CHLOROTHALONIL
EXPLANATION
First draft prepared by Dr. E. Arnold,
Health and Welfare Canada, Ottawa, Canada
Chlorothalonil has been reviewed by seven previous Joint Meetings
from 1974 to 1987 (Annex 1, 1975ab, 1978ab, 1980ab, 1982ab, 1984,
1985a, 1986ac, 1987b, and 1988b). Data considered on chlorothalonil
itself include metabolism and biotransformation studies in mice, rats
and dogs; pharmacological studies in mice and rats; acute toxicity in
several species; short-term toxicity studies in mice, rats and dogs;
in vivo and in vitro genotoxicity studies; teratology studies in
rats and rabbits; and a number of long-term toxicity/oncogenicity
studies in mice and rats. Data on the metabolite 4-hydroxy-2,5,6-
trichloroisophthalonitrile have also been considered including
genotoxicity, reproduction and carcinogenicity studies. In 1987, a
temporary ADI of 0-0.003 mg/kg bw based on toxicity data for
chlorothalonil, not the 4-hydroxy derivative, was extended. However,
because of concern for the demonstrated oncogenicity in rats and
pending completion of an ongoing oncogenicity study in rats, a high
safety factor was used. The carcinogenicity study in rats together
with further metabolism data have now been submitted.
EVALUATION FOR ACCEPTABLE DAILY INTAKE
BIOLOGICAL DATA
Biochemical aspects
Metabolism
Oral administration
Rats
A single oral dose of 14C-chlorothalonil at 50 mg/kg bw in
aqueous (0.75% methyl cellulose) suspension was given to nine
germ-free Sprague-Dawley male rats in a volume of 10 ml/kg bw. A
tenth rat was given the vehicle alone as a control. Urine and faeces
were collected at 24 hour intervals to 96 hours. At study termination
at 96 hours, blood was collected and the kidneys and the rest of the
carcass were saved. All samples were frozen pending analysis.
Urinary excretion of radioactivity accounted for 2.36-4.02% (mean
3.12%) of the administered dose while faecal excretion accounted for
61.3-95.6% (mean 84.4%). Blood levels accounted for 0.0098-0.0229%
(mean 0.0143%) of the dose in 8 of the 9 rats and 0.0517% in the
remaining rat. The kidney contained 0.037-0.066% of the dose in all
rats. Carcass radioactivity accounted for 0.177-0.285% of the dose in
the 8 rats with similar blood levels and 1.78% in outlier rat.
Urinary excretion of test material occurred mainly in the first 24
hours after dosing except in one rat which showed almost equal
excretion at 0-24 and 24-48 hours. Faecal excretion also was
predominantly in the first 24 hours except in the one rat in which
peak excretion was observed in the period 24-48 hours.
Eight 0-24 hour and four 24-48 hour urine samples were analyzed
for methylated thiols. No methylated thiols were detected in the 0-24
hour urine of 6/8 rats or in 24-48 hour urine from 2/4 rats. No
monothiol derivative was detected in any urine sample. The dithiol
derivative was detected only in one 24-48 hour sample. The trithiol
derivative was detected in two samples of 0-24 hour urine and two of
24-48 hour urine representing urine from three rats. The rat which
excreted trithiols in both periods was also the rat which excreted the
dithiol derivative. The highest concentration of thiols detected
accounted for 0.053% of the administered dose (Magee et al., 1990).
Dogs
Two male beagle dogs were given 14C-chlorothalonil by gelatin
capsule at an average dose of 49.9 mg/kg bw. Urine was collected for
24 hour periods for 10 days and faeces for 24 hour periods for 12
days. Recovery of test material during the study was virtually
complete (mean 100.1% of the administered dose). Most of the material
was excreted in the faeces (mean 99.6% of the administered dose) with
only small amounts in urine and cage washings (0.2% and 0.3%,
respectively). However, white particles were observed in the 0-24
hour faeces which may have been unabsorbed material. Urine samples
were analyzed for mono-, di-, and trithiols but none were detected at
3 ng, the limit of sensitivity of the method (Savides et al.,
1989a).
