CHLOROTHALONIL
EXPLANATION
Chlorothalonil was evaluated for acceptable intake by the Joint
Meeting in 1974 and further reviewed in 1977, 1979, 1981, 1983 and
1985 (Annex 1, FAO/WHO, 1975a, 1978a, 1980a, 1982a, 1984 and 1986a).
In 1974 a temporary ADI of 0.03 mg/kg bw was estimated. In 1981 the
Meeting reduced the temporary ADI to 0.005 mg/kg bw due to the absence
of adequate metabolic data and assuming total conversion of the parent
compound to a more toxic metabolite, the 4-hydroxy derivative. This
value was further reduced by the 1985 JMPR to 0.0005 mg/kg bw because
of concern for oncogenicity. The 1985 Meeting recommended that any
available data on the on-going carcinogenicity studies in rats and
mice be submitted for evaluation when available, and required further
metabolism data to identify the change in metabolic pattern with
increasing dose, as well as further characterization of the GSH
conjugation occurring in the G.I. tract and kidney. The oncogenicity
study in mice and the metabolism studies required by the 1985 JMPR,
plus several additional studies, including two 90-day studies in rats
(one with chlorothalonil and the other with the monoglutathione
conjugate of chlorothalonil), several mutagenicity studies and three
histopathological re-evaluations have been made available to the 1987
JMPR and have been summarized in this monograph addendum.
EVALUATION FOR ACCEPTABLE INTAKE
BIOLOGICAL DATA
Biochemical aspects
Metabolism
Rats
The absorption, tissue accumulation and excretion of radiolabel
were studied in male Sprague-Dawley rats given a single oral dose of
either 0, 5, 50 and 200 mg/kg bw 14C-chlorothalonil (96-98% pure) in
water, uniformly labelled in the benzene ring. Rats were fasted from
12 hours before dosing to termination and were probably without water
supply between dosing and termination. In 3 separate experiments, 5
animals/sex/group were sacrificed 2, 9 and 24 hours after dosing, in 8
different tissues, in carcasses, in blood, in contents of stomach,
small and large intestine and in cage washings at termination.
The total recovery of radioactivity was 86-104% of the
administered dose at all sampling times for all dose levels. In rats
of all three dose groups sacrificed 2 hours after dosing, 76 to 80% of
the administered radioactivity was found in the small intestinal
content. Less than 1% of the administered dose was found in blood,
liver, kidney, lung, heart, fat and muscle. Nine hours after dosing
87-90% of the administered dose was found in the large intestinal
content. After 24 hours from dosing 88-95% of the administered dose
was still in the large intestinal content. Faecal excretion, however,
was very minor in this study, possibly due to the animal fasting.
Recovery of radioactivity in the urine of rats killed 24 hours after
dosing was also minor (0.4 to 0.6%). Urine output was minimal during
this period due to the fact that the animals were probably without
water supply during the time between dosing and termination.
Concentration of chlorothalonil µg equivalents in blood and
tissues of rodents dosed with 50 and 200 mg/kg bw and killed at 24
hours was proportionately higher than expected when compared with rats
of the low dose group. The authors of the study interpreted these
results as the "saturation of an absorption and/or elimination
mechanism in male rats between doses of 50 and 200 mg/kg".
The HPLC analysis of methanol extracts of faeces showed at least
7 radioactive peaks. Two of these peaks had identical retention times
as chlorothalonil and 4-hydroxy-2,3,5-trichloroisophthalonitrole. A
higher proportion of chlorothalonil was apparently metabolized by the
5 mg/kg bw dose rats than by the animals of the two higher dose groups
(Lee et al., 1982).
In another study excrete (urine and faeces) and tissues from male
Sprague-Dawley rats administered single oral doses of 5 or
200 mg/kg bw 14C-chlorothalonil (96-98% pure, labelled in the
benzene ring) in water were analysed by HPLC to determine the extent
of metabolism and the identity of the compounds to which
chlorothalonil was metabolized. Rats were starved for 12 hours before
and 24 hours after dosing and were given drinking water ad libitum.
Urine samples and faeces were collected at 2, 9, 24, 36, 48, 60, 72,
84 and 96 hours after dosing. The total mean recovery of the
administered radioactivity in faeces and urine 96 hours after dosing
was 77% and 8.6% for the low dose animals, and 62% and 4.7% for the
high dose rats, respectively. Maximum elevation was between 24 and 36
hours for faeces in all groups, between 2 and 9 hours for urine of the
low dose group and between 9 and 24 hours for urine of the high dose
group. At 200 mg/kg bw, the urine radioactivity increased and then
decreased slower than at 5 mg/kg bw. Extraction of radioactivity with
methanol from faeces was dose- and time-dependent. It was higher at
the 200 mg/kg bw dose and at early times. HPLC analysis of faeces
extracts indicated that chlorothalonil more extensively metabolized
after the low level than after the high dose level. With the low dose
level, several peaks were present in the HPLC profile, 3 of which
corresponded to those seen in the HPLC profile obtained from faeces of
high dose animals. The majority of radioactivity at both dose levels
was unextracted and apparently bound to faecal components. In urine
only about 15% of the radioactivity was lost during purification. HPLC
analysis of purified urine showed at least 3 peaks at both dose
levels, 64% of the radioactivity being in the form of highly polar
metabolite(s). These three metabolites recovered in urine were 0.05,
0.3 and 3.5% (high dose urine) and 0.08, 0.6 and 4.5% (low dose urine)
of the administered dose, respectively (Marciniszyn et al., 1983).
