primarily in triacylglycerols in fat,
    were about 0.3 ppm, corresponding to about 2-3% of the applied dose
    (Yackovich & Miller, 1983b).

         The pharmacokinetics of haloxyfop and its methyl ester was
    investigated in eight male volunteers, who received a single oral dose
    of 0.2 mg/kg bw haloxyfop as the sodium salt or a single dermal dose
    of 0.8 mg/kg bw haloxyfop methyl ester in xylene and surfactants.
    After oral administration, the compound was rapidly absorbed, with a
    half-life of 0.9 h, and excreted with a half-life of about six days.
    Urine was the principal route of elimination, containing about 79% of
    the applied dose. Slow skin absorption was observed after dermal
    application, with a half-life of 37 h. The urinary excretion indicated
    that only about 3% of the applied dose had been absorbed As the
    elimination half-life was also about six days, the elimination
    kinetics is independent of the route of administration. Moreover, the
    similarity of the excretion patterns of haloxyfop and its methyl ester
    suggest that the ester is rapidly hydrolysed to the parent acid in man
    as well (Nolan  et al., 1985a).

         The experimental data indicated that these esters are rapidly
    hydrolysed  in vivo to the acid form of haloxyfop (Smith  et al.,
    1983) and that the toxicity of the esters and acid forms are
    equivalent on a molar basis (Herman  et al., 1983b); however, the
    ester moiety may alter the rate and extent of percutaneous absorption
    -- an important factor with respect to the exposure of applicators. An
    additional study was therefore carried out in which a single dermal
    dose of a 12.5% haloxyfop EE-containing formulation, corresponding to
    0.77 mg/kg bw of haloxyfop ethoxy ethyl ester, was applied topically
    to the forearms of three male volunteers. The clearance from plasma
    and the urinary excretion were similar to those seen in the study of
    haloxyfop methyl ester (Nolan  et al., 1985a), indicating that the
    haloxyfop ethyl ester is also rapidly hydrolysed to the acid. The
    dermal absorption was estimated to be about 1% of the applied dose
    (Nolan  et al., 1985b).

    (b)  Biotransformation

         In male and female B6C3F1 mice treated with a single oral dose of
    5 mg/kg bw 14C-haloxyfop (acid), as the sodium salt, unchanged
    parent compound was found in plasma, bile, and urine samples, and a
    glucuronide conjugate was identified (Smith  et al., 1984).

         One male and one female rat were given a single oral dose of
    2.5 mg/kg bw 14C-haloxyfop (acid) administered as the sodium salt or
    14C-haloxyfop methyl ester in corn oil. More than 97% of the 14C
    activity in plasma, erythrocytes, and liver was identified as the
    parent acid. No peak was observed at the retention time of the methyl
    ester 48 h after treatment, suggesting that rapid hydrolysis of the
    ester had occurred in the gastrointestinal tract, probably before
    absorption. Haloxyfop acid and one major and two minor species were
    detected in the urine of male and female rats. The urine of males
    given haloxfop contained 75%, of the 14C activity, and that of males
    given the methyl ester contained 60%; the urine of females given
    haloxfop contained 94% and that of females given the methyl ester
    contained 89% of the radiolabel excreted in the form of the parent
    acid. Bile from both male and female rats also contained haloxyfop
    (parent acid), representing about 50% of the radiolabel found in bile
    plus a conjugate (Smith  et al., 1982).

         Male and female Fischer 344 rats received a single oral dose of
    2.5 mg/kg bw 14C haloxyfop methyl ester in corn oil, and analysis of
    plasma at various times showed the presence of haloxyfop (acid) at the
    levels that would be expected after administration of an equimolar
    dose of haloxyfop itself. Furthermore, as in the study conducted by
    Smith  et al. (1982), no methyl ester was detected in blood 24 h
    after treatment (Waechter  el al., 1982).

         Male Fischer 344 rats were given a single oral dose of 1.4 or
    2.5 mg/kg bw 14C-haloxfop ethoxy ethyl ester in corn oil. No ester
    was seen in any blood sample extracted under either acidic or
    non-acidic conditions, and the ester appears to be rapidly hydrolysed,
    presumably in the gastrointestinal tract or at the latest in the
    blood. Analysis of non-acidified blood extracts from rats given the
    ethoxy ethyl ester by gavage showed that 80% of the 14C activity
    represented a conjugate of haloxyfop (acid) and about 20% corresponded
    to the parent acid (Smith  et al., 1983).

         In male beagle dogs that received a single oral dose of 2.4 mg/kg
    bw 14C-haloxyfop (acid) as the sodium salt, virtually all of the
    radiolabel in the faeces but less than 25% of that in bile represented
    unchanged haloxyfop. It may therefore be assumed that the metabolites
    in the bile were conjugates that hydrolysed back to the parent acid in
    the gastrointestinal tract (Nolan  et al., 1987).

    2.  Toxicological studies

    (a)  Acute toxicity

         The results of studies of the acute toxicity of haloxyfop (acid)
    and its methyl and ethoxy ethyl esters are summarized in Table 1.
    Signs of acute toxicity consisted of rapid shallow breathing,
    prostration, and lethargy. The oral LD50 values for haloxyfop (acid)
    and its esters are similar in rats and mice, indicating that they have
    a common metabolic pathway. A comparison of the LD50 values after
    oral and intraperitoneal administration also reveals similar values,
    indicating extensive gastrointestinal absorption. These results are in
    good agreement with the findings of the various pharmacokinetic
    studies.

         The marked difference in the oral LD50 value for haloxyfop EE
    in rats reported by Carreon  et al. (1982) (520 mg/kg bw) and Jones
    (1983a,b) (2870 mg/kg bw) may be due to greater gastrointestinal
    absorption when the test material is formulated in a 10% acetone-corn-
    oil solution instead of distilled water.

    (b)  Short-term toxicity

    Mice

         Groups of 10 male and 10 female B6C3F1 mice (15 controls of each
    sex) were fed diets containing concentrations of haloxyfop (acid)
    (purity, 96%) corresponding to 0, 0.002, 0.02, 0.2, or 2 mg/kg bw per
    day for 13 weeks. Further groups of 10-12 mice of each sex were given
    doses of 0, 0.02, or 2 mg/kg bw per day for 36 weeks, with an
    interruption of one week between week 13 and 14. In the 13-week study,
    treatment-related effects were seen only at the highest dose. Reduced
    body weight was seen only in females. Males had slightly increased

    serum alkaline phosphatase activity. Haematological parameters were
    not influenced by treatment. At necropsy, alterations were observed
    grossly only in the liver, consisting of slight enlargement and
    darkening in animals of each sex, accompanied by increased liver
    weights. Histopathological changes were confined to the liver and
    again to the highest dose group; they consisted of hepatocellular
    enlargement and greater cytoplasmic homogeneity (lack of cytoplasmic
    microvesiculation) than in controls.

         In the 36-week study, treatment had no effect on behaviour,
    body-weight development, food consumption, or haematological
    parameters. In males at the highest dose, increased alkaline
    phosphatase activity was observed. In animals at this dose, the livers
    were slightly enlarged and darkened and their weights were increased,
    and the kidney weights of males were decreased. Histopathological
    alterations were observed in the livers of animals of each sex and in
    the kidneys of males. The changes in the liver consisted of
    hepatocellular enlargement, increased cytoplasmic homogeneity, and
    slightly increased eosinophilia. The kidneys of the males at the
    highest dose showed decreased cytoplasmic vacuolation of the proximal
    convoluted tubular cells in comparison with controls. The NOAELs were
    0.2 mg/kg bw per day in the 13-week study and 0.02 mg/kg bw per day in
    the 36-week study Gorzinski  et al., 1982a).

    Rats

         In a four-week study, groups of five CDF Fischer 344 rats of each
    sex (control group, 10 rats of each sex) received haloxyfop (acid)
    (purity, 99.99 or 99%) in the diet providing concentrations of 0,
    0.01, 0.1, 1, and 10 mg/kg bw per day. The doses were selected on the
    basis of the results of a one-week palatability and a two-week dietary
    study (Tollett  et al., 1981). Effects on the liver were indicated by
    an increase in serum alkaline phosphatase activity in males at the
    highest dose. An increase in liver weights at 1 and 10 mg/kg bw per
    day was most pronounced in males. Histopathological changes observed
    at > 1 mg/kg bw per day in animals of each sex consisted of
    hepatocellular swelling and increased eosinophilic staining of the
    cytoplasm. A slight decrease in mature sperm (spermatozoa) was seen in
    the testes and epididymides of some rats at 10 mg/kg bw per day. The
    NOAEL was 0.1 mg/kg bw per day, biased on biochemical, histological,
    and weight changes in the liver (Gorzinski  et al., 1982b).

        Table 1.  Acute toxicity of haloxyfop (acid) and its methyl and ethoxy ethyl esters
                                                                                                                                              

    Test materiala        Species     Sex              Route              LD50 or LC50          Purity     Reference
                                                                          (mg/kg bw or (%)
                                                                          mg/litre air)
                                                                                                                                              

    Haloxyfop (acid)      Rat         Male             Oral               340                   NR         Carreon et al. (1980)
                                      Female                              550
    Haloxyfop EEb         Rat         Male, female     Oral               520                   95         Carreon et al. (1982)
    Haloxyfop EE          Rat         Male             Oral               700                   94.7       Jones (1983a)
                                      Female                              900
    Haloxyfop EEc         Rat         Male, female     Oral               2870                  98.8       Jones (1983b)
    Haloxyfop ME          Rat         Male             Oral               300                   98.6       Mizell & Lomax (1989a)
                                      Female                              620
    Haloxyfop (acid)      Rabbit      Male, female     Dermal             > 5000                NR         Carreon et al. (1980)
    Haloxyfop EE          Rat         Male, female     Dermal             > 5000                95         Carreon et al. (1982)
    Haloxyfop EE          Rat         Male, female     Dermal             > 2000                94.7       Jones (1982a)
    Haloxyfop EE          Rat         Male, female     Dermal             > 2000                98.8       Jones (1982b)
    Haloxyfop ME          Rat         Male, female     Dermal             > 2000                98.6       Mizell et al. (1989)
    Haloxyfop EEd         Rat         Male, female     Intraperitoneal    670                   96.5       Jones el al. (1986)
    Haloxyfop MEe         Rat         Male, female     Intraperitoneal    220                   98.6       Mizell et al. (1989)
    Haloxyfop EE          Mouse       Male, female     Oral               610                   97         Oda (1986)
    Haloxyfop ME          Mouse       Male, female     Oral               710                   98.6       Mizell & Lomax (1989b)
                                                                                                                                              

    NR, not reported
    a   Haloxyfop EE, haloxyfop ethoxy ethyl ester; haloxyfop ME, haloxyfop methyl ester
    b   Administered as a 10% solution in a 10% acetone-corn oil (1:9) solution
    c   Formulated in distilled water
    d   Formulated in arachis oil
    e   Administered as a 25% solution in corn oil
             Groups of 80 male CDF Fischer 344 were fed diets providing doses
    of 0, 0.1, or 1 mg/kg bw per day for four weeks and were then allowed
    to recover for six weeks. Groups of 7-10 rats per dose were sacrificed
    at two-week intervals during both the treatment and the recovery
    period to study the onset and reversibility of hepatic changes.
    Treatment-related effects appeared at both doses, consisting of
    increased liver weights (transient at 0.1 mg/kg bw per day)
    accompanied by histopathological changes (hepatocellular enlargement
    and cytoplasmic eosinophilia) at the highest dose. After the recovery
    period, only the relative liver weights of animals at the high dose
    were still increased, but the histological changes remained. In
    animals sacrificed after four and six weeks of recovery, no changes
    were seen. The serum levels of haloxyfop were approximately
    proportional to the dose. The plasma clearance half-life of six days
    after a single oral dose was similar to the serum clearance half-lives
    of eight days for both doses after repeated oral administration
    (Herman  et al., 1983a).