In order to confirm the results of the above study, a second
study in dogs was carried out. Three male beagle dogs were given
14C-chlorothalonil suspended in 0.75% aqueous methylcellulose by
gavage at a nominal dose level of 50 mg/kg bw (estimate of actual dose
level based on the recovery data: 28-63 mg/kg bw). The dog which
received the highest amount (63 mg/kg) regurgitated about 16% of the
dose one hour and fifteen minutes after dosing. Two of these dogs
were the same ones dosed in the above study which had received control
diet for approximately 5 months between the studies. For collection
of urine the dogs were catheterized for 4, 10 and 24 hours,
respectively. Subsequent urine samples were collected from the time
of catheter removal to 24 hours and then at 24 hour intervals until 8
days after dosing. Faeces were collected every 24 hours.
Faecal radioactivity in the first 24 hours after dosing accounted
for 53.9-96.4% of the nominal administered dose or 76.2-98.1% of the
estimated actual dose. A further amount of 0.53-8.6% of the nominal
dose (0.96-6.8% of estimated actual dose) was recovered in faeces on
days 2-3. Urinary excretion accounted for 0.66-1.17% of the nominal
dose (0.73-1.86% of the estimated actual dose) and was predominantly
recovered in the first 24 hours. Results from catheterization
indicated that most of the excretion occurred during the first 10
hours after dosing.
Aliquots of urine from two of the dogs were analyzed to generate
chromatographic profiles. For both dogs the profiles of 4 to 10 hour
samples were sufficiently similar to permit pooling. The 12 and 14
hour samples from one of the dogs were pooled and the sample collected
from 10-24 hours in the other dog was also analyzed. No methylated
thiols were detected in any of the samples. Less than half of the
radiolabel in urine was extractable in acidified ethyl acetate
suggesting that the metabolites were more polar than those observed in
the rat (Savides et al., 1990a).
Monkeys
Four male Chinese rhesus monkeys (Macaca mulatta) were given
uniformly benzene ring labelled 14C-chlorothalonil, suspended in 0.75%
aqueous methylcellulose, orally by gavage at a dose of 50 mg/kg bw.
An indwelling catheter was placed in the saphenous vein of the lower
leg to permit periodic blood sample collection. Urine was collected
using an external catheter device for 48 hours after dosing and then
the catheters were removed and the monkeys were housed in metabolism
cages for 96 hours after dosing and urine and faeces were collected at
72 and 96 hours.
Blood concentration vs. time plots indicated considerable
variability between animals. Peak blood levels were seen 3-18 hours
after dosing. In two of the monkeys blood levels rose rapidly; in one
of these the blood level declined rapidly while in the other blood
levels declined rather slowly. The other two animals took longer to
attain peak blood levels and the blood levels declined at an
intermediate rate. In these latter two animals higher peak blood
levels were reached than in the two which reached the peak rapidly.
Blood elimination half life was 6.9, 7.6, 19.8 and 35.0 hours in the
four animals. Area under the blood concentration curve was
52,000-158,000 ng-eq hr/ml for the 0-30 sampling interval which was
compared to 94,000 ng-eq hr/ml following the same dose in the rat.
Over 96 hours a total of 1.75-4.13% of the administered dose was
excreted in urine and 52.3-91.6% in faeces. In 3/4 monkeys, urine
excreted in the period 24-48 hours contained the highest amount of
test material. The other animal excreted most of the radioactivity
12-48 hours after dosing. The same 3/4 monkeys had peak faecal
excretion also in the 48 hour sample. The other monkey had high
excretion in the first 12 hours and virtually complete faecal
excretion by 48 hours after dosing.
Extraction of urine with acidified ethyl acetate recovered 32-
65% (mean 49%) of the radiolabel in the urine. Under similar
conditions, about 75% of the radiolabel was extractable in rat urine.
This suggests that in monkeys the radiolabel is excreted as more polar
metabolites than in the rat. Monothiols were not detected in any of
the urine samples. Dithiol was detected in urine of only one of the
monkeys while trithiols were detected in all urine samples. The total
amount of thiols excreted by monkeys was 0.001 to 0.01% of the
administered dose. This was compared to the excretion in the rat in
which 1.63% of the administered dose was excreted in the form of di-
and trithiols (Savides et al., 1990b).