The biliary excretion of radioactivity after a single oral dose
of 5 mg/kg bw 14C-chlorothalonil (95.5% radiochemically pure) was
studied in 8 male and 4 female Sprague-Dawley rats. Chlorothalonil
uniformly labelled in the benzene ring was administered as a
suspension in 0.75% methylcellulose with a mean particle size of 3.8
microns. Bile was collected by cannulation in 60 min fractions for 48
hours; blood and urine samples were collected at 6, 24 and 48 hours
and faeces at 24 and 48 hours after dosing. Total mean recovery of
radioactivity in this study was 83 to 97%. In 48 hours, male and
female rats excreted 21.1% and 16.7% of the administered dose in bile
and 7.6% and 11.7% in urine, respectively. Peak concentrations of
radioactivity in bile were reached within 2 hours after dosing for
both sexes. Levels of radioactivity in blood were higher at 6 than 24
or 48 hours after dosing for both sexes. Faeces contained 50 and 61%
of the administered radioactivity in male and female rats,
respectively.
It was concluded by the authors of the study that absorption of
chlorothalonil is particle size-dependant and that approximately 34%
of the administered dose was absorbed by both male and female animals
(Marciniszyn et al., 1985a).
The enzymatic and non-enzymatic conjugation of gluthathione with
14C-chlorothalonil (99% radiochemically pure) labelled in the
benzene ring was investigated in vitro. The isolation and
identification of the gluthathione conjugates as metabolites of
chlorothalonil in the bile of rats administered 14C-chlorothalonil
was also investigated. HPLC analysis showed that on incubation of
14C-chlorothalonil with reduced gluthathione (GSH) under aqueous
conditions both in the presence and absence of GSH S-transferase, 3 or
4 polar products were formed. These products were tentatively
identified as gluthathione conjugates of chlorothalonil, formed
apparently in the following stepwise manner, chlorothalonil ->
monoconjugate -> diconjugate -> triconjupte. Mono- and diconjugates
of chlorothalonil were also synthesized under organic conditions.
These were apparently identical, by TLC and HPLC analysis, to those
formed under aqueous conditions, both before and after peptide bond
cleavage.
Based on the limited preliminary data presented (polarity,
molecular weight and chromatography), the authors of the study
suggested that the major metabolite in the bile of rats dosed orally
with 5 mg/kg bw 14C-chlorothalonil was the di-gluthathione conjugate
of chlorothalonil (Savides et al., 1985a).
The urinary metabolites of chlorothalonil were studied in 4 male
Sprague-Dawely rats given a single oral dose of 200 mg/kg bw
14C-chlorothalonil (98.6% radiochemically pure) uniformly labelled
in the benzene ring. Chlorothalonil was given as a homogeneous
suspension in 0.75% methylcellulose in water, with a particle size of
3.2 microns. The pooled urine samples collected from 0 to 24 hours
after dosing contained 2.3% of the administered dose. Two metabolites
were identified by gas chromatography/mass spectrometry (GC/MS)
analysis: dithiodichloroisophthalonitrile and
trithiochioroisophthalonitrile, in both the free sulfhydryl and the
methylated form.
Extraction of urine with ethylacetate removed 15.4% of the
radioactivity present in urine. GC/MS analysis of these ethyl acetate
extracts identified trimethylthiomonochloroisophthalonitrile and
dimethylthiodichloroisophthalonitrile in a 1:1 ratio. Acidification of
urine and further extraction with ethylacetate removed an additional
54.4% of the urine radioactivity. When this acid extract was applied
onto a C18 Sep-Pak cartridge, 30% of the radiolabel was eluted with
methylene chloride and 70% with methanol. GC/MS analysis of the
methylene chloride eluate resulted in identification of the trimethyl
and dimethyl thiols. GC/MS analysis of the fraction eluted with
methanol before and after methylation with diazomethane indicated that
both methylated and non-methylated thiols were present in the urine.
It was concluded by the authors of the study that thiol metabolites of
chlorothalonil were formed from its conjugation with gluthathione
(Marciniszyn et al., 1985b).
The biliary excretion of radioactivity was studied in groups of 6
male Sprague-Dawley rats administered a single dose of 1.5, 5, 50 or
200 mg/kg bw 14C-chlorothalonil (98% radiochemically pure) uniformly
labelled in the aromatic ring as a suspension with a mean particle
size of 3.6 to 5.0 microns in 0.75% methylcellulose in water. Rat bile
duct was cannulated and bile was collected in 1 hour fractions for 48
hours after dosing. Blood, urine and faeces were also collected at
various times after dosing and at termination. During the 48 hours
after a single dose of 1.5, 5, 50 and 200 mg/kg bw, biliary excretion
was 22.5, 16.4, 16.3 and 7.7% of the administered dose, respectively.
Profiles of radioactivity excretion after the two low doses were
quantitatively different from those obtained after the two high doses.
The authors of the study interpreted these results as indicative of a
change in metabolism occurring between 5 and 50 mg/kg bw, possibly due
to saturation of biliary excretion.
Mean urinary excretion in the 48 hours after dosing was 8.0, 8.2
and 7.6% of the administered dose at 1.5, 5 and 50 mg/kg bw,
respectively, and only 4.7% at the high dose level of 200 mg/kg bw.
Excretion of radioactivity in urine within 6 hours after dosing was
inversely related to the dose administered. Total recovery of
radioactivity in this study was 89-99% in the 3 low dose groups and
74% after the 200 mg/kg bw dose. After doses of 1.5, 5, 50 and
200 mg/kg bw, rats absorbed 32, 25.7, 25.9 and 15.5% of the
administered dose, respectively. It was concluded by the authors of
the sponsor report that enterohepatic circulation or reabsorption of
biliary metabolites from the gastrointestinal tract did not contribute
significantly to the amount of radiolabel in the kidney. Based on a
one-compartment model for chlorothalonil absorption and excretion and
using several assumptions, it was calculated that the rate of
absorption of the 200 mg/kg bw dose was only twice as fast as that of
the 50 mg/kg bw dose (Marciniszyn et al., 1986a).
