898. Haloxyfop (Pesticide residues in food: 1995 evaluations Part II Toxicological & Environmental)

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 material^a 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 EE^b Rat Male, female Oral 520 95 Carreon et al. (1982)
Haloxyfop EE Rat Male Oral 700 94.7 Jones (1983a)
Female 900
Haloxyfop EE^c 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 EE^d Rat Male, female Intraperitoneal 670 96.5 Jones el al. (1986)
Haloxyfop ME^e 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 ß-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 ß-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).

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 F_1a and F_1b litters. The F₁ rats were
maintained on the test diet for 137 days and then mated to produce the
F_2a, F_2b, and F_2c litters. The F₂ generation was fed the diet
for 64 days before being mated to produce the F_3a and F_3b
generations. The F_2c 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 F_1b litters were
reduced, and the body weights of F₁ adults, particularly females
(chosen from F_1b litters), were slightly lower throughout the
pre-breeding period; reduced mean body weights were also observed tor
F_2a pups throughout lactation and for F_2b 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 F_2b and F_2c 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 F_2b 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 F_2c 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 F_2a 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 F_1b
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 F_2b
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 F_1a and F_1b litters and F₁ males
and females to produce the F_2a and F_2b 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 F_1a and F_1b neonates;
the differences in the F_1b litters were statistically significant,
whereas a significant reduction was observed in the F_1b litters only
at the highest dose. The reduction in pup weight may be explained by
the slightly larger F_1a litters at 0.065 mg/kg bw. The weights of
F_2a 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/plate^a 99 Negative Domoradzki (1981)
TA100, TA1535, TA1537,
TA1538

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

Chromosomal aberration Human lymphocytes 100-1000 mg/ml^a 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/ml^c 99.4 Negative^e Bhaskar Gollapudi &
166.7 - 1667 mg/ml^d 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 ß
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 ß-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 ß-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 ß-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 material^a Species Sex Route Result Purity (%) Reference

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

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

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 Irritation^c 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 Bühler 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 ß-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 ß-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 ß-oxidase, hepatic carnitine acetyltrans-
ferase, and catalase were also reversed; however, the activity of
fatty acid ß-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 ß-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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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, Schütz, 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