Dermal administration
Rats
Four groups of 5 male CD Sprague-Dawley (Charles River) rats were
treated dermally with 14C-chlorothalonil in acetone at a dose of about
5 mg/kg bw. Urinary excretion in the first 48 hours after dosing
accounted for about 3% of the administered dose (range 2.52-4.0%) with
approximately equal amounts excreted after 0-24 and 24-48 hours.
There was considerable variability between the four groups with
respect to thiol excretion in urine. Total thiols represented 0.07,
0.01, 0.007 and 0.001% of the administered dose in the four groups.
Monothiol was detected in the 0-24 hour sample of one group only.
Dithiols were detected in urine from two of the four groups. Trithiol
was detected in all urine samples. These data were compared to the
results of oral studies. Urinary excretion was greater following oral
than following dermal treatment (8% ± 3%) and there was at least a 20
fold difference between the amount of the administered dose excreted
as thiols (Savides et al., 1989b).
Special in vitro study on metabolism
Liver and kidney mitochondrial preparations were prepared from
male CD Sprague-Dawley (Charles River) rats. The preparations were
incubated with one of the following sulfur analogs of chlorothalonil:
the monothiol analog, the dithiol analog, the monoglutathione analog
and the diglutathione analog. With the dithiol analog no increase in
oxygen consumption was observed following the addition of adenosine
diphosphate (ADP) to either liver or kidney mitochondrial preparations
indicating complete inhibition of state 3 respiration. The monothiol
analog also affected oxygen uptake by liver mitochondria but had no
effect on kidney mitochondria. Neither of the glutathione analogs had
an effect on oxygen uptake (Savides et al., 1988).
Toxicological studies
Short-term studies
Dogs
Renal pathology data from a two year dog study, evaluated by the
JMPR in 1974, were re-examined. In this study 8 males and 8 females
were given diets containing 0, 60 or 120 ppm of chlorothalonil. Four
dogs/sex/group were sacrificed at one year and the remaining four
dogs/sex/group at two years. Renal tubule vacuolation was observed in
all of the females except one high dose dog at two years. In males
the lesion was observed in 0/4, 0/4 and 3/4 dogs at one year and 2/4,
0/4 and 1/4 dogs at two years at 0, 60 and 120 ppm, respectively. A
second pathologist examining these tissues concluded that the observed
lesion was probably an artifact of fixation. The NOAEL of 120 ppm,
originally established in 1974, was confirmed by this re-examination
of the data.
Long-term/Oncogenicity studies
Rats
Groups of 55 weanling Charles River Fischer 344 rats/sex/dose
level were given diets containing chlorothalonil at dose levels to
provide nominal intakes of 2, 4, 15 or 175 mg/kg bw/day for 99-125
weeks. Additionally, groups of 10 rats/sex/dose were given the same
diets for one year to serve as interim sacrifice animals. The target
lower dose levels for the study were 1.5 and 3 mg/kg bw/day but the
possibility of reduced bioavailability in the diet due to binding
dictated the use of the higher (2.0 and 4.0 mg/day) levels. The diet
was presented twice a week and was frozen prior to use to minimize
binding and increase bioavailability. Achieved concentrations provided
dose levels of at least 1.5, 3.3, 14.7 and 173 mg/kg bw/day.
At the 175 mg/kg bw/day dose level discolored urine was observed
during the first year of the study, which occurred at a lower
incidence during the second year. Labored breathing was observed in
a few animals in each group, which was increased at the end of the
study in the high dose group. Survival was reduced in males at 175
mg/kg bw/day and this group was sacrificed at 99 weeks. The other
groups of males were sacrificed at 111 weeks at which time survival in
the 15 mg/kg bw/day group was lower than in controls. All groups of
females were sacrificed at 125 weeks. The females at 175 mg/kg bw/day
had lower survival than controls at termination.