The absorption, tissue distribution and excretion of
radioactivity was investigated in male Sprague-Dawley rats
administered orally 5 successive daily doses of 14C-chlorothalonil
(98.4% radiochemically pure) labelled in the benzene ring and
suspended in 0.75% methylcellulose. Groups of 20 animals each received
1.5, 5, 50 or 160 mg/kg bw and were killed 2, 9, 24, 96, and 168 hours
after the 5th dose and at termination. Four additional groups of 2
rats each were treated similarly and used for blood radioactivity
determination at 2 and 24 hours after the 1st, 3rd and 5th dose
administration. Some contamination of urine samples by loose faeces
may have occurred during the first 24 hours after the first dose in
this study.
The major excretion of radiolabel was through the faeces in all
groups. During the 5-day dosing period and the 168 hours after the 5th
dose administration rats excreted 82-85% of the administered dose in
faeces and 5 to 6.7% in urine. At the 50 and 160 mg/kg bw dose levels
the percent of administered dose excreted in urine was at least 25%
less than that excreted at 1.5 or 5 mg/kg bw. Radioactivity in blood
was consistently higher at 6 than at 24 hours after each
administration. The blood concentrations 6 hours after the first dose
and 6 hours after the 5th dose were similar (71 and 90 ng/ml,
respectively). A plateau in blood radioactivity was reached apparently
after one dose administration and maintained through 5
administrations. The maximum concentration of radiolabel in kidneys
(< 0.2%) after the 5th dose administration was reached at 2 hours for
all dose levels. Seven days after the 5th dose administration kidneys
contained 14, 16, 23 and 25% of these maximum concentrations,
respectively. Maximum concentrations of radioactivity in kidneys after
50 and 160 mg/kg bw/day were not proportional to that found after the
2 lower dose levels. Liver contained 6 to 7 fold less radioactivity
than kidneys.
It was concluded by the authors of the sponsor report that rats
responded to high doses (50 and 160 mg/kg bw/day) of chlorothalonil in
a different manner than to low doses (< 5 mg/kg bw/day) (Marciniszyn
et al., 1986b).
The effects of the renal secretion inhibitor probenecid on
plasma, urine and kidney levels of chlorothalonil was studied in 3
groups of male Sprague-Dawely rats given a single oral dose of 4.7 or
52 mg/kg bw 14C-chlorothalonil (99% radiochemically pure) labelled
in the benzene ring and present as a suspension of 3.7 microns
particle size in 0.75% methylcellulose. In 3 different experiments
rats were injected i.p. with 139 (Expt. 1 and 2) or 244 (Expt. 3)
mg/kg bw probenecid in corn oil 1 hour before chlorothalonii
administration (52 mg/kg bw in Expt. 1 and 2, and 4.7 mg/kg bw in
Expt. 3 control rats were given 14C-chloronthalonil and corn oil but
no probenecid. Rats of Expt. 1 and 3 were terminated six hours and rats
of Expt. 2 two hours after chlorothalonil administration.
In probenecid-treated rats of Expt. 1 and 2, the mean plasma
level of radioactivity was 146 and 156% of that of controls, the mean
urinary excretion of radioactivity as a percent of the administered
dose was 50 and 40% of that of controls and radiolabel content in
kidneys (as µg equiv./g of kidney) was 70 and 58% of that of controls,
respectively. In Expt. 3, where a lower chlorothalonil/higher
probenecid dosage was used, the% of the administered dose of
chlorothalonil present in the kidneys 6 hours from termination was
even lower than was found in Expt. 1 and 2 (48% of that of controls
compared with 73 and 69% of that of controls in Experiment 1 and 2,
respectively). The authors of the study reported that GC/MS analysis
of these extracts after methylation revealed the presence of the
trimethylthiol derivative of chlorothalonil in urine of treated and
control animals (6.2 and 8.3% of the urinary radioactivity,
respectively). A small amount of the dimethylthiol derivative was also
present in control urine. It was concluded by the authors of the study
that probenecid pretreatment decreased the active secretion by the
kidney of the majority of the urinary metabolites of chlorothalonil
(Savides et al., 1985b).
The effect of multiple dosing with chlorothalonil on the
excretion of thiol metabolites in urine was studied in 4 groups of 20
Sprague-Dawely rats orally administered 1.5, 5, 50 or 60 mg/kg bw/day
14C-chlorothalonil for 5 consecutive days. Four rats from each dose
level were sacrificed at 2, 9, 24, 96 and 168 hours after the final
(5th) dose administration. Urine samples were collected at 24 hour
intervals after each dose administration, acidified and extracted with
ethylacetate. The extractability of radiolabel from acidified urine
decreased both with increasing dose level and with repetitive dosing,
ranging from 84% (on day 1 at the 1.5 mg/kg bw/day dose level) to 49%
(on day 5 at 160 mg/kg bw/day). GC/MS analysis of urine samples
identified dithiol (dithiodichloroisophthalonitrile) and trithiol
(trithiochloroisophthalonitrile) metabolites of chlorothalonil in all
samples analyzed. The amount of the dithiol excreted in the urine on
day 1 as a percent of the total urine radioactivity was 5.2, 9 and
15.4% at 5, 50 and 160 mg/kg bw/day. In contrast, the amount of the
trithiol excreted in the same urine was approximately the same at all
3 dose levels (15.7, 16 and 16.8% of total urine radioactivity,
respectively). The absolute amounts of both thiols excreted in 4
subsequent days of treatment at 50 and 160 mg/kg bw/day decreased
dramatically. The ratio between the absolute amounts of trithiol and
dithiol in urine at 50 and 160 mg/kg bw/day also increased with
multiple dosing. This ratio was also inversely related to the dose in
day 1 urine (3, 1.8 and 1.1 at 5, 50 and 160 mg/kg bw/day,
respectively). GC/MS analysis of the non-extractable phase of urine
from rats of the 160 mg/kg bw/day group gave spectra which were
tentatively identified as those of cysteinyltrichloroisophthalonitrile
and cysteinyltrichlorocyanobenzoic acid. The 4-and 5-hydroxy and the
4- and 5-dechlorinated analog of chlorothalonil were not found to be
present in any of the urine samples that were analyzed.