         Groups of 15 Fischer 344 rats of each sex received doses of 0,
    0.002, 0.02, 0.2, or 2 mg/kg bw per day haloxyfop (acid) (purity, 96%)
    for 16 weeks, and groups of 12 received doses of 0, 0.02, or 2 mg/kg
    bw per day for 37 weeks. In the 16-week study, treatment had no effect
    on behaviour, body weight, food consumption, haematological
    parameters, or the results of urinalysis. As a viral infection
    (symptoms consistent with sialodacryoadenitis) was observed in animals
    in all groups a few days before the scheduled termination of the
    study, it was extended for an additional three weeks. Male rats at the
    highest dose showed a slight increase in alkaline phosphatase
    activity; a dose-related increase in liver weight was observed in
    males at 0.2 and 2 mg/kg bw, and testicular weights were slightly
    decreased at 2 mg/kg bw. In females, increased liver weights were
    observed only at the highest dose. At this dose, the kidneys of
    animals of each sex were darkened or greenish. Histopathological
    examinations of most organs revealed changes in the liver consisting
    of enlarged hepatocytes and a slightly increased degree of cytoplasmic
    homogeneity in males at 0.2 and 2 mg/kg bw and an increased degree of
    cytoplasmic homogeneity in females only at the highest dose. The
    darkened appearance of the kidney was not seen as differences in
    pigmentation of the renal tubular epithelial cells on microscopic
    examination.

         In the 37-week study, the same viral infection affected animals
    in all dose groups. No significant difference was observed in body
    weights between treated and untreated animals at termination of the
    study. The treatment did not change the results of haematology or
    urinalysis. Significant differences in clinical chemical parameters at
    the highest dose consisted of a slight increase in alkaline
    phosphatase activity, particularly in males, and an increase in serum

    alanine aminotransferase activity, particularly in females. Gross
    pathological changes observed in the liver and kidney of males at the
    highest dose consisted of slight liver enlargement and discolouration
    of the kidneys. The liver weights were increased at this dose in
    animals of each sex. Histopathological examination of the livers
    revealed hepatocellular enlargement and increased cytoplasmic
    homogeneity at the highest dose in males and females. In the kidneys,
    slight, chronic, progressive glomerulonephropathy occurred at a higher
    incidence in males at the highest dose than in controls. Variable
    accumulation of brown pigment in renal tubular epithelial cells was
    seen in all treated animals and was extensive in males at the highest
    dose. The pigment did not react with stains used to detect the
    presence of iron, calcium, phosphate, or bilirubin or lipofuscin
    pigment but probably represented a form of ceroid or lipofuscin
    pigments. No other histopathological alteration, including
    degenerative changes, was found in the kidneys. The NOAEL in the
    16-week study was 0.02 mg/kg bw per day for males and 0.2 mg/kg bw per
    day for females, based on changes in liver weight and histopathology.
    The NOAEL in the 37-week study was 0.02 mg/kg bw per day, based on
    biochemical, macroscopic, and microscopic changes in the liver
    (Gorzinski  et al., 1982c).

         Groups of 10 male Fischer 344 rats were fed diets containing
    haloxyfop ethoxy ethyl ester (purity, 99.3%) at concentrations
    providing daily doses of 0, 0.00028, 0.0028, or 0.028 mmol/kg bw per
    day, equivalent to 0, 0.1,1, and 10 mg/kg bw per day haloxyfop (acid).
    The treatment had no effect on body weight, food consumption, or the
    results of urinalysis. Clinical chemical examinations revealed a
    treatment-related increase in serum alkaline phosphatase activity,
    which was statistically significant only at the high dose, and
    dose-dependent decreases in cholesterol concentrations, which were
    statistically significant at the two higher doses; a slight but
    statistically significant increase in albumin and a slight decrease in
    globulin levels were also found at these doses. At the highest dose,
    minor decreases in packed cell volume, haemoglobin levels, and red
    blood cell count were observed. Organ weight changes consisted of an
    increase in liver weights at the two higher doses and a decrease in
    testicular weights at the highest dose. Treatment-related gross
    pathological alterations were limited to the livers of animals at the
    two highest doses and consisted of an increase in liver size. The
    enlarged livers and increased liver weights at the two higher doses,
    equivalent to 1 and 10 mg/kg bw haloxyfop (acid), respectively, were
    qualitatively and quantitatively similar to the results seen with
    haloxyfop (acid). The NOAEL was 0.00028 mmol/kg bw per day haloxyfop
    ethoxy ethyl ester, equivalent to 0.1 mg/kg bw per day haloxyfop
    (acid), based on a reduction in cholesterol concentration
    (Herman  et al., 1983b).

         A sample of the optically-active R-enantiomer form of haloxyfop
    (purity, 99.4%) was given in the diet to groups of 10 male and 10
    female Fischer 344 rats, at concentrations providing doses of 0,
    0.065, 0.2, or 2 mg/kg bw per day, for 16 weeks. Two additional groups
    of 10 rats of each sex were kept on diets providing levels of 0 or
    2 mg/kg bw per day for the same period and were then allowed to
    recover for four weeks. Females at the highest dose had slightly
    increased food consumption and body-weight gain. Males at the highest
    dose had small decreases in packed cell volume, haemoglobin, and
    erythrocyte count, which were minimal although statistically
    significant, after the recovery period. No further effects were seen
    on erythrocyte morphology or histopathological evaluation of the bone
    marrow. Alkaline phosphatase activity and potassium levels were
    increased in animals of each sex at the highest dose. Cholesterol
    levels were decreased in males at 0.2 and 2 mg/kg bw and in females at
    2 mg/kg bw. All of these parameters returned to normal during the
    recovery period. Liver weights were increased in males and females at
    the highest dose, and relative liver weights were increased in males
    at 0.2 mg/kg bw, even after the recovery period. Testicular weight was
    decreased at the highest dose, and the decrease remained after the
    recovery period. Gross pathological examination reveal no alterations;
    microscopic alterations were confined to the livers of males at the
    highest dose and consisted of enlarged centrilobular hepatocytes and
    increased eosinophilic staining of hepatocytes. No such effects were
    observed at the end of the recovery period. The NOAEL was 0.065 mg/kg
    bw per day, based on the reduction in cholesterol levels (Barna-Lloyd
     et al., 1989).

    Dogs

         Groups of two beagle dogs of each sex were fed diets containing
    haloxyfop (acid) (purity, > 99%) for five weeks, at doses selected on
    the basis of the results of a range-finding and palatability study
    (Barna-Lloyd, 1983a) of 0, 5, 15, and 45 mg/kg bw per day. Bloody
    faeces were seen sporadically in both males and one female at the high
    dose. As a duodenal ulcer was also found in one male dog and as
    ulcerative lesions of the gastric and duodenal mucosa were found in
    the range-finding study, a causative relationship to the treatment
    cannot be excluded. Both males at the high dose showed clinical signs
    of illness towards the end of the study, resulting in a diagnosis of
    pneumonia; this effect was considered not to be a direct result of
    treatment but to be due to aspiration of feed particles. The treatment
    resulted in marked weight loss in both males and in one female at the
    high dose and in animals of each sex at the intermediate dose. Food
    consumption was reduced only at the high dose in animals of each sex.
    Alterations in haematological parameters were observed in these
    animals, consisting of decreases in packed cell volume, erythrocyte
    count, and haemoglobin values, particularly in males; the platelet

    count was markedly reduced in most animals at this dose. Alanine
    transaminase and serum alkaline phosphatase activities were markedly
    increased in animals of each sex at the high dose, and cholesterol
    concentrations were reduced in animals of each sex at 15 and 45 mg/kg
    bw. Bromsulphthalein retention in serum, determined as an indicator of
    the excretory function of the liver, was markedly increased in animals
    of each sex at the high dose. The relative liver and kidney weights
    were increased in males at the intermediate and high doses and in
    females at the high dose. The only pathological lesion probably
    attributable to treatment was the duodenal ulcer found in one male at
    the high dose; histopathological examination confirmed the presence of
    a focal, sharply demarcated ulcer. The NOAEL was 5 mg/kg bw per day,
    based on reduced body-weight gain, changes in biochemical parameters,
    and changes in liver and kidney weights (Barna-Lloyd  et al., 1983b).