Body weights were lower than the controls in both sexes at 175
mg/kg bw/day and in males at 15 mg/kg bw/day. A slight but consistent
depression in body weight was also seen at the nominal 4 mg/kg bw/day
level but the difference was not considered to be biologically
significant. Food consumption was reduced in both sexes at 175 mg/kg
bw/day during week 1 and increased on a "g/kg bw" basis for the
remainder of the study. There were no treatment-related effects on
haematological parameters or ophthalmology. At 175 mg/kg bw/day both
sexes showed a number of changes in clinical chemistry: increased
phosphorus and cholesterol levels and reduced alkaline phosphatase and
alanine amino transferase levels. Males at 175 and 15 mg/kg bw/day
had increased BUN levels. Males at 175 mg/kg bw/day showed increased
urinary volume and reduced specific gravity at 18 and 23 months.
Absolute kidney weights were increased in males and females at 175
mg/kg bw/day at 12 months and to a lesser extent in females given
nominal levels of 4 or 15 mg/kg bw/day. At terminal sacrifice only
the females at 175 mg/kg bw/day were affected.
Chronic progressive nephropathy was observed in all groups but
was more severe at 175 mg/kg bw/day and to a lesser extent at 15 mg/kg
bw/day than in the low dose groups. Focal epithelial hyperplasia in
kidney was observed in all groups but was significantly increased in
incidence at 175 mg/kg bw/day and slightly increased in incidence in
females at 4 and 15 mg/kg bw/day. Epithelial hyperplasia and
hyperkeratosis of the non-glandular stomach were seen at doses of 4
mg/kg bw/day and higher and were dose-related in incidence and
severity. In the kidney, tubular carcinomas were observed in both
sexes at 175 mg/kg/ bw/day (7/55 males and 11/55 females) but in no
other group. Tubular adenomas were seen in 1/55, 1/54, 1/54, 3/54 and
17/55 males at 0, 2, 4, 15 and 175 mg/kg bw/day, respectively, and in
24/55 females at 175 mg/kg bw/day. In the stomach, non-glandular
papillomas were seen in 0, 0, 3, 2 and 5 males and 1, 1, 2, 4 and 7
females at 0, 2, 4, 15 and 175 mg/kg bw/day, respectively. No
squamous cell carcinomas were observed in males but one was seen in
each the control and 15 mg/kg bw/day groups and 3 in the 175 mg/kg
bw/day females. The NOEL for non-neoplastic effects in this study was
1.5 mg/kg bw/day. There was a possible slight increase in incidence of
kidney and stomach neoplasms at 15 mg/kg bw/day but no tumorigenicity
was observed at the nominal 2 and 4 mg/kg bw/day levels (actual 1.5
and 3.3 mg/kg bw/day) (Wilson and Killeen, 1989).
COMMENTS
A study in germ-free rats was conducted to test the hypothesis
that at least part of the metabolism of chlorothalonil in the rat is
carried out by the microflora in the gastrointestinal tract. In germ-
free rats, urinary excretion of radioactivity accounted for about 3%
of the administered dose, about half the amount observed in urine of
non-germ-free rats dosed at the same level. Thiols in urine accounted
for about 0.05% of the administered dose in germ-free rats compared to
1.6% of the administered dose in non-germ-free rats. These data
support the theory that the microflora of the gastrointestinal tract
are involved in the metabolism of chlorothalonil in the rat.
Studies in dogs and monkeys also indicated lower urinary
excretion of radioactivity than with non-germ-free rats: about 1% of
the administered dose in the dog and 1.8-4.1% of the administered dose
in the monkey. No thiols were detected in dog urine and only small
amounts in monkey urine (0.001-0.01% of the administered dose).
An in vitro study with rat liver and kidney mitochondria
demonstrated a toxic effect on mitochondria from both tissues with the
dithiol metabolites of chlorothalonil, but no effects with glutathione
conjugates.
In an oncogenicity study in rats, kidney and stomach tumours were
observed at 175 mg/kg bw/day. There were slight increases in kidney
tubule adenomas and nonglandular stomach papillomas at 15 mg/kg bw/day
but there was no evidence of tumorigenicity at lower dose levels.
Focal epithelial hyperplasia in kidney and epithelial hyperplasia and
hyperkeratosis of the nonglandular stomach were observed in a dose-
related incidence. The NOAEL for non-neoplastic lesions in the kidney
and stomach was 1.5 mg/kg bw/day. The NOAEL for neoplastic lesions
was 3.3 mg/kg bw/day.