It was concluded by the authors of the study that the glutathione
pathway is specifically involved in chlorothalonil metabolism and that
a second pathway becomes increasingly involved in chlorothalonil
metabolism with successive dose administrations (Savides et al.,
1986a).
The in vitro metabolism of 14C-chlorothalonil by mucosal
cells from the stomach and/or small intestine of Sprague-Dawely rats
was investigated quantitatively using an HPLC/LSC technique. During
the incubation of chlorothalonil with stomach squamous or stomach
glandular cells and with intestinal cells, several products were
formed (2 in stomach incubations and 5 in intestinal incubations)
which were more polar than chlorothalonil. In all incubations two
compounds were found with elution times corresponding to those of the
di- and monogluthathione conjugates of chlorothalonil. It was
speculated that bacteria, which were probably present in the
incubations, may have contributed to chlorothalonil metabolism
(Savides et al., 1986c).
The distribution of radioactivity was investigated in kidney
organelles of CD Sprague-Dawely rats administered a single oral dose
of 50 mg/kg bw 14C-chlorothalonil (97% radiochemically pure)
labelled in the benzene ring. Chlorothalonil was given as a suspension
of 5 microns particle size in 0.75% methylcellulose in water. Six
hours after dosing rats were terminated and kidneys were removed,
homogenized and fractionated by ultracentrifugation for radiolabel
assay. The radiolabel was distributed in all fractions of kidney
homosenate: 0.2% in the nuclear fraction, 6.3% in cellular debris, 7%
in heavy mitochondrial fraction, 3.2% in light mitochondrial-lysosomal
fraction, 2% in microspinal fraction and 81.2% in soluble fraction.
The total organelle fraction contained 18.7% of the radioactivity
present in the homosenate (Savides et al., 1987).
An in vitro gut sac apparatus was used to study the absorption
of chlorothalonil through the intestinal wall of Sprague-Dawely rats.
Five hundred microliters of a 3.4 microns particle size suspension of
14C-chlorothalonil (97.1% radiochemically pure) in 0.75%
methylcellulose, corresponding to approximately 2.5 mg test material,
were instilled into each of 3 rat intestinal segments sealed at both
ends (gut sac) and incubated at 37°C for 6 hours in Krebbs Henseleit
buffer gassed with 95% O2/5% CO2.
At the end of the incubation the mean recovery of radioactivity
in mucosal compartment, serosal compartment and intestinal segment was
71, 5.8 and 1.3%, respectively. HPLC analysis of the buffer of the
serosal side of the gut sac showed that none of the transferred
material was chlorothalonil. It was suggested by the authors of the
study that following an oral dose of chlorothalonil to rats,
chlorothalonil is converted to polar metabolites in the
gastrointestinal tract and that these metabolites, not chlorothalonil,
are absorbed (Savides et al., 1986d).
The effect of the Gamma-Glutamyl Transpeptidase inhibitor AT-125
on the metabolism of 14C-chlorothalonil (97.1% radiochemically pure)
in male Sprague-Dawley rats is being invest in a pilot study still in
progress. A group of 3 rats were administered intraperitoneally
10 mg/kg bw AT-125 in saline, 1 hour before a single oral dose of
560 mg/&kg bw 14C-chlorothanil in 0.75% methylcellulose suspension.
Three control rats received saline and chlorothalonil. Data available
from an interim report indicated that AT-125 inhibited the in vivo
activity of Gamma-Glutamyl Transpeptidase for at least 12 hours, since
during this period percent extractability of radiolabel from urine of
rats pretreated with AT-125 was significantly (20%) lower than that
from urine of controls (> 70%). The major (42% of the total
radiolabel in urine) non-extractible metabolite excreted in urine of
AT-125-pretreated animals was tentatively identified by HPLC analysis
as the diglutathione conjugate of chlorothalonil. It was suggested by
the authors of the study that almost 60% of the radiolabel excreted in
the urine samples analysed were multiple glutathione conjugates of
chlorothalonil (Marciniszyn and Killeen, 1987a).
The covalent binding of radiolabel to kidney DNA was studied in 4
male Sprague-Dawley rats administered by gavage a single dose of
49 mg/kg bw 14C-chlorothalonil (99% radiochemically pure) as a
3.8 micron particle size suspension in 0.75% methylcellulose. Three
control groups of 4 rats each were given either a single oral dose of
methylcellulose solution or a single i.p. injection of 27 mg/kg bw
14C-dimethylnitrosamine (14C-DMNA) in saline (positive control) or
saline alone. Rats were terminated 6 hours after treatment and kidneys
were removed, homogenized and assayed for protein and DNA binding
after purification by chromatography or hydroxylapatite/ethanol
precipitation. Both compounds were found to bind covalently to
proteins (2259 ± 627 and 2112 ± 293 dpm/mg, of protein for
14C-chlorothalonil and 14C-DMNA, respectively). DNA fractions from
14C-DMNA-treated rats contained 2.9-15.5% of the radioactivity
recovered from chromatography (recovery was 78-93%). DNA from
14C-chlorothalonil-dosed rats contained 0.02 to 1.4% of the
recovered radioactivity. A mean covalent binding index of 245 ± 91 was
calculated for 14C-DMNA-administered animals. No radioactivity was
found bound to purified DNA of 14C-chlorothalonil treated rats
(Marciniszyn et al., 1987).
Special studies on metabolites
The concentration of radioacitivity in blood and kidneys was
studied in male Sprague-Dawley rats administered a single oral or
intraperitoneal dose of the mono-glutathione conjugate of
14C-chlorothalonil (91.3% radiochemically pure) uniformly labelled
in the benzene ring. The conjugate was administered as a 0.75%
methylcellulose suspension at a dosage of 115 mg/kg bw (equivalent to
57 mg/kg bw chlorothalonil).