         Groups of four male and four female beagle dogs were fed diets
    providing doses of 0, 2, 5, or 20 mg/kg bw per day of haloxyfop (acid)
    (purity, 99.8%) for 13 weeks. Survival, food consumption and the
    results of urinalysis and ophthalmoscopy were not affected by the
    treatment. At the highest dose, decreased body-weight gain was found
    in animals of each sex, resulting in reduced overall body-weight gain
    only in males. Males and females at the highest dose had decreased
    erythrocyte counts, haemoglobin concentrations, haematocrit values,
    and platelet levels. A decrease in cholesterol concentration was
    observed at the intermediate and high doses. Slightly reduced levels
    of total and free triiodothyronine, and free thyroxin were also seen
    at these doses, accompanied by decreases in thyroid and parathyroid
    weights. At the highest dose, liver weights were increased in animals
    of each sex and kidney weights were increased, particularly in males.
    Histopathological examination revealed alterations in the liver,
    thyroid, and testis of animals at doses > 5 mg/kg bw. In the liver,
    slight hepatocellular enlargement was noted at the highest dose in
    animals of each sex and at the intermediate dose in females; this
    change appeared to be associated with glycogen deposition. The
    microscopic alterations in the thyroid glands consisted of a slight
    decrease in follicular size and hypertrophy of follicular epithelial
    cells at the two higher doses. The decreased amount of colloid in
    thyroid follicles seen at these doses suggests that the changes were
    an adaptive mechanism that may be associated with an increased rate of
    colloid turnover. Alterations in the testes observed at the two higher
    doses included a slight decrease in tubular size and more multi-
    nucleated spermitids in the tubular lumens. As these changes may
    reflect normal variation in maturation, a relationship with treatment
    is questionable. As significant increases in hepatic peroxisomal fatty
    acid -oxidation or peroxisomal volume density have been reported in
    animals fed dietary levels of haloxyfop as low as 0.5 mg/kg bw (rats)
    and 1 mg/kg bw (mice) (Stott  et al., 1985a,b; see section
    ( g)(ii)), similar studies were conducted in these dogs. Electron
    microscopic examinations of hepatocytes of animals in the control and

    high-dose groups indicated no change in peroxisomal volume density or
    structural changes in peroxisomes or other organelles. Hepatic
    peroxisomal fatty acid -oxidation (substrate, palmitoyl-coenzyme A)
    was, however, induced  in vitro at the intermediate and high doses.
    The NOAEL was 2 mg/kg bw per day, based on changes in biochemical
    parameters and organ weights and histopathological effects on the
    liver and thyroids (Dietz  et al., 1987).

         Groups of six beagle dogs of each sex were fed diets containing
    targeted doses of 0, 0.05, 0.5, and 5 mg/kg bw per day of haloxyfop
    (acid) (purity, 99.9%) for 12 months. Treatment had no effect on
    general condition, body-weight development, food consumption, organ
    weights, or the results of urinalysis. Treatment-related effects
    occurred in animals of each sex only at the highest dose.
    Haematological examination revealed slightly decreased packed cell
    volume and haemoglobin in males at the intermediate and high doses,
    with no clear dose-response relationship; moreover, the packed cell
    volumes of concurrent controls were reported to be unusually high in
    comparison with those of historical controls, resulting in significant
    decreases of this parameter. The same explanation may be valid for the
    haemoglobin values. These haematological changes were therefore
    considered not to be biologically relevant. Changes in some clinical
    chemical parameters were inconsistent, except that serum cholesterol
    levels were decreased in animals of each sex at the highest dose. No
    gross pathological or histopathological lesions attributable to
    treatment were seen. The NOAEL was 0.5 mg/kg bw per day, based on
    reduced cholesterol levels (Barna-Lloyd  et al., 1984).

    Monkeys

         Groups of four male and four female cynomolgus monkeys were given
    haloxyfop (acid) (purity, 99%) at doses of 0, 2, 10, or 30 mg/kg bw
    per day by nasogastric intubation for 13 weeks. The doses were
    selected on the basis of the results of a four-week study of liver
    peroxisome density (Gerbig  et al., 1985; see section ( g)(ii)).
    Treatment had no effect on body weight, food consumption, or the
    results of ophthalmoscopy. Erythrocyte count, haemoglobin
    concentration, and haematocrit were slightly reduced in animals at the
    highest dose, the effects being less pronounced in females than in
    males; no effects were seen in bone marrow or peripheral blood. At
    this dose, the cholesterol concentration was decreased in animals of
    each sex. The changes in organ weights consisted of an increase in
    kidney weights at the two higher doses and increased liver and
    decreased thyroid weights at the highest dose. Microscopic examination
    revealed hepatocellular hypertrophy associated with an increased
    content of cytoplasmic lipid. Less severe hepatocyte enlargement was
    also noted in single animals at the intermediate dose. Electron
    microscopic examination of the hepatocytes confirmed a slightly higher

    cytoplasmic lipid volume density in monkeys at the highest dose. The
    thyroid glands of some animals in different dose groups were decreased
    in size, and the size and weight differences observed in females at
    the high dose were associated with a decrease in the size of thyroid
    follicles and an increase in the size of the epithelial cells lining
    the follicles (hypertrophy). These effects were not accompanied by
    cell necrosis and were probably due to a physiological increase in
    colloid turnover. The incidence of microscopic changes in the thyroids
    of males at the high dose was not different from that in the controls
    or other treatment groups. Quantitative ultrastructural evaluation of
    centrilobular hepatocytes from male and female monkeys given 0 and
    30 mg/kg bw revealed no difference in peroxisomal volume density but
    indicated a slight increase in smooth endoplasmic reticulum in those
    treated with the high dose. There was no significant difference in
    hepatic peroxisomal activity between control and treated monkeys. The
    NOAEL was 2 mg/kg bw per day, based on organ weight changes and
    histopathological alterations in the liver (Yano  et al., 1987).

    (c)  Long-term toxicity and carcinogenicity

    Mice

         Groups of 70 male and 70 female B6C3F1 mice were maintained on
    diets providing haloxyfop (acid) (purity, 99.6%) at doses of 0, 0.03,
    0.065, or 0.6 mg/kg bw per day. Ten mice of each sex per group were
    sacrificed at six and 12 months, and the remaining 50 animals of each
    sex were killed after 24 months. Treatment had no effect on behaviour,
    mortality, body-weight development, food consumption, or haemato-
    logical or clinical chemical parameters. The liver weights of males at
    the highest dose were slightly increased after six and 12 months of
    treatment, and histopathological examination showed slightly altered
    cytoplasmic tinctorial properties of centrilobular hepatocytes and a
    decrease in cytoplasmic vacuolization in male and female mice at this
    dose. The liver weights were not increased at termination of the
    study. Gross pathological examination showed an increased number of
    treated mice with one or more liver masses or nodules, with incidences
    in males of 22, 30, 32, and 34% and in females of 12, 14, 26, and 26%
    at 0, 0.03, 0.065, and 0.6 mg/kg bw per day, respectively.
    Histological examination revealed an increase in the incidence of
    hepatic foci in males at 0.03 and 0.6 mg/kg bw in comparison with
    concurrent controls, with incidences of 14, 24,10, and 24%,
    respectively. Dose-dependent increases in the incidences of
    hepatocellular tumours were seen in male and female mice at 0.065 and
    0.6 mg/kg bw, which were statistically significant in males at the
    highest dose: The incidences of primary hepatocellular tumours (benign
    or malignant) were 26, 30, 40, and 54% in males and 14, 18, 26, and
    30% in females, respectively. The incidence of benign hepatocellular
    adenoma in males at the highest dose was 38%, and the incidence of

    malignant, non-metastatic hepatocellular tumours in females was 16%;
    these incidences were significantly increased in comparison with
    controls. The ranges of incidence of primary hepatocellular neoplasms
    in historical control mice of this strain were reported to be 23-60%
    in males and 9-32% in females; the ranges of incidence were 12-52% and
    7-24% for adenoma and 5-24% and 0-10% for carcinoma (adapted from
    Billington, 1995). The NOAEL was 0.03 mg/kg bw per day, based on a
    dose-related increase in the incidence of hepatocellular tumours in
    animals of each sex at 0.065 and 0.6 mg/kg bw per day (Tollett
     et al., 1985) A review of the carcinogenicity of haloxyfop in mice
    was also available (Swenberg, 1994; see Appendix).

    Rats

         Groups of 50 male and 50 female Fischer 344 rats were fed diets
    containing haloxyfop (acid) (purity, 99.7%) at targeted doses of 0,
    0.01, 0.03, 0.065, and 0.1 mg/kg bw per day for males and 0, 0.01,
    0.03, 0.065, and 1 mg/kg bw per day for females, for two years. Groups
    of 20 rats of each sex per dose were sacrificed at six and 12 months
    of treatment. At these times, treatment-related effects were seen in
    the liver and kidney at doses > 0.065 mg/kg bw. The only change in
    clinical chemical parameters was a slight increase in serum alanine
    aminotransferase activity in males fed the two highest doses and
    sacrificed after one year. Increased liver weights were observed at
    doses > 0.065 mg/kg bw at both interim sacrifices. After six
    months, slightly increased relative liver weights were found in
    females at 0.065 mg/kg bw and in males at 0.1 mg/kg bw, whereas
    females at the highest dose had increased absolute and relative liver
    weights. At the one-year interim sacrifice, females at this dose had
    increased relative liver weights. At both interim sacrifices, the
    livers of males and females at the highest dose showed an increase in
    hepatocyte size and eosinophilic staining of the cytoplasm in the
    centrilobular region. Changes in the kidney consisted of slight dark
    discolouration of the renal cortex in animals at the highest dose at
    both interim sacrifices and at doses > 0.065 mg/kg bw after one
    year. The discolouration appeared to be due to an increased quantity
    of pigment granules in the proximal convoluted tubules, which stained
    for iron and in the periodic acid-Schiff reaction (Tollett  et al.,
    1983).

         At the time of terminal sacrifice, no effects had been seen on
    mortality, body weight, food consumption, organ weights (including
    liver weights), or the results of haematological and clinical chemical
    tests, urinalysis, or histopathology, so that the alterations seen at
    interim sacrifice were not confirmed. Histopathological changes in the
    kidney included an increased quantity of renal pigment in the proximal
    convoluted tubules of females at the high dose, accompanied by a
    slight decrease in kidney weights. The pigmentation was similar to
    that found at the interim sacrifices. A single male at 0.1 mg/kg bw
    and two females at 1 mg/kg bw had focal accumulation of hepatocytes of

    normal appearance in the pancreas. Although the biological
    significance of these findings is questionable, this alteration has
    been identified as a treatment-related effect in rats (Reddy  et al.,
    1984). There was no increase in the incidence of neoplasia. The NOAEL
    was 0.065 mg/kg bw per day, based on transient changes in liver
    weights and histopathological alterations in the liver and kidney at
    higher doses (Yano  et al., 1984).