The metabolism of chlorothalonil in the rat differs from that in
the monkey and dog. This difference is related to a considerable
degree to the gut flora in the rat. This fact suggests that the dog
or the monkey may be more suitable models than the rat for predicting
the metabolism of chlorothalonil by man.
The existing data on reproduction were considered to be
unsatisfactory. However, a new rat reproduction study is presently
underway.
A two-year study in dogs was evaluated in 1974. No treatment-
related effects on the kidney were observed at 24 months in four males
and four females given 120 ppm chlorothalonil in the diet. Since the
present rat oncogenicity study demonstrates a NOAEL and the metabolism
data assist in elucidating the mechanism, concern regarding
carcinogenicity has been alleviated. Since the dog is now considered
to be a more relevant model than the rat, the dog study has been
selected as the most suitable basis for estimating an ADI. Therefore,
a safety factor 100 was applied to the NOAEL in the study (3.0 mg/kg
bw/day) to estimate of the ADI.
TOXICOLOGICAL EVALUATION
Level causing no toxicological effect
Mouse: 15 ppm in the diet, equal to 1.6 mg/kg bw/day
Rat: 1.5 mg/kg bw/day
Dog: 120 ppm in the diet, equivalent to 3.0 mg/kg bw/day
Estimate of acceptable daily intake for humans
0-0.03 mg/kg bw
Studies which will provide information valuable to
the continued evaluation of the compound
- Reproduction study in rats known to be in progress
- Observations in humans
REFERENCES
Magee, T.A., Savides, M.C., Marciniszyn, J.P., and Killeen, J.C., Jr.,
(1990). Study to evaluate the metabolic pathway of chlorothalonil
(14C-ASC-2787) in germ-free rats. Unpublished report no.
3060-88-0219-AM-001 from Ricerca Inc., Painesville, Ohio, USA.
Submitted to WHO by Fermenta ASC, Mentor, Ohio, USA.
Savides, M.C., Marciniszyn, J.P., and Killeen, J.C., Jr., (1988). A
study to evaluate the effects of sulfur-containing analogs of
chlorothalonil on mitochondrial function. Unpublished report no.
1479-87-0037-AM-001 from Ricerca, Inc., Painesville, Ohio, USA.
Submitted to WHO by Fermenta ASC, Mentor, Ohio, USA.
Savides, M.C., Marciniszyn, J.P.,and Killeen, J.C.,Jr., (1989a). Study
to compare the metabolism of chlorothalonil in dogs with its
metabolism in rats following oral administration of
14C-chlorothalonil. Unpublished report no. 1626-88-0008-AM-001 from
Ricerca, Inc., Painesville, Ohio, USA. Submitted to WHO by Fermenta
ASC, Mentor, Ohio, USA.
Savides, M.C., Marciniszyn, J.P., and Killeen, J.C., Jr., (1989b).
Study to determine the metabolic pathway for chlorothalonil following
dermal application to rats. Unpublished report no. 1625-87-0057-AM-001
from Ricerca Inc., Painesville, Ohio, USA. Submitted to WHO by
Fermenta ASC, Mentor, Ohio, USA.
Savides, M.C., Marciniszyn, J.P., and Killeen, J.C., Jr., (1990a).
Study of the urinary excretion of radiolabel by catheterized dogs
following oral administration of 14C-chlorothalonil by gavage.
Unpublished report no. 3086-89-0041-AM-001 from Ricerca, Inc.,
Painesville, Ohio, USA. Submitted to WHO by Fermenta ASC, Mentor,
Ohio, USA.
Savides, M.C., Marciniszyn, J.P., and Killeen, J.C., Jr. (1990b).
Study to evaluate the urinary metabolites of chlorothalonil from male
rhesus monkeys. Unpublished report no. 3349-89-0179-AM-001 from
Ricerca, Inc., Painesville, Ohio, USA. Submitted to WHO by Fermenta
ASC, Mentor, Ohio, USA.
Wilson, N.H., and Killeen, J.C., Jr. (1989). A tumorigenicity study of
technical chlorothalonil in rats. Unpublished report no.
1102-84-0103-TX-007 from Ricerca, Inc., Painesville, Ohio, USA.
Submitted to WHO by Fermenta ASC, Mentor, Ohio, USA.