Six hours after dosing the mean blood concentrations of
radiolabel were 10 times higher in i.p. dosed rats than in orally
administered animals. The mean percent of the administered dose found
in kidneys at this time was 0.02 and 3.2% in orally and i.p.
administered rats, respectively. Extractability of radioactivity in
urine with acidified ethylacetate was 55 and 86% in orally and i.p.
dosed rats, respectively. The urine of orally dosed rats were shown by
GC/MS analysis of the methylated extracts to contain 9% of the
radioactivity as the trithiol derivative of chlorothalonil and 5.1% as
the dithiol derivative. In contrast, the urine of i.p. dosed rats
contained less than 1% of the radiolabel as the dimethylthiol but not
trimethylthiol derivative. When the results were compared with those
of a previous study where 54 mg/kg bw chlorothalonil was administered
orally to rats, the percentages of the administered dose found in
kidney, blood and urine were similar in the two studies.
It was concluded by the authors of the study that the addition of
a glutathione molecule to the chlorothalonil ring reduced the acute
toxicity of chlorothalonil when administered intraperitoneally. It was
also suggested that orally administered mono-glutathione conjugate of
chlorothalonil is further conjugated with glutathione in the
gastrointestinal tract prior to absorption (Savides et al., 1986b).
Toxicological studies
Special studies on carcinogenicity
Mice
Groups of 60 male Charles River CD-1 mice were administered diets
containing 0, 10, 40, 175, and 750 ppm technical chlorothalonil (98.0%
pure) for up to two years. At week 16 the 10 ppm dosage was changed to
15 ppm to assure a minimum dose of 1.5 mg/kg bw/day throughout the
study. Ten mice/group underwent haematological examination at 12, 18
and 24 months. All mice terminated on schedule, in extremis or found
dead were necropsied. The absolute and relative weights of brain and
kidney of the animals terminated at interim and terminal sacrifices
were measured. The kidneys, stomach and related lymph nodes from all
animals were examined microscopically.
No compound-related effects were noted on the distribution of
overt signs of toxicity, mortality, body weight of food consumption
throughout the study. At 12 months, but not at 18 and 24 months,
animals at 750 ppm had a statistically significant decrease in
haemoglobin and hematocrit values, which were not considered to be
treatment-related. No treatment-related changes or masses were found
by macroscopic examination of mice sacrificed or dying during the
study. At interim sacrifice a statistically significant increase of
relative kidney to body weight was noted in mice at 750 ppm when
compared to controls. At terminal sacrifice absolute kidney weight and
relative kidney to body or kidney to brain weights of mice at 750 ppm
were significantly higher than in controls.
Histopathological examination of the kidneys, stomach, renal and
gastric lymph nodes performed by a different laboratory on all the
animals used in those study showed treatment-related non-neoplastic
changes in both organs. The reported renal changes included an
increase in the incidence and severity of epithelial hyperplasia in
the proximal convoluted tubules and in the incidence of karyomegaly
(increased nuclear size) at 175 and 750 ppm and, possibly, an increase
in the incidence of tubular hypertrophy at 750 ppm. The treatment
related changes seen in the stomach included an increased incidence of
hyperkeratosis and squamous epithelial hyperplasia of the forestomach
at 40, 175 and 750 ppm chlorothalonil. Treatment-related neoplasms
were not seen in the kidneys at any dose level tested. Only two mice,
one at 40 ppm and the other at 175 ppm dietary level, had tubular
adenoma. The incidence of all tumors (papilloma and squamous cell
carcinoma) in the forestomach was 2/60, 0/60, 0/60, 1/60 and 4/60 at
0, 15, 40, 175 and 750 ppm, respectively. No treatment-related
microscopic alterations were seen in either renal or gastric lymph
nodes.
Based on the non-neoplastic gastric lesions, the NOEL of
chlorothalonil for Charles River CD-1 mice in this study was 15 ppm,
equal to 1.57 mg/kg bw/day. Based on the neoplastic changes, the NOEL
in this study was considered by the authors of the study to be at
least 175 ppm, equal to 21.3 mg/kg bw/day (Wilson and Killeen, 1987a).
A histopathological re-evaluation of the kidneys was conducted by
Dr. Busey (Wilson et al., 1986) for the mouse oncogenicity study by
Wilson et. al. (1983). The re-evaluation confirmed the treatment-
related higher incidence of tubular adenomas and carcinomas observed
in males, but not in female CD-1 mice, during the original evaluation.
The incidences of these tumors found in male mice by the two authors are
compared in Table 1.
This re-evaluation showed also compound-related, not clearly
dose-related non neoplastic lesions of the kidney in both male and
female mice at all three dose levels tested. These changes were not
found during the original evaluation and included epithelial
hyperplasia and tubular hypertrophy. The incidence of epithelial
hyperplasia was 0/60, 31/60, 36/60 and 30/60 in males and 0/60, 8/60,
4/60 and 17/60 in females at 0, 750, 1500 and 3000 ppm, respectively.
This change was present in 13/15 mice with tubular tumor. In the
remaining two animals with tumor, autolysis of the renal tissue
prevented the comparison. It was stated by the pathologist (personal
communication from Dr. W.M. Busey, Experimental Pathology
Laboratories, Inc., Herndon, VA, USA, 1986, submitted to WHO by
Fermenta Plant Protection Co., Painesville, OH, USA) that this
epithelial hyperplasia was qualitatively similar to the hyperplasia
seen in kidneys of a previous subchronic mouse study. The authors of
the Sponsor report considered this change preneoplastic in nature.
Detection of this change by the pathologist, however, was reported to
be highly dependent upon the prior knowledge of its potential presence
(Wilson et al., 1986).