    (d)  Reproductive toxicity

    Rats

         Groups of 30 male and 30 female Fischer 344 rats were maintained
    on diets providing haloxyfop (acid) (purity, 99.6%) at doses of 0,
    0.005, 0.05, or 1 mg/kg bw per day over three generations. The F0
    animals were maintained on the test diet for 102 days and were then
    mated to produce the F1a and F1b litters. The F1 rats were
    maintained on the test diet for 137 days and then mated to produce the
    F2a, F2b, and F2c litters. The F2 generation was fed the diet
    for 64 days before being mated to produce the F3a and F3b
    generations. The F2c rats were maintained on the test diet for 61
    days and were then cross-mated with untreated stock males and females
    to further investigate the reduced fertility observed in previous
    matings.

         Treatment had no influence on the behaviour, appearance, or
    mortality of the animals. In some generations, the dose of 1 mg/kg bw
    resulted in reduced body weights in comparison with controls during
    various periods: the body weights of pups in the F1b litters were
    reduced, and the body weights of F1 adults, particularly females
    (chosen from F1b litters), were slightly lower throughout the
    pre-breeding period; reduced mean body weights were also observed tor
    F2a pups throughout lactation and for F2b adults of each sex
    during treatment. Males at 0.05 mg/kg bw also had lowered mean body
    weights towards the end of treatment. The reduced body-weight gain was
    usually accompanied by reduced food consumption.

         Fertility (the number of pregnant females in relation to the
    number of females mated) was reduced in treated F1 adults producing
    the F2b and F2c litters in comparison with the controls, but no
    clear dose-response relationship was observed and the fertility
    indices in the controls varied markedly (by 53-90%.) The lowest
    fertility index was 40% in the F1 adults producing the F2b litter,
    which is lower than the range in the controls. In the absence of a
    dose-response relationship and of a consistent pattern over the
    generations, these effects may not be related to treatment. To further
    investigate this question, rats of the F2c generation were
    cross-mated with untreated males and females. No effect on fertility
    was seen, as the fertility indices were 80-93%.

         Microphthalmia and/or anophthalmia were observed in control and
    treated animals. The incidence in the controls was 0-1%. The highest
    incidences occurred in F2a pups, with 0.6% in controls, 2% at
    0.005 mg/kg bw, 1.4% at 0.05 mg/kg bw, and 1.4% at 1 mg/kg bw. As
    there was no dose-response relationship and no consistent pattern over
    the generations, the increased incidences are not considered to be
    related to treatment. The range of incidence of this lesion in fetuses
    of historical controls was reported to be 0-4%.

         Liver weights (absolute and/or relative) were increased and
    kidney weights decreased in most generations at the highest dose,
    particularly in adults. Histopathological examination of F1b
    weanlings revealed no treatment-related alterations. F1 adult males
    at the highest dose had enlarged hepatocytes, often with altered
    tinctorial properties. The convoluted tubules of animals at the
    highest dose had increased pigmentation, suggested to be due to a form
    of lipofuscin (Tollett  et al., 1983). No gross or histopathological
    changes were found in reproductive organs that would explain the
    reduced fertility observed in F1 adults. The NOAEL for reproductive
    toxicity was 1 mg/kg bw per day, based on reduced fertility indices in
    some instances. The NOAEL for body-weight gain depression was
    0.05 mg/kg bw per day, based on reduced body-weight gain in the F2b
    generation (Jeffries  et al., 1985).

         Groups of 30 male and 30 female Sprague-Dawley rats were
    maintained on diets providing haloxyfop (acid) (purity, 99.4%) at
    doses of 0, 0.01, 0.065, or 1 mg/kg bw per day for eight (F0
    generation) or 11 weeks (F1 generation). The F0 males and females
    were mated twice to produce the F1a and F1b litters and F1 males
    and females to produce the F2a and F2b litters. Feeding of the two
    highest doses resulted in a dose-related reduction in the body weight
    and body-weight gain in 14- and 21-day-old F1a and F1b neonates;
    the differences in the F1b litters were statistically significant,
    whereas a significant reduction was observed in the F1b litters only
    at the highest dose. The reduction in pup weight may be explained by
    the slightly larger F1a litters at 0.065 mg/kg bw. The weights of
    F2a neonates at 21 days were also decreased after treatment at the
    highest dose. Neither food consumption nor reproductive parameters
    were affected by treatment. Gross pathological examination of F0 and
    F1 adults revealed enlarged livers in some F0 males and females at
    the highest dose. The NOAEL was 0.065 mg/kg bw per day, based on
    neonatal body-weight changes at 1 mg/kg bw per day (Nitschke  et al.,
    1985).

    (e)  Developmental toxicity

    Rats

         Groups of 30 female Fischer 344 rats received 0, 0.1, 1.0, or
    7.5 mg/kg bw per day haloxyfop (sodium salt) by gavage on days 6-15 of
    gestation. Maternal toxicity, evidenced by slightly reduced
    body-weight gain and reduced food consumption, was seen in animals at
    the highest dose early in gestation (days 6-8). The incidence of
    malformations was not increased. Variations included a significant
    increase in the incidence of delayed ossification in vertebrae
    (thoracic centra) and a slight increase in the incidence of unfused
    thoracic centra at the highest dose. The delay in skeletal development
    may indicate an embryotoxic effect, probably consequent to maternal
    toxicity. The NOAEL was 1 mg/kg bw per day, based on maternal and
    embryotoxicity at the higher dose (Hayes  et al., 1983).

         Groups of female Wistar rats were fed diets containing haloxyfop
    ethoxy ethyl ester (purity, 98.2%) by gavage at doses of 0, 5, 10, or
    50 mg/kg bw per day on days 6-16 of gestation No signs of maternal
    toxicity were observed. The two higher doses resulted in an increased
    number of resorptions and fewer live fetuses per litter; vaginal
    haemorrhage was also observed at these doses. Mean fetal weight was
    reduced at the highest dose. Examination of external and soft tissues
    revealed a dose-dependent increase in the incidence of cachectic
    fetuses and an increased incidence of ureterohydronephrosis in all
    treated groups. The effects at the lowest dose were less pronounced
    and sometimes not significantly different from the control.
    Retardation of ossification was observed at the two higher doses
    (Machera, 1993).

    Rabbits

         Groups of 30 female New Zealand white rabbits received 0, 1.0,
    7.5, or 20 mg/kg bw per day haloxyfop (sodium salt) by gavage on days
    6-18 of gestation. Five animals at the high dose, one animal at the
    intermediate dose, and one animal at the low dose died. No other
    effects on general appearance or body-weight development were observed
    that indicated maternal toxicity. At the highest dose, there was an
    increased incidence of resorptions. Some fetal alterations were more
    frequent in treated than in control groups. The incidences of severe
    forelimb flexure were 1% at the intermediate dose, 2% at the high
    dose, and 0% in controls; the incidence of forelimb flexure in
    historical controls was reported to be 3%. Microphthalmia was not
    observed in any control fetus, but occurred at incidences of 0.5% at
    the intermediate dose and 1% at the high dose; this lesion was not
    found in historical controls in the 10 studies considered. A
    significant increase in the incidence of crooked hyoid bone

    (considered to be a minor skeletal variant) was observed at the
    highest dose. The absence of major external malformations in the
    concurrent control group is unusual in view of the means of about 4%
    of fetuses (range, 1-7%) and 21% of litters (range, 7-42%) reported in
    historical controls. The incidences of total major malformations in
    this study were reported to be 0, 17, 16 and 24% of litters at 0, 1,
    7.5, and 20 mg/kg bw, respectively, in good agreement with the mean
    value of 20% (Hayes  et al., 1983).

         A further study was conducted to determine whether the small
    increase in the incidence of malformations-in particular
    microphthalmia-observed in the previous study could be confirmed.
    Groups of 25-30 female rabbits received haloxyfop (sodium salt)
    (purity, 99.6%) by gavage at doses of 0, 3.0. 7.5, or 15 mg/kg bw per
    day on days 6-18 of gestation. Four dams at the highest dose died: two
    died due to an intubation error, and two others were found dead on
    days 25 and 28 of gestation having shown no previous signs of
    toxicity. No other signs of maternal toxicity were observed, and no
    treatment-related signs of embryo- or fetotoxicity were noted. The
    incidences of malformations were 2, 3, 2, and 2% of fetuses at 0, 3.0,
    7.5, and 15 mg/kg bw group, respectively; the mean percentage of major
    malformations in historical controls was 4% of fetuses (range, 1-7%).
    The malformations were mainly skeletal changes, such as fused and
    forked ribs, and vertebral and soft tissue changes, such as single
    hydrocephalus or coarctation of the pulmonary artery. No
    microphthalmia was observed (Hanley  et al., 1985).

         The NOAEL in these two studies was 7.5 mg/kg bw per day with
    respect to maternal toxicity and 15 mg/kg bw per day for embryo- and
    fetotoxicity.

    (f)  Genotoxicity

         The results of studies of the genotoxicity of haloxyfop are shown
    in Table 2.

    (g)  Special studies

    (i)  Dermal and ocular irritation and dermal sensitization

         Studies on the irritating and sensitizing potential of haloxfop
    and its esters are summarized in Table 3.

        Table 2.  Results of tests for the genotoxicity of haloxyfop and its R enantiomer
                                                                                                                                              

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

    Haloxyfop (acid)
    In vitro

    Reverse mutation           S. typhimurium TA98,        2-2000 mg/platea          99          Negative      Domoradzki (1981)
                               TA100, TA1535, TA1537,
                               TA1538

    Forward mutation,          Chinese hamster ovary       25-2000 mg/mlb            NR          Negative      Myhr (1982)
      hprt locus               cells

    Chromosomal aberration     Human lymphocytes           100-1000 mg/mla           99.4        Negative      Bootman et al. (1986)

    Unscheduled DNA            Rat hepatocytes             0.3  10-9-0.3           99          Negative      Domoradzki (1980)
                                                           10-3 mol/litre

    In vivo
    Micronucleus formation     Rat bone marrow             Single oral dose of       99.6        Negative      Bhaskar Gollapudi et al.
                                                           0, 30, 100, or 300                                  (1982)
                                                           mg/kg bw
    R-Enantiomer

    Reverse mutation           S. typhimurium TA98,        5-1580 mg/plate           99.4        Negative      Bhaskar Gollapudi &
                               TA100, TA1535, TA1537                                                           Samson (1989)
                                                                                                                                              

    Table 2.  (cont'd).
                                                                                                                                              

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

    Chromosomal aberration     Rat lymphocytes             50-500 mg/mlc             99.4        Negativee     Bhaskar Gollapudi &
                                                           166.7 - 1667 mg/mld                                 Linscombe (1989)

    Unscheduled DNA            Rat hepatocytes             5-500 mg/ml               99.4        Negative      McClintock & Bhaskar
                                                                                                               Gollapudi (1989)
                                                                                                                                              

    NR, not reported
    a   Toxic at highest doses
    b   Sharp increase in toxicity at 600-2000 mg/ml
    c   With metabolic activation: no mitotic activity at doses > 1667 mg/ml
    d   Without metabolic activation; no mitotic activity at 5000 mg/ml
    e   Positive response at 1667 mg/ml not confirmed in an experiment conducted at concentrations of 750-1667 mg/ml
        (ii)  Peroxisome proliferation

         Haloxyfop (acid) (purity, > 99%) was administered in the food to
    groups of 21-25 Fischer 344 rats and B6C3F1 mice of each sex at doses
    of 0, 0.1, 0.5, 1.0, or 10 mg/kg bw per day for a four-week challenge
    period. At the end of the challenge period, three animals of each sex
    were examined by electron microscopy and eight of each sex in anenzyme
    assay; the remainder were kept on normal diet for a study of recovery.
    In the enzyme assays, the activities of hepatic peroxisomal enzymes
    were determined  in vitro, including those involved in the 
    oxidation of fatty acids, hepatic carnitine acetyltransferase,
    glycerophosphate dehydrogenase, and catalase. The treatment had no
    effect on behaviour or general condition in either species. Body
    weight and food consumption were reduced in male rats at 10 mg/kg bw,
    and a dose-related increase in the liver weights of rats of each sex
    was observed at this dose and also in males at 1 mg/kg bw; in females,
    only the relative liver weights were increased at 10 mg/kg bw. In
    mice, marked liver weight increases were seen at 10 mg/kg bw in
    animals of each sex, and the protein content of livers of mice at the
    high dose was increased. Microscopic changes were observed in the
    livers of rats and mice at 0.5, 1.0, or 10 mg/kg bw, consisting of
    hepatocellular enlargement and/or alteration in cytoplasmic staining
    (increased eosinophilia). These changes affected the entire hepatic
    lobule in male rats at 10 mg/kg bw, whereas in the other groups the
    changes were found only in the centrilobular region. The microscopic
    changes in mice of each sex were similar to those in male rats. A few
    rats and mice at 10 mg/kg bw also showed a slight increase in the
    presence of mitotic figures and necrosis of individual hepatocytes.

         Quantitative evaluation of electron micrographs of hepatocytes
    from mice and rats showed increased peroxisome volume densities in
    animals of each sex at 10 mg/kg bw, which were 12 times higher than in
    controls in male rats, eight times higher in male mice, three times
    higher in female rats, and 14 times higher in female mice. Less
    pronounced increases were also found at 1.0 mg/kg bw in male mice and
    male and female rats and at 0.5 mg/kg bw in male rats. Other changes
    seen in hepatocytes from rats at the high dose included irregular
    peroxisomal shape and the occurrence of filamentous nucleoids.

         Various increases were observed in the activities of the enzymes
    related to peroxisomal fatty acid catabolism. Palmitoyl-CoA was used
    as the substrate in the assay for fatty acid -oxidase. The specific
    activity of this enzyme, which generates a major portion of
    peroxisomal hydrogen peroxide  in vitro, was induced in mice and rats
    of each sex at 10 mg/kg bw. The degree of induction in male rats was
    greater (393% of controls) than that in females (109% of controls).
    Dose-related increases in fatty acid -oxidase activity were also seen
    in male rats at 0.5 and 1 mg/kg bw, attaining activities corresponding
    to 160 and 294% of that of controls, respectively. In mice, induction
    of fatty acid -oxidase resulted in activities of 337% in male and

        Table 3.  Dermal and ocular irritation and sensitization due to haloxyfop (acid) and its methyl and ethoxy ethyl esters
                                                                                                                                              

    Test materiala      Species         Sex              Route               Result                Purity (%)     Reference
                                                                                                                                              

    Haloxyfop (acid)    Rabbit          Male, female     Skin                No irritation         NR             Carreon et al. (1980)
                                                         Eye                 Irritationa

    Haloxyfop EE        Rabbit          Female           Skin                No irritation         95             Carreon et al. (1982)
                                        Male, female     Eye                 Irritationb

    Haloxyfop EE        Rabbitf         Female           Skin                No irritation         94.7           Jones (1982c)

    Haloxyfop EE        Rabbit          Female           Skin                Irritation            98.8           Jones (1982d)

    Haloxyfop ME        Rabbit          Male, female     Skin                No irritation         98.6           Mizell (1989a)

    Haloxyfop ME        Rabbit          Male, female     Eye                 Irritationc           98.6           Mizell (1989b)

    Haloxyfop EE        Guinea-pig      Male, female     Various             No sensitization      95             Carreon et al. (1982);
                                                                             (up to 100%                          Jones (1982e, 1983c)
                                                                             concentration)

    Haloxyfop ME        Guinea-pig      Male             Bhler test         No sensitization      98.6           Mizell (1989c)
                                                         (modified)          (up to 100%
                                                                             concentration)
                                                                                                                                              

    Table 3.  (cont'd).
                                                                                                                                              

    Test materiala      Species         Sex              Route               Result                Purity (%)     Reference
                                                                                                                                              

    Haloxyfop ME        Guinea-pig      NR               Magnusson/          No sensitization      Technical      Jones (1994)
                                                         Kligman             (up to 100%
                                                         maximization        concentration)
                                                         test
                                                                                                                                              

    NR, not reported; EE, ethoxy ethyl; ME, methoxy ethyl
    a   In unwashed eyes, conjunctival redness, swelling, discharge, reddening of the iris, and opacity covering up
        to 100% of the cornea in most animals; effects lasting < 21 days. In washed eyes, similar signs but reversible
        by 10 days after exposure.
    b   In unwashed eyes, conjunctival redness and swelling, discharge, and transient reddening of the iris in single
        animals; all signs disappeared within three days after exposure.
    c   Conjuctival redness and discharge; effects reversible within 24 h after treatment; all eyes unwashed
        257% in female mice at 10 mg/kg bw in comparison with controls,
    whereas at lower doses no induction was observed. The specific
    activity of catalase, the peroxisomal enzyme responsible for the
    destruction of hydrogen peroxide, was induced in both rats and mice
    but to a lesser extent. Catalase activity, measured as the hydrolysis
    of hydrogen peroxide substrate, was 145% and 174% of the control
    activity in male rats ingesting 1 and 10 mg/kg bw, respectively,
    whereas in females at 10 mg/kg bw the catalase activity was only 134%
    of controls. In mice at 10 mg/kg bw, catalase activity was about 206%
    of the control value in animals of each sex. An increase in the
    capacity to transfer fatty acid -oxidase-generated acyl-coenzyme A
    groups out of the peroxisomes into mitochondria was indicated by
    increases in hepatic carnitine acetyltransferase activities. These
    dose-related increases in male rats at 0.5, 1.0, and 10 mg/kg bw
    resulted in values 2-22-fold higher than those of the controls. In
    females, only a fourfold increase was found at the high dose. The
    hepatic carnitine acetyltransferase activity in mice of each sex at 1
    and 10 mg/kg bw was increased by about two- and 14-fold in comparison
    with controls, respectively. At 0.5 mg/kg bw, the activity was
    slightly increased in female mice only. Weak increases in
    glycerophosphate dehydrogenase activity were observed at 10 mg/kg bw
    in male rats and in mice of each sex, attaining statistical
    significance only in male rats at the high dose (Stott  et al.,
    1985a). The NOAEL was 0.1 mg/kg bw per day with respect to changes in
    biochemical parameters and histological changes in the liver
    indicative of peroxisomal proliferation.

         In a subsequent study, the reversibility of the effects observed
    in the previous study was determined after a two- to four-week
    recovery period. After the two-week recovery, male rats and mice of
    each sex at the high dose had increased liver weights, altered hepatic
    morphology (enlarged hepatocytes, altered tinctorial properties), and
    elevated fatty acid -oxidase, hepatic carnitine acetyltransferase,
    and glycerophosphate dehydrogenase (male rats) activities. The
    magnitude of these changes was, however, lower than at the end of
    treatment. The liver weights of male rats at the high dose were still
    greater than those of the controls after either recovery period. In
    mice, the liver weights were still increased after two weeks' recovery
    in males and females at 10 mg/kg bw and in females at 1 mg/kg bw. The
    histopathological changes were similar to those found immediately
    after treatment, consisting of increased hepatocyte size and/or
    alterations in hepatocellular cytoplasmic staining in some groups.
    Microscopic changes were found in male rats at 1 and 10 mg/kg bw after
    two weeks' recovery, whereas only males treated with 10 mg/kg bw still
    had slight changes after four weeks' recovery. A similar pattern of
    reversibility was found in mice, in which the histopathological
    changes were present after two weeks' recovery in males and females at
    10 mg/kg bw; none were seen after the four-week recovery period. The

    activities of fatty acid -oxidase, hepatic carnitine acetyltrans-
    ferase, and catalase were also reversed; however, the activity of
    fatty acid -oxidase was still elevated in male rats at the high dose
    (168% of control) after the four-week recovery period. The increased
    liver protein concentrations observed in mice after treatment were not
    seen after two weeks' recovery. No data are available on the
    reversibility of these effects after long-term exposure
    (Stott  et al., 1985b).

         Haloxyfop (acid) (purity, 99.6%) as the sodium salt was given to
    groups of two male cynomolgus monkeys by nasogastric intubation at
    doses of 0, 5, or 20 mg/kg bw for four weeks. Two additional monkeys
    were given 20 mg/kg bw per day over four weeks and then maintained on
    normal diet for at least four weeks. There was no apparent effect on
    the behaviour, general appearance, activity, body weight, food
    consumption, or haematological parameters. A reduction in serum
    cholesterol concentrations was observed at the high dose but the
    values had returned to normal by the end of the four-week recovery
    period. There were no changes in organ weights and no histopatho-
    logical alterations. Electron microscopy of the livers of the controls
    and animals at the high dose revealed no treatment-related differences
    in the peroxisomes or other intracellular components of the liver in
    the treated monkeys (Gerbig  et al., 1985).