Table 1. Comparison of the incidences of renal tubular adenomas and
carcinomas found in male mice by Brown (Wilson et al., 1983)
and Busey (Wilson et al., 1986)
Diagnosis Dose level (ppm)
0* 750* 1500* 3000*
Brown:
Tubular adenoma 0 4 4 2
Tubular carcinoma 0 2 0 2
Total animals with these tumors: 0 6+ 4 4
Busey:
Tubular adenoma 0 3 3 4
Tubular carcinoma 0 3 1 1
Total animals with these tumors: 0 6+ 4 5
* The kidneys from 60 animals/group/sex were examined.
+ p < 0.05, when compared to controls.
Rats
(ongoing study)
Groups of 65 male and female Fischer 344 rats are being given
diets containing 0, 2, 4, 15 and 175 mg/kg bw/day chlorothalonil.
Potential effects of chlorothalonil administration on body weight and
histopathology of selected tissues from all animals (kidneys, stomach,
renal and gastric lymph nodes) are being or will be evaluated.
Histopathological evaluation of rats which died or were killed
in extremis during the first year of the study or underwent the
one-year interim sacrifice on December 11 and 12, 1986, is ongoing.
All surviving animals will be terminated after 30 months of
chlorothalonil administration.
By week 56 of this study the mean body weights of the high dose
(175 mg/kg bw/ day) male and female rats were lower than those of the
animals of the relative control groups. In addition, during this
period dark yellow urine was noted in 50 male and 38 female rats of
the high dose group, but not in controls or in low/intermediate dose
animals.
A final report of this study is scheduled to be completed in
December 1988 (Wilson & Killeen, 1987b).
A histopathological re-evaluation by Busey (Wilson et al.,
1986b) of the kidneys of the oncogenicity study in Fischer 344 rats by
McGee & Brown (1985) confirmed the original finding of treatment
related tubular adenomas and carcinomas in both male and female rats.
When the single diagnosis of tubular adenomas and carcinomas made in
the two evaluations are compared, an overall 65% (45/69) agreement was
observed. A comparison of the incidences of renal tumors found in male
and female rats by the two pathologists is reported in Table 2.
Treatment-related, non-neoplastic lesions of the renal tubules
which were not observed in the original evaluation were found during
this re-evaluation in both male and female rats. These changes were
epithelial hyperplasia and tubular hypertrophy in both sexes and
cortical cyst(s) in males. The incidence of epithelial hyperplasia was
0/60, 32/60, 30/60 and 36/60 in males and 5/60, 35/60, 39/60 and 48/60
in females at 0, 40, 80 and 175 mg/kg bw/day, respectively. The
occurrence of tubular hyperplasia and that of tubular tumors appeared
to be highly correlated. In only one animal of the high dose group,
out of 69 rats with tubular adenoma or carcinoma, no tubular
hyperplasia was found. In three other cases autolysis of the renal
tissue prevented the comparison. It was concluded by the pathologist
(personal communication from Dr. W.M. Busey, Experimental Pathology
Laboratories, Inc., Herndon, VA, USA, 1986, submitted to WHO by
Fermenta Plant Protection Co., Painesville, OH, USA) that the
epithelial hyperplasia observed in this study was qualitatively
similar to that seen in recent subchronic and old chronic studies with
chlorothalonil in rats. This tubular hyperplasia was considered by the
authors of the Sponsor report to be preneoplastic in nature. They
reported, however, that detection of this change by the pathologist
was highly dependent upon prior knowledge of its potential presence
(Wilson et al., 1986).
Table 2. Comparison of the incidences of renal tubular adenomas and carcinomas found
in male and female rats by Brown (Mcgee & Brown, 1985) & Busey (Wilson et al.
1986b).
Dose level (mg/kg bw/day)
0 40 80 175
Diagnosis M* F* M F M F M F
Brown:
Tubular adenoma 0 0 2 2 4 4 11 9
Tubular carcinoma 0 0 5 1 2 2 7 11
Anaplastic renal carcinoma 0 0 0 0 1 0 0 3
Total animals with these tumors: 0 0 7+ 3 7+ 6+ 18++ 23++
Busey:
Tubular adenoma 0 0 3 3 5 10 7 15
Tubular carcinoma 0 0 4 1 2 0 13 11
Total animals with these tumors: 0 0 7+ 4 7+ 10+ 18++ 23++
* 60 animals/group/sex were examined.
+ p < 0.05, when compared to controls.
++ p < 0.01, when compared to controls.
Special studies in mutagenicity
The ability of chlorothalonil (98.8% pure) to induce chromosomal
aberrations in Chinese hamster ovary cells was tested in the presence
and absence of metabolic activation by liver S9 mix from
Aroclor-pretreated rats. A longer incubation time was used in the
assay without metabolic activation due to cell cycle delay.
Triethylenemelanine and cyclophosphamide were used as the positive
control in the assay without and with activation, respectively. A
statistically significant treatment-related, apparently dose-dependant
increase in the number of cells with structural aberrations was found
in the absence but not in the presence of metabolic activation when
compared with the solvent control. The authors of the Sponsor report
suggested that the positive result without metabolic activation may be
of no biological significance to the intact mammalian organism since
chlorothalonil was negative in in vivo chromosomal aberration assays
and metabolism studies in rats suggest that only metabolites are
absorbed through the gastrointestinal tract (Mizens et al., 1986c).
The mutagenic potential of a number of compounds related to
chlorothalonil has been evaluated in the Ames test with and without
metabolic activation with S9 from kidney of male Fischer rats. These
compounds were 2,5-dichloro-4,6-bismercaptoisophtaonitrile,
S-(2,4-dicyano-3,5,6-trichlorophenyl) glutathione, 5-chloro-2,4,6-
trimercap- toisophtalonitrile, S,S'-(2,4-dicyano-3.6-dichlorophenyl)
dicysteine and S,S',S"-(2,4-dicyano-6-chlorophenyl)-tricysteine (purity
ranging 90.5-97.5%). Four other compounds were used as positive controls.