         In a study of possible species differences in sensitivity to
    hepatic peroxisomal induction by haloxyfop, the results of studies in
    which peroxisomal induction had been investigated in rats, mice, dogs,
    and monkeys were reviewed, and liver weights, light microscopy
    findings in liver, hepatic peroxisomal volume density, and serum
    cholesterol levels were compared (Barnard  et al., 1986). Samples of
    liver were collected for electron microscopy from three dogs of each
    sex per group in the 12-month dietary study in beagle dogs at doses of
    0, 0.05, 0.5, and 5 mg/kg bw per day for 12 months (Barna-Lloyd
     et al., 1984; see short-term studies); and from two male monkeys at
    each dose in the four-week study at oral doses of 0, 5, and 20 mg/kg
    bw per day (Gerbig  et al., 1985, see short-term studies). Electron
    microscopic and morphometric point-counting techniques were used to
    quantify peroxisomal volume density, and the results were expressed as
    percent of hepatocellular cytoplasm occupied by peroxisomes.
    Examination of liver sections from controls, from dogs at 5 mg/kg bw,
    and from monkeys at 20 mg/kg bw showed that haloxyfop at these doses
    had no effect. The results of these studies are in good agreement with
    the lack of organ weight changes and histopathological alterations and
    indicate that the livers of dogs and monkeys are less sensitive than
    those of mice and rats to the inducing effects of haloxyfop.

    (iii)  Stereochemical inversion

         Groups of four Fischer 344 rats of each sex were given an oral
    dose of 11 mg/kg bw 14C-haloxyfop (racemic material), and urine and
    faecal samples were collected at 24-h intervals for 10 days. The
    elimination half-life in female rats was estimated to be about 16.3 h,
    and most of the dose that was excreted in the urine was present as the
    parent compound; > 98% of the haloxyfop present in the urine and
    faeces of female rats corresponded to the R-enantiomer. In male rats,
    the elimination half-life was about 70 h, and most of the dose was
    excreted in faeces. Again, most of the excreted haloxyfop in urinary
    and faecal samples was the R-enantiomer. Thus, stereochemical
    inversion is rapid and nearly complete in the rat (Bartels & Smith
    1988, 1989).

    Comments

         After oral administration of the racemic mixture to rats, 98% of
    the parent compound was in the form of the R-isomer, indicating rapid
    conversion of the S-enantiomer  in vivo.

         Various studies on the fate of haloxyfop (acid) and its esters
    have been conducted in mice, rats, dogs, monkeys, and humans.
    Haloxyfop is rapidly absorbed and eliminated primarily as the
    unchanged parent compound after oral administration. A marked sex
    difference in the excretion pattern was noted in rats, the principal
    route of excretion being the faeces in males and the urine in females.
    The major route of elimination was also faecal in mice and dogs (only
    males studied), whereas in monkeys (only males studied) the main
    elimination route was the urine. The plasma elimination half-lives
    were lowest in female rats (one day) and longest in male rats (six
    days). Comparative pharmacokinetic investigations with haloxyfop
    (acid) and its esters in rats showed very similar patterns of
    absorption and excretion. The esters were rapidly converted to the
    parent acid. Pharmacokinetic studies in man after oral administration
    revealed rapid absorption. The plasma elimination half-life was about
    six days. Urine was the main excretory route; dermal absorption was
    estimated to be about 3% of an applied dose.

         After a single oral dose to rats, haloxyfop and its esters showed
    similar, moderate toxicity. The LD50 was about 300 mg/kg bw for the
    acid and its methyl ester and about 500 mg/kg bw for the ethoxyethyl
    ester. The WHO has classified haloxyfop as 'moderately hazardous'.

         Two short-term studies were conducted in mice. In one study, the
    diet provided doses of 0, 0.002, 0.02, 0.2, or 2 mg/kg bw haloxyfop
    per day over 13 weeks; in the other study, the doses were 0, 0.02, or
    2 mg/kg bw per day over 36 weeks. At 2 mg/kg bw per day in both
    studies, effects in the liver consisted of increases in serum alkaline
    phosphatase activity (in males), increased liver weights,
    hepatocellular enlargement, and increased eosinophilia. In addition,
    in the 36-week study, the kidneys of high-dose males showed a decrease
    in cytoplasmic vacuolation. The NOAELs were 0.2 mg/kg bw per day in
    the 13-week study and 0.02 mg/kg in the 36-week study.

         Two short-term dietary studies were conducted in rats. In the 16-
    and 37-week studies, the doses were 0, 0.002, 0.02, 0.2, or 2 mg/kg bw
    per day and 0, 0.02, or 2 mg/kg bw per day, respectively. At 2 mg/kg
    bw per day in both studies, changes including an increase in serum
    alkaline phosphatase activity, liver enlargement, an increase in liver
    weight, a slight decrease in testicular weights, and histopathological
    changes in the liver (hepatocellular enlargement and increased
    cytoplasmic homogeneity) were observed. In male rats, treatment-
    related effects on the liver were observed at 0.2 mg/kg bw per day in
    the 16-week study. The NOAEL was therefore 0.02 mg/kg bw per day in
    both studies.

         In a 13-week study in dogs, doses of 0, 2, 5, or 20 mg/kg bw per
    day haloxyfop were given in the diet. At 5 mg/kg bw per day and above,
    changes in biochemical parameters (reduced thyroid hormone levels,
    decreased cholesterol concentration), decreased thyroid and
    parathyroid weights, and histopathological changes in the liver
    (hepatocellular enlargement) and thyroid gland (decrease in follicular
    size, hypertrophy of follicular epithelial cells) were observed. At
    20 mg/kg bw per day, decreased body-weight gain, reductions in various
    haematological parameters and increases in liver and kidney weights
    were found. The NOAEL was 2 mg/kg bw per day.

         In a 12-month dietary study in dogs at doses of 0, 0.05, 0.5, or
    5 mg/kg bw haloxyfop per day, a decrease in the serum cholesterol
    concentration was found in animals of each sex at 5 mg/kg bw per day.
    The NOAEL was 0.5 mg/kg bw per day.

         In a 13-week study in monkeys at doses of 0, 2, 10, or 30 mg/kg
    bw haloxyfop per day, oral administration of 30 mg/kg bw per day
    resulted in a decrease in the cholesterol concentration, an increase
    in liver and kidney weights, a decrease of thyroid weights,
    hepatocellular hypertrophy, increased content of cytoplasmic lipid,
    and, in females, a decrease in the size of the thyroid follicles and
    hypertrophy of the follicular epithelial cells. At 10 mg/kg bw,
    increased kidney weights and slight hepatocellular enlargement were
    observed. The NOAEL was 2 mg/kg bw per day.

         A two-year dietary study of carcinogenicity in mice at doses of
    0, 0.03, 0.065, or 0.6 mg/kg bw haloxyfop per day resulted in a
    dose-dependent increase in the incidence of hepatocellular tumours at
    0.065 and 0.6 mg/kg bw per day in animals of each sex. Statistically
    significant increases in the incidence of adenomas in males and
    carcinomas in females were observed at 0.6 mg/kg bw per day. Other
    histopathological effects on the liver, including altered cytoplasmic
    staining properties of centrilobular hepatocytes and a decrease in
    cytoplasmic vacuolization, were observed at 0.6 mg/kg bw per day. The
    NOAEL was 0.03 mg/kg bw per day on the basis of an increased incidence
    of liver tumours at higher doses.

         In a two-year study of carcinogenicity, rats were given diets
    providing doses of 0, 0.01, 0.03, 0.065, or 0.1 mg/kg bw per day for
    males and 0, 0.001, 0.03, 0.065, or 1 mg/kg bw per day for females.
    Treatment-related effects on the liver at 0.1 mg/kg bw per day and
    above included transient organ weight increases, hepatocellular
    enlargement and increased eosinophilia. There was no evidence of
    carcinogenic potential. The NOAEL was 0.065 mg/kg bw per day.

         In a study of developmental toxicity in rats at oral doses of 0,
    0.1, 1, or 7.5 mg/kg bw haloxyfop (sodium salt) per day, maternal
    toxicity was observed at 7.5 mg/kg bw per day, as evidenced by reduced
    body-weight gain and reduced food consumption. In the fetus,
    ossification was delayed at 7.5 mg/kg bw per day. No evidence for
    teratogenicity was observed. The NOAEL for maternal toxicity and
    embryotoxicity was 1 mg/kg bw per day.

         In two studies of developmental toxicity in rabbits at doses of
    0, 1, 7.5, or 20 mg/kg bw per day and 0, 3, 7.5, or 15 mg/kg bw per
    day, maternal deaths occurred at 15 mg/kg bw per day and above.
    Embryotoxicity was seen at 20 mg/kg bw per day, as indicated by an
    increased incidence of resorptions. The results gave no evidence of
    teratogenic potential. The NOAEL was 7.5 mg/kg bw per day for maternal
    toxicity and 15 mg/kg bw per day for embryotoxicity.

         In a two-generation study, rats were treated with diets providing
    doses of 0, 0.01, 0.065, or 1 mg/kg bw haloxyfop per day. Reductions
    in body-weight gains were observed in weanlings of the F1 and F2
    generations at 1 mg/kg bw per day. Reproduction was not affected by
    the treatment. The NOAEL was 1 mg/kg bw per day for reproduction and
    0.065 mg/kg bw per day for body-weight changes in neonates.

         Haloxyfop has been adequately tested for genotoxicity in a range
    of tests  in vivo and  in vitro. The Meeting concluded that it is
    not genotoxic.

         In order to study the mechanism of hepatocarcinogenesis in mice,
    several short-term studies were conducted to investigate the effect of
    haloxyfop on hepatocellular peroxisomes. Peroxisome induction was
    evidenced by histopathological and electron microscopic changes and
    increased activities of enzymes involved in the -oxidation of fatty
    acids and of hepatic carnitine acetyltransferase in mice at a dose of
    1 mg/kg bw per day and in rats at 0.5 mg/kg bw per day. The increase
    in liver tumour incidence observed at 0.065 mg/kg bw per day in the
    long-term study of carcinogenicity in mice indicates that peroxisome
    proliferation is not an important part of the mechanism by which this
    non-genotoxic compound is carcinogenic.

         An ADI of 0-0.0003 mg/kg bw was established on the basis of the
    NOAEL of 0.03 mg/kg bw per day in the two two-year studies in mice,
    using a safety factor of 100.

    Toxicological evaluation

     Levels that cause no toxic effect

    Mouse:    0.03 mg/kg bw per day (two-year study of toxicity and
              carcinogenicity)

    Rat:      0.065 mg/kg bw per day (two-year study of toxicity and
              carcinogenicity and study of reproductive toxicity)
              1 mg/kg bw per day (maternal, embryo-, and fetotoxicity in
              study of developmental toxicity)

    Rabbit:   7.5 mg/kg bw per day (maternal toxicity in study of
              developmental toxicity)

    Dog:      0.5 mg/kg bw per day (12-month study of toxicity)

    Monkey:   2 mg/kg bw per day (13-week study of toxicity)

     Estimate of acceptable daily intake for humans

         0-0.0003 mg/kg bw

        Toxicological criteria for setting guidance values for dietary and non-dietary exposure to haloxyfop
                                                                                                                           

    Exposure                     Route, study type species                     Result, Remarks
                                                                                                                           

    Short-term (1- 7 days)       Skin, irritation, rabbit                      Irritating
                                 Eye, irritation, rabbit                       Irritating
                                 Skin, sensitization, guinea-pig (Buehler      No sensitization
                                 and Magnusson/Kligman)
                                 Oral, toxicity, rat                           LD50 = 340-550 mg/kg bw per day
                                 Dermal, toxicity, fat, rabbit                 LD50 > 2000 mg/kg bw per day

    Medium-term (1-26 weeks)     Repeated dietary, one month, dog              NOAEL = 5 mg/kg bw per day, reduced
                                                                               body-weight gain, reduced cholesterol
                                                                               and increased liver and kidney weights
                                 Repeated dietary, 16 weeks, rat               NOAEL = 0.02 mg/kg bw per day,
                                                                               increase in liver weights and
                                                                               histopathological changes in liver
                                 Oral, developmental toxicity, rat             NOAEL = 1 mg/kg bw per day,
                                                                               reduction in maternal body-weight gain,
                                                                               delayed ossification
                                 Dietary, reproductive toxicity rat            NOAEL = 0.065 mg/kg bw per day,
                                                                               reduced neonatal body-weight gain

    Long-term (> oneyear)        Repeated dietary, two years, toxicity and     NOAEL = 0.03 mg/kg bw per day;
                                 carcinogenicity, mouse                        increased incidence of liver tumours
                                                                                                                           

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         Europe, Wantage, Oxon, United Kingdom.

    Jones, J.R. (1982c) DOWCO 453 EE Tech Lot NP2: A primary skin
         irritation and corrosivity study in the rabbit. Report
         No 3230-50/136. Unpublished report prepared by Hazleton
         Laboratories Europe Ltd, North Yorkshire, United Kingdom.
         Submitted to WHO by Dow Elanco Europe, Wantage, Oxon,
         United Kingdom.

    Jones, J.R. (1982d) DOWCO 453 EE Tech Lot OP2: A primary skin
         irritation and corrosivity study in the rabbit. Report
         No. 3228-50/128. Unpublished report prepared by Hazleton
         Laboratories Europe Ltd, North Yorkshire, United Kingdom
         Submitted to WHO by Dow Elanco Europe, Wantage, Oxon,
         United Kingdom

    Jones, J.R (1982e) DOWCO 456 EE: Delayed contact hypersensitivity
         study in the guinea pig. Report No. 2977-50/107. Unpublished
         report prepared by Hazleton Laboratories Europe Ltd, Harrogate,
         United Kingdom. Submitted to WHO by Dow Elanco Europe, Wantage,
         Oxon, United Kingdom.

    Jones, J.R. (1983a) DOWCO 453 EE Tech Lot NP2: Acute oral toxicity
         study (LD50) in the rat. Report No. 3263-50/134. Unpublished
         report prepared by Hazleton Laboratories Europe Ltd, North
         Yorkshire, United Kingdom. Submitted to WHO by Dow Elanco Europe,
         Wantage, Oxon, United Kingdom.

    Jones, J.R. (1983b) DOWCO 453 EE Tech Lot OP2: Acute oral toxicity
         study (LD50) in the rat. Report No. 3372-50/126. Unpublished
         report prepared by Hazleton Laboratories Europe Ltd, North
         Yorkshire, United Kingdom. Submitted to WHO by Dow Elanco Europe,
         Wantage, Oxon, United Kingdom.

    Jones, J.R. (1983c) DOWCO 453 EE Tech. Lot NP2: Delayed contact
         hypersensitivity study in the guinea pig (Buehler test). Report
         No. 3394-50/143. Unpublished report prepared by Hazelton
         Laboratories Europe Ltd, Harrogate, United Kingdom. Submitted to
         WHO by Dow Elanco Europe, Wantage, Oxon, United Kingdom.

    Jones, J.R. (1994) Haloxyfop-R Methyl Ester Technical:
         Magnusson/Kligman maximisation study in the guinea pig. Study
         No 291/52. Unpublished report prepared by Safepharm Laboratories,
         Derby, United Kingdom. Submitted to WHO by Dow Elanco Europe,
         Wantage, Oxon, United Kingdom.

    Jones, J.R., Blackwell, M.P. & Collier, T.A. (1986) DOWCO 453 EE
         Ester: Acute parenteral (intraperitoneal) toxicity test in the
         rat. Project No. 44/85. Unpublished report prepared by Safepharm
         Laboratories, Ltd, Derby, United Kingdom. Submitted to WHO by Dow
         Elanco Europe, Wantage, Oxon, United Kingdom.

    Machera, K. (1993) Developmental toxicity of haloxyfop ethoxyethyl
         ester in the rat.  Bull. Environ. Contain. Toxicol., 51, 625-632

    McClintock, M.L. & Bhaskar Gollapudi, B. (1989) Evaluation of XRD-535
         in the rat hepatocyte unscheduled DNA synthesis (UDS) assay.
         Study No. TXT: DR-0298-5651-004. Unpublished report prepared by
         Dow Chemical Lake Jackson Research Center, Freeport, Texas, USA.
         Submitted to WHO by Dow Elanco Europe, Wantage, Oxon,
         United Kingdom.

    Mizell, M.J. (1989a) XRD-535 Methyl Ester: Primary dermal irritation
         study in New Zealand white rabbits. Study No. DR-0217-5704-001 B
         Unpublished report prepared by Dow Chemical Company, Midland,
         Michigan, USA. Submitted to WHO by Dow Elanco Europe, Wantage,
         Oxon, United Kingdom.

    Mizell, M.J. (1989b) XRD-535 Methyl Ester: Primary eye irritation
         study in New Zealand white rabbits. Study No. DR-0217-5704-001C.
         Unpublished report prepared by Dow Chemical, Midland, Michigan,
         USA. Submitted to WHO by Dow Elanco Europe, Wantage, Oxon,
         United Kingdom.

    Mizell, M.J. (1989c) XRD-535 Methyl Ester: Dermal sensitization
         potential in the Hartley albino guinea pig. Study
         No. DR-0217-5704-001 E; M-005152-001E. Unpublished report
         prepared by Dow Chemical Company, Midland, Michigan, USA.
         Submitted to WHO by Dow Elanco Europe, Wantage, Oxon,
         United Kingdom.

    Mizell, M.J. & Lomax, L.G. (1989a) XRD-535 Methyl Ester: Acute oral
         toxicity study in Fischer 344 rats. Study No. DR-0217-5704-001A.
         Unpublished report prepared by Dow Chemical, Midland, Michigan,
         USA. Submitted to WHO by Dow Elanco Europe, Wantage, Oxon,
         United Kingdom.

    Mizell, M.J. & Lomax, L.G. (1989b) XRD-535 Methyl Ester: Acute oral
         toxicity study in B6C3F1 mice. Study No. DR-0217-5704-001G.
         Unpublished report prepared by Dow Chemical, Midland, Michigan,
         USA. Submitted to WHO by Dow Elanco Europe, Wantage, Oxon,
         United Kingdom.

    Mizell, M.J., Schuetz, D.J. & Johnson, K.A. (1989a) XRD-535 Methyl
         Ester: Acute dermal toxicity study in Fischer 344 rats. Study
         No. DR- 0217-5704-001D. Unpublished report prepared by Dow
         Chemical, Midland, Michigan, USA. Submitted to WHO by Dow Elanco
         Europe, Wantage, Oxon, United Kingdom.

    Mizell, M.J., Eisenbrandt, D.L. & Battjes, J.E. (1989b) XRD-535 Methyl
         Ester: Acute intraperitoneal toxicity study in Fischer 344 rats.
         Study No. DR- 0217-5704-001F. Unpublished report prepared by Dow
         Chemical Company, Midland, Michigan, USA. Submitted to WHO by Dow
         Elanco Europe, Wantage, Oxon, United Kingdom.

    Mylar, B.C. (1982) Mutagenicity evaluation of XRD0453 in the CHO HGPRT
         forward mutation assay. Study No. 20989; Dow Report
         No. HET K 131381-(21). Unpublished report prepared by Litton
         Bionetics, Inc., Kensington, Maryland, USA. Submitted to WHO by
         Dow Elanco Europe, Wantage, Oxon, United Kingdom.

    Nitschke, K.D., Hayes, W.C., Berdasco, N.M., Yano, B.L. & Rao, K.S.
         (1985) DOWCO 453) two-generation dietary reproduction study in
         Sprague-Dawley rats. Study No. HET K 131381-039. Unpublished
         report prepared by Dow Chemical, Midland, Michigan, USA.
         Submitted to WHO by Dow Elanco Europe, Wantage, Oxon,
         United Kingdom.

    Nolan, R.J., Campbell, J.T., Tollett, J.T. & Saunders, J.H. (1985a)
         DOWCO 453: Pharmacokinetics in human volunteers following a
         single oral or dermal dose. Study No. HET K 131381-037.
         Unpublished report prepared by Dow Chemical, Midland, Michigan,
         USA. Submitted to WHO by Dow Elanco Europe, Wantage, Oxon,
         United Kingdom.

    Nolan, R.J., Campbell, R.A. & Venable, J.R. (1985b) DOWCO 453:
         Pharmacokinetics of the ethoxy ethyl ester of DOWCO 453 in human
         volunteers following a single dermal dose. Study
         No. HET DR-0210-0416-007. Unpublished report prepared by Dow
         Chemical, Midland, Michigan, USA. Submitted to WHO by Dow Elanco
         Europe, Wantage, Oxon, United Kingdom.