The Salmonella typhimurium tester strains TA98, TA100, TA1535, TA1537
and TA1538 were used. In all these studies, there was no significant
increase (doubling) over solvent control values in the number of
revertants for any of the five tester strains used either with or
without metabolic activation (Mizens et al., 1985a, 1985b, 1986a,
1986b and 1987).
Short-term studies
Rats
Two groups of 15 male Fischer 344 rats were administered either
single daily doses of 75 mg/kg bw chlorothalonil technical (97.9%
pure) or equimolar doses (150 mg/kg bw) of its monoglutathione
conjugate (92.5% pure) in aqueous 0.5% methylcellulose suspensions by
gavage for 93 days. A control group of 15 animals was administered the
vehicle by gavage for 93 days. The homogeneity and stability of the
two test compounds in the vehicle were controlled throughout the
study.
One rat dosed with chlorothalonil was considered moribund and was
sacrificed on the 73rd day of the study. Dark yellow urine was
reported irregularly in all but one animal administered
chlorothalonil. Statistically significant lower (5 to 9%) mean body
weights were noted from week 4 for rats treated with chlorothalonil
when compared to controls. Absolute food consumption was lower for
chlorothalonil-treated animals during the first week of the study due
to gastric irritation. Significantly lower mean SGPT values were
observed in both treatment groups when compared to controls after 7 or
13 weeks of treatment. A statistically significant increase in kidney
weight was noted in both treated groups when compared to the control
group at termination.
The histopathological examination of the kidneys showed
treatment-related changes in rats of both treated groups. These
changes included vacuolar degeneration (which was absent in control
animals), proliferative interstitial fibrosis, tubular ectasis and
tubular casts. Important foci of tubular necrosis have been found in
all treated and non-treated animals. The origin of this change was
unknown. Both macroscopic and microscopic alterations were observed in
the forestomach of rats treated with chlorothalonil but not in those
administered the monoglutathione conjugate or in control animals.
These changes were hyperplasia of the squamous epithelium,
hyperkeratosis and gastritis, often associated with ulcerations and
erosions.
In this study the urine of 10 rats/treated group were analysed on
days 1 and 4 at the end of weeks 2, 4, 8 and 12 for the presence of
the dithiol and trithiol metabolites of chlorothalonil. GC/MS analysis
of the urine extracts provided unequivocal identification of these
products. The chlorothalonil-treated rats excreted the trithiol
metabolite on days 1 and 4 and at weeks 2 and 4, and the
monoglutathione conjugate-treated animals excreted the trithiol on
days 1 and 4 and at week 4. The dithiol metabolite was only detected
in chlorothalonil-treated animals on day 1. The total amount of
trithiol metabolite excreted by chlorothalonil-treated animals was
approximately five times greater than the total amount of trithiol
found in the urine of monoglutathione conjugate-treated rats.
It was concluded by the authors of the study that the presence of
the trithiol metabolite in urine of animals of both groups was
consistent with the involvement of glutathione conjugation in the
metabolism of both chlorothalonil and the monoglutathione conjugate of
chlorothalonil (Ford & Killeen, 1987a).
Groups of 90 male Fischer 344 rats were administered a diet
containing 0 or 175 mg/kg bw/day chlorothalonil (97.9% pure) for up to
91 days. Body weight, food and compound consumption were measured, and
clinical observations were made weekly throughout the study. Ten
animals/group were sacrificed at day 4 and 7 and at the end of week 2,
4, 6, 8, 10, 12 and after 91 days of treatment and brain and kidney
weights were recorded. Urine samples were collected prior to sacrifice
for determination of thiol metabolites of chlorothalonil. For each
animal the right and the left kidney were evaluated microscopically by
two independent laboratories using two different fixation and staining
procedures.
All animals were sacrificed on schedule and no treatment-related
clinical changes were observed throughout the study. A
treatment-related statistically significant decrease (3-9%) in mean
body weight values was observed throughout the study in treated
animals when compared to controls. Mean food consumption, both
absolute (g/animal/day) and relative (g/kg/day) to body weight, was
significantly lower in chlorothalonil-treated rats than in controls on
days 4 and 7. A statistically significant treatment-related increase
in kidney weight, both absolute (5 to 24%) and relative to body (9 to
33%) or brain (6 to 28%) weight, was observed in chlorothalonil-
treated rats when compared to controls. Macroscopic examination of the
stomach showed treatment-related changes of the non-glandular mucosa of
the forestomach (multifocal erosions from day 7 to week 8 and thickening
from week 4 to 13). No other macroscopic changes were observed which
could be attributed to chlorothalonil administration.
Histopathological examination showed treatment-related changes in
both the fore-stomach and kidneys. The stomach alterations observed
remained substantially identical throughout the study and included
slight to moderately severe squamous epithelial hyperplasia and
hyperkeratosis in all treated animals. Ulcers were also found in all
treated rats at day 4 and erosions of the forestomach in several
treated animals from day 7 of study. A change in pattern of the
chlorothalonil-dependent microscopic renal lesions with increasing
time of chlorothalonil treatment was noted by each of the two
different laboratories involved in the study. According to one report
(Report 1), during the first week of treatment (days 4 and 7) all
treated rats, but no control animal, had tubular epithelial
degeneration and vacuolation with nuclear pyknosis, loss of brush
border, karyomegaly. Moderate epithelial regeneration was also present
at day 7. The second laboratory report (Report 2) reported an
important vacuolar degeneration (but no epithelial regeneration) at
day 4 and 7 in treated but not in untreated rats. At the end of week 2
for both reports the kidney morphology of treated rats was not
markedly different from that of controls. The alterations present in
treated but not in control rats included minimal to moderate tubular
hypertrophy in some rats (Report 1) or vacuolar degeneration, tubular
extasis and foci of basophilic tubules in a few cases (Report 2).