    Nolan, R.J., Dryzga, M.D., Yano, B.L. & Bartels, M.J. (1987)
         Haloxyfop: Pharmacokinetics following oral administration to male
         beagle dogs. Study No. HET K 131381-061. Unpublished report
         prepared by Dow Chemical, Midland, Michigan, USA. Submitted to
         WHO by Dow Elanco Europe, Wantage, Oxon, United Kingdom.

    Oda, S. (1986) Acute oral toxicity study of DOWCO 453 EE in mice.
         Study No. B-862. Unpublished report prepared by K Research Center
         Inc, Tokyo, Japan. Submitted to WHO by Dow Elanco Europe,
         Wantage, Oxon, United Kingdom

    Reddy, J.K., Rao, M.S., Qureshi, S.A., Reddy, M.K., Scarpelli, D.G. &
         Lalwani, N.D. (1984) Induction and origin of hepatocytes in rat
         pancreas.  J. Cell Biol., 98, 2082-2090.

    Smith, F.A., Hermann, E.A., Dryzga, M.D. & Ramsey, J.C (1982) The
         pharmacokinetics of 14C-DOWCO 453 in Fischer 344 rats. Study
         No. HET K 131381-(26). Unpublished report prepared by Dow
         Chemical, Midland, Michigan, USA. Submitted to WHO by Dow Elanco
         Europe, Wantage, Oxon, United Kingdom.

    Smith, F.A., Veenstra, G.E., Freshour, N.L. & Hermann, E.A. (1983)
         DOWCO 453 Ethoxy Ethyl Ester: Pharmacokinetics in Fischer 344
         rats following oral administration. Study No. HET DR-0210-
         0416-004. Unpublished report prepared by Dow Chemical, Midland,
         Michigan, USA. Submitted to WHO by Dow Elanco Europe, Wantage,
         Oxon, United Kingdom.

    Smith, F.A., Kropscott, B.E. & Kastl, P.E. (1984) The pharmacokinetics
         of DOWCO 453 in the B6C3F1 mouse. Study No. HET K 131381-036.
         Unpublished report prepared by Dow Chemical, Midland, Michigan,
         USA. Submitted to WHO by Dow Elanco Europe, Wantage, Oxon,
         United Kingdom.

    Stott, W.T., Yano, B.L., Barnard, D. Williams, D.M., Hannah, M.A.,
         Cieszlak, F.S., Albee, R. & Smith, K. (1985a) The proliferation
         of hepatocellular peroxisomes in rats and mice ingesting
         haloxyfop. Study No. HET K 131381-44/K 131381-46. Unpublished
         report prepared by Dow Chemical, Midland, Michigan, USA.
         Submitted to WHO by Dow Elanco Europe, Wantage, Oxon,
         United Kingdom.

    Stott, W.T., Yano, B.L., Hannah, M.A., Battjes, I.E. & Cieszlak, F.S.
         (1985b) The proliferation of hepatocellular peroxisomes in rats
         and mice ingesting haloxyfop: 2- and 4-Week recovery. Study
         No. HET K 131381-046B. Unpublished report prepared by Dow
         Chemical, Midland, Michigan, USA. Submitted to WHO by Dow Elanco
         Europe, Wantage, Oxon, United Kingdom.

    Swenberg, J.A. (1994) Expert report: on the toxicity mid
         carcinogenicity of haloxyfop. Unpublished report dated 27
         September 1994. Submitted to WHO by Dow Elanco Europe, Wantage,
         Oxon, United Kingdom.

    Tollett, J.T., Blogg, C.D., Wade, C.E., Morden, M.C, Schtz, D.J.,
         Dittenberger, D.A., Hermann, E.A & Gorzinski, S.J. (1981)
         DOWCO 453: Results of a 1-week palatability study and a 2-week
         probe study in feed in CDF Fischer 344 rats. Study No. HET
         K-131381-(2). Unpublished report prepared by Dow Chemical,
         Midland, Michigan, USA. Submitted to WHO by Dow Elanco Europe,
         Wantage, Oxon, United Kingdom.

    Tollett, J.T., Yano, B.L., Campbell, R.A. & Herman, J.K. (1983) DOWCO
         453 Acid: Results of a 6-month and 1-year interim sacrifice of a
         2-year dietary toxicity-oncogenicity study in CDF Fischer 344
         rats. Study No. HET K-1313181(18a). Unpublished report prepared
         by Dow Chemical, Midland, Michigan, USA. Submitted to WHO by Dow
         Elanco Europe, Wantage, Oxon, United Kingdom.

    Tollett, J.T., Yano, B.L., Cieszlak, F.S. & Battjes, J.E. (1985) DOWCO
         453: Two-year dietary chronic toxicity-oncogenicity study in
         B6C3F1 mice. Study No. HET K 131381(22). Unpublished report
         prepared by Dow Chemical, Midland, Michigan, USA. Submitted to
         WHO by Dow Elanco Europe, Wantage, Oxon, United Kingdom.

    Yano, B.L., Campbell, R.A. & Tollett, J.T. (1984) DOWCO 453: Two-year
         chronic toxicity-oncogenicity study in CDF Fischer 344 rats.
         Study No. HET K 131381(18B). Unpublished report prepared by Dow
         Chemical, Midland, Michigan, USA. Submitted to WHO by Dow Elanco
         Europe, Wantage, Oxon, United Kingdom.

    Yano, B.L., Marler, R.J., Sorrells, R.M., Hannah, M.A., Stott W.T.,
         Landenberger, B.D., Williams, D.M. Weiss, S.K. (1987) Haloxyfop:
         13-Week oral toxicity study in cynomolgus monkeys. Study
         No. 131381-058 (T-1242). Unpublished report prepared by Dow
         Chemical, Midland, Michigan, USA. Submitted to WHO by Dow Elanco
         Europe, Wantage, Oxon, United Kingdom.

    Yackovich, P.R. & Miller, J.H. (1983a) Fate of DOWCO 453 fed to laying
         hens. Study No. GH-C1635. Unpublished report prepared by Dow
         Chemical, Midland, Michigan, USA. Submitted to WHO by Dow Elanco
         Europe, Wantage, Oxon, United Kingdom.

    Yackovich, P.R. & Miller, J.H. (1983b) The fate of DOWCO 453 fed to
         lactating goats. Study No. GH-C1624. Unpublished report prepared
         by Dow Chemical, Midland, Michigan, USA. Submitted to WHO by Dow
         Elanco Europe, Wantage, Oxon, United Kingdom.

    Waechter, J.M., Campbell, R.A., Dryzga, M.D. & Ramsey, J.C. (1982)
         DOWCO 453 Methyl Ester: Pharmacokinetics in Fischer 344 rats
         following oral administration. Study No. HET K 132986-003.
         Unpublished report prepared by Dow Chemical, Midland, Michigan,
         USA. Submitted to WHO by Dow Elanco Europe, Wantage, Oxon,
         United Kingdom.

    Appendix: Expert report

         An additional evaluation of the long-term carcinogenicity was
    made, including a re-evaluation of the slides from liver tumours,
    further quantitative data, particularly with respect to historical
    controls, and the results of numerous statistical analyses (Swenberg,
    1994). The conclusions are summarized here.

         In addition to transient changes in liver weights, the
    non-neoplastic histopathological changes included altered tinctorial
    properties in hepatocytes at 0.6 mg/kg bw and an increased incidence
    of foci of cellular alteration in livers of male mice at 0.03 and
    0.6 mg/kg. bw. No increase was found at the intermediate dose of
    0.065 mg/kg bw. The incidence at 0.03 and 0.6 mg/kg bw was 24%, in
    comparison with 14% in the concurrent control group; the range of the
    incidence of hepatocellular foci in male historical controls is 0-20%
    (11 studies). Thus, the incidence seen in the carcinogenicity studies
    slightly exceeded the historical control range. Swenberg concluded
    that the incidence of liver foci is similar to that of historical
    controls from the laboratory and that it is not affected by exposure
    to haloxyfop. The absence of a dose-response relationship supports
    that conclusion.

         With respect to neoplastic findings, numerous statistical
    analyses were conducted, with particular attention to the increases in
    tumour incidence in comparison with concurrent and historical
    controls; possible increases in tumour multiplicity and decreases in
    tumour latency were also considered in the evaluation. Using both the
    original statistical methods and an improved method for tumours with a
    high background incidence, the incidence of hepatocellular adenomas
    and adenomas or carcinomas significantly increased in males at
    0.6 mg/kg bw, and a significant increase in the incidence of
    hepatocellular carcinomas was identified in females. A statistically
    significant linear trend was identified for the same end-points. When
    the trend test was conducted excluding the data for the high dose, no
    significant trend was seen for any type or combination of hepatic
    tumours.

         Statistical comparisons with historical control groups from 11
    studies were also conducted. Only the incidence of hepatocellular
    carcinomas in females at 0.6 mg/kg bw was statistically significantly
    increased when the original statistical methods were used, and there
    was a trend for this group. When the additional statistical methods
    were used, no significant increase was seen. Moreover, the incidence
    of liver rumours (adenomas and carcinomas combined) in the concurrent
    male controls was found to be unusually low, as 5/11 of the historical
    control groups had statistically higher incidences. A further
    statistical analysis included a comparison of the data on liver
    tumours in each dose group with each of the 11 historical control

    groups. This analysis showed a significant increase in the incidence
    of adenomas in males at the high dose in 2/11 comparisons and an
    increased incidence of adenomas or carcinomas in 3/11. The incidence
    of adenomas was significantly increased in females at the high dose in
    1/11 comparisons, that of carcinomas in 6/11, and that of adenomas or
    carcinomas in 2/11. Comparisons for males at the low and intermediate
    doses revealed both increases and decreases, with no consistent
    pattern. Females at the low dose ha d a significantly increased
    incidence of carcinomas in 1/11 comparisons. Comparisons for females
    at the intermediate dose showed a significantly increased incidence of
    adenomas in 1/11 comparisons and an increased incidence of adenomas or
    carcinomas in 2/11 comparisons.

         Swenberg concluded that the data are equivocal with regard to a
    carcinogenic effect in males at the highest dose, while there appears
    to be a carcinogenic effect for females at this dose. No carcinogenic
    response was found at the intermediate dose in this analysis. A small
    increase in tumour multiplicity was also seen in females at the high
    dose, with 1.6 tumours per animal, in comparison with 1.0 tumour per
    animal in the control group. The treatment had no effect on the
    lifespan of animals bearing hepatic tumours.
    


    See Also:
       Toxicological Abbreviations