After 4 or more weeks of treatment, treated rats had epithelial
hyperplasia and tubular hypertrophy and, after 91 days, also clear
cell hyperplasia according to Report 1. The treatment-related changes
after 4 weeks reported in Report 2 were-tubular ectasis, foci of
basophilic tubules and proliferative interstitial fibrosis. Analysis
of 24 hour urine samples from treated animals were shown to contain
small amounts (less than 0.1% of the daily administered dose) of the
trithiol metabolite of chlorothalonil.
It was concluded by the authors of the study that the
administration of 175 mg/kg bw/day chlorothalonil in the diet to
Fischer 344 rats induced degenerative changes in the kidneys,
ultimately resulting in epithelial hyperplasia and tubular hypertrophy
(Ford and Killeen, 1987b).
COMMENTS
Several metabolism studies on the oncogenicity study in mice
required by the 1985 JMPR and some additional data have been submitted
and evaluated.
14C-chlorothalonil administered orally as a suspension is
incompletely (15-35%) and rapidly absorbed by the g.i. tract.
Absorption probably occurs mainly through the small intestine and it
is proportionately higher after a small dose than after a large dose.
Chlorothalonil is rapidly distributed to the kidney, where it is
covalently bound to proteins but not to DNA.
Approximately 8-12% of an oral dose is excreted in the urine,
17-21% in the bile and 50-61% remains unabsorbed in the faeces.
Chlorothalonil metabolites are actively secreted by the kidney.
Identification by GC-MS of the urinary metabolites indicates that
chlorothalonil is conjugated with glutathione. The 4-hydroxy
derivative of chlorothalonil is not a metabolite in non-ruminants. The
data available suggest saturable absorption and excretion and a change
in metabolism at high doses (between 5 and 50 mg/kg body weight) or
after successive doses.
In a 90-day study, where the toxicity of chlorothalonil and that
of its monoglutathione conjugate were compared, both compounds induced
microscopic changes of the renal tubules. In another short-term study
the chlorothalonil-dependent renal lesions showed a change in pattern
with time.
In a recent tumorigenicity study in mice chlorothalonil,
administered via the diet, was not oncogenic to the kidneys but
induced a few papillomas and squamous cell carcinomas in the
forestomach which, however, were not considered to be indicative of an
oncogenic potential for man.
Treatment-related non-neoplastic lesions were seen in the kidneys
and in the forestomach. Based on these non-neoplastic gastric lesions
the NOAEL in this study was 15 ppm, equal to 1.6 mg/kg bw/day. The
absence in this study of treatment-related renal tumors in mice given
750 ppm chlorothalonil in the diet is at variance with the results of
a previous mouse oncogenicity study.
Chlorothalonil induced chromosomal aberrations in Chinese hamster
ovary cells in vitro, in the absence but not in the presence of
metabolic activation by rat kidney S9 mix. However, in a previous
chromosomal aberration study in vivo and in several other studies
in vitro and in vivo the compound was not mutagenic.
The meeting was informed that a tumorigenicity feeding study in
rats is being currently conducted and the final report is expected by
December 1988. In consideration of this ongoing study, which is being
conducted at lower doses than previous rat or mouse studies, and of
the new metabolism data, the meeting agreed that a temporary ADI based
on toxicity data for chlorothalonil, not the 4-hydroxy derivative,
should be extended. Moreover, because of concern for the demonstrated
oncogenicity in rodents, a high safety factor was used.
TOXICOLOGICAL EVALUATION
LEVEL CAUSING NO TOXICOLOGICAL EFFECT
Mouse: 15 ppm in the diet, equal to 1.6 mg/kg bw/day
Rat: 60 ppm in the diet, equivalent to 3 mg/kg bw/day
Dog: 60 ppm in the diet, equivalent to 1.5 mg/kg bw/day
ESTIMATE OF TEMPORARY ACCEPTABLE DAILY INTAKE FOR MAN
0-0.003 mg/kg bw.
STUDIES WITHOUT WHICH THE DETERMINATION OF A FULL ADI IS IMPRACTICABLE
To be submitted to WHO by 1989
1. The ongoing oncogenicity study in rats.
2. Further studies on the mechanism of the organ-specific
toxicity of chlorothalonil to the kidney in order to confirm
the metabolic pathway responsible for nephrotoxicity.
STUDIES WHICH WILL PROVIDE INFORMATION VALUABLE IN THE CONTINUED
EVALUATION OF THE COMPOUND.
Observations in man.
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Histopathologic re-evaluation of renal tissue from a mouse
tumorigenicity study with chlorothalonil (5TX.79-0102). Unpublished
report No. 764-5TX-85-0072-002 from SDS Biotech Corp., Painesville,
OH, USA. Study performed by Busey W.M., Experimental Pathology
Laboratories, Inc., Herndon, VA, USA. Submitted to WHO by Fermenta
Plant Protection Co., Painesville, OH, USA.
See Also:
Toxicological Abbreviations
Chlorothalonil (EHC 183, 1996)
Chlorothalonil (HSG 98, 1995)
Chlorothalonil (ICSC)
Chlorothalonil (WHO Pesticide Residues Series 4)
Chlorothalonil (Pesticide residues in food: 1977 evaluations)
Chlorothalonil (Pesticide residues in food: 1981 evaluations)
Chlorothalonil (Pesticide residues in food: 1983 evaluations)
Chlorothalonil (Pesticide residues in food: 1985 evaluations Part II Toxicology)
Chlorothalonil (Pesticide residues in food: 1990 evaluations Toxicology)
Chlorothalonil (Pesticide residues in food: 1992 evaluations Part II Toxicology)
Chlorothalonil (IARC Summary & Evaluation, Volume 30, 1983)
Chlorothalonil (IARC Summary & Evaluation, Volume 73, 